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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Inhibition of cell proliferation is an important biologic function of interferons (IFNs), which has been exploited in therapeutic treatment of certain hematologic malignancies. However, the molecular mechanism was not clear. We have recently shown that IFNs (alpha/beta and gamma) inhibit protein kinase C (PKC)-dependent (such as PDGF and phorbol ester) but not PKC-independent (such as epidermal growth factor) activation of Raf-1 and mitogen-activated protein kinases (MAPK/ERKs) in fibroblasts (Xu et al, Mol Cell Biol 14:8018, 1994), suggesting a novel mechanism by which IFNs execute their antiproliferative function. Monocytes/macrophages are primary targets in vivo for IFN-gamma, the major activity of macrophage-activating factor. In the present study, mechanism of IFN-gamma-induced antiproliferative action in macrophages in response to colony-stimulating factor-1 (CSF-1) has been investigated. Our results show that antiproliferative effect of IFN-gamma overrode mitogenic effect of CSF-1 and phorbol ester, as measured by early gene expression, DNA synthesis and cell proliferation. Although activation, phosphorylation, and turnover of the CSF-1 receptor and CSF-1-induced increase in diacylglycerol production remained normal, IFN-gamma blocked CSF-1-stimulated activation of mitogen-activated protein kinases, Raf-1 kinase, increase in GTP-bound Ras and tyrosine phosphorylation, and activation of protein kinase C delta (PKC-delta). PKC-delta was required for CSF-1-induced mitogenic signaling and a primary target for IFN-gamma-induced inhibition. Interestingly, although phorbol myristate acetate stimulated Ras activation, PKC-delta did not appear to be an upstream activator of Ras. These studies clearly indicated that IFN-gamma specifically inhibits PKC-delta activation, resulting in blockage of the early events of mitogenesis in macrophages in response to CSF-1.
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PMID:Blockage of the early events of mitogenic signaling by interferon-gamma in macrophages in response to colony-stimulating factor-1. 870 28

Growth factor stimulation of the mitogen-activated protein (MAP) kinase pathway in fibroblasts is inhibited by cyclic AMP (cAMP) as a result of inhibition of Raf-1. In contrast, cAMP inhibits neither nerve growth factor-induced MAP kinase activation nor differentiation in PC12 pheochromocytoma cells. Instead, in PC12 cells cAMP activates MAP kinase. Since one of the major differences between the Ras/Raf/MAP kinase cascades of these cell types is the expression of B-Raf in PC12 cells, we compared the effects of cAMP on Raf-1 and B-Raf. In PC12 cells maintained in serum-containing medium, B-Raf was refractory to inhibition by cAMP, whereas Raf-1 was effectively inhibited. In contrast, both B-Raf and Raf-1 were inhibited by cAMP in serum-starved PC12 cells. The effect of cAMP is thus dependent upon growth conditions, with B-Raf being resistant to cAMP inhibition in the presence of serum. These results were extended by studies of Rat-1 fibroblasts into which B-Raf had been introduced by transfection. As in PC12 cells, B-Raf was resistant to inhibition by cAMP in the presence of serum, whereas Raf-1 was effectively inhibited. In addition, the expression of B-Raf rendered Rat-1 cells resistant to the inhibitory effects of cAMP on both growth factor-induced activation of MAP kinase and mitogenesis. These results indicate that Raf-1 and B-Raf are differentially sensitive to inhibition by cAMP and that B-Raf expression can contribute to cell type-specific differences in the regulation of the MAP kinase pathway. In contrast to the situation in PC12 cells, cAMP by itself did not stimulate MAP kinase in B-Raf-expressing Rat-1 cells. The activation of MAP kinase by cAMP in PC12 cells was inhibited by the expression of a dominant negative Ras mutant, indicating that cAMP acts on a target upstream of Ras. Thus, it appears that a signaling component upstream of Ras is also require for cAMP stimulation of MAP kinase in PC12 cells.
Mol Cell Biol 1995 Oct
PMID:Differential regulation of Raf-1 and B-Raf and Ras-dependent activation of mitogen-activated protein kinase by cyclic AMP in PC12 cells. 756 4

In GN4 rat liver epithelial cells, angiotensin II (Ang II) and other agonists which activate phospholipase C stimulate tyrosine kinase activity in a calcium-dependent, protein kinase C (PKC)-independent manner. Since Ang II also produces a proliferative response in these cells, we investigated downstream signaling elements traditionally linked to growth control by tyrosine kinases. First, Ang II, like epidermal growth factor (EGF), stimulated AP-1 binding activity in a PKC-independent manner. Because increases in AP-1 can reflect induction of c-Jun and c-Fos, we examined the activity of the mitogen-activated protein (MAP) kinase family members Erk-1 and -2 and the c-Jun N-terminal kinase (JNK), which are known to influence c-Jun and c-Fos transcription. Ang II stimulated MAP kinase (MAPK) activity but only approximately 50% as effectively as EGF; again, these effects were independent of PKC. Ang II also produced a 50- to 200-fold activation of JNK in a PKC-independent manner. Unlike its smaller effect on MAPK, Ang II was approximately four- to sixfold more potent in activating JNK than EGF was. Although others had reported a lack of calcium ionophore-stimulated JNK activity in lymphocytes and several other cell lines, we examined the role of calcium in GN4 cells. The following results suggest that JNK activation in rat liver epithelial cells is at least partially Ca(2+) dependent: (i) norepinephrine and vasopressin hormones that increase inositol 1,4,5-triphosphate stimulated JNK; (ii) both thapsigargin, a compound that produces an intracellular Ca(2+) signal, and Ca(2+) ionophores stimulated a dramatic increase in JNK activity (up to 200-fold); (iii) extracellular Ca(2+) chelation with ethylene glycol tetraacetic acid (EGTA) inhibited JNK activation by ionophore and intracellular chelation with 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl-ester (BAPTA-AM) partially inhibited JNK activation by Ang II or thapsigargin; and (iv) JNK activation by Ang II was inhibited by pretreatment of cells with thapsigargin and EGTA, a procedure which depletes intracellular Ca(2+) stores. JNK activation following Ang II stimulation did not involve calmodulin; either W-7 nor calmidizolium, in concentrations sufficient to inhibit Ca(2+)/calmodulin-dependent kinase II, blocked JNK activation by Ang II. In contrast, genistein, in concentrations sufficient to inhibit Ca(2+)-dependent tyrosine phosphorylation, prevented Ang II and thapsigargin-induced JNK activation. In summary, in GN4 rat liver epithelial cells, Ang II stimulates JNK via a novel Ca(2+)-dependent pathway. The inhibition by genistein suggest that Ca(2+)-dependent tyrosine phosphorylation may modulate the JNK pathway in a cell type-specific manner, particularly in cells with a readily detectable Ca(2+)-regulated tyrosine kinase.
Mol Cell Biol 1995 Nov
PMID:Angiotensin II stimulates calcium-dependent activation of c-Jun N-terminal kinase. 756 68

Although substantial evidence supports a critical role for the activation of Raf-1 and mitogen-activated protein kinases (MAPKs) in oncogenic Ras-mediated transformation, recent evidence suggests that Ras may activate a second signaling pathway which involves the Ras-related proteins Rac1 and RhoA. Consequently, we used three complementary approaches to determine the contribution of Rac1 and RhoA function to oncogenic Ras-mediated transformation. First, whereas constitutively activated mutants of Rac1 and RhoA showed very weak transforming activity when transfected alone, their coexpression with a weakly transforming Raf-1 mutant caused a greater than 35-fold enhancement of transforming activity. Second, we observed that coexpression of dominant negative mutants of Rac1 and RhoA reduced oncogenic Ras transforming activity. Third, activated Rac1 and RhoA further enhanced oncogenic Ras-triggered morphologic transformation, as well as growth in soft agar and cell motility. Finally, we also observed that kinase-deficient MAPKs inhibited Ras transformation. Taken together, these data support the possibility that oncogenic Ras activation of Rac1 and RhoA, coupled with activation of the Raf/MAPK pathway, is required to trigger the full morphogenic and mitogenic consequences of oncogenic Ras transformation.
Mol Cell Biol 1995 Nov
PMID:Activation of Rac1, RhoA, and mitogen-activated protein kinases is required for Ras transformation. 756 96

Adhesion to extracellular matrix mediates cell cycle progression in mid-late G1; this effect involves an integrin-dependent organization of the cytoskeleton and a consequent change in cell shape. In an effort to identify potential signal-transducing agents that are associated with integrin-dependent shape changes, we looked for kinase activities that were stimulated by long-term adhesion of G0-synchronized NIH-3T3 cells to fibronectin-coated dishes. Several kinase activities were stimulated by this procedure, two of which migrated at 42 and 44 kDa and phosphorylated myelin basic protein in vitro. Blotting with anti-phosphotyrosine and anti-mitogen-activated protein (MAP) kinase antibodies identified these enzymes as ERK 1 and ERK 2. In contrast to the rapid and transient activation of these MAP kinases by platelet-derived growth factor, stimulation of MAP kinase activity by fibronectin was gradual, persistent, and associated with cell spreading rather than cell attachment itself. Cytochalasin D blocked the activation of MAP kinase activity that was induced by the binding of cells to fibronectin. Moreover, MAP kinase was also activated by adhesion of cells to vitronectin and type IV collagen; these effects were also associated with cell spreading. These results distinguish the regulation of G1 phase MAP kinase activity by soluble mitogens and extracellular matrix. They also implicate MAP kinase in shape-dependent cell cycle progression.
Mol Biol Cell 1995 Mar
PMID:Integrin-dependent activation of MAP kinase: a link to shape-dependent cell proliferation. 761 63

A novel pp90rsk Ser/Thr kinase (referred to as RSK3) was cloned from a human cDNA library. The RSK3 cDNA encodes a predicted 733-amino-acid protein with a unique N-terminal region containing a putative nuclear localization signal. RSK3 mRNA was widely expressed (but was predominant in lung and skeletal muscle). By using fluorescence in situ hybridization, the human RSK3 gene was localized to band q27 of chromosome 6. Hemagglutinin epitope-tagged RSK3 was expressed in transiently transfected COS cells. Growth factors, serum, and phorbol ester stimulated autophosphorylation of recombinant RSK3 and its kinase activity toward several protein substrates known to be phosphorylated by RSKs. However, the relative substrate specificity of RSK3 differed from that reported for other isoforms. RSK3 also phosphorylated potential nuclear target proteins including c-Fos and histones. Furthermore, although RSK3 was inactivated by protein phosphatase 2A in vitro, the enzyme was not activated by ERK2/mitogen-activated protein (MAP) kinase. In contrast, the kinase activity of another epitope-tagged RSK isoform (RSK-1) was significantly increased by in vitro incubation with ERK2/MAP kinase. Finally, we used affinity-purified RSK3 antibodies to demonstrate by immunofluorescence that endogenous RSK3 undergoes serum-stimulated nuclear translocation in cultured HeLa cells. These results provide evidence that RSK3 is a third distinct isoform of pp90rsk which translocates to the cell nucleus, phosphorylates potential nuclear targets, and may have a unique upstream activator. RSK3 may therefore subserve a discrete physiologic role(s) that differs from those of the other two known mammalian RSK isoforms.
Mol Cell Biol 1995 Aug
PMID:RSK3 encodes a novel pp90rsk isoform with a unique N-terminal sequence: growth factor-stimulated kinase function and nuclear translocation. 762 30

Nerve growth factor (NGF) and epidermal growth factor (EGF) elicit contrasting actions on PC12 pheochromocytoma cells; NGF causes neuronal differentiation, and EGF induces proliferation. However, ectopic expression of the Src homology 2 (SH2) and SH3-containing oncogenic adaptor protein v-Crk in PC12 cells results in EGF-inducible neuronal differentiation (Hempstead, B. L., Birge, R. B., Fajardo, J. E., Glassman, R., Mahadeo, D., Kraemer, R., and Hanafusa, H. (1994) Mol. Cell. Biol. 14, 1964-1971). Here we show that v-Crk complexes with both the tyrosine-phosphorylated EGF receptor and the Ras guanine nucleotide exchange factor SOS in PC12 cells and is involved in an pathway analogous to that of Grb2. Expression of v-Crk results in an enhanced and sustained activation of Ras and mitogen-activated protein (MAP) kinase following EGF or NGF stimulation, implying that v-Crk can couple divergent tyrosine kinase pathways to Ras. To investigate the causal relationship between EGF receptor binding, MAP kinase activation, and neurite outgrowth, we stably expressed two v-Crk SH2 point mutants, v-Crk(R273N) and v-Crk(H294R) in PC12 cells. Mutations within the SH2 domain of v-Crk block binding of v-Crk to the tyrosine phosphorylated EGF receptor, compromise v-Crk's ability to cause EGF-dependent neurite outgrowth, and act in a dominant negative manner for NGF-induced neurite outgrowth. However, the kinetics of MAP kinase activation in EGF- or NGF-treated v-Crk-(R273N)PC12 cells was comparable with that in v-CrkPC12 cells. These data are consistent with a model in which v-Crk regulates the strength of a tyrosine kinase signal leading to prolonged activation of Ras and MAP kinase. However, the experiments with the SH2 mutants suggest that sustained activation, by itself, may not be sufficient to switch the fate of v-CrkPC12 cells from proliferation toward differentiation.
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PMID:v-Crk modulation of growth factor-induced PC12 cell differentiation involves the Src homology 2 domain of v-Crk and sustained activation of the Ras/mitogen-activated protein kinase pathway. 765 47

p44erk1 is a member of a family of tyrosyl-phosphorylated and mitogen-activated protein (MAP) kinases that participate in cell cycle control. A full-length erk1 cDNA was isolated from a human hepatoma cell line (Hep G2) library. The erk1 cDNA clone shared approximately 96% predicted amino acid identity with partial sequences of rodent erk1 cognates, and the erk1 gene was assigned to human chromosome 16 by hybrid panel analysis. Human erk1 expressed in Escherichia coli as a glutathione S-transferase fusion (GST-Erk1) protein was substantially phosphorylated on tyrosine in vivo. It underwent further autophosphorylation in vitro (up to 0.01 mol of P per mol) at the regulatory Tyr-204 site and at additional tyrosine and serine residues. Threonine autophosphorylation, presumably at the regulatory Thr-202 site, was also detected weakly when the recombinant kinase was incubated in the presence of manganese, but not in the presence of magnesium. Before and after cleavage of the GST-Erk1 protein with thrombin, it exhibited a relatively high level of myelin basic protein phosphotransferase activity, which could be reduced eightfold by treatment of the kinase with the protein-tyrosine phosphatase CD45, but not by treatment with the protein-serine/threonine phosphatase 2A. The protein-tyrosine kinase p56lck catalyzed phosphorylation of GST-Erk1 at two autophosphorylations sites, including Tyr-204, and at a novel site. A further fivefold stimulation of the myelin basic protein phosphotransferase activity of the GST-Erk1 was achieved in the presence of a partially purified MAP kinase kinase from sheep platelets. Under these circumstances, there was primarily an enhancement of the tyrosine phosphorylation of GST-Erk1. This MAP kinase kinase also similarly phosphorylated a catalytically compromised version of GST-Erk1 in which Lys-71 was converted to Ala by site-directed mutagenesis.
Mol Cell Biol 1993 Aug
PMID:Molecular cloning, expression, and characterization of the human mitogen-activated protein kinase p44erk1. 768 43

Previous studies have shown that the noncatalytic carboxy-terminal tail of the p70 S6 kinase (amino acids 422 to 525) contains an autoinhibitory pseudosubstrate domain that is phosphorylated in situ during activation and in vitro by mitogen-activated protein kinases. The present study shows that a recombinant p70 deleted of the carboxy-terminal tail (p70 delta CT104) nevertheless exhibits a basal and serum-stimulated 40S kinase activity and susceptibility to inhibition by wortmannin very similar to those of the parent, full-length p70 kinase. Carboxy-terminal deletion reduces the extent of maximal inhibition produced by rapamycin, from > 95% in the full-length p70 to 60 to 80% in p70 delta CT104, without altering the sensitivity to rapamycin inhibition (50% inhibitory concentration of 2 nM). Serum activation of p70 delta CT104, as with the parent, full-length p70, is accompanied by an increase in 32P content (about twofold) in situ and a slowing in electrophoretic mobility; both modifications are inhibited by pretreatment with wortmannin or rapamycin. 32P-peptide maps of p70 delta CT104 show multisite phosphorylation, and wortmannin and rapamycin appear to cause preferential dephosphorylation of the same subset of sites. Thus, it is likely that activation of the kinase requires phosphorylation of p70 at sites in addition to those previously identified in the carboxy-terminal tail. Evidence that the carboxy-terminal tail actually functions as a potent intramolecular inhibitor of kinase activity in situ is uncovered by deletion of a short acidic segment (amino acids 29 to 46) from the p70 amino-terminal noncatalytic region. Deletion of amino acids 29 to 46 causes a >95% inhibition of p70 activity despite continue phosphorylation of the carboxy-terminal tail in situ; additional deletion of the carboxy-terminal tail (yielding p70 delta 29-46/ delta CT104) increases activity 10-fold, to a level approaching that of p70 delta CT104. Deletion of residues 29 to 46 also abolishes completely the sensitivity of p70 to inhibition by rapamycin but does not alter the susceptibility to activation by serum of inhibition by wortmannin. Although the mechanisms underlying the effects of the delta 29-46 deletion are not known, they are not attributable to loss of the major in situ p70 phosphorylation site at Ser-40. Thus, activation of the p70 S6 kinase involves multiple, independent inputs directed at different domains of the p70 polypeptide. Disinhibition from the carboxy-terminal tail requires, in addition to its multisite phosphorylation, an activating input dependent on the presence of amino acids 29 to 46; this p70-activating input may be the same as that inhibited by rapamycin but is distinct from that arising from the wortmannin-inhibitable phosphatidylinositol 3-kinase. In addition, as exemplified by the rapamycin-resistant but mitogen- and wortmannin-sensitive p70 delta 29-46/ delta CT104 mutant, a further activating input, which probably involves site-specific phosphorylation in the segment between amino acids 46 to 421, is necessary.
Mol Cell Biol 1995 May
PMID:Multiple independent inputs are required for activation of the p70 S6 kinase. 773 16

Ceramide and ceramide-1-phosphate are sphingolipid analogues of diacylglycerol and phosphatidate, respectively, and they are putative second messengers of agonist-stimulated sphingomyelin metabolism. The interactions of exogenous cell-permeable ceramides and ceramide-1-phosphates in modifying DNA synthesis and signal transduction were investigated in Rat-1 fibroblasts. C2- and C8-Ceramide-1-phosphates (N-acetylsphingosine-1-phosphate and N-octanoylsphingosine-1-phosphate, respectively) at 1-10 microM stimulated DNA synthesis and cell division. This effect was blocked by cell-permeable ceramides. C2-Ceramide stimulated the conversion of exogenous C8-ceramide-1-phosphate to C8-ceramide, with very little production of sphingosine or sphingosine-1-phosphate. This mechanism may be partly responsible for preventing the stimulation of DNA synthesis. Unlike phosphatidate or lyso-phosphatidate, concentrations of C8-ceramide-1-phosphate that stimulated DNA synthesis did not inhibit adenylate cyclase activity, nor did they increase the activities of phospholipase D or mitogen-activated protein kinases (42- and 44 kDa isoforms). Although ceramide-1-phosphate can be considered as an analogue of phosphatidate, the effects of this compound on signal transduction differ considerably from those of phosphatidate. This work demonstrates that short-chain ceramide-1-phosphates can be used as novel external agonists that can stimulate DNA synthesis. This effect can be counteracted by short-chain ceramides.
Mol Pharmacol 1995 May
PMID:Short-chain ceramide-1-phosphates are novel stimulators of DNA synthesis and cell division: antagonism by cell-permeable ceramides. 774 76


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