Posttranslational modification of proteins with fatty acids in platelets

Direct modification of proteins by fatty acid can occur as cotranslational N-myristoylation of an N-terminal glycine residue or as posttranslational thioesterification of cysteine residue(s). Platelets provide an excellent model system for studying the posttranslational type of modification in the absence of active protein synthesis and in the absence of protein synthesis-related protein modifications with lipids. Using this model system it was shown that thioesterification of proteins with fatty acid is less specific for palmitate than it was thought earlier and that other saturated, mono- and even polyunsaturated long chain fatty acids can also participate. The chain length and the extent of unsaturation of the protein-linked fatty acid moiety can, very likely, modulate hydrophobic protein-membrane lipid and protein-protein interactions. CD9, HLA class I glycoprotein, glycoproteins Ib, IX and IV, P-selectin and alpha subunits of G proteins have been demonstrated unequivocally as S-fatty acid acylated platelet proteins.     

Immunohistochemical localization of G protein beta1, beta2, beta3, beta4, beta5, and gamma3 subunits in the adult rat brain

The regional distributions of the G protein beta subunits (Gbeta1-beta5) and of the Ggamma3 subunit were examined by immunohistochemical methods in the adult rat brain. In general, the Gbeta and Ggamma3 subunits were widely distributed throughout the brain, with most regions containing several Gbeta subunits within their neuronal networks. The olfactory bulb, neocortex, hippocampus, striatum, thalamus, cerebellum, and brainstem exhibited light to intense Gbeta immunostaining. Negative immunostaining was observed in cortical layer I for Gbeta1 and layer IV for Gbeta4. The hippocampal dentate granular and CA1-CA3 pyramidal cells displayed little or no positive immunostaining for Gbeta2 or Gbeta4. No anti-Gbeta4 immunostaining was observed in the pars compacta of the substantia nigra or in the cerebellar granule cell layer and Purkinje cells. Immunoreactivity for Gbeta1 was absent from the cerebellar molecular layer, and Gbeta2 was not detected in the Purkinje cells. No positive Ggama3 immunoreactivity was observed in the lateral habenula, lateral septal nucleus, or Purkinje cells. Double-fluorescence immunostaining with anti-Ggamma3 antibody and individual anti-Gbeta1-beta5 antibodies displayed regional selectivity with Gbeta1 (cortical layers V-VI) and Gbeta2 (cortical layer I). In conclusion, despite the widespread overlapping distributions of Gbeta1-beta5 with Ggamma3, specific dimeric associations in situ were observed within discrete brain regions.     

Posttranslational modifications of the 5'-AMP-activated protein kinase beta1 subunit

The AMP-activated protein kinase (AMPK) consists of catalytic alpha and noncatalytic beta and gamma subunits and is responsible for acting as a metabolic sensor for AMP levels. There are multiple genes for each subunit and the rat liver AMPK alpha1 and alpha2 catalytic subunits are associated with beta1 and gamma1 noncatalytic subunits. We find that the isolated gamma1 subunit is N-terminally acetylated with no other posttranslational modification. The isolated beta1 subunit is N-terminally myristoylated. Transfection of COS cells with AMPK subunit cDNAs containing a nonmyristoylatable beta1 reduces, but does not eliminate, membrane binding of AMPK heterotrimer. The isolated beta1 subunit is partially phosphorylated at three sites, Ser24/25, Ser182, and Ser108. The Ser24/25 and Ser108 sites are substoichiometrically phosphorylated and can be autophosphorylated in vitro. The Ser-Pro site in the sequence LSSS182PPGP is stoichiometrically phosphorylated, and no additional phosphate is incorporated into this site with autophosphorylation. Based on labeling studies in transfected cells, we conclude that alpha1 Thr172 is a major, although not exclusive, site of both basal and stimulated alpha1 phosphorylation by an upstream AMPK kinase.     

Biochemical and structural analysis of the IgE binding sites on ara h1, an abundant and highly allergenic peanut protein

Allergy to peanut is a significant IgE-mediated health problem because of the high prevalence, potential severity, and chronicity of the reaction. Ara h1, an abundant peanut protein, is recognized by serum IgE from >90% of peanut-sensitive individuals. It has been shown to belong to the vicilin family of seed storage proteins and to contain 23 linear IgE binding epitopes. In this communication, we have determined the critical amino acids within each of the IgE binding epitopes of Ara h1 that are important for immunoglobulin binding. Surprisingly, substitution of a single amino acid within each of the epitopes led to loss of IgE binding. In addition, hydrophobic residues appeared to be most critical for IgE binding. The position of each of the IgE binding epitopes on a homology-based molecular model of Ara h1 showed that they were clustered into two main regions, despite their more even distribution in the primary sequence. Finally, we have shown that Ara h1 forms a stable trimer by the use of a reproducible fluorescence assay. This information will be important in studies designed to reduce the risk of peanut-induced anaphylaxis by lowering the IgE binding capacity of the allergen.     

The G protein alpha s subunit incorporates [3H]palmitic acid and mutation of cysteine-3 prevents this modification

We investigated whether alpha s could be acylated by palmitate by transfecting COS cells with the cDNA for the wild-type, long form of alpha s and metabolically labeling with [3H]palmitate or [35S]methionine. Cells were separated into particulate and soluble fractions and immunoprecipitated with a specific peptide antibody. [3H]Palmitate was incorporated into both endogenous and transfected alpha s. Inhibition of protein synthesis with cycloheximide did not block the radiolabeling of alpha s with [3H]palmitate. Hydroxylamine treatment caused a release of the tritium radiolabel, demonstrating that the incorporation was through a thioester bond. The tritium radiolabel was base-labile and comigrated with [3H]palmitate on thin-layer chromatography. The third residue of the wild-type alpha s was mutated from a cysteine to an alanine by site-directed mutagenesis. This mutant was expressed in COS cells and localized to the particulate fraction as determined by immunoprecipitation of the [35S]methionine-labeled cells. The cysteine-3 mutant did not undergo radiolabeling with [3H]palmitate, indicating that this residue is crucial for the modification.     

KID-1, a protein kinase induced by depolarization in brain

Membrane depolarization leads to changes in gene expression that modulate neuronal plasticity. Using representational difference analysis, we have identified a previously undiscovered cDNA, KID-1 (kinase induced by depolarization), that is induced by membrane depolarization or forskolin, but not by neurotrophins or growth factors, in PC12 pheochromocytoma cells. KID-1 is an immediate early gene that shares a high degree of sequence similarity with the family of PIM-1 serine/threonine protein kinases. Recombinant KID-1 fusion protein is able to catalyze both histone phosphorylation and autophosphorylation. KID-1 mRNA is present in a number of unstimulated tissues, including brain. In response to kainic acid and electroconvulsive shock-induced seizures, KID-1 is induced in specific regions of the hippocampus and cortex.     

The Cdk-associated protein Cks1 functions both in G1 and G2 in Saccharomyces cerevisiae

The CKS1 gene of Saccharomyces cerevisiae encodes a small essential protein shown to interact genetically and physically with the Cdc28 protein kinase. To investigate the specific functions of the CKS1 gene product, conditional temperature-sensitive mutant alleles were generated. The mutations were found to impair the ability of cells to undergo both the G1/S-phase and G2/M-phase transitions of the cell cycle, as well as the ability to bud. Mutants were not defective, however, in their ability to activate Cdc28 kinase as assayed in vitro on the substrate histone H1. It is likely, therefore, that Cks1 mediates a more specialized function of the Cdc28 kinase such as its ability to form specific multimeric complexes or to localize properly in cellular compartments.     

Subunit of glycosylation-inhibiting factor is an abundant protein that binds to certain glycoproteins and sugars

The adherence of Staphylococcus aureus to biomaterials used in orthopaedic surgery (polymethylmethacrylate, fresh bone, steel and titanium alloys) and to glass was studied in vitro at 1, 2, 6, 24 and 48 h of incubation. Nonslime-producing strains (72, 80 and 510) and slime-producing variants of these strains were used. An automated and fast method of ATP-bioluminiscence was applied to determine bacterial viability. The lowest adherence corresponded to polymethylmethacrylate and bone, and the highest to metals. Significant adherence was detected in all cases after 6 h and was strain dependent, being lowest for strain 72. In most cases, adherence of nonslime-producing variants was not significant compared with controls, and slime-producing were more adherent than nonslime-producing variants. These differences were maximal at 6 h or 48 h, depending on the strain and the material. The findings suggest that the appearance of slime-producing cells within a given nonslime-producing bacterial population may jeopardise postoperative immune systems and antibiotic efficacy as a consequence of biofilm formation on implants and prostheses.     

Somatostatin receptor-mediated signaling in smooth muscle. Activation of phospholipase C-beta3 by Gbetagamma and inhibition of adenylyl cyclase by Galphai1 and Galphao

In COS-7 cells, all five cloned somatostatin receptors are coupled via inhibitory G proteins to activation of an unidentified phospholipase C-beta (PLC-beta) isozyme and inhibition of adenylyl cyclase. In the present study, intestinal smooth muscle cells (SMC) that express only one receptor type, sstr3, and possess a full complement of G proteins and PLC-beta isozymes were used to identify the PLC-beta isozyme and the G proteins coupled to it and to adenylyl cyclase. Somatostatin-14 bound with high affinity to intestinal SMC; stimulated D-myo-inositol-1,4,5-trisphosphate (IP3) formation, Ca2+ release, and contraction; and inhibited forskolin-stimulated cAMP formation in a pertussis toxin-sensitive fashion. Somatostatin also stimulated phosphoinositide hydrolysis in plasma membranes. Only those somatostatin analogs that shared a high affinity for sstr3 receptors elicited muscle contraction. IP3 formation, Ca2+ release, and contraction in permeabilized SMC and phosphoinositide hydrolysis in plasma membranes were inhibited (approximately 80%) by pretreatment with antibodies to PLC-beta3 but not other PLC-beta isozymes, and by antibodies to Gbeta but not Galpha. Inhibition of cAMP formation was partially blocked by antibody to Galphai1 or Galphao and additively blocked by a combination of both antibodies. Somatostatin-stimulated [35S]GTPgammaS-Galpha complexes in plasma membranes were bound selectively by Galphai1 and Galphao antibodies. We conclude that in smooth muscle sstr3 is coupled to Gi1 and Go; the alpha subunits of both G proteins mediate inhibition of adenylyl cyclase, while the betagamma subunits mediate activation of PLC-beta3.     

Activation of Rap1, antagonist to ras, by Crk-C3G

Rap1 was identified as gene whose overexpression suppressed transformation by ras. Rap1 belongs to the Ras family. The amino acid sequences of Rap1 and Ras show 55% identity to each other. Due to this high sequence similarity, Rap1 binds to effector molecules of Ras, however, Rap1 does not activate them. Thus, Rap1 functions are antagonistic to Ras in the cells. C3G was identified as a Crk SH3-binding guanine nucleotide exchange factor. Biochemical and cell biological analyses revealed that C3G is a Rap1 activator. Since it has been considered that Crk transduces signals from tyrosine kinases, this finding suggests that the activity of Rap1 is also under the control of tyrosine kinases. Overexpression of C3G in ras-transformed cells caused the morphology of the cells to revert to that of normal cells. Moreover, a mutant cell line that was resistant to EGF-dependent transformation was isolated. In the cell line a mutation was found in crk gene that was the cause of the resistance. These findings suggest that Crk-C3G-Rap1 pathway may function as an anti-transformation machinery.     

Anandamide, an endogenous cannabinomimetic substance, modulates rat brain protein kinase C in vitro

Anandamide (AnNH, N-arachidonoyl-ethanolamine) has been recently proposed as the endogenous ligand for mammalian brain cannabinoid receptor. Non-cannabinoid receptor-mediated, intracellular actions have been also found for this novel mediator. Here we present evidence for the modulation by anandamide of rat brain protein kinase C (PKC) activity in vitro. The ethanolamide of arachidonic acid (AA) was more active than the free acid in increasing phosphatidylserine (PS)-induced PKC activation (EC50 = 40 microM), but inhibited dioleylglycerol-induced potentiation of both Ca(2+)- and Ca2+/PS-induced PKC activation (IC50 = 8 microM and 30 microM, respectively). A dual modulatory action of anandamide on PKC, exerted by binding to the diacylglycerol regulatory site, is hypothesized in rat brain.     

Biochemical and immunological analysis of an abundant form of glutathione S-transferase, in mouse testis

One of the major forms of glutathione S-transferase (designated as Ft transferase) has been identified and purified to near homogeneity from mouse testis. The purification was achieved by ammonium sulfate fractionation, DEAE cellulose chromatography, hydroxylapatite chromatography and the preparative isoelectric focusing. Purified Ft transferase has an isoelectric point of 4.9 +/- 0.3 and was shown to be a homodimer with a native molecular weight of about 50000. Immunologically, antisera to Ft transferase do not crossreact with F2 or F3 transferase. However, a weak cross reactivity was observed between the antisera to F3 transferase and FT transferase. Biochemical properties of purified Ft transferase are similar to those transferases isolated from mouse liver. Tissue distributions of the multiple forms of glutathione S-transferase were examined by column isoelectric focusing of various mouse tissue homogenates. It was found that mouse Ft transferase is present only in testis as a major form and in brain as a minor form, but not in other tissues that were examined.     

Activation of PI 3-kinase by G protein betagamma subunits

We have reported that fMLP-induced activation of pertussis toxin-sensitive GTP-binding proteins in THP-1 cells potentiates the insulin-induced accumulation of PtdIns(3,4,5)P3, a product of phosphoinositide 3-kinase (T. Okada et al., Biochem. J. 317, 475-480, 1996). The synergism in PtdIns(3,4,5)P3 accumulation was observed in Chinese hamster ovary cells expressing both insulin and fMLP receptors. In rat adipocytes, which represent the physiological target cells of insulin, receptor-mediated activation of GTP-binding protein by adenosine and prostaglandin E2 potentiated the insulin-induced PtdIns(3,4,5)P3 accumulation. In cell-free systems, the activity of the p85/p110beta subtype of phosphoinositide 3-kinase was, while that of p85/p110alpha was not, stimulated by the betagamma subunits of the GTP-binding proteins. We propose here a hypothesis that the p85/p110beta subtype is under the control of both the insulin receptors and the GTP-binding protein-coupled receptors in intact cell systems.     

3H-morphine-6beta-glucuronide binding in brain membranes and an MOR-1-transfected cell line

Morphine-6beta-glucuronide (M6G) is a potent morphine metabolite. In an effort to further explore its mechanisms of action, we synthesized 3H-M6G of high specific activity and examined its binding. Although its affinity toward traditional mu receptors is similar to morphine in binding assays in brain and in Chinese hamster ovary cells stably transfected with MOR-1, M6G is >100-fold more potent than morphine in analgesic assays. This apparent discrepancy cannot be explained by differing intrinsic activities of the two drugs because both agents are partial agonists with similar efficacies in adenylyl cyclase assays in the transfected cell lines. Behavioral studies have implied the possibility of a distinct M6G receptor. Detailed binding studies in brain tissue reveal evidence for heterogeneity. Nonlinear regression analysis of 3H-M6G saturation studies reveals two components. The lower-affinity component (K(D) = 1.93 +/- 0.6 nM) corresponds to labeling of traditional mu receptors. In addition, 3H-M6G labels another site of low abundance with very high affinity (K(D) = 68 +/- 7 pM). Competition studies indicate that both sites are relatively mu selective. However, several compounds clearly distinguish between the two sites. These binding studies support the concept of a unique M6G receptor responsible for its analgesic activity.     

MP1 encodes an abundant and highly antigenic cell wall mannoprotein in the pathogenic fungus Penicillium marneffei

We characterized the inhibitory activity of several acetylenic and olefinic compounds on cytochrome P450 (CYP)-derived arachidonic acid omega-hydroxylation and epoxidation using rat renal cortical microsomes and recombinant CYP proteins. Among the acetylenic compounds, 6-(2-propargyloxyphenyl)hexanoic acid (PPOH) and N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide were found to be potent and selective inhibitors of microsomal epoxidation with IC50 values of 9 and 13 microM, respectively. On the other hand, 17-octadecynoic acid inhibited both omega-hydroxylation and epoxidation of arachidonic acid with IC50 values of 7 and 5 microM, respectively. The olefinic compounds N-methylsulfonyl-12, 12-dibromododec-11-enamide (DDMS) and 12, 12-dibromododec-11-enoic acid (DBDD) exhibited a high degree of selectivity inhibiting microsomal omega-hydroxylation with an IC50 value of 2 microM, whereas the IC50 values for epoxidation were 60 and 51 microM for DDMS and DBDD, respectively. Studies using recombinant rat CYP4A isoforms showed that PPOH caused a concentration-dependent inhibition of omega-hydroxylation and 11, 12-epoxidation by CYP4A3 or CYP4A2 but had no effect on CYP4A1-catalyzed omega-hydroxylase activity. On the other hand, DDMS inhibited both CYP4A1- and CYP4A3- or CYP4A2-catalyzed arachidonic acid oxidations. Inhibition of microsomal activity by PPOH, but not DDMS, was time- and NADPH-dependent, a result characteristic of a mechanism-based irreversible inhibitor. These studies provide information useful for evaluating the role of the CYP-derived arachidonic acid metabolites in the regulation of renal function and blood pressure.     

MCC, a cytoplasmic protein that blocks cell cycle progression from the G0/G1 to S phase

The MCC gene was isolated from the human chromosome 5q21 by positional cloning and was found to be mutated in several colorectal tumors. In this study, we prepared specific antibodies and detected the MCC gene product as a cytoplasmic 100-kDa phosphoprotein in mouse NIH3T3 cells. Immunoelectron microscopic analysis showed that the MCC protein is associated with the plasma membrane and membrane organelles in mouse intestinal epithelial cells and neuronal cells. The amount of the MCC protein remained constant during the cell cycle progression of NIH3T3 cells, while its phosphorylation state changed markedly in a cell cycle-dependent manner, being weakly phosphorylated in the G0/G1 and highly phosphorylated during the G1 to S transition. Overexpression of the MCC protein blocked the serum-induced cell cycle transition from the G1 to S phase, whereas a mutant MCC, initially identified in a colorectal tumor, did not exhibit this activity. These results suggest that the MCC protein may play a role in the signaling pathway negatively regulating cell cycle progression.     

Activation of G1 progression, JNK mitogen-activated protein kinase, and actin filament assembly by the exchange factor FGD1

Cdc42 has been shown to control bifurcating pathways leading to filopodia formation/G1 cell cycle progression and to JNK mitogen-activated protein kinase activation. To dissect these pathways further, the cellular effects induced by a Cdc42 guanine nucleotide exchange factor, FGD1, have been examined. All exchange factors acting on the Rho GTPase family have juxtaposed Dbl homology (DH) and pleckstrin homology (PH) domains. We report here that FGD1 triggers G1 cell cycle progression and filopodia formation in Swiss 3T3 fibroblasts as well as JNK mitogen-activated protein kinase activation in COS cell transfection assays. FGD1-induced filopodia formation is Cdc42-dependent, and both the DH and PH domains are essential. Although expression of the FGD1 DH domain alone does not activate Cdc42 and induce filopodia, it does trigger both the JNK cascade in COS cells and G1 progression in quiescent Swiss 3T3 cells. We conclude that FGD1 can trigger G1 progression independently of actin polymerization or integrin adhesion complex assembly. Furthermore, since FGD1 activates JNK and G1 progression in a Cdc42-independent manner, it must have additional, as yet unidentified, targets.     

Activation of brain B-Raf protein kinase by Rap1B small GTP-binding protein

Rap1 small GTP-binding protein has the same amino acid sequence at its effector domain as that of Ras. Rap1 has been shown to antagonize the Ras functions, such as the Ras-induced transformation of NIH 3T3 cells and the Ras-induced activation of the c-Raf-1 protein kinase-dependent mitogen-activated protein (MAP) kinase cascade in Rat-1 cells, whereas we have shown that Rap1 as well as Ras stimulates DNA synthesis in Swiss 3T3 cells. We have established a cell-free assay system in which Ras activates bovine brain B-Raf protein kinase. Here we have used this assay system and examined the effect of Rap1 on the B-Raf activity to phosphorylate recombinant MAP kinase kinase (MEK). Recombinant Rap1B stimulated the activity of B-Raf, which was partially purified from bovine brain and immunoprecipitated by an anti-B-Raf antibody. The GTP-bound form was active, but the GDP-bound form was inactive. The fully post-translationally lipid-modified form was active, but the unmodified form was nearly inactive. The maximum B-Raf activity stimulated by Rap1B was nearly the same as that stimulated by Ki-Ras. Rap1B enhanced the Ki-Ras-stimulated B-Raf activity in an additive manner. These results indicate that not only Ras but also Rap1 is involved in the activation of the B-Raf-dependent MAP kinase cascade.