A novel human gene similar to the protein kinase (PK) coding domain of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) codes for a serine-threonine PK and is expressed in melanoma cells

The large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) is a multifunctional protein that contains a serine-threonine protein kinase (PK) activity (Nelson, J. W., Zhu, J. , Smith, C. C., Kulka, M., and Aurelian, L. (1996) J. Biol. Chem. 271, 17021-17027). Phylogenetic analyses indicated that ICP10 PK belongs to a distinct subfamily of growth factor receptor serine-threonine PKs that are characterized by their ability to function with a limited number of conserved catalytic motifs (Hunter, J. C. R., Smith, C. C., and Aurelian, L. (1995) Int. J. Onc. 7, 515-522). Here, we report the isolation and characterization of a novel gene, designated H11, that contains an open reading frame of 588 nucleotides, which encodes a protein similar to ICP10 PK. The H11 protein has Mn(2+)-dependent serine-threonine-specific PK activity as determined with a GST-H11 fusion protein and by immununocomplex PK/immunoblotting assays of 293 cells transfected with a H11 eukaryotic expression vector. PK activity is ablated by mutation of Lys(113) within the presumtive catalytic motif II (invariant Lys). 293 cells stably transfected with H11 acquire anchorage-independent growth. Endogenous H11 RNA and the H11 phosphoprotein are expressed in melanoma cell lines and primary melanoma tissues at levels higher than in normal melanocytes and in benign nevi. Melanoma cell proliferation is inhibited by treatment with antisense oligonucleotides that inhibit H11 translation, suggesting that H11 expression is associated with cell growth.     

The transmembrane helical segment but not the invariant lysine is required for the kinase activity of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10).

The large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) is a chimera consisting, at the amino terminus, of a Ser/Thr protein kinase (PK) with features of a signal peptide and a transmembrane (TM) helical segment, and at the carboxy-terminus, of the ribonucleotide reductase (Chung et al., 1989, 1990). Membrane immunofluorescence of ICP10 transformed cells with antibodies to synthetic peptides located upstream or downstream of the TM indicates that ICP10 is a membrane-spanning protein. Site-directed and deletion mutants were used to further characterize ICP10-PK. Mutation of Gly106 in catalytic motif I or of the invariant Lys in catalytic motif II, and deletion of both motifs (amino acids 106-178) did not eliminate kinase activity. PK activity was retained by the invariant Lys mutant expressed in bacteria and following protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to membrane filters. Both ICP10 and the invariant Lys mutant bound 14C-labeled rho-fluorosulfonylbenzoyl 5'-adenosine, an ATP affinity analog. The deletion mutant had 4-fold lower kinase activity than ICP10-PK, and it was insensitive to Mn2+, suggesting that these motifs are involved in Mn2+ activation of kinase activity. PK activity was lost by deletion of the TM segment (amino acid residues 85-106).     

The herpes simplex virus type 2 R1 protein kinase (ICP10 PK) blocks apoptosis in hippocampal neurons, involving activation of the MEK/MAPK survival pathway.

Herpes simplex virus type 1 (HSV-1) and HSV-2 trigger or counteract apoptosis by a cell-specific mechanism. Our studies are based on previous findings that the protein kinase (PK) domain of the large subunit of HSV-2 ribonucleotide reductase (ICP10) activates the Ras/MEK/MAPK pathway (Smith et al., J. Virol. 74:10417, 2000). Because survival pathways can modulate apoptosis, we used cells that are stably or transiently transfected with ICP10 PK, an HSV-2 mutant deleted in ICP10 PK (ICP10DeltaPK) and the MEK-specific inhibitor U0126 to examine the role of ICP10 PK in apoptosis. Apoptosis was induced by staurosporine or D-mannitol in human (HEK293) cells or HEK293 cells stably transfected with the ICP10 PK-negative mutant p139 (JHL15), as determined by morphology, DNA fragmentation, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL), caspase-3 activation, and poly(ADP-ribose) polymerase (PARP) cleavage. HEK293 cells stably transfected with ICP10 (JHLa1) were protected from apoptosis. ICP10 but not p139 protected neuronally differentiated PC12 cells from death due to nerve growth factor withdrawal, and apoptosis (determined by TUNEL) and caspase-3 activation were seen in primary hippocampal cultures infected with ICP10DeltaPK but not with HSV-2 or a revertant virus [HSV-2(R)]. The data indicate that ICP10 has antiapoptotic activity under both paradigms and that it requires a functional PK activity. The apoptotic cells in primary hippocampal cultures were neurons, as determined by double immunofluorescence with fluorescein-labeled dUTP (TUNEL) and phycoerythrin-labeled antibodies specific for neuronal proteins (TuJ1 and NF-160). Protection from apoptosis was associated with MEK/MAPK activation, as evidenced by (i) increased levels of activated (phosphorylated) MAPK in HSV-2- but not ICP10DeltaPK-infected cultures and (ii) inhibition of MAPK activation by the MEK-specific inhibitor U0126. MEK and MAPK were activated by infection with UV-inactivated but not antibody-neutralized HSV-2, suggesting that activation requires cellular penetration but is independent of de novo viral protein synthesis.     

The PK Domain of the Large Subunit of Herpes Simplex Virus Type 2 Ribonucleotide Reductase (ICP10) Is Required for Immediate-Early Gene Expression and Virus Growth

The large subunit of herpes simplex virus (HSV) ribonucleotide reductase (RR), RR1, contains a unique amino-terminal domain which has serine/threonine protein kinase (PK) activity. To examine the role of the PK activity in virus replication, we studied an HSV type 2 (HSV-2) mutant with a deletion in the RR1 PK domain (ICP10ΔPK). ICP10ΔPK expressed a 95-kDa RR1 protein (p95) which was PK negative but retained the ability to complex with the small RR subunit, RR2. Its RR activity was similar to that of HSV-2. In dividing cells, onset of virus growth was delayed, with replication initiating at 10 to 15 h postinfection, depending on the multiplicity of infection. In addition to the delayed growth onset, virus replication was significantly impaired (1,000-fold lower titers) in nondividing cells, and plaque-forming ability was severely compromised. The RR1 protein expressed by a revertant virus [HSV-2(R)] was structurally and functionally similar to the wild-type protein, and the virus had wild-type growth and plaque-forming properties. The growth of the ICP10ΔPK virus and its plaque-forming potential were restored to wild-type levels in cells that constitutively express ICP10. Immediate-early (IE) genes for ICP4, ICP27, and ICP22 were not expressed in Vero cells infected with ICP10ΔPK early in infection or in the presence of cycloheximide, and the levels of ICP0 and p95 were significantly (three- to sevenfold) lower than those in HSV-2- or HSV-2(R)-infected cells. IE gene expression was similar to that of the wild-type virus in cells that constitutively express ICP10. The data indicate that ICP10 PK is required for early expression of the viral regulatory IE genes and, consequently, for timely initiation of the protein cascade and HSV-2 growth in cultured cells.     

Pyruvate kinase type M2 is phosphorylated at tyrosine residues in cells transformed by Rous sarcoma virus

Chicken embryo cells (CECs) contain pyruvate kinase (PK) type M2 (M2-PK). Transformation of CECs by Rous sarcoma virus (RSV) leads to a reduction in the affinity of PK for the substrate phosphoenolpyruvate. In vitro, M2-PK can be phosphorylated at tyrosine residues by pp60v-src, the transforming protein of RSV. To study tyrosine phosphorylation of M2-PK in intact RSV-transformed cells, the protein was immunoprecipitated from 32P-labeled normal and RSV-SR-A-transformed CECs. Phosphoamino acid analysis of immunoprecipitated M2-PK revealed that M2-PK of both normal and transformed CECs contained phosphoserine and small amounts of phosphothreonine. Only M2-PK of transformed CECs contained phosphotyrosine in addition. For enzyme kinetic studies M2-PK was partially purified by chromatography upon DEAE-Sephacel and hydroxyapatite. A decreased affinity for phosphoenolpyruvate was observed 3 h after the onset of transformation using the temperature-sensitive mutant of RSV, ts-NY 68. The kinetic changes were correlated with tyrosine phosphorylation of M2-PK, but there is no direct evidence that they are caused by post-translational modification of the enzyme.     

Protein kinase activity associated with the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10).

The large subunit of the herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (RR1) is demonstrated to possess serine/threonine-specific kinase activity. Computer-assisted sequence analysis identified regions within the amino terminus of ICP10 that are homologous to the catalytic domain of known protein kinases (PKs). An in vitro kinase assay confirmed the ability of ICP10, immunoprecipitated from either HSV-2-infected cells or from cells transfected with an ICP10 expression vector, to autophosphorylate and transphosphorylate exogenous substrates in the presence of ATP and Mg2+. The HSV-1 RR1 was shown to be negative for PK activity under these conditions. PK activity was localized to a 57-kDa amino-terminal region within ICP10 that lacked RR activity.     

Protein kinase C phosphorylation of desmin at four serine residues within the non-alpha-helical head domain

We reported that phosphorylation by either cAMP-dependent protein kinase or protein kinase C (Ca2+/phospholipid-dependent enzyme) in vitro induces disassembly of the desmin filaments (Inagaki, M., Gonda, Y., Matsuyama, M., Nishizawa, K., Nishi, Y., and Sato, C. (1988) J. Biol. Chem. 263, 5970-5978). For this subunit protein, Ser-29, Ser-35, and Ser-50 within the non-alpha-helical head domain were shown to be the sites of phosphorylation for cAMP-dependent protein kinase (Geisler, N., and Weber, K. (1988) EMBO J. 7, 15-20). In the present work, we identified the sites of desmin phosphorylated in vitro by other protein kinase which affects the filament structure. The protein kinase C-phosphorylated desmin was hydrolyzed with trypsin, and the phosphorylated peptides were isolated by reverse-phase chromatography. Sequential analysis of the purified phosphopeptides, together with the known primary sequence, revealed that Ser-12, Ser-29, Ser-38, and Ser-56 were phosphorylated by protein kinase C. All four sites are located within the non-alpha-helical head domain of desmin. Ser-12, Ser-38, and Ser-56, specifically phosphorylated by protein kinase C, have arginine residues at the carboxyl-terminal side (Arg-14, Arg-42, and Arg-59, respectively). Ser-29 phosphorylated by both protein kinase C and cAMP-dependent protein kinase has arginine residues at the amino and carboxyl termini (Arg-27 and Arg-33). These findings support the view that the head domain-specific phosphorylation strongly influences desmin filament structure; however, each protein kinase differed with regard to site recognition on this domain.     

A novel protein phosphatase is a binding partner for the protein kinase domains of UNC-89 (Obscurin) in Caenorhabditis elegans

Mutation of the Caenorhabditis elegans gene unc-89 results in disorganization of muscle A-bands. unc-89 encodes a giant polypeptide (900 kDa) containing two protein kinase domains, PK1 and PK2. Yeast two-hybrid screening using a portion of UNC-89 including PK2, yielded SCPL-1 (small CTD phosphatase-like-1), which contains a C terminal domain (CTD) phosphatase type domain. In addition to the PK2 domain, interaction with SCPL-1 required the putative autoinhibitory sequence, and immunoglobulin (Ig) and fibronectin type 3 (Fn3) domains lying N-terminal of the kinase domain. SCPL-1 also interacts with PK1, and it similarly requires the kinase domain and upstream Fn3 and Ig domains. Analogous regions from the two other giant kinases of C. elegans, twitchin and TTN-1, failed to interact with SCPL-1. The interaction between SCPL-1 and either Ig-Fn3-PK2 or Fn3-Ig-PK1 was confirmed by biochemical methods. The scpl-1b promoter is expressed in the same set of muscles as unc-89. Antibodies to SCPL-1 localize to the M-line and a portion of the I-band. Bacterially expressed SCPL-1 proteins have phosphatase activity in vitro with properties similar to previously characterized members of the CTD phosphatase family. RNA interference knockdown results in a defect in the function of egg-laying muscles. These studies suggest a new role for the CTD phosphatase family, that is, in muscle giant kinase signaling.     

Structural and functional linkages between subunit interfaces in mammalian pyruvate kinase

Mammalian pyruvate kinase (PK) is a four-domain enzyme that is active as a homo-tetramer. Tissue-specific isozymes of PK exhibit distinct levels of allosteric regulation. PK expressed in muscle tissue (M1-PK) shows hyperbolic steady-state kinetics, whereas PK expressed in kidney tissue (M2-PK) displays sigmoidal kinetics. Rabbit M1 and M2-PK are isozymes whose sequences differ in only 22 out of 530 residues per subunit, and these changes are localized in an inter-subunit interface. Previous studies have shown that a single amino acid mutation to M1-PK at either the Y (S402P) or Z (T340 M) subunit interface can confer a level of allosteric regulation that is intermediate to M1-PK and M2-PK. In an effort to elucidate the roles of the inter-subunit interaction in signal transmission and the functional/structural connectivity between these interfaces, the S402P mutant of M1-PK was crystallized and its structure resolved to 2.8 A. Although the overall S402P M1-PK structure is nearly identical with the wild-type structure within experimental error, significant differences in the conformation of the backbone are found at the site of mutation along the Y interface. In addition, there is a significant change along the Z interface, namely, a loss of an inter-subunit salt-bridge between Asp177 of domain B and Arg341 of domain A of the opposing subunit. Concurrent with the loss of the salt-bridge is an increase in the degree of rotational flexibility of domain B that constitutes the active site. Comparison of previous PK structures shows a correlation between an increase in this domain movement with the loss of the Asp177: Arg341 salt-bridge. These results identify the structural linkages between the Y and Z interfaces in regulating the interconversion of conformational states of rabbit M1-PK.     

The herpes simplex virus type 2 gene ICP10PK protects from apoptosis caused by nerve growth factor deprivation through inhibition of caspase-3 activation and XIAP up-regulation

The herpes simplex virus type 2 (HSV-2) protein ICP10PK has anti-apoptotic activity in virus-infected hippocampal cultures through activation of the Ras/Raf-1/MEK/ERK pathway. To exclude the possible contribution of other viral proteins to cell fate determination, we examined the survival of primary hippocampal cultures and neuronally differentiated PC12 cells transfected with ICP10PK from apoptosis caused by nerve growth factor (NGF) withdrawal. NGF deprivation caused apoptosis in cultures mock-transfected or transfected with the kinase-negative ICP10 mutant p139(TM), but not in ICP10PK-transfected cultures. In one clone (PC47), ICP10PK inhibited caspase-3 activation through up-regulation/stabilization of adenylate cyclase (AC), activation of PKA and MEK, and the convergence of the two pathways on extracellular signal-regulated kinase activation. The anti-apoptotic proteins Bag-1 and Bcl-2 were stabilized and the pro-apoptotic protein Bad was phosphorylated (inactivated). In another clone (PC70), ICP10PK inhibited apoptosis through MEK-dependent up-regulation of the anti-apoptotic protein XIAP (that inhibits the activity of processed caspase-3) and down-regulation of the apoptogenic protein Smac/DIABLO. This may be cell-type specific, but the baculovirus p35 protein did not potentiate the neuroprotective activity of ICP10PK in PC12 cells, suggesting that ICP10PK inhibits both caspase activation and activity. The data indicate that ICP10PK inhibits apoptosis independent of other viral proteins and is a promising neuronal gene therapy platform.     

Protein-kinase-C-catalyzed phosphorylation of the microtubule-binding domain of microtubule-associated protein 2 inhibits its ability to induce tubulin polymerization

It has previously been demonstrated that microtubule-associated protein 2 (MAP2) is a good substrate for the purified protein kinase C [Tsuyama, S., Bramblett, G. T., Huang, K.-P. & Flavin, M. (1986) J. Biol. Chem. 261, 4110-4116; Akiyama, T., Nishida, E., Ishida, J., Saji, N., Ogawara, H., Hoshi, M., Miyata, Y. & Sakai, H. (1986) J. Biol. Chem. 261, 15648-15651]. We have shown here that phosphorylation of MAP2, catalyzed by protein kinase C, reduces the ability to induce tubulin polymerization. MAP2 is divided into two domains by digestion with alpha-chymotrypsin; the microtubule-binding and the non-binding (projection) domains. The limited chymotryptic digestion following phosphorylation of MAP2 by protein kinase C has shown that both the domains of MAP2 were phosphorylated by protein kinase C, 50-60% of the incorporated phosphates being detected in the microtubule-binding domain. Polypeptide fragments, containing the microtubule-binding domain of MAP2, were purified by DEAE-cellulose column chromatography after chymotryptic digestion of MAP2. The purified microtubule-binding fragments were competent to polymerize tubulin, and served as good substrates for protein kinase C. The phosphorylation of the microtubule-binding fragments by protein kinase C reduced their ability to induce tubulin polymerization. These results suggest that the ability of MAP2 to induce tubulin polymerization is inhibited by phosphorylation of the microtubule-binding domain catalyzed by protein kinase C.     

Erythropoietin induces cytosolic protein phosphorylation and dephosphorylation in erythroid cells.

Erythropoietin, the prime regulator of red blood cell growth and differentiation, causes rapid changes in the phosphorylation of several integral plasma membrane proteins (Choi, H-S., Wojchowski, D. M., and Sytkowski, A. J. (1987) J. Biol. Chem. 262, 2933-2936; Choi, H-S., Bailey, S. C., Donahue, K. A., Vanasse, G. J., and Sytkowski, A. J. (1990) J. Biol. Chem. 265, 4143-4148). In the present study we have demonstrated that erythropoietin's signal is transduced rapidly to the cytosol resulting in specific phosphorylation/dephosphorylation events. Erythropoietin treatment of Rauscher murine erythroleukemia cells previously labeled with [32P]orthophosphate results in a rapid increase in phosphorylation of two cytosolic proteins, designated pp96 and pp80, and a decrease in phosphorylation of another protein, designated pp90. The relative molecular mass and pI of pp80 are virtually identical to those reported for the protein kinase C substrate p80, or "MARCKS protein." Treatment of the cells with 12-O-tetradecanoylphorbol-13-acetate also increases pp80 but not pp96 phosphorylation, suggesting that erythropoietin triggers a protein kinase C-dependent pathway to pp80 and a protein kinase C-independent pathway to pp96. The effect of erythropoietin on pp96 phosphorylation was also shown in nontransformed erythroid cells isolated from the spleens of phenylhydrazine-treated mice. In contrast, almost no 32P labeling of pp80 or pp90 was detected, and pp80 and pp90 protein were nearly absent from these normal cells. These differences in expression and phosphorylation of erythropoietin-sensitive phosphoproteins may be related to the growth factor independence or dependence of the erythroid cells.     

Differential and common recognition of the catalytic sites of the cGMP-dependent and cAMP-dependent protein kinases by inhibitory peptides derived from the heat-stable inhibitor protein

Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein of cAMP-dependent protein kinase (Cheng, H.-C., Kemp, B. E., Pearson, R. B., Smith, A. J., Misconi, L., Van Patten, S. M., and Walsh, D. A. (1986) J. Biol. Chem. 261, 989-992) were tested as inhibitors of cGMP-dependent protein kinase. The peptides themselves were not substrates. cGMP-dependent protein kinase activity was assayed using histone H2B and two synthetic peptide substrates. Consistent with previous observations of other peptide inhibitors of this enzyme (Glass, D. B. (1983) Biochem. J. 213, 159-164), the inhibitory peptides had no effect on the phosphorylation of histone H2B, but they competitively inhibited cGMP-dependent phosphorylation of the two peptide substrates. The parent inhibitor peptide, PKI(5-24)amide, and a series of analogs had Ki (or IC50) values for cGMP-dependent protein kinase in the range of 15-190 microM. In contrast to their effects on the cAMP-dependent protein kinase, the inhibitory peptides were substantially less potent with cGMP-dependent protein kinase, and potency was reduced by the presence of the NH2-terminal residues (residues 5-13). We conclude that the two protein kinases share a recognition of the basic amino acid cluster within the pseudosubstrate region of the peptide, but that the cGMP-dependent protein kinase does not recognize additional NH2-terminal determinants that make the inhibitor protein extremely potent toward the cAMP-dependent enzyme. Even- when tested at high concentrations and with peptide substrates, the native inhibitor protein did not inhibit cGMP-dependent protein kinase under assay conditions in which the peptides derived from it were inhibitory. Thus, the native inhibitor protein appears to have structural features which block interaction with the cGMP-dependent enzyme and enhance its selectivity for cAMP-dependent protein kinase.     

The effector domain of myristoylated alanine-rich C kinase substrate binds strongly to phosphatidylinositol 4,5-bisphosphate

Both the myristoylated alanine-rich protein kinase C substrate protein (MARCKS) and a peptide corresponding to its basic effector domain, MARCKS-(151-175), inhibit phosphoinositide-specific phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP(2)) in vesicles (Glaser, M., Wanaski, S., Buser, C. A., Boguslavsky, V., Rashidzada, W., Morris, A., Rebecchi, M., Scarlata, S. F., Runnels, L. W., Prestwich, G. D., Chen, J., Aderem, A., Ahn, J., and McLaughlin, S. (1996) J. Biol. Chem. 271, 26187-26193). We report here that adding 10-100 nm MARCKS-(151-175) to a subphase containing either PLC-delta or -beta inhibits hydrolysis of PIP(2) in a monolayer and that this inhibition is due to the strong binding of the peptide to PIP(2). Two direct binding measurements, based on centrifugation and fluorescence, show that approximately 10 nm PIP(2), in the form of vesicles containing 0.01%, 0.1%, or 1% PIP(2), binds 50% of MARCKS-(151-175). Both electrophoretic mobility measurements and competition experiments suggest that MARCKS-(151-175) forms an electroneutral complex with approximately 4 PIP(2). MARCKS-(151-175) binds equally well to PI(4,5)P(2) and PI(3,4)P(2). Local electrostatic interactions of PIP(2) with MARCKS-(151-175) contribute to the binding energy because increasing the salt concentration from 100 to 500 mm decreases the binding 100-fold. We hypothesize that the effector domain of MARCKS can bind a significant fraction of the PIP(2) in the plasma membrane, and release the bound PIP(2) upon interaction with Ca(2+)/calmodulin or phosphorylation by protein kinase C.     

Ligands of macrophage scavenger receptor induce cytokine expression via differential modulation of protein kinase signaling pathways

Our previous works demonstrated that ligands of macrophage scavenger receptor (MSR) induce protein kinases (PKs) including protein-tyrosine kinase (PTK) and up-regulate urokinase-type plasminogen activator expression (Hsu, H. Y., Hajjar, D. P., Khan, K. M., and Falcone, D. J. (1998) J. Biol. Chem. 273, 1240--1246). To continue to investigate MSR ligand-mediated signal transductions, we focus on ligands, oxidized low density lipoprotein (OxLDL), and fucoidan induction of the cytokines tumor necrosis factor-alpha (TNF) and interleukin 1 beta (IL-1). In brief, in murine macrophages J774A.1, OxLDL and fucoidan up-regulate TNF production; additionally, fucoidan but not OxLDL induces IL-1 secretion, prointerleukin 1 (proIL-1, precursor of IL-1) protein, and proIL-1 message. Simultaneously, fucoidan stimulates activity of interleukin 1-converting enzyme. We further investigate the molecular mechanism by which ligand binding-induced PK-mediated mitogen-activated protein kinase (MAPK) in regulation of expression of proIL-1 and IL-1. Specifically, fucoidan stimulates activity of p21-activated kinase (PAK) and of the MAPKs extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), and p38. Combined with PK inhibitors and genetic mutants of Rac1 and JNK in PK activity assays, Western blotting analyses, and IL-1 enzyme-linked immunosorbent assay, the role of individual PKs in the regulation of proIL-1/IL-1 was extensively dissected. Moreover, tyrosine phosphorylation of pp60Src as well as association between pp60Src and Hsp90 play important roles in fucoidan-induced proIL-1 expression. We are the first to establish two fucoidan-mediated signaling pathways: PTK(Src)/Rac1/PAK/JNK and PTK(Src)/Rac1/PAK/p38, but not PTK/phospholipase C-gamma 1/PKC/MEK1/ERK, playing critical roles in proIL-1/IL-1 regulation. Our current results indicate and suggest a model for MSR ligands differentially modulating specific PK signal transduction pathways, which regulate atherogenesis-related inflammatory cytokines TNF and IL-1.     

The pp60v-src tyrosine kinase desensitizes epidermal growth factor binding to 3T3 fibroblasts by two distinct protein kinase C-independent mechanisms

Protein kinase C (Ca2+/phospholipid-dependent enzyme) is known to phosphorylate the epidermal growth factor receptor and reduce its affinity for epidermal growth factor. Transformation of 3T3 fibroblasts by the oncogenic tyrosine kinase pp60v-src is accompanied by an elevation of cellular diacylglycerol and partial activation of protein kinase C (Wolfman, A., Wingrove, T. G., Blackshear, P. J., and Macara, I. G. (1987) J. Biol. Chem. 262, 16546-16552). We therefore asked whether pp60v-src can down-modulate the epidermal growth factor receptor. We report that within 15 min of activating temperature-sensitive pp60v-src, binding of 125I-labeled epidermal growth factor to 3T3 cells falls at least 50%. Two distinct processes control the down-modulation by pp60v-src. The first is rapid and transient, while the second requires protein synthesis and persists long after inactivation of pp60v-src. Surprisingly, both mechanisms seem to be protein kinase C-independent. Both operate by decreasing the affinity of the epidermal growth factor receptor for its ligand.     

Signal transduction through the vasoactive intestinal peptide receptor stimulates phosphorylation of the tyrosine kinase pp60c-src.

The present study demonstrates that signal transduction through a receptor lacking intrinsic tyrosine protein kinase activity involves a rapid and potent phosphorylation of a non-receptor tyrosine protein kinase in the membranes. Vasoactive intestinal peptide (VIP) stimulates phosphorylation of a membrane protein with a M.W. of 56 KD (pp60) in the cultured chick embryonic retinal pigment epithelium. VIP stimulates phosphorylation of the pp60 with such efficiency and potency that the maximal phosphorylation has been observed at the earliest time (3 minutes at 1 x 10(-6)M VIP) and the lowest concentration (1 x 10(-11)M for 20 minutes) examined. Western blot analysis with a monoclonal antibody anti-pp60src (GD11, Parsons et al., J. Virol. 51, 272-282, 1984) indicates that the pp60 is the pp60c-src, a normal cell oncogene product with intrinsic tyrosine protein kinase activity.