Rapid and selective detection of fatty acylated proteins using omega-alkynyl-fatty acids and click chemistry.
Progress in understanding the biology of protein fatty acylation has been impeded by the lack of rapid direct detection and identification methods. We first report that a synthetic omega-alkynyl-palmitate analog can be readily and specifically incorporated into GAPDH or mitochondrial 3-hydroxyl-3-methylglutaryl-CoA synthase in vitro and reacted with an azido-biotin probe or the fluorogenic probe 3-azido-7-hydroxycoumarin using click chemistry for rapid detection by Western blotting or flat bed fluorescence scanning. The acylated cysteine residues were confirmed by MS. Second, omega-alkynyl-palmitate is preferentially incorporated into transiently expressed H- or N-Ras proteins (but not nonpalmitoylated K-Ras), compared with omega-alkynyl-myristate or omega-alkynyl-stearate, via an alkali sensitive thioester bond. Third, omega-alkynyl-myristate is specifically incorporated into endogenous co- and posttranslationally myristoylated proteins. The competitive inhibitors 2-bromopalmitate and 2-hydroxymyristate prevented incorporation of omega-alkynyl-palmitate and omega-alkynyl-myristate into palmitoylated and myristoylated proteins, respectively. Labeling cells with omega-alkynyl-palmitate does not affect membrane association of N-Ras. Furthermore, the palmitoylation of endogenous proteins including H- and N-Ras could be easily detected using omega-alkynyl-palmitate as label in cultured HeLa, Jurkat, and COS-7 cells, and, promisingly, in mice. The omega-alkynyl-myristate and -palmitate analogs used with click chemistry and azido-probes will be invaluable to study protein acylation in vitro, in cells, and in vivo.


Expression of a prenylation-deficient Rab4 interferes with propagation of insulin signaling through insulin receptor substrate-1.
Rab proteins are small GTP-binding proteins of the Ras superfamily that function in the regulation of vesicle transport processes. The Rab4 isoform has been implicated in insulin action. For instance, overexpression of a prenylation-deficient form of Rab4 has been shown to inhibit insulin-dependent GLUT4 translocation. Other steps affected by Rab4 in the cascade of events resulting from insulin receptor activation have not been elucidated. In the present studies, we measured effects on insulin-signaling proteins in 3T3-L1 adipocytes transiently expressing cytoplasmic forms of Rab4 and Rab5. Expression of a mutant Rab4 lacking a prenylation site resulted in reduced insulin-dependent phosphorylation ofcytoplasmic and internal membrane-associated insulin receptor substrate-1, leading to decreased insulin receptor substrate-1-associated phosphatidylinositol 3'-OH kinase activation and decreased Akt activation. These effects were not observed upon introduction of a similar mutant form of Rab5. These data indicate that Rab4 or a Rab4-associated protein is involved at one or more steps in propagating the insulin signal, in addition to any role it may play in the regulation of GLUT4 vesicle translocation. Our results support models of insulin signaling in which regulation of internal membrane trafficking plays a role in transduction of the insulin signal.


Preventing apoptotic cell death by a novel small heat shock protein.
NCBI database analysis indicated that the human C1orf41 protein (small heat shock-like protein-Hsp16.2) has sequence similarity with small heat shock proteins (sHsps). Since sHsps have chaperone function, and so prevent aggregation of denatured proteins, we determined whether Hsp16.2 could prevent the heat-induced aggregation of denatured proteins. Under our experimental conditions, recombinant Hsp16.2 prevented aggregation of aldolase and glyceraldehyde-3-phosphate dehydrogenase, and protected Escherichia coli cells from heat stress indicating its chaperone function. Hsp16.2 also formed oligomeric complexes in aqueous solution. Hsp16.2 was found to be expressed at different levels in cell lines and tissues, and was mainly localized to the nucleus and the cytosol, but to a smaller extent, it could be also found in mitochondria. Hsp16.2 could be modified covalently by poly(ADP ribosylation) and acetylation. Hsp16.2 over-expression prevented etoposide-induced cell death as well as the release of mitochondrial cytochrome c and caspase activation. These data suggest that Hsp16.2 can prevent the destabilization of mitochondrial membrane systems and could represent a suitable target for modulating cell death pathways.


Regulation of the p53 pathway by ubiquitin and related proteins.
The p53 tumour suppressor protein is subject to many levels of control, including modification with ubiquitin and related proteins such as SUMO and NEDD8. These modifications regulate p53 at a number of levels, including control of protein turnover, alterations in sub-cellular localization and changes in the ability to regulate gene expression. Numerous E3 ligases that can mediate these modifications of p53 have been described, some of which promote conjugation with more than one ubiquitin-like protein. Understanding the complexity of this mechanism of p53 regulation will help in the development of therapeutic drugs that function to modulate these events.


Kaposi's sarcoma-associated herpesvirus-encoded protein kinase and its interaction with K-bZIP.
The oncogenic herpesvirus, Kaposi's sarcoma-associated herpesvirus, also identified as human herpesvirus 8, contains genes producing proteins that control transcription and influence cell signaling. Open reading frame 36 (ORF36) of this virus encodes a serine/threonine protein kinase, which is designated the viral protein kinase (vPK). Our recent efforts to elucidate the role of vPK in the viral life cycle have focused on identifying viral protein substrates and determining the effects of vPK-mediated phosphorylation on specific steps in viral replication. The vPK gene was transcribed into 4.2-kb and 3.6-kb mRNAs during the early and late phases of viral reactivation. vPK is colocalized with viral DNA replication/transcription compartments as marked by a polymerase processivity factor, and K-bZIP, a protein known to bind the viral DNA replication origin (Ori-Lyt) and to regulate viral transcription. The vPK physically associated with and strongly phosphorylated K-bZIP at threonine 111, a site also recognized by the cyclin-dependent kinase Cdk2. Both K-bZIP and vPK were corecruited to viral promoters targeted by K-bZIP as well as to the Ori-Lyt region. Phosphorylation of K-bZIP by vPK had a negative impact on K-bZIP transcription repression activity. The extent of posttranslational modification of K-bZIP by sumoylation, a process that influences its repression function, was decreased by vPK phosphorylation at threonine 111. Our data thus identify a new role of vPK as a modulator of viral transcription.


Biosynthesis of GlyCAM-1, a mucin-like ligand for L-selectin.
L-selectin, a member of the selectin family of leukocyte-endothelial adhesion proteins, mediates the initial attachment of lymphocytes to lymph node high endothelial venules during lymphocyte recirculation. One of the endothelial-associated ligands for L-selectin is GlyCAM-1, a mucin-like glycoprotein, which presents novel sulfated, sialylated and fucosylated O-glycans. In order to understand the generation of these glycans, we have examined the biosynthesis of GlyCAM-1 in lymph node organ culture. Using peptide-specific antibodies, lectins, and recombinant L-selectin, we detected the following species of GlyCAM-1: unglycosylated (< 28 kDa); modified with GalNAc only (28-33 kDa); modified with sialic acid, fucose, and sulfate but lacking L-selectin reactivity (40-50 kDa); and mature (L-selectin-reactive) ligand (50-60 kDa). Pulse-chase labeling at 15 degrees C suggested that GalNAc is added in a pre-Golgi compartment. Treatment with brefeldin A almost completely blocked sulfation, indicating that this modification occurs in the trans-Golgi network. Two distinct sialylation events occurred in the presence of brefeldin A, while fucosylation was partially blocked. We conclude that sialylation precedes both fucosylation and sulfation during biosynthesis. This ordering will help to identify the critical acceptor structures recognized by lymph node glycosyltransferases and sulfotransferases.


Inhibition of protein palmitoylation, raft localization, and T cell signaling by 2-bromopalmitate and polyunsaturated fatty acids.
The ability of the Src family kinases Fyn and Lck to participate in signaling through the T cell receptor is critically dependent on their dual fatty acylation with myristate and palmitate. Here we identify a palmitate analog, 2-bromopalmitate, that effectively blocks Fyn fatty acylation in general and palmitoylation in particular. Treatment of COS-1 cells with 2-bromopalmitate blocked myristoylation and palmitoylation of Fyn and inhibited membrane binding and localization of Fyn to detergent-resistant membranes (DRMs). In Jurkat T cells, 2-bromopalmitate blocked localization of the endogenous palmitoylated proteins Fyn, Lck, and LAT to DRMs. This resulted in impaired signaling through the T cell receptor as evidenced by reductions in tyrosine phosphorylation, calcium release, and activation of mitogen-activated protein kinase. We also examined the ability of long chain polyunsaturated fatty acids (PUFAs) to inhibit protein fatty acylation. PUFAs have been reported to inhibit T cell signaling by excluding Src family kinases from DRMs. Here we show that the PUFAs arachidonic acid and eicosapentaenoic acid inhibit Fyn palmitoylation and consequently block Fyn localization to DRMs. We propose that inhibition of protein palmitoylation represents a novel mechanism by which PUFAs exert their immunosuppressive effects.


Effect of the statin atorvastatin on intracellular signalling by the prostacyclin receptor in vitro and in vivo.
Prostacyclin plays a central role within the vasculature. We have previously established that the prostacyclin receptor (IP) undergoes isoprenylation, a lipid modification obligate for its function. The aim of the current study was to investigate the effect of the hydroxy methyl glutaryl co-enzyme A reductase inhibitor atorvastatin on signalling and function of the IP expressed in mammalian whole cells and in platelets isolated from patients undergoing therapeutic intervention with atorvastatin. Initially, the effect of atorvastatin on signalling by the human (h) and mouse (m) IP overexpressed in human embryonic kidney 293 cells and the hIP endogenously expressed in human erythroleukaemic 92.1.7 cells was investigated. Atorvastatin significantly reduced IP-mediated cAMP generation (IC(50) 6.6-11.1 microm) and [Ca(2+)](i) mobilization (IC(50) 7.2-16.4 microm) in a concentration-dependent manner, but had no effect on signalling by the nonisoprenylated beta(2) adrenergic receptor or the alpha or beta isoforms of the human thromboxane A(2) receptor (TP). Moreover, atorvastatin significantly reduced IP-mediated crossdesensitization of signalling by TP alpha (IC(50) 10.4 microm), but not by TP beta. In contrast to the whole-cell data, atorvastatin therapy did not interfere with IP-mediated cAMP generation or IP-induced inhibition of TP-mediated aggregation of platelets isolated from human volunteers undergoing therapeutic intervention with atorvastatin (10-80 mg per daily dose). In conclusion, while data generated in whole cells indicated that atorvastatin significantly impairs signalling by both the hIP and mP, the in vivo clinical data indicated that, at the administered therapeutic dose, atorvastatin does not significantly compromise IP signalling and function in humans.


Roles for negative cell regulator 14-3-3sigma in control of MDM2 activities.
The 14-3-3sigma, upregulated by p53 in response to DNA damage, can have a positive-feedback impact driving p53 activities and is a human cancer epithelial marker downregulated in various tumors. However, the precise roles of 14-3-3sigma during tumorigenesis are not well characterized. Here, we show that 14-3-3sigma is a critical regulator of murine double minute oncogene (MDM2). 14-3-3sigma interacts with MDM2 at the RING domain. The C-terminal region of 14-3-3sigma binds to MDM2 very efficiently. Importantly, 14-3-3sigma overexpression leads to destabilization of MDM2 through enhancing MDM2 self-ubiquitination and accelerating turnover rate. Conversely, loss of 14-3-3sigma results in a significant increase in MDM2 protein. Moreover, live-cell images indicated that 14-3-3sigma can affect the location of MDM2 from the nucleus to the cytoplasm, and that MDM2-mediated cytoplasmic localization of p53 can be reversed by the presence of 14-3-3sigma. Significantly, we further showed that 14-3-3sigma causes MDM2 downregulation, thereby stabilizing p53 and inhibiting tumor growth in animal tumors. Also, 14-3-3sigma blocks MDM2-mediated retinoblastoma degradation and p53 NEDDylation. Our results provide evidence that 14-3-3sigma is a pivotal MDM2 regulator involved in blocking a variety of activities of MDM2.


Activation of H-ras61L-specific signaling pathways does not require posttranslational processing of H-ras.
We have previously demonstrated that H-ras61L retained transforming activity when lacking C-terminal lipid modifications, provided that plasma membrane localization was restored by an N-terminal transmembrane domain. Since several ras-activated pathways contribute to the transformed phenotype, we utilized a novel set of transmembrane domain-anchored H-ras derivatives to examine if lipids are required for activation of any specific signaling pathways. We demonstrate here that H-ras61L-induced activation of the Raf/MEK/MAPK pathway, including recruitment of Raf to the plasma membrane and activation of Raf and MAPK, does not require C-terminal processing of H-ras61L. Biochemical fractionation experiments confirm the localization of TM-ras derivatives to the plasma membrane, as well as the ras-mediated recruitment of c-Raf-1. Changes in the actin cytoskeleton, controlled by H-ras61L-mediated activation of the Rac/ Rho pathway, as well as PI 3-kinase activation, can also occur in the absence of C-terminal lipid modifications. Finally, downstream events, such as the induction of the immediate-early gene c-fos or neurite outgrowth in PC12 cells, are stimulated by the expression of plasma membrane-anchored, nonlipidated H-ras6lL. These results demonstrate that H-ras can be functionally targeted to the plasma membrane using a transmembrane domain sequence and that several signal transduction pathways downstream of H-ras can be activated without the presence of normal lipid modifications.


The dynamic role of GRP78/BiP in the coordination of mRNA translation with protein processing.
The role of GRP78/BiP in coordinating endoplasmic reticular (ER) protein processing with mRNA translation was examined in GH3 pituitary cells. ADP-ribosylation of GRP78 and eukaryotic initiation factor (eIF)-2alpha phosphorylation were assessed, respectively, as indices of chaperone inactivation and the inhibition of translational initiation. Inhibition of protein processing by ER stress (ionomycin and dithiothreitol) resulted in GRP78 deribosylation and eIF-2 phosphorylation. Suppression of translation relative to ER protein processing (cycloheximide) produced approximately 50% ADP-ribosylation of GRP78 within 90 min without eIF-2 phosphorylation. ADP-ribosylation was reversed in 90 min by cycloheximide removal in a manner accelerated by ER stressors. Cycloheximide sharply reduced eIF-2 phosphorylation in response to ER stressors for about 30 min; sensitivity returned as GRP78 became increasingly ADP-ribosylated. Reduced sensitivity of eIF-2 to phosphorylation appeared to derive from the accumulation of free, unmodified chaperone as proteins completed processing without replacements. Prolonged (24 h) incubations with cycloheximide resulted in the selective loss of the ADP-ribosylated form of GRP78 and increased sensitivity of eIF-2 phosphorylation in response to ER stressors. Brefeldin A decreased ADP-ribosylation of GRP78 in parallel with increased eIF-2 phosphorylation. The cytoplasmic stressor, arsenite, which inhibits translational initiation through eIF-2 phosphorylation without affecting the ER, also produced ADP-ribosylation of GRP78.


Interference of active site specific reagents in plasminogen-streptokinase active site formation.
We have recently observed slow, non-Michaelis-Menten kinetics of activation of native cat plasminogen by catalytic concentrations of streptokinase. In order to understand the reasons for this phenomenon, we undertook to study the formation of the plasminogen-streptokinase activator complex under the same plasminogen activation conditions. The results obtained in this study show that the potential active site in both cat and human plasminogen is capable of binding strongly the specific substrates (S) p-nitrophenyl p-guanidinobenzoate (NPGB) and H-D-valyl-L-leucyl-L-lysyl-p-nitroanilide, through the active site is incapable of hydrolyzing these substrates. Binding studies support these and the following conclusions. Streptokinase binds to this zymogen-substrate complex to create the ternary plasminogen-S-streptokinase complex, which then slowly converts to an acylated plasminogen-streptokinase form. This acylation reaction is 550 times slower than acylation of the preformed plasminogen-streptokinase complex by NPGB. The same reaction also occurs with human plasminogen, though the acylation reaction is 10 times faster than when the cat zymogen is used. NPGB binds specifically to plasminogen but not to streptokinase. These studies proved that inhibition of cat plasminogen activation by streptokinase occurs at the level of activator complex formation. We conclude from our studies that streptokinase binding to both cat and human plasminogen occurs at the potential active site of the zymogen. Consequently, it is probable that plasminogen activation in vivo is inhibited by binding of active site specific inhibitors to plasminogen.


Age-related changes in human bone proteoglycan structure. Impact of osteogenesis imperfecta.
Proteoglycans (PGs) are a family of molecules that undergo extensive post-translational modifications that include addition of glycosaminoglycan (GAG) chains as well as N- and O-linked oligosaccharides to the protein core. PG composition and structure have been reported to alter with age. To test whether the post-translational modifications to PGs can serve as in vitro surrogate end point markers for chronological age, the extent of GAG modifications was determined for PGs derived from normal human bone cells of 14 donors (age range, fetal to 60 years). Isolated cells were steady state radiolabeled with (35)SO(4)(2-) and [(3)H]GlcN. For biglycan and decorin, iduronate content was linearly correlated with age (increased 1.5x between fetal and age 60 years). For the syndecan-like heparan sulfate PG, the N-sulfation of post-natal cells increased over 3.5-fold until reaching a plateau during the 4th decade of life. The amount of O-linked oligosaccharides was also found to decrease as a function of increasing normal donor age, whereas the specific activity of the metabolic precursor pool remained constant regardless of donor age. These age-related changes in post-translational modifications were then used to demonstrate that osteoblasts derived from patients with osteogenesis imperfecta did not exhibit facets of a pre-mature aging, but rather were arrested in a fetal-like phenotypic state. A growth matrix rich in thrombospondin altered PG metabolism in osteoblastic cells, resulting in the production and secretion of the fetal-like (rich in O-linked oligosaccharides) forms of decorin and biglycan. This effect was qualitatively different from the effect of transforming growth factor-beta, which predominantly altered GAGs rather than O-linked oligosaccharides. No other Arg-Gly-Asp protein (fibronectin, vitronectin, type I collagen, osteopontin, and bone sialoprotein) showed any detectable effect on PG metabolism in bone cells. These results indicate that a proper matrix stoichiometry is critical for metabolism of PGs.


cDNA sequence, in vitro synthesis, and intramitochondrial lipoylation of H-protein of the glycine cleavage system.
H-protein is a component of the glycine cleavage system loosely associated with the mitochondrial inner membrane and has lipoic acid as a prosthetic group. cDNA clones encoding H-protein were isolated from a bovine liver cDNA library with an oligonucleotide probe from the amino acid sequence of the NH2-terminal region. DNA sequence analysis and deduced amino acid sequence showed that the cDNAs encoded an H-protein precursor of 173 amino acids including a 48-amino acid presequence. Calculated molecular mass of the precursor and mature protein without lipoic acid were 18,790 and 13,846, respectively. Northern blot analysis indicated a major mRNA component of 1.1 kilobases and a minor component of 0.6 kilobase. The result is consistent with the presence of two adenylation signals in the 3'-untranslated region of the cDNA. In vitro transcription and translation of the H-protein cDNA produced a 19-kDa protein recognized by antibody raised to chicken H-protein. Bovine liver H-protein precursor has no lipoic acid prosthetic group. When incubated with isolated bovine liver mitochondria, the precursor was imported into mitochondria, processed to its mature form with a molecular mass of 14 kDa, and lipoylated at lysine 59. These results indicate that lipoylation is not required for the import of H-protein precursor into mitochondria and H-protein is lipoylated in mitochondria which probably contain the physiologically active form of lipoic acid as well as the enzyme(s) responsible for the attachment.


SUMOylation inhibits SF-1 activity by reducing CDK7-mediated serine 203 phosphorylation.
Steroidogenic factor 1 (SF-1) is an orphan nuclear receptor selectively expressed in the adrenal cortex and gonads, where it mediates the hormonal stimulation of multiple genes involved in steroid hormone biosynthesis. SF-1 is the target of both phosphorylation and SUMOylation, but how these modifications interact or contribute to SF-1 regulation of endogenous genes remains poorly defined. We found that SF-1 is selectively SUMOylated at K194 in Y1 adrenocarcinoma cells and that although SUMOylation does not alter the subcellular localization of SF-1, the modification inhibits the ability of SF-1 to activate target genes. Notably, whereas SF-1 SUMOylation is independent of S203 phosphorylation and is unaffected by adrenocorticotropin (ACTH) treatment, loss of SUMOylation leads to enhanced SF-1 phosphorylation at serine 203. Furthermore, preventing SF-1 SUMOylation increases the mRNA and protein levels of multiple steroidogenic enzyme genes. Analysis of the StAR promoter indicates that blockade of SF-1 SUMOylation leads to an increase in overall promoter occupancy but does not alter the oscillatory recruitment dynamics in response to ACTH. Notably, we find that CDK7 binds preferentially to the SUMOylation-deficient form of SF-1 and that CDK7 inhibition reduces phosphorylation of SF-1. Based on these observations, we propose a coordinated modification model in which inhibition of SF-1-mediated transcription by SUMOylation in adrenocortical cancer cells is mediated through reduced CDK7-induced phosphorylation of SF-1.


The role of G protein methylation in the function of a geranylgeranylated beta gamma isoform.
The gamma subunit of heterotrimeric G proteins is isoprenylated and methylated on its carboxyl terminal cysteine residue. While retinal transducin is farnesylated, all other gamma subunits are modified by geranylgeranylation. An immobilized form of pig liver esterase (iPLE) is able to hydrolyze the methyl ester of a geranylgeranylated beta gamma isoform (beta 1 gamma 2). Since methylation is the only reversible reaction in the isoprenylation pathway, it could be a site of regulation of G protein activity. With both the methylated and demethylated beta 1 gamma 2 now available, the role of methylation for a geranylgeranylated heterotrimeric G protein may be addressed. Here, it is reported that methylation has no effect on the ability of beta gamma to interact with an alpha subunit, as probed by ADP-ribosylation studies with pertussis toxin, and has a small effect (less than 2-fold) on the ability of geranylgeranylated beta gamma to activate phosphatidylinositol-specific phospholipase C (PIPLC) and phosphoinositide 3 kinase (PI3K). In binding studies, demethylation only slightly decreased the ability of beta 1 gamma 2 to adhere to azolectin vesicles. Therefore, methylation of heterotrimeric G proteins appears to have only a minor effect in signal transduction processes which can be correlated to a decrease in hydrophobicity of the beta gamma subunit.


Sumoylation and the function of CCAAT enhancer binding protein alpha (C/EBP alpha).
CCAAT enhancer binding protein alpha (C/EBP alpha) is the founding member of a family of basic region/leucine zipper (bZIP) transcription factors and is a master regulator of granulopoiesis. It is expressed at high levels throughout myeloid differentiation and binds to the promoters of multiple myeloid-specific genes at different stages of myeloid maturation. Profound hematopoietic abnormalities occur in mice nullizygous for C/EBP alpha including a selective early block in the differentiation of granulocytes. Mutations in C/EBP alpha are present in a subset of patients with AML presenting with a normal karyotype. These mutations can result in the expression of a 30 kDa dominant negative C/EBP alpha isoform, which contributes to loss of C/EBP alpha function. The molecular basis for this observation remains unknown. In addition to phosphorylation, C/EBP alpha is modified, post-translationally by a small ubiquitin-related modifier (SUMO) at a lysine residue (K159), which lies within the growth inhibitory region of the C/EBP alpha protein. Sumoylation at K159 in the C/EBP alpha protein prevents association of the SWI/SNF chromatin remodeling complex with C/EBP alpha, thereby hampering transactivation. In this review, the functional implications of post-translational modification, particularly sumoylation, of C/EBP alpha in normal granulopoiesis and leukemia are considered.


v-Src suppresses SHPS-1 expression via the Ras-MAP kinase pathway to promote the oncogenic growth of cells.
We investigated the effect of cell transformation by v-src on the expression and tyrosine phosphorylation of SHPS-1, a putative docking protein for SHP-1 and SHP-2. We found that transformation by v-src virtually inhibited the SHPS-1 expression at mRNA level. While nontransforming Src kinases including c-Src, nonmyristoylated forms of v-Src had no inhibitory effect on SHPS-1 expression, transforming Src kinases including wild-type v-Src and chimeric mutant of c-Src bearing v-Src SH3 substantially suppressed the SHPS-1 expression. In cells expressing temperature sensitive mutant of v-Src, suppression of the SHPS-1 expression was temperature-dependent. In contrast, tyrosine phosphorylation of SHPS-1 was rather activated in cells expressing c-Src or nonmyristoylated forms of v-Src. SHPS-1 expression in SR3Y1 was restored by treatment with herbimycin A, a potent inhibitor of tyrosine kinase, or by the expression of dominant negative form of Ras. Contrary, active form of Mekl markedly suppressed SHPS-1 expression. Finally, overexpression of SHPS-1 in SR3Y1 led to the drastic reduction of anchorage independent growth of the cells. Taken together, our results suggest that the suppression of SHPS-1 expression is a pivotal event for cell transformation by v-src, and the Ras-MAP kinase cascade plays a critical role in the suppression.


Palmitoylation of SARS-CoV S protein is necessary for partitioning into detergent-resistant membranes and cell-cell fusion but not interaction with M protein.
Coronaviruses are enveloped RNA viruses that generally cause mild disease in humans. However, the recently emerged coronavirus that caused severe acute respiratory syndrome (SARS-CoV) is the most pathogenic human coronavirus discovered to date. The SARS-CoV spike (S) protein mediates virus entry by binding cellular receptors and inducing fusion between the viral envelope and the host cell membrane. Coronavirus S proteins are palmitoylated, which may affect function. Here, we created a non-palmitoylated SARS-CoV S protein by mutating all nine cytoplasmic cysteine residues. Palmitoylation of SARS-CoV S was required for partitioning into detergent-resistant membranes and for cell-cell fusion. Surprisingly, however, palmitoylation of S was not required for interaction with SARS-CoV M protein. This contrasts with the requirement for palmitoylation of mouse hepatitis virus S protein for interaction with M protein and may point to important differences in assembly and infectivity of these two coronaviruses.


Size variations in the mucin-type domain of hamster oviductin: identification of the polypeptide precursors and characterization of their biosynthetic maturation.
The recent sequencing of a cDNA clone for hamster oviductin revealed that this zona pellucida-associated glycoprotein is a particularly intriguing chimeric molecule because it encloses regions of significant similarity with chitinase-related proteins as well as a carboxyterminal mucin-type domain. This domain contains contiguous Ser/Thr-rich repeated stretches of 15 amino acids; similar units are also found in the deduced sequence of human oviductin. Such structural domains constitute a central feature of mucins. We amplified this region from 16 hamster oviductin cDNA clones and identified three length variants. In order to elucidate the biosynthetic maturation of the glycoprotein, a high-titer antiserum against synthetic peptides derived from internal sequences of hamster oviductin was produced and used in pulse-chase experiments. Two major and one minor polypeptide precursors were identified from tunicamycin-treated cell lysates and in vitro translated products from oviductal poly(A)+ RNA. Their apparent molecular masses correlate with the predicted lengths of the three size variants identified by polymerase chain reaction (PCR) amplification. Using glycosylation and transport inhibitors, we sought to dissect the posttranslational sequential steps leading to the final maturation of hamster oviductin and proposed a compartmental model for its biosynthesis. The polypeptide precursors are rapidly converted in the endoplasmic reticulum into an N- and O-glycosylated premature form of 80-90 kDa (time < 20 min), which is further O-glycosylated and sulfated in the trans-Golgi network, giving rise to the secreted species of 160-350 kDa. The polymorphism in the heavily O-glycosylated region of hamster oviductin is predicted to increase the heterogeneity of the glycoprotein. Such changes may alter the putative biological function of the different variants mediated by their mucin-type domain, such as protection of the embryo and/or adhesion-related phenomena.


Palmitoylation of the sphingosine 1-phosphate receptor S1P is involved in its signaling functions and internalization.
The lipid mediator sphingosine 1-phosphate (S1P) regulates several cellular processes through binding to its receptors (S1P(1)-S1P(5)), which are heterotrimeric G protein-coupled receptors. Here, we report that all S1P receptors are palmitoylated. In S1P(1), three Cys residues in the cytoplasmic tail are palmitoylated. We examined the roles of palmitoylation of S1P(1) using model cells in which wild-type S1P(1) or a non-palmitoylated mutant S1P(1) was overproduced. Compared with wild-type S1P(1), the non-palmitoylated S1P(1) exhibited binding affinity similar to the natural ligand S1P but lower to the synthetic ligand FTY720 phosphate (FTY720-P), the active form of the immunomodulator FTY720. However, downstream signaling of non-palmitoylated S1P(1) was similarly affected by S1P and FTY720-P stimulation. Moreover, upon stimulation with S1P, internalization of the mutant non-palmitoylated S1P(1) was retarded, compared with that of the wild-type protein. This effect was much more pronounced with FTY720-P stimulation. Similar differences were observed for the phosphorylation of S1P(1) and its mutant. These findings may provide insights into the molecular mechanisms of the pharmacological effects of FTY720. Finally, palmitoylation of wild-type S1P(1) increased upon treatment with S1P, suggesting that S1P(1) undergoes a palmitoylation/depalmitoylation cycle after stimulation by its ligands.


Orientation of palmitoylated CaVbeta2a relative to CaV2.2 is critical for slow pathway modulation of N-type Ca2+ current by tachykinin receptor activation.
The G(q)-coupled tachykinin receptor (neurokinin-1 receptor [NK-1R]) modulates N-type Ca(2+) channel (Ca(V)2.2 or N channel) activity at two distinct sites by a pathway involving a lipid metabolite, most likely arachidonic acid (AA). In another study published in this issue (Heneghan et al. 2009. J. Gen Physiol. doi:10.1085/jgp.200910203), we found that the form of modulation observed depends on which Ca(V)beta is coexpressed with Ca(V)2.2. When palmitoylated Ca(V)beta2a is coexpressed, activation of NK-1Rs by substance P (SP) enhances N current. In contrast, when Ca(V)beta3 is coexpressed, SP inhibits N current. However, exogenously applied palmitic acid minimizes this inhibition. These findings suggested that the palmitoyl groups of Ca(V)beta2a may occupy an inhibitory site on Ca(V)2.2 or prevent AA from interacting with that site, thereby minimizing inhibition. If so, changing the orientation of Ca(V)beta2a relative to Ca(V)2.2 may displace the palmitoyl groups and prevent them from antagonizing AA's actions, thereby allowing inhibition even in the presence of Ca(V)beta2a. In this study, we tested this hypothesis by deleting one (Bdel1) or two (Bdel2) amino acids proximal to the alpha interacting domain (AID) of Ca(V)2.2's I-II linker. Ca(V)betas bind tightly to the AID, whereas the rigid region proximal to the AID is thought to couple Ca(V)beta's movements to Ca(V)2.2 gating. Although Bdel1/beta2a currents exhibited more variable enhancement by SP, Bdel2/beta2a current enhancement was lost at all voltages. Instead, inhibition was observed that matched the profile of N-current inhibition from Ca(V)2.2 coexpressed with Ca(V)beta3. Moreover, adding back exogenous palmitic acid minimized inhibition of Bdel2/beta2a currents, suggesting that when palmitoylated Ca(V)beta2a is sufficiently displaced, endogenously released AA can bind to the inhibitory site. These findings support our previous hypothesis that Ca(V)beta2a's palmitoyl groups directly interact with an inhibitory site on Ca(V)2.2 to block N-current inhibition by SP.


Regulation of SNAP-25 trafficking and function by palmitoylation.
The SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) protein SNAP-25 (25 kDa synaptosome-associated protein) is essential for regulated exocytosis in neuronal and neuroendocrine cells. Whereas the majority of SNARE proteins contain transmembrane domains, SNAP-25 is instead anchored to membranes by the palmitoylation of a central cysteine-rich region. In this review, we discuss the mechanisms of SNAP-25 palmitoylation and how this modification regulates the intracellular trafficking and exocytotic function of this essential protein.


Ubc9 is essential for viability of higher eukaryotic cells.
Ubc9 is an enzyme involved in the conjugation of SUMO-1 (small ubiquitin related modifier 1) to target proteins. The SUMO-1 conjugation system is well conserved from yeasts to higher eukaryotes, but many SUMO-1 target proteins reported recently in higher eukaryotic cells, including IkappaBalpha, MDM2, p53, and PML, are not present in yeasts. To determine the physiological roles of SUMO-1 conjugation in higher eukaryotic cells, we constructed a conditional UBC9 mutant of chicken DT40 cells containing the UBC9 transgene under control of a tetracycline-repressible promoter and characterized their loss of function phenotypes. Ubc9 disappeared 3 days after the addition of tetracycline and the increase in viable cell number stopped 4 days after the addition of drug. In contrast to the cases of ubc9 mutants of budding and fission yeasts, which show defects in progression of G2 or early M phase and in chromosome segregation, respectively, we did not observe accumulation of cells in G2/M phase or a considerable increase in the frequency of chromosome missegregation upon depletion of Ubc9 but we did observe an increase in the number of cells containing multiple nuclei, indicating defects in cytokinesis. A considerable portion of the Ubc9-depleted cell population was committed to apoptosis without accumulating in a specific phase of the cell cycle, suggesting that chromosome damages are accumulated in Ubc9-depleted cells, and apoptosis is triggered without activating checkpoint mechanisms under conditions of SUMO-1 conjugation system impairment.


A targeted proteomic analysis of the ubiquitin-like modifier nedd8 and associated proteins.
Nedd8 is a small ubiquitin-like protein that can be conjugated to substrate-proteins in a process known as neddylation. Although neddylation plays a critical regulatory role in cell proliferation and development, the spectrum of Nedd8 substrates and its interaction network remain poorly understood. To explore the neddylation pathway at the proteome level, we have affinity purified Nedd8 modified and associated proteins from HEK293 cells stably expressing GST-Nedd8 and employed LC-MS/MS for subsequent protein identification. A total of 496 GST-Nedd8 modified and associated proteins have been identified, including all of the eight cullin family members (i.e., Cul-1, -2, -3, -4A, -4B, -5, -7, and Parc) that are involved in the neddylation and ubiquitin-proteasome degradation pathway. In addition, a group of proteins involved in transcription, DNA repair and replication, cell cycle regulation and chromatin organization, and remodeling have been copurified and identified. Apart from protein identification, the neddylation sites of cullins were determined by MS/MS analysis, which agree well with previous mutagenesis studies. Furthermore, MS analyses revealed that Nedd8 K11, K22, K48, and K60 can form chains in vivo, whereas Nedd8 K22 and K48 can be neddylated in vitro. These results present the first molecular evidence for in vitro and in vivo polyneddylation, suggesting that chain formation of ubiquitin and ubiquitin-like proteins may be a general phenomenon for these modifications. Although much remains to be explored for the biological significance of the observations, this work provides critically important information regarding Nedd8 chain assembly and its interaction network. The vast amount of proteomic information obtained here can provide clues on the biological role of Nedd8 and lay the foundation for an in-depth analysis of the regulation of the Nedd8 pathway.


Golgi targeting of human guanylate-binding protein-1 requires nucleotide binding, isoprenylation, and an IFN-gamma-inducible cofactor.
Human guanylate-binding protein-1 (hGBP-1) is a large GTPase, similar in structure to the dynamins. Like many smaller GTPases of the Ras/Rab family, it is farnesylated, suggesting it may dock into membranes and perhaps play a role in intracellular trafficking. To date, however, hGBP-1 has never been associated with a specific intracellular compartment. Here we present evidence that hGBP-1 can associate with the Golgi apparatus. Redistribution from the cytosol to the Golgi was observed by immunofluorescence and subcellular fractionation after aluminum fluoride treatment, suggesting that it occurs when hGBP-1 is in its GTP-bound state. Relocalization was blocked by a farnesyl transferase inhibitor. The C589S mutant of hGBP-1, which cannot be farnesylated, and the previously uncharacterized R48P mutant, which cannot bind GTP, both failed to localize to the Golgi. These two mutants had a dominant-negative effect, preventing endogenous wild-type hGBP-1 from efficiently redistributing after aluminum fluoride treatment. Furthermore, hGBP-1 requires another IFN-gamma-induced factor to be targeted to the Golgi, because constitutively expressed hGBP-1 remained cytosolic in cells treated with aluminum fluoride unless the cells were preincubated with IFN-gamma. Finally, two nonhydrolyzing mutants of hGBP-1, corresponding to active mutants of Ras family proteins, failed to constitutively associate with the Golgi; we propose three possible explanations for this surprising result.


Fatty acylation of the rat asialoglycoprotein receptor. The three subunits from active receptors contain covalently bound palmitate and stearate.
Rat hepatic asialoglycoprotein receptors (ASGP-Rs) are hetero-oligomers composed of three homologous glycoprotein subunits, designated rat hepatic lectins (RHL) 1, 2, and 3. ASGP-Rs mediate the endocytosis and degradation of circulating glycoconjugates containing terminal N-acetylgalactosamine or galactose, including desialylated plasma glycoproteins. We have shown in permeable rat hepatocytes that the ligand binding activity of one subpopulation of receptors (designated State 2 ASGP-Rs) can be decreased or increased, respectively, by ATP and palmitoyl-CoA (Weigel, P. H., and Oka, J. A. (1993) J. Biol. Chem. 268, 27186-27190). We proposed that a reversible and cyclic acylation/deacylation process may regulate ASGP-R activity during endocytosis, receptor-ligand dissociation, and receptor recycling. In the accompanying paper (Zeng, F-Y., and Weigel, P. H. (1995) J. Biol. Chem. 270, 21388-21395), we show that the ligand binding activity of affinity-purified State 2 ASGP-Rs is decreased by treatment with hydroxylamine under mild conditions consistent with these ASGP-Rs being fatty acylated in vivo. In this study, we used a chemical method to determine the presence of covalently-bound fatty acids in individual ASGP-R subunits. The affinity-purified ASGP-R preparations were separated by SDS-polyacrylamide gel electrophoresis under nonreducing conditions, and the gel slices containing individual RHL subunits were treated with alkali to release covalently bound fatty acids, which were subsequently analyzed by gas chromatography and confirmed by gas chromatography-mass spectrometry. Both stearic and palmitic acids were detected in all three receptor subunits. Pretreatment of ASGP-Rs with hydroxylamine before SDS-polyacrylamide gel electrophoresis reduced the content of both fatty acids by 66-80%, indicating that most of these fatty acids are attached to cysteine residues via thioester linkages. Furthermore, when freshly isolated hepatocytes were cultured in the presence of [3H]palmitate, all three RHL subunits in affinity-purified ASGP-Rs were metabolically labeled. We conclude that RHL1, RHL2, and RHL3 are modified by fatty acylation in intact cells.


Rheb binds tuberous sclerosis complex 2 (TSC2) and promotes S6 kinase activation in a rapamycin- and farnesylation-dependent manner.
Recently the tuberous sclerosis complex 2 (TSC2) tumor suppressor gene product has been identified as a negative regulator of protein synthesis upstream of the mTOR and ribosomal S6 kinases. Because of the homology of TSC2 with GTPase-activating proteins for Rap1, we examined whether a Ras/Rap-related GTPase might be involved in this process. TSC2 was found to bind to Rheb-GTP in vitro and to reduce Rheb GTP levels in vivo. Over-expression of Rheb but not Rap1 promoted the activation of S6 kinase in a rapamycin-dependent manner, suggesting that Rheb acts upstream of mTOR. The ability of Rheb to induce S6 phosphorylation was also inhibited by a farnesyl transferase inhibitor, suggesting that Rheb may be responsible for the Ras-independent anti-neoplastic properties of this drug.


The HOOK-domain between the SH3 and the GK domains of Cavbeta subunits contains key determinants controlling calcium channel inactivation.
Ca(v)beta subunits of voltage-gated calcium channels contain two conserved domains, a src-homology-3 (SH3)-domain and a guanylate kinase-like (GK)-domain. The SH3-domain is split, with its final (fifth) beta-strand separated from the rest of the domain by an intervening sequence termed the HOOK-domain, whose sequence varies between Ca(v)beta subunits. Here we have been guided by the recent structural studies of Ca(v)beta subunits in the design of specific truncated constructs, with the goal of investigating the role of the HOOK-domain of Ca(v)beta subunits in the modulation of inactivation of N-type calcium channels. We have coexpressed the beta subunit constructs with Ca(v)2.2 and alpha(2)delta-2, using the N-terminally palmitoylated beta(2a) subunit, because it supports very little voltage-dependent inactivation, and made comparisons with beta(1b) domains. Deletion of the variable region of the beta(2a) HOOK-domain resulted in currents with a rapidly inactivating component, and additional mutation of the beta(2a) palmitoylation motif further enhanced inactivation. The isolated GK-domain of beta(2a) alone enhanced current amplitude, but the currents were rapidly and completely inactivating. When the beta(2a)-GK-domain construct was extended proximally, by including the HOOK-domain and the epsilon-strand of the SH3-domain, inactivation was about four-fold slower than in the absence of the HOOK domain. When the SH3-domain of beta(2a) truncated prior to the HOOK-domain was coexpressed with the (HOOK+epsilonSH3+GK)-domain of beta(2a), all the properties of beta(2a) were restored, in terms of loss of inactivation. Furthermore, removal of the HOOK sequence from the (HOOK+epsilonSH3+GK)-beta(2a) construct increased inactivation. Together, these results provide evidence that the HOOK domain is an important determinant of inactivation.


Studies on G-protein alpha.betagamma heterotrimer formation reveal a putative S-prenyl-binding site in the alpha subunit.
The alpha and betagamma subunits of heterotrimeric G-proteins contain specific lipid modifications, which are required for their biological function. However, the relevance of these modifications to the interactions within the heterotrimeric G-protein is not fully understood. In order to explore the role of the S-prenyl moiety of the isoprenylated betagamma dimer of retinal transducin, betagamma(t), in the formation of the heterotrimeric complex with the corresponding N-acylated alpha subunit, alpha(t), we employed purified fully processed subunits, which are soluble in aqueous solutions without detergents. Pertussis-toxin-mediated [(32)P]ADP-ribosylation of alpha(t) is strongly stimulated (approximately 10-fold) in the presence of betagamma(t) and can thus serve as a measure for heterotrimer formation. Using this assay, preincubation of alpha(t) with S-prenyl analogues containing farnesyl or geranylgeranyl moieties was found to inhibit heterotrimer formation in a dose-dependent manner. The inhibition was competitive and reversible, as indicated by its reversal upon increase of the betagamma(t) dimer concentration or by removal of the S-prenyl analogue using gel filtration. The competitive nature of the inhibition is supported by the marked attenuation of the inhibition when the S-prenyl analogue was added to alpha(t) together with or after betagamma(t). The inhibition does not involve interaction with the alpha(t) acyl group, since an S-prenyl analogue inhibited the [(32)P]ADP-ribosylation of an unlipidated alpha(t) mutant. These data suggest the existence of a hitherto unrecognized S-prenyl-binding site in alpha(t), which is critical for its interaction with prenylated betagamma(t).


Phosphonocarboxylate inhibitors of Rab geranylgeranyl transferase disrupt the prenylation and membrane localization of Rab proteins in osteoclasts in vitro and in vivo.
Nitrogen-containing bisphosphonate drugs such as risedronate act by inhibiting farnesyl diphosphate synthase, thereby disrupting protein prenylation in osteoclasts. We recently found that an anti-resorptive phosphonocarboxylate analogue of risedronate, 3-PEHPC (previously referred to as NE10790), selectively prevents prenylation of Rab GTPases in vitro by specifically inhibiting Rab geranylgeranyl transferase. In this study, we demonstrate that unprenylated Rab6 could be detected in J774 cells after treatment with 3-PEHPC or risedronate for as little as 4 h, and reached 50% after 24 h. Furthermore, treatment of J774 cells or osteoclasts with either 3-PEHPC or risedronate disrupted membrane association of several Rab family proteins. Like risedronate, the effects of 3-PEHPC are likely to be restricted to osteoclasts in vivo, since both risedronate and 3-PEHPC inhibited Rab prenylation in osteoclasts, but not in general bone marrow cells, when administered to rabbits in vivo. Analysis of two new phosphonocarboxylate analogues of 3-PEHPC (3-PEPC and 2-PEPC) revealed that, first, the geminal hydroxyl group is not essential for inhibition of Rab prenylation by phosphonocarboxylates, but does contribute to their anti-resorptive potency, most likely by enhancing their affinity for bone mineral. Second, the position of the nitrogen in the side chain of phosphonocarboxylates is crucial for their ability to inhibit Rab prenylation and hence to inhibit bone resorption. In addition, there is a good correlation between the ability of the phosphonocarboxylates to inhibit Rab prenylation and to inhibit bone resorption in vitro, indicating that these compounds are a new class of pharmacological agents that inhibit bone resorption by specifically preventing prenylation of Rab proteins. Furthermore, although phosphonocarboxylates are analogues of bisphosphonates, the structure-activity relationships of phosphonocarboxylates for inhibiting Rab geranylgeranyltransferase appear to differ from the structure-activity relationships of bisphosphonates for inhibiting farnesyl diphosphate synthase.


ADP-ribosylation of proteins in brain regions of rats during postnatal development.
Post-translational modifications of chromatin-bound proteins play an important role in the regulation of eukaryotic gene expression. Processes such as acetylation, methylation, phosphorylation and ADP-ribosylation may alter the interaction of these proteins with DNA and consequently affect chromatin conformation and the binding of enzymes and other molecules involved in the regulation of gene expression. In the present study the process of ADP-ribosylation of chromosomal proteins (histone and non-histone proteins) in some rat brain regions during postnatal development was investigated; also the effect of epidermal growth factor (EGF) on this process in fetal brain slices was studied. It has been found that the process of ADP-ribosylation of total histones extracted from rat cerebral cortex and cerebellum at 1, 10 and 30 days of age, increases from 1 to 10 days of age (i.e. the period of maximal cell proliferation) and decreases thereafter, while the process of ADP-ribosylation of non-histone proteins (NHPs) sharply decreases during the same developmental period. The addition of EGF to fetal brain slices causes a significant increase of ADP-ribosylation of total histones (particularly of the histone H1 fraction) and also of NHPs and microsomal proteins. This result is in agreement with the effect of EGF as a mitogen factor, previously shown in astroglial cell cultures.


Regulation of AMPA receptor extrasynaptic insertion by 4.1N, phosphorylation and palmitoylation.
The insertion of AMPA receptors (AMPARs) into the plasma membrane is an important step in the synaptic delivery of AMPARs during the expression of synaptic plasticity. However, the molecular mechanisms regulating AMPAR insertion remain elusive. By directly visualizing individual insertion events of the AMPAR subunit GluR1 in rodents, we found that the protein 4.1N was required for activity-dependent GluR1 insertion. Protein kinase C (PKC) phosphorylation of the serine 816 (S816) and S818 residues of GluR1 enhanced 4.1N binding to GluR1 and facilitated GluR1 insertion. In addition, palmitoylation of GluR1 C811 residue modulated PKC phosphorylation and GluR1 insertion. Finally, disrupting 4.1N-dependent GluR1 insertion decreased surface expression of GluR1 and the expression of long-term potentiation. Our study uncovers a previously unknown mechanism that governs activity-dependent GluR1 trafficking, reveals an interaction between AMPAR palmitoylation and phosphorylation, and underscores the functional importance of 4.1N in AMPAR trafficking and synaptic plasticity.


Refined crystal structures of red and green fluorescent proteins from the button polyp Zoanthus.
The three-dimensional structures of the wild-type red (zRFP574) and green (zGFP506) fluorescent proteins (FP) from the button polyp Zoanthus have been determined at 1.51 and 2.2 A resolution, respectively. In addition, the crystal structures of a zGFP506 variant (zGFP506_N66D) with replacement of the first chromophore-forming residue (Asn66 to Asp) have been determined in the transitional 'green' and mature 'red' states at 2.4 and 2.2 A, respectively. The monomers of these proteins adopt the typical fold of the green fluorescent protein (GFP) family, consisting of an 11-stranded beta-barrel with a chromophore embedded in the middle of an internal alpha-helix directed along the beta-barrel axis. Post-translational modification of the chromophore-forming sequence Asn66-Tyr67-Gly68 within zGFP506 results in a typical GFP-like coplanar two-ring structure consisting of a five-membered imidazolinone heterocycle with the phenolic ring of Tyr67 in a cis orientation to the C(alpha)-N(67) bond. A novel post-translational modification of the chromophore-forming sequence Asp66-Tyr67-Gly68 in zRFP574 expands the protein maturation beyond the green-emitting form and results in decarboxylation of the Asp66 side chain. It is suggested that electrostatic conflict between the closely spaced negatively charged side chains of the chromophore Asp66 and the proximal catalytic Glu221 is most likely to be the trigger for the chain of reactions resulting in the observed decarboxylation. The chromophore structures of wild-type zGFP506 and of its mutant zGFP506_N66D in the 'green' and 'red' states support this suggestion. The beta-barrel frames of zRFP574 and zGFP506 reveal the presence of a water-filled pore leading to the chromophore Tyr67, similar to that observed previously in TurboGFP. An analysis of the residue composition at two inter-monomer interfaces in the tetrameric biological unit of zRFP574 and zGFP506, as well as of zYFP538 from the same species, has revealed a group of highly conserved residues that are apparently responsible for oligomerization. These residues present initial useful targets for rational mutagenesis aimed at designing monomeric forms of the fluorescent proteins, which are more suitable for practical applications.


An S-acylation switch of conserved G domain cysteines is required for polarity signaling by ROP GTPases.
Rho GTPases are master regulators of cell polarity. For their function, Rhos must associate with discrete plasma membrane domains. Rho of Plants (ROPs) or RACs comprise a single family. Prenylation and S-acylation of hypervariable domain cysteines of Ras and Rho GTPases are required for their function; however, lipid modifications in the G domain have never been reported. Reversible S-acylation involves the attachment of palmitate (C16:0) or other saturated lipids to cysteines through a thioester linkage and was implicated in the regulation of signaling. Here we show that transient S-acylation of Arabidopsis AtROP6 takes place on two conserved G domain cysteine residues, C21 and C156. C21 is relatively exposed and is accessible for modification, but C156 is not, implying that its S-acylation involves a conformational change. Fluorescence recovery after photobleaching beam-size analysis shows that S-acylation of AtROP6 regulates its membrane-association dynamics, and detergent-solubilization studies indicate that it regulates AtROP6 association with lipid rafts. Site-specific acylation-deficient AtROP6 mutants can bind and hydrolyze GTP but display compromised effects on polar cell growth, endocytic uptake of the tracer dye FM4-64, and distribution of reactive oxygen species. These data reveal an S-acylation switch that regulates Rho signaling.


Polyunsaturated fatty acids block platelet-activating factor-induced phosphatidylinositol 3 kinase/Akt-mediated apoptosis in intestinal epithelial cells.
We have shown earlier that platelet-activating factor (PAF) causes apoptosis in enterocytes via a mechanism that involves Bax translocation to mitochondria, followed by caspase activation and DNA fragmentation. Herein we report that, in rat small intestinal epithelial cells (IEC-6), these downstream apoptotic effects are mediated by a PAF-induced inhibition of the phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (Akt) signaling pathway. Treatment with PAF results in rapid dephosphorylation of Akt, phosphoinositide-dependent kinase-1, and the YXXM p85 binding motif of several proteins and redistribution of Akt-pleckstrin homology domain-green fluorescent protein, i.e., an in vivo phosphatidylinositol (3,4,5)-trisphosphate sensor, from membrane to cytosol. The proapoptotic effects of PAF were inhibited by both n-3 and n-6 polyunsaturated fatty acids but not by a saturated fatty acid palmitate. Indomethacin, an inhibitor of prostaglandin biosynthesis, did not influence the baseline or PAF-induced apoptosis, but 2-bromopalmitate, an inhibitor of protein palmitoylation, inhibited all of the proapoptotic effects of PAF. Our data strongly suggest that an inhibition of the PI 3-kinase/Akt signaling pathway is the main mechanism of PAF-induced apoptosis in enterocytes and that polyunsaturated fatty acids block this mechanism very early in the signaling cascade independently of any effect on prostaglandin synthesis, and probably directly via an effect on protein palmitoylation.


Alternative O-glycosylation/O-phosphorylation of the murine estrogen receptor beta.
Estrogen receptor beta, a homologue to estrogen receptor alpha, is a new member of the steroid hormone receptor family. Recently, we documented that estrogen receptor alpha, like other transcription factors, is modified by O-linked N-acetylglucosamine (O-GlcNAc), a ubiquitous transitory posttranslational modification on nuclear and cytoplasmic proteins. Here, we report that estrogen receptor beta is alternatively modified by either O-GlcNAc or O-phosphate. Lectin chromatography of in vitro translated protein first suggested that murine estrogen receptor beta (mER-beta) is O-GlcNAcylated. Structural characterization of the carbohydrate moieties on mER-beta, overexpressed in insect Sf9 cells, confirmed the presence of O-GlcNAc. mER-beta, overexpressed in mammalian cells, is also O-GlcNAcylated. The major site of O-GlcNAc on mER-beta from Sf9 cells is Ser(16) near the N-terminus. Concomitant analyses also documented the O-phosphorylation of mER-beta at Ser(16). MALDI-TOF mass spectrometry showed alternative occupancy of this locus by these two abundant and dynamic posttranslational modifications. The localization of a major O-GlcNAc/O-phosphate site in proximity of the transactivation domain and as part of a PEST region (target sequences for rapid protein degradation) on mER-beta suggests that these modifications may play a role in regulating estrogen receptor beta transactivation and turnover.


Cell-type-specific and site-specific N-glycosylation of type I and type II human tissue plasminogen activator.
Tissue plasminogen activator (t-PA) is an important initiator of fibrinolysis. The t-PA polypeptide has four potential N-glycosylation sites of which three are occupied in type I (Asn-117, -184, and -448) and two in type II (Asn-117 and -448). In an effort to elucidate the factors controlling the expression of N-linked oligosaccharides on this polypeptide, we have used a combination of sequential exoglycosidase digestion, methylation analysis, and controlled acetolysis to determine the oligosaccharide structures at each of the N-glycosylation sites of type I and type II t-PA when isolated from a human colon fibroblast cell strain and from a Bowes melanoma cell line. Our results suggest the following: (i) type I and type II t-PA are N-glycosylated in an identical way at Asn-117 and Asn-448, when isolated from the same cell line; (ii) Asn-117 is predominantly associated with oligomannose-type structures in all cases; (iii) Asn-184 and Asn-448 are predominantly associated with complex-type structures when t-PA is isolated from fibroblast cells, but with both complex- and oligomannose-type structures when isolated from melanoma cells; (iv) fibroblast cell derived t-PA is associated with both neutral and sialylated oligosaccharides, while melanoma cell derived t-PA is also associated with sulfated oligosaccharides, which are located exclusively at Asn-448 of type II t-PA; (v) no complex-type structures occur in common between t-PA from the two cell lines. These results indicate that the t-PA glycoprotein is secreted by each cell line as a set of glycoforms, each glycoform being unique with respect to the nature and disposition of oligosaccharides on a common polypeptide. Further, the two cell lines express no glycoform in common, despite expressing the same t-PA polypeptide. The implications of these results for both the control of oligosaccharide processing in different cell lines and the genetic engineering of mammalian glycoproteins are discussed.


Functional and structural roles of conserved cysteine residues in the carboxyl-terminal domain of the follicle-stimulating hormone receptor in human embryonic kidney 293 cells.
The carboxyl-terminal segment of G protein-coupled receptors has one or more conserved cysteine residues that are potential sites for palmitoylation. This posttranslational modification contributes to membrane association, internalization, and membrane targeting of proteins. In contrast to other members of the glycoprotein hormone receptor family (the LH and thyroid-stimulating hormone receptors), it is not known whether the follicle-stimulating hormone receptor (FSHR) is palmitoylated and what are the effects of abolishing its potential palmitoylation sites. In the present study, a functional analysis of the FSHR carboxyl-terminal segment cysteine residues was carried out. We constructed a series of mutant FSHRs by substituting cysteine residues with alanine, serine, or threonine individually and together at positions 629 and 655 (conserved cysteines) and 627 (nonconserved). The results showed that all three cysteine residues are palmitoylated but that only modification at Cys629 is functionally relevant. The lack of palmitoylation does not appear to greatly impair coupling to G(s) but, when absent at position 629, does significantly impair cell surface membrane expression of the partially palmitoylated receptor. All FSHR Cys mutants were capable of binding agonist with the same affinity as the wild-type receptor and internalizing on agonist stimulation. Molecular dynamics simulations at a time scale of approximately 100 nsec revealed that replacement of Cys629 resulted in structures that differed significantly from that of the wild-type receptor. Thus, deviations from wild-type conformation may potentially contribute to the severe impairment in plasma membrane expression and the modest effects on signaling exhibited by the receptors modified in this particular position.


Vitamin K-dependent carboxylase activity, prothrombin mRNA, and prothrombin production in two cultured rat hepatoma cell lines.
The presence of under-gamma-carboxylated forms of plasma prothrombin is a marker for human primary hepatocellular carcinoma. A rat hepatoma cell line (7777) which was previously shown to secrete undercarboxylated prothrombin when grown as a solid tumor has now been grown in monolayer culture. This cell line has a decreased activity of the microsomal vitamin K-dependent carboxylase when compared to a control (H4IIEC3) hepatoma line, does not increase intracellular prothrombin concentrations in response to vitamin K depletion, and secretes undercarboxylated prothrombin even when grown in vitamin K supplemented media. Prothrombin gene expression in the 7777 cell line, as measured by prothrombin mRNA levels, was not altered in the 7777 cell line. This cell line appears to be a model for assessing the cellular alterations responsible for undercarboxylated prothrombin excretion by human hepatocellular tumors.


Regulation by cAMP of post-translational processing and subcellular targeting of endothelial nitric-oxide synthase (type 3) in cardiac myocytes.
Cardiac myocytes express the nitric-oxide synthase isoform originally identified in endothelial cells, termed eNOS or NOS3, where it plays a role in regulating myocyte responsiveness to both adrenergic and muscarinic cholinergic autonomic nervous system agonists. eNOS in endothelial cells has been shown to undergo extensive post-translational processing, and in cardiac myocytes as well as endothelial cells, eNOS has been shown to be targeted to plasmalemmal caveolae, a process that is dependent on myristoylation and palmitoylation. Other post-translational modifications essential for the correct subcellular targeting of eNOS have not been described previously. We demonstrate, using [35S]methionine pulse-chase experiments, that native eNOS in adult rat ventricular myocytes is initially translated as a nonpalmitoylated 150-kDa isoform, which is associated with cytosolic and intracellular membrane-enriched fractions. This is subsequently processed to a palmitoylated 135-kDa isoform, which is found only in a sarcolemma-enriched membrane fraction. Forskolin, an agent that elevates intracellular cAMP, rapidly inhibited processing of the 150-kDa isoform to the 135-kDa isoform and transport of eNOS to the sarcolemma, effects paralleled by protein kinase A-dependent phosphorylation of the larger eNOS isoform. Forskolin also decreased palmitoylation of the 135-kDa isoform, although it did not accelerate depalmitoylation of sarcolemmal eNOS, as determined by pulse-chase experiments with [3H]palmitate. Thus, post-translational processing of a 150-kDa isoform of myocyte eNOS appears to be necessary for intracellular trafficking of the enzyme to sarcolemmal caveolae. Both the post-translational processing and subcellular targeting of eNOS appear to be modified by changes in intracellular cAMP, an effect that may have important implications for cardiac myocyte responsiveness to autonomic agonists in vivo.


Redox-mediated modification of PLZF by SUMO-1 and ubiquitin.
Earlier, we reported that the transcriptional repressor promyelocytic leukemia zinc-finger protein (PLZF) is sumoylated at position K242, and the sumoylation regulated its biological function. Here, we show that the sumoylation site can be modified by ubiquitin. The stability and nuclear localization of PLZF were regulated by the antagonistic relationship between sumoylation and ubiquitination. We observed the antagonistic effects of ubiquitin and SUMO-1 on PLZF under oxidative stress induced by serum deprivation. Thus, the choice between modification of PLZF by SUMO or ubiquitin was determined by the intracellular level of ROS, which was generated by serum deprivation that inactivated the SUMO-conjugating enzymes Uba2 and Ubc9, and resulted in decrease of sumoylation. The ubiquitination was increased under these conditions. The expression of BID, a known transcriptional target protein of PLZF, was decreased, and the consequent apoptosis was induced by the ROS generated during serum starvation. On the basis of these results, we propose that PLZF post-translational modification is controlled by intracellular ROS, and the biological function of PLZF is regulated by sumoylation and ubiquitination.


Herpes simplex virus protein UL11 but not UL51 is associated with lipid rafts.
The UL11 and UL51 gene products of herpes simplex virus (HSV) are membrane-associated tegument proteins that are incorporated into the HSV virion. UL11 and UL51 are conserved throughout the herpesvirus family. Both UL11 and UL51, either singly or in combination, are involved in virion envelopment and/or egress. Both proteins are fatty acylated: UL11 is both acylated by myristoic and palmitoic acids and UL51 is monoacylated by palmitoic acid. Using confocal microscopy and sucrose gradient fractionations in transfected or HSV-infected cells, we found that HSV-2 UL11 but not UL51 was associated with lipid rafts. The dual acylation of UL11 was necessary for lipid raft association, as mutations in the myristoylation or palmitoylation sites prevented lipid raft association. These differences in lipid raft association may contribute to the functional differences between UL11 and UL51.


The proprotein convertase (PC) PCSK9 is inactivated by furin and/or PC5/6A: functional consequences of natural mutations and post-translational modifications.
PCSK9 is the ninth member of the proprotein convertase (PC) family. Some of its natural mutations have been genetically associated with the development of a dominant form of familial hyper- or hypocholesterolemia. The exact mechanism of action of PCSK9 is not clear, although it is known to enhance the intracellular degradation of the low density lipoprotein (LDL) receptor in acidic compartments, likely the endosomes/lysosomes. We analyzed the post-translational modifications of PCSK9 and show that it is sulfated within its prosegment at Tyr38. We also examined the susceptibility of PCSK9 to proteolytic cleavage by the other members of the PC family. The data show that the natural gain-of-function mutations R218S, F216L, and D374Y associated with hypercholesterolemia result in total or partial loss of furin/PC5/6A processing at the motif RFHR218 downward arrow. In contrast, the loss-of-function mutations A443T and C679X lead either to the lack of trans-Golgi network/recycling endosome localization and an enhanced susceptibility to furin cleavage (A443T) or to the inability of PCSK9 to exit the endoplasmic reticulum (C679X). Furthermore, we report the presence of both native and furin-like cleaved forms of PCSK9 in circulating human plasma. Thus, we propose that PCSK9 levels are finely regulated by the basic amino acid convertases furin and PC5/6A. The latter may reduce the lifetime of this proteinase and its ability to degrade the cell-surface LDL receptor, thereby regulating the levels of circulating LDL cholesterol.


Role of variant prealbumin in the pathogenesis of familial amyloidotic polyneuropathy: fate of normal and variant prealbumin in the circulation.
According to recent studies on protein chemistry and genetic engineering, replacement of the Val30 residue of prealbumin by methionine is believed to play a critical role in the formation of amyloid deposit and the pathogenesis of familial amyloidotic polyneuropathy (FAP). However, only limited information is available concerning the behavior of prealbumin in the circulation. To obtain the molecular insight into the mechanism of amyloid deposition, it is indispensable to know the fates of normal and variant prealbumin in vivo. Thus, the fates of prealbumin samples from normal and FAP patients were studied in normal rats as well as in animals that were challenged with acute inflammation induced by turpentine. The effect of in vitro photooxidation of prealbumin samples on their behavior was also examined in vivo. Kinetic analysis revealed no appreciable difference between prealbumin samples from normal and FAP patients. These results suggest that factors other than the rate of transfer of the variant form prealbumin from plasma to an extravascular compartment may play a critical role in the pathogenesis of amyloid deposition in FAP patients.


O-linked GlcNAc modification of cardiac myofilament proteins: a novel regulator of myocardial contractile function.
In addition to O-phosphorylation, O-linked modifications of serine and threonine by beta-N-acetyl-D-glucosamine (GlcNAc) may regulate muscle contractile function. This study assessed the potential role of O-GlcNAcylation in cardiac muscle contractile activation. To identify specific sites of O-GlcNAcylation in cardiac myofilament proteins, a recently developed methodology based on GalNAz-biotin labeling followed by dithiothreitol replacement and light chromatography/tandem mass spectrometry site mapping was adopted. Thirty-two O-GlcNAcylated peptides from cardiac myofilaments were identified on cardiac myosin heavy chain, actin, myosin light chains, and troponin I. To assess the potential physiological role of the GlcNAc, force-[Ca(2+)] relationships were studied in skinned rat trabeculae. Exposure to GlcNAc significantly decreased calcium sensitivity (pCa50), whereas maximal force (F(max)) and Hill coefficient (n) were not modified. Using a pan-specific O-GlcNAc antibody, it was determined that acute exposure of myofilaments to GlcNAc induced a significant increase in actin O-GlcNAcylation. This study provides the first identification of O-GlcNAcylation sites in cardiac myofilament proteins and demonstrates their potential role in regulating myocardial contractile function.


PIASy-mediated repression of the androgen receptor is independent of sumoylation.
PIASy, a member of the protein inhibitor of activated STAT (PIAS) family, represses the transcriptional activity of the androgen receptor (AR). In this report, we investigate the mechanism of PIASy-mediated repression of AR. We show that AR binds to the RING-finger like domain of PIASy. PIASy contains two transcriptional repression domains, RD1 and RD2. RD1, but not RD2, is required for PIASy-mediated repression of AR. We show that the RD1 domain binds HDAC1 and HDAC2 and that HDAC activity is required for PIASy-mediated AR repression. PIAS proteins possess small ubiquitin-related modifier (SUMO) E3 ligase activity. Conjugation of SUMO-1 to AR has been implicated in the regulation of AR activity. We examine if the SUMO ligase activity of PIASy is required for PIASy to repress AR. We show that a mutant PIASy, defective in promoting sumoylation, retains the ability to repress AR transcription. In addition, mutation of all the known sumoylation acceptor sites of AR does not affect the transrepression activity of PIASy on AR. Our results suggest that PIASy may repress AR by recruiting histone deacetylases, independent of its SUMO ligase activity.


Effect of propeptide mutations on post-translational processing of factor IX. Evidence that beta-hydroxylation and gamma-carboxylation are independent events.
Post-translational processing of Factor IX includes glycosylation, cleavage of the signal peptide and propeptide, vitamin K-dependent carboxylation of specific glutamic acid residues to form gamma-carboxyglutamic acid, and beta-hydroxylation of aspartic acid at residue 64 to form beta-hydroxyaspartic acid. The human Factor IX cDNA coding sequence was modified in the propeptide region (residue -18 to -1) using oligonucleotide-directed site-specific mutagenesis, and the altered Factor IX cDNA was expressed in Chinese hamster ovary cells. The effects of the mutations on proteolytic processing, gamma-carboxylation, and beta-hydroxylation were assessed by direct structural analysis. After purification, the molecular weight of each of the recombinant Factor IX species and its NH2-terminal amino acid sequence were shown to be identical to those of plasma Factor IX. gamma-Carboxyglutamic acid and beta-hydroxyaspartic acid analyses revealed that recombinant wild-type Factor IX contained 9.2 gamma-carboxyglutamic acid and 0.3 beta-hydroxyaspartic acid residues/molecule compared with 11.4 gamma-carboxyglutamic acid and 0.39 beta-hydroxyaspartic acid residues in plasma Factor IX. When the 18-residue propeptide was deleted or when the cells were grown in the presence of sodium warfarin, secreted Factor IX contained no detectable gamma-carboxyglutamic acid but 0.36 and 0.40 residues of beta-hydroxyaspartic acid, respectively. Point mutations leading to substitution of alanine for phenylalanine at residue -16 or glutamic acid for alanine at residue -10 contained 0.2 and 1.7 gamma-carboxyglutamic acid residues, respectively, and 0.2 residues of beta-hydroxyaspartic acid. These data confirm that the propeptide mutations made do not interfere with proteolytic processing and that the Factor IX propeptide contains a recognition site that designates the adjacent glutamic acid-rich domain for gamma-carboxylation. In contrast, beta-hydroxylation of aspartic acid 64 is an independent process which does not require vitamin K and is mediated through a hydroxylation recognition site in the mature Factor IX, not in the propeptide.


Sequential and gamma-secretase-dependent processing of the betacellulin precursor generates a palmitoylated intracellular-domain fragment that inhibits cell growth.
Betacellulin (BTC) belongs to the family of epidermal growth factor (EGF)-like growth factors that are expressed as transmembrane precursors and undergo proteolytic ectodomain shedding to release soluble mature ligands. BTC is a dual-specificity ligand for ErbB1 and ErbB4 receptors, and can activate unique signal-transduction pathways that are beneficial for the function, survival and regeneration of pancreatic beta-cells. We have previously shown that BTC precursor (proBTC) is cleaved by ADAM10 to generate soluble ligand and a stable, transmembrane remnant (BTC-CTF). In this study, we analyzed the fate of the BTC-CTF in greater detail. We demonstrated that proBTC is cleaved by ADAM10 to produce BTC-CTF, which then undergoes intramembrane processing by presenilin-1- and/or presenilin-2-dependent gamma-secretase to generate an intracellular-domain fragment (BTC-ICD). We found that the proBTC cytoplasmic domain is palmitoylated and that palmitoylation is not required for ADAM10-dependent cleavage but is necessary for the stability and gamma-secretase-dependent processing of BTC-CTF to generate BTC-ICD. Additionally, palmitoylation is required for nuclear-membrane localization of BTC-ICD, as demonstrated by the redistribution of non-palmitoylated BTC-ICD mutant to the nucleoplasm. Importantly, a novel receptor-independent role for BTC-ICD signaling is suggested by the ability of BTC-ICD to inhibit cell growth in vitro.


Crm1p mediates regulated nuclear export of a yeast AP-1-like transcription factor.
The yeast AP-1-like transcription factor, Yap1p, activates genes required for the response to oxidative stress. Yap1p is normally cytoplasmic and inactive, but will activate by nuclear translocation if cells are placed in an oxidative environment. Here we show that Yap1p is a target of the beta-karyopherin-like nuclear exporter, Crm1p. Yap1p is constitutively nuclear in a crm1 mutant, and Crm1p binds to a nuclear export sequence (NES)-like sequence in Yap1p in the presence of RanGTP. Recognition of Yap1p by Crm1p is inhibited by oxidation, and this inhibition requires at least one of the three cysteine residues flanking the NES. These results suggest that Yap1p localization is largely regulated at the level of nuclear export, and that the oxidation state affects the accessibility of the Yap1p NES to Crm1p directly. We also show that a mutation in RanGAP (rna1-1) is synthetically lethal with crm1 mutants. Yap1p export is inhibited in both rna1-1 and prp20 (RanGNRF) mutant strains, but Yap1p rapidly accumulates at the nuclear periphery after shifting rna1-1, but not other mutant cells to the non-permissive temperature. Thus, disassembly of export complexes in response to RanGTP hydrolysis may be required for release of substrate from a terminal binding site at the nuclear pore complex (NPC).


Direct demonstration of geranylgeranylation and farnesylation of Ki-Ras in vivo.
It has recently been reported that Ki-Ras protein can be modified in vitro by farnesylation or geranylgeranylation. However, a previous analysis of Ki-Ras prenylation in vivo found only farnesylated Ki-Ras. In this report it is shown that under normal conditions, Ki-Ras is farnesylated in vivo and when cells are treated with the farnesyl transferase inhibitors B956 or B957, farnesylation is inhibited and Ki-Ras becomes geranylgeranylated in a dose dependent manner. These results have strong implications in the design of anticancer drugs based on inhibition of prenylation.


Three-dimensional organization of promyelocytic leukemia nuclear bodies.
Promyelocytic leukemia nuclear bodies (PML-NBs) are mobile subnuclear organelles formed by PML and Sp100 protein. They have been reported to have a role in transcription, DNA replication and repair, telomere lengthening, cell cycle control and tumor suppression. We have conducted high-resolution 4Pi fluorescence laser-scanning microscopy studies complemented with correlative electron microscopy and investigations of the accessibility of the PML-NB subcompartment. During interphase PML-NBs adopt a spherical organization characterized by the assembly of PML and Sp100 proteins into patches within a 50- to 100-nm-thick shell. This spherical shell of PML and Sp100 imposes little constraint to the exchange of components between the PML-NB interior and the nucleoplasm. Post-translational SUMO modifications, telomere repeats and heterochromatin protein 1 were found to localize in characteristic patterns with respect to PML and Sp100. From our findings, we derived a model that explains how the three-dimensional organization of PML-NBs serves to concentrate different biological activities while allowing for an efficient exchange of components.


Nerve growth factor induces a succession of increases in isoprenylated methylated small GTP-binding proteins of PC-12 pheochromocytoma cells.
Pheochromocytoma (PC-12) cells exposed to nerve growth factor (NGF) acquire a sympathetic neuron-like phenotype. This NGF-response is blocked by methylation inhibitors and can be mimicked by the farnesylated methylated small GTP-binding protein p21ras. The implicated involvement of prenylation, methylation and a small GTP-binding protein in the NGF-response has been studied by directly measuring 3H-mevalonic acid (MVA)-metabolites incorporated into proteins, protein carboxy [methyl-3H]ester formation and levels of [alpha-32P]GTP-binding proteins in NGF-induced PC-12 cells. We demonstrate that NGF induces a 2-3-fold increase in 21-24 kDa methylated membrane proteins that incorporate 3H-MVA-metabolites, and bind GTP. Levels of [alpha-32P]GTP-binding in these proteins were increased by 2-3-fold. Methylation and membrane association of the small GTP-binding proteins were blocked by lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, which also enhanced their labeling by 3H-MVA-metabolites. Cycloheximide reduced the levels of [methyl-3H] labeled 21-24 kDa proteins and of the overlapping [alpha-32P]GTP binding-proteins. About 70% of the [methyl-3H]-groups found in these proteins were recovered from two dimensional gel blots in nine distinct spots of [alpha-32P]GTP-binding proteins. Taken together these results strongly suggest that in PC-12 cells, NGF induces an increase in the synthesis of prenylated methylated small GTP-binding proteins. The efficacy of lovastatin blockage of protein methylation and enhancement of 3H-MVA-metabolites incorporation into GTP-binding proteins was lower in NGF-induced cells than in controls. This suggests that NGF also induces an increase in HMG-CoA reductase activity. At the early phase of the NGF response in PC-12 cells (15 min-1 h), the levels of two small GTP-binding proteins (molecular mass of 21-22 kDa and 23-24 kDa) were increased. Thus, at least two proteins, of which one but not the other may be p21ras, appear to be involved in the early response. After a lag period of 24 h with NGF, a second more robust phase of increase in methylated small GTP-binding proteins was apparent. This relatively late response, which was almost completed within 24 h, may reflect involvement of small GTP-binding proteins in neurite-outgrowth and in the functional activity of the differentiated cells. Many small GTP-binding proteins were increased during the second phase, precluding electrophoretic separation of all of them. 3 proteins, however, were well separated (one 23-24 kDa protein and two 21-22 kDa proteins).(ABSTRACT TRUNCATED AT 400 WORDS)


Farnesylation of Cenp-F is required for G2/M progression and degradation after mitosis.
Farnesyl transferase inhibitors induce G2/M cell cycle delays that cannot be explained by inhibition of the Ras GTPase. Recently, the kinetochore protein Cenp-F has been shown to be farnesylated. Here, we show that ectopic expression of the kinetochore targeting domain of Cenp-F delays progression through G2/M. Significantly, this is dependent on the CAAX farnesylation motif. We also show that localisation of Cenp-F to the nuclear envelope at G2/M and kinetochores in prometaphase is dependent both on its CAAX motif and farnesyl transferase activity. Strikingly, farnesyl transferase activity is also required for Cenp-F degradation after mitosis. Thus, these observations suggest that farnesylation of Cenp-F is required not only for its localisation to the nuclear envelope and kinetochores but also for timely progression through G2/M and its degradation after mitosis. In addition, these observations raise the possibility that the anti-proliferative effects induced by farnesyl transferase inhibitors may be due to inhibition of Cenp-F function and/or turnover.


Fine mapping of posttranslational modifications of the linker histone H1 from Drosophila melanogaster.
The linker histone H1 binds to the DNA in between adjacent nucleosomes and contributes to chromatin organization and transcriptional control. It is known that H1 carries diverse posttranslational modifications (PTMs), including phosphorylation, lysine methylation and ADP-ribosylation. Their biological functions, however, remain largely unclear. This is in part due to the fact that most of the studies have been performed in organisms that have several H1 variants, which complicates the analyses. We have chosen Drosophila melanogaster, a model organism, which has a single H1 variant, to approach the study of the role of H1 PTMs during embryonic development. Mass spectrometry mapping of the entire sequence of the protein showed phosphorylation only in the ten N-terminal amino acids, mostly at S10. For the first time, changes in the PTMs of a linker H1 during the development of a multicellular organism are reported. The abundance of H1 monophosphorylated at S10 decreases as the embryos age, which suggests that this PTM is related to cell cycle progression and/or cell differentiation. Additionally, we have found a polymorphism in the protein sequence that can be mistaken with lysine methylation if the analysis is not rigorous.


Synthesis of functionalized rab GTPases by a combination of solution- or solid-phase lipopeptide synthesis with expressed protein ligation.
Prenylated proteins with non-native functionalities are generally very difficult to obtain by recombinant or enzymatic means. The semisynthesis of preparative amounts of prenylated Rab guanosine triphosphatases (GTPases) from recombinant proteins and synthetic prenylated peptides depends largely on the availability of functionalised prenylated peptides corresponding to the proteins' native structure or modifications thereof. Here, we describe and compare solution-phase and solid-phase strategies for the generation of peptides corresponding to the prenylated C terminus of Rab7 GTPase. The solid-phase with utilisation of a hydrazide linker emerges as the more favourable approach. It allows a fast and practical synthesis of pure peptides and gives a high degree of flexibility in their modification. To facilitate the analysis of semisynthetic proteins, the synthesised peptides were equipped with a fluorescent group. Using the described approach, we introduced fluorophores at several different positions of the Rab7 C terminus. The position of the incorporated fluorescent groups in the peptides did not influence the protein-ligation reaction, as the generated peptides could be ligated onto thioester-tagged Rab7. However, it was found that the positioning of the fluorescent group had an influence on the functionality of the Rab7 proteins; analysis of the interaction of the semisynthetic Rab7 proteins with REP (Rab escort protein) and GDI (guanosine diphosphate dissociation inhibitor) molecules revealed that modification of the peptide side chains or of the C-terminal isoprenoid did not significantly interfere with complex formation. However, functionalisation of the C terminus was found to have an adverse effect on complex formation and stability, possibly reflecting low structural flexibility of the Rab GDI/REP molecules in the vicinity of the lipid-binding site.


ADP-ribosylation of Rhizobium meliloti glutamine synthetase III in vivo.
The control of glutamine synthetase (GS), the first enzyme in the main pathway used by Rhizobium meliloti to assimilate ammonia, is central to cellular nitrogen metabolism. R. meliloti is unusual in having three distinct types of GS, including a unique GS, GSIII, that differs considerably from both GSI, which resembles other bacterial GS proteins and GSII, which resembles the GS found in eukaryotes. We show here that GSIII can be post-translationally modified in vivo by ADP-ribosylation at an arginine residue. 32PO4 attached to GSIII during bacterial growth as part of the modifying group could be removed by treatment with snake venom phosphodiesterase or by turkey erythrocyte ADP-ribosylarginine hydrolase. Treatment of modified GSIII with hydroxylamine at neutral pH releases a chromophore that has the retention time of ADP-ribose when analyzed by reversed-phase high performance liquid chromatography. ADP-ribosylation inhibits GSIII activity.


Constitutively active Rheb induces oncogenic transformation.
Rheb (Ras-homolog enriched in brain) is a component of the phosphatidylinositol 3-kinase (PI3K) target of rapamycin (TOR) signaling pathway, functioning as a positive regulator of TOR. Constitutively active mutants of Rheb induce oncogenic transformation in cell culture. The transformed cells are larger and contain more protein than their normal counterparts. They show constitutive phosphorylation of the ribosomal protein S6 kinase and the eukaryotic initiation factor 4E-binding protein 1, two downstream targets of TOR. The TOR-specific inhibitor rapamycin strongly interferes with transformation induced by constitutively active Rheb, suggesting that TOR activity is essential for the oncogenic effects of mutant Rheb. Rheb-induced transformation is also dependent on a C-terminal farnesylation signal that mediates localization to a cellular membrane. An engineered N-terminal myristylation signal can substitute for the farnesylation. Immunofluorescence localizes wild-type and mutant Rheb to vesicular structures in the cytoplasm, overlapping with the endoplasmic reticulum.


Site-specific mutagenesis identifies three cysteine residues in the cytoplasmic tail as acylation sites of influenza virus hemagglutinin.
The hemagglutinin (HA) of influenza virus is a type I transmembrane glycoprotein which is acylated with long-chain fatty acids. In this study we have used oligonucleotide-directed mutagenesis of cloned cDNA and a simian virus 40 expression system to determine the fatty acid binding site in HA and to examine possible functions of covalently linked fatty acids. The results show that the HA is acylated through thioester linkages at three highly conserved cysteine residues located in the cytoplasmic domain and at the carboxy-terminal end of the transmembrane region, whereas a cysteine located in the middle of the membrane-spanning domain is not acylated. Mutants lacking fatty acids at individual or all three attachment sites acquire endoglycosidase H-resistant oligosaccharide side chains, are cleaved into HA1 and HA2 subunits, and are transported to the plasma membrane at rates similar to that of wild-type HA. All mutants are membrane bound and not secreted into the medium. These results exclude transport signal and membrane-anchoring functions of covalently linked fatty acids for this integral membrane glycoprotein. Furthermore, lack of acylation has no obvious influence on the biological activities of HA: cells expressing fatty acid-free HA bind to and, after brief exposure to mildly acidic pH, fuse with erythrocytes; the HA-induced polykaryon formation is not impaired, either. Other possible functions of covalently linked fatty acids in integral membrane glycoproteins which cannot be examined in conventional cDNA expression systems are discussed.


Galectin-1 is a novel structural component and a major regulator of h-ras nanoclusters.
The organization of Ras proteins into nanoclusters on the inner plasma membrane is essential for Ras signal transduction, but the mechanisms that drive nanoclustering are unknown. Here we show that epidermal growth factor receptor activation stimulates the formation of H-Ras.GTP-Galectin-1 (Gal-1) complexes on the plasma membrane that are then assembled into transient nanoclusters. Gal-1 is therefore an integral structural component of the H-Ras-signaling nanocluster. Increasing Gal-1 levels increases the stability of H-Ras nanoclusters, leading to enhanced effector recruitment and signal output. Elements in the H-Ras C-terminal hypervariable region and an activated G-domain are required for H-Ras-Gal-1 interaction. Palmitoylation is not required for H-Ras-Gal-1 complex formation, but is required to anchor H-Ras-Gal-1 complexes to the plasma membrane. Our data suggest a mechanism for H-Ras nanoclustering that involves a dual role for Gal-1 as a critical scaffolding protein and a molecular chaperone that contributes to H-Ras trafficking by returning depalmitoylated H-Ras to the Golgi complex for repalmitoylation.


INF2 is an endoplasmic reticulum-associated formin protein.
In addition to its ability to accelerate filament assembly, which is common to formins, INF2 is a formin protein with the unique biochemical ability to accelerate actin filament depolymerization. The depolymerization activity of INF2 requires its actin monomer-binding WASP homology 2 (WH2) motif. In this study, we show that INF2 is peripherally bound to the cytoplasmic face of the endoplasmic reticulum (ER) in Swiss 3T3 cells. Both endogenous INF2 and GFP-fusion constructs display ER localization. INF2 is post-translationally modified by a C-terminal farnesyl group, and this modification is required for ER interaction. However, farnesylation is not sufficient for ER association, and membrane extraction experiments suggest that ionic interactions are also important. The WH2 motif also serves as a diaphanous autoregulatory domain (DAD), which binds to the N-terminal diaphanous inhibitory domain (DID), with an apparent dissociation constant of 1.1 muM. Surprisingly, the DID-DAD interaction does not inhibit the actin nucleation activity of INF2; however, it does inhibit the depolymerization activity. Point mutations to the DAD/WH2 inhibit both the DID-DAD interaction and depolymerization activity. Expression of GFP-INF2 containing these DAD/WH2 mutations causes the ER to collapse around the nucleus, with accumulation of actin filaments around the collapsed ER. This study is the first to show the association of an actin-assembly factor with the ER.


Altered pattern of Cul-1 protein expression and neddylation in human lung tumours: relationships with CAND1 and cyclin E protein levels.
The Cul-1 protein is the scaffold element of SCF complexes that are involved in the proteasomal degradation of numerous proteins regulating cell cycle progression. Owing to this central role in cell growth control, aberrant expression of the components of SCF is thought to play a role during tumourigenesis. Nothing is known about Cul-1 expression in human tumours. In this study, we have analysed its status in a series of 128 human lung carcinomas, comprising 50 non-small cell lung cancers (NSCLCs; 29 squamous cell carcinomas and 21 adenocarcinomas) and 78 neuroendocrine (NE) lung tumours (24 typical and atypical carcinoids, 19 large cell NE carcinomas and 35 small cell lung carcinomas), using immunohistochemistry. We report for the first time an altered pattern of Cul-1 expression in human tumours; indeed, we show that Cul-1 expression is up-regulated in 40% (51/128) of all lung tumours as compared to normal lung tissues, including 34% (17/50), 75% (18/24) and 30% (16/54) of NSCLCs, carcinoids and high grade neuroendocrine lung carcinomas, respectively. Furthermore, we demonstrate that high levels of Cul-1 protein are associated with a low KI67 proliferative index (p = 0.005) and with a decrease in the cyclin E oncoprotein (p = 0.0003), one of the major targets of SCF complexes. These data suggest that up-regulation of Cul-1 could protect cells from hyperproliferative signals through cyclin E down-regulation. Cul-1 is modified by neddylation, a post-translational modification that grafts ubiquitin-like Nedd8/Rub1 residues and controls Cul-1 activity. We also provide evidence that neddylated forms of Cul-1 are specifically expressed in high-grade NE lung tumours and are associated with down-regulation of the Cul-1 inhibitor CAND1 (p = 0.03) and a high level of cyclin E (p = 0.0002). These data support the notion that alterations in the Cul-1 neddylation/deneddylation pathway could contribute to the development of these highly aggressive lung tumours.


Tyrosine sulfation of statherin.
Tyrosylprotein sulfotransferase (TPST), responsible for the sulfation of a variety of secretory and membrane proteins, has been identified and characterized in submandibular salivary glands (William et al. Arch Biochem Biophys 1997; 338: 90-96). In the present study we demonstrate the sulfation of a salivary secretory protein, statherin, by the tyrosylprotein sulfotransferase present in human saliva. Optimum statherin sulfation was observed at pH 6.5 and at 20 mm MnCl(2). Increase in the level of total sulfation was observed with increasing statherin concentration. The K(m)value of tyrosylprotein sulfotransferase for statherin was 40 microM. Analysis of the sulfated statherin product on SDS-polyacrylamide gel electrophoresis followed by autoradiography revealed (35)S-labelling of a 5 kDa statherin. Further analysis of the sulfated statherin revealed the sulfation on tyrosyl residue. This study is the first report demonstrating tyrosine sulfation of a salivary secretory protein. The implications of this sulfation of statherin in hydroxyapatite binding and Actinomyces viscosus interactions are discussed.