p21ras and protein kinase C function in distinct and interdependent signaling pathways in C3H 10T1/2 fibroblasts.

Both p21ras and protein kinase C (PKC) are believed to function downstream of plasma membrane-associated tyrosine kinases in cellular signal transduction pathways. However, it has remained controversial whether they function in the same pathway and, if so, what their relative position and functional relationship in such a pathway are. We investigated the possibilities that p21ras and PKC function either upstream or downstream of each other in a common linear pathway or that they function independently in colinear signal pathways. Either decreased expression of endogenous normal ras in fibroblasts transfected with an inducible antisense ras construct or overexpression of a mutant ras gene reduced the capacity of the phorbol ester tetradecanoyl phorbol acetate to trigger expression of the tetradecanoyl phorbol acetate-responsive and ras-dependent reporter gene osteopontin (OPN). PKC depletion decreased basal OPN mRNA levels, and the overexpression of ras restored OPN expression to the level of non-PKC-depleted cells. We propose a model in which ras and PKC function in distinct and interdependent signaling pathways.     

Activation of the mitogen-activated protein kinase pathway in Triton X-100 disrupted NIH-3T3 cells by p21 ras and in vitro by plasma membranes from NIH 3T3 cells.

We describe a novel Triton-disrupted mammalian cell system wherein the pathways for activation of mitogen-activated protein (MAP) kinases (MAPKs) are capable of direct biochemical manipulation in vitro. MAPKs p42mapk and p44mapk are activated in signal transduction cascade(s) initiated by occupancy of plasma membrane receptors for peptide growth factors, hormones, and neurotransmitters. One likely activation pathway for MAPKs consists of sequential activations of c-ras, c-raf-1, and a protein-tyrosine/threonine kinase, MAP kinase kinase. Triton-disrupted cells retained capacity for activation of the pathway by both peptide growth factors and by addition of GTP-loaded p21 rasVal12. Incubation of disrupted cells with an antibody that neutralized the function of c-ras (Y13-259) abolished receptor-mediated stimulation of MAPK as did acute addition of 200 microM azatyrosine. Activation of the pathway was reconstituted in a cell-free system using high-speed supernatants generated from Triton-disrupted cells together with purified plasma membranes from parental cells and as a heterogeneous system using purified plasma membranes from v-ras-transformed cells. These systems will allow biochemical dissection in vitro of the interaction(s) between c-ras and the MAPK pathway in mammalian cells.     

Insulin-induced p21ras activation does not require protein kinase C, but a protein sensitive to phenylarsine oxide

Insulin treatment of fibroblasts overexpressing the insulin receptor causes a rapid accumulation of the GTP-bound form of p21ras. We have studied the involvement of protein kinase C (PKC) in, and the effect of phenylarsine oxide (PAO), a putative inhibitor of tyrosine phosphatase activity on, this process. Activation of p21ras was not observed when the cells were stimulated with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and pretreatment with TPA for 16 h, sufficient to down-regulate PKC activity, did not abolish p21ras activation by insulin. These results show that PKC is not involved in the insulin-induced activation of p21ras. Pretreatment of the cells with PAO for 5 min completely blocked insulin-induced p21ras activation. Addition of 2,3-dimercaptopropanol prevented this inhibition by PAO. Also, addition of PAO after insulin stimulation could reverse the activation of p21ras. Since PAO did not affect overall phosphorylation of the insulin receptor beta-chain, we conclude that a PAO-sensitive protein is involved in the induction of p21ras activation by insulin.     

p42 mitogen-activated protein kinase and p90 ribosomal S6 kinase are selectively phosphorylated and activated during thrombin-induced platelet activation and aggregation.

Human platelets provide an excellent model system for the study of phosphorylation events during signal transduction and cell adhesion. Platelets are terminally differentiated cells that exhibit rapid phosphorylation of many proteins upon agonist-induced activation and aggregation. We have sought to identify the kinases as well as the phosphorylated substrates that participate in thrombin-induced signal transduction and platelet aggregation. In this study, we have identified two forms of mitogen-activated protein kinase (MAPK), p42mapk and p44mapk, in platelets. The data demonstrate that p42mapk but not p44mapk becomes phosphorylated on serine, threonine, and tyrosine during platelet activation. Immune complex kinase assays, gel renaturation assays, and a direct assay for MAPK activity in platelet extracts all support the conclusion that p42mapk but not p44mapk shows increased kinase activity during platelet activation. The activation of p42mapk, independently of p44mapk, in platelets is unique since in other systems, both kinases are coactivated by a variety of stimuli. We also show that platelets express p90rsk, a ribosomal S6 kinase that has previously been characterized as a substrate for MAPK. p90rsk is phosphorylated on serine in resting platelets, and this phosphorylation is enhanced upon thrombin-induced platelet activation. Immune complex kinase assays demonstrate that the activity of p90rsk is markedly increased during platelet activation. Another ribosomal S6 protein kinase, p70S6K, is expressed by platelets but shows no change in kinase activity upon platelet activation with thrombin. Finally, we show that the increased phosphorylation and activity of both p42mapk and p90rsk does not require integrin-mediated platelet aggregation. Since platelets are nonproliferative cells, the signal transduction pathways that include p42mapk and p90rsk cannot lead to a mitogenic signal and instead may regulate cytoskeletal or secretory changes during platelet activation.     

Effect of troponin C on the cooperativity in Ca2+ activation of cardiac muscle

The presence of protein kinase C (PKC), a key enzyme in signal transduction, has not been investigated in fungal cells. The phorbol ester TPA, an activator of PKC, may be used as an indicator of the presence and role of PKC in Phycomyces blakesleeanus spores. Activation of spore germination by acetate was prevented by 6 nM TPA. The TPA analog 4 alpha PDD, an ineffective activator of PKC, did not affect spore germination. 3 mM dbcAMP, on the other hand, reversed the inhibition of germination caused by TPA. TPA-stimulated protein kinase activity was detected in spores. The possible relationship between PKC and the increased levels of cAMP that accompany the induction of spore germination is discussed.     

Differential regulation of the p72-74 RAF-1 kinase in 3T3 fibroblasts expressing ras or src oncogenes

The c-raf-1 protooncogene encodes a p72-74 serine/threonine-specific kinase that has been implicated in growth factor-mediated signal transduction and malignant transformation. Here, we compared the effects of Ha-c-ras and v-src oncogenes on the regulation of p72-74 RAF-1 kinase in NIH3T3 cells. In both serum-starved and platelet-derived growth factor-treated v-src-transformed cells, the RAF-1 kinase was constitutively activated, displaying characteristic retarded mobility in electrophoretic gels and elevated activity in in vitro kinase assays. In contrast, the RAF-1 protein from quiescent ras-transformed cells did not exhibit constitutively shifted gel mobility or elevated kinase activity but did respond normally with regard to platelet-derived growth factor- and phorbol myristate acetate-induced changes in p72-74 RAF-1 phosphorylation and kinase activity. 3T3 cells transformed by ras, however, contained elevated levels of p72-74 RAF-1 protein (as determined by immunoblotting), suggesting an indirect influence on this kinase. Quantitative differences in the levels and subcellular distribution of immunodetectable protein kinase C enzymes did not account for the differences between src- and ras-transformed 3T3 cells with regard to regulation of the RAF kinase. These findings in serum-deprived 3T3 cells demonstrate that expression of a ras oncogene can be insufficient for full activation of the p72-74 RAF-1 kinase, implying necessity for an additional growth factor-mediated stimulus.     

Effect of a dominant inhibitory Ha-ras mutation on mitogenic signal transduction in NIH 3T3 cells.

We used a dominant inhibitory mutation of c-Ha-ras which changes Ser-17 to Asn-17 in the gene product p21 [p21(Asn-17)Ha-ras] to investigate ras function in mitogenic signal transduction. An NIH 3T3 cell line [NIH(M17)] was isolated that displayed inducible expression of the mutant Ha-ras gene (Ha-ras Asn-17) via the mouse mammary tumor virus long terminal repeat and was growth inhibited by dexamethasone. The effect of dexamethasone induction on response of quiescent NIH(M17) cells to mitogens was then analyzed. Stimulation of DNA synthesis by epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) was completely blocked by p21(Asn-17) expression, and stimulation by serum, fibroblast growth factor, and platelet-derived growth factor was partially inhibited. However, the induction of fos, jun, and myc by EGF and TPA was not significantly inhibited in this cell line. An effect of p21(Asn-17) on fos induction was, however, demonstrated in transient expression assays in which quiescent NIH 3T3 cells were cotransfected with a fos-cat receptor plasmid plus a Ha-ras Asn-17 expression vector. In this assay, p21(Asn-17) inhibited chloramphenicol acetyltransferase expression induced by EGF and other growth factors. In contrast to its effect on DNA synthesis, however, Ha-ras Asn-17 expression did not inhibit fos-cat expression induced by TPA. Conversely, downregulation of protein kinase C did not inhibit fos-cat induction by activated ras or other oncogenes. These results suggest that ras proteins are involved in at least two parallel mitogenic signal transduction pathways, one of which is independent of protein kinase C. Although either pathway alone appears to be sufficient to induce fos, both appear to be necessary to induce the full mitogenic response.     

Inhibition of connexin43 gap junctional intercellular communication by TPA requires ERK activation

The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), is a potent inhibitor of gap junctional intercellular communication (GJIC). This inhibition requires activation of protein kinase C (PKC), but the events downstream of this kinase are not known. Since PKC can activate extracellular signal regulated kinases (ERKs) and these also downregulate GJIC, we hypothesized that the inhibition of GJIC by TPA involved ERKs. TPA treatment (10 ng/ml for 30 min) of WB-F344 rat liver epithelial cells strongly activated p42 and p44 ERK-1 and -2, blocked gap junction-mediated fluorescent dye-coupling, and induced connexin43 hyperphosphorylation and gap junction internalization. These effects were completely prevented by inhibitors of PKC (bis-indolylmaleimide I; 2 microM) and ERK activation (U-0126; 10 microM). These data suggest that ERKs are activated by PKC in response to TPA treatment and are downstream mediators of the gap junction effects of the phorbol ester.     

Phosphorylation of p42/44(MAPK) by various signal transduction pathways activates cytosolic phospholipase A(2) to variable degrees

Arachidonic acid has been implicated to play a role in physiological and pathophysiological processes and is selectively released by the 85-kDa cytosolic phospholipase A(2) (cPLA(2)). The activity of cPLA(2) is regulated by calcium, translocating the enzyme to its substrate, and by phosphorylation by a mitogen-activated protein kinase (MAPK) family member and a MAPK-activated protein kinase. In this study, the signal transduction pathways in growth factor-induced phosphorylation of p42/44(MAPK) and cPLA(2) activation were investigated in Her14 fibroblasts. p42/44(MAPK) in response to epidermal growth factor was not only phosphorylated via the Raf-MEK pathway but mainly through protein kinase C (PKC) or a related or unrelated kinase in which the phosphorylated p42/44(MAPK) corresponded with cPLA(2) activity. Serum-induced phosphorylation of p42/44(MAPK) also corresponded with cPLA(2) activity but is predominantly mediated via Raf-MEK and partly through PKC or a related or unrelated kinase. In contrast, activation of PKC by phorbol ester did not result in increased cPLA(2) activity, while p42/44(MAPK) is phosphorylated, mainly via Raf-MEK and through MEK. Moreover, p42/44(MAPK) phosphorylation is present in quiescent and proliferating cells, and p42/44(MAPK) is entirely phosphorylated via Raf-MEK, but it only corresponds to cPLA(2) activity in the former cells. Collectively, these data show that p42/44(MAPK) in proliferating, quiescent, and stimulated cells is phosphorylated by various signal transduction pathways, suggesting the activation of different populations of p42/44(MAPK) and cPLA(2).     

CD2 antigen mediated activation of the guanine nucleotide binding proteins p21ras in human T lymphocytes.

T cell stimulation via the TCR complex (TCR/CD3 complex) results in activation of the guanine nucleotide binding proteins encoded by the ras protooncogenes (p21ras). In the present study we show that the activation state of p21ras in T lymphocytes can also be controlled by triggering of the CD2 Ag. The activation state of p21ras is controlled by GTP levels on p21ras. In T cells stimulation of protein kinase C is able to induce an accumulation of "active" p21ras-GTP complexes due to an inhibitory effect of protein kinase C stimulation on the intrinsic GTPase activity of p21ras. The regulatory effect of protein kinase C on p21ras GTPase activity appears to be mediated via regulation of GAP, the GTPase activating protein of p21ras. In the present report, we demonstrate that the TCR/CD3 complex and the CD2 Ag control the accumulation of p21ras-GTP complexes via a regulatory effect on p21ras GTPase activity. The TCR/CD3 complex and CD2 Ag are also able to control the cellular activity of GAP. These data demonstrate that p21ras is part of the signal transduction responses controlled by the CD2 Ag, and reveal that the TCR/CD3 complex and CD2 Ag control the activation state of p21ras via a similar mechanism.     

Signal transduction events in mammalian cells in response to ionizing radiation

Ionizing radiations elicit a variety of biological effects in mammalian cells. In recent years altered signal transduction has been recognized as a key cellular response to ionizing radiation. Several oncogenes, the products of which are components of signal transduction pathways and which are over-expressed in many tumors, are specifically induced in cells exposed to radiation. It has also become evident that the oncogene ras and the serine/threonine protein kinase oncogenes raf and PKC confer radio-resistance to tumor cells. Modulation of these genes or their activity by natural compounds may offer a strategy to treat cancer by enhancing radiation-induced apoptosis of tumor cells.     

Serum-induced translocation of mitogen-activated protein kinase to the cell surface ruffling membrane and the nucleus

The mitogen-activated protein (MAP) kinase signal transduction pathway represents an important mechanism by which growth factors regulate cell function. Targets of the MAP kinase pathway are located within several cellular compartments. Signal transduction therefore requires the localization of MAP kinase in each sub-cellular compartment that contains physiologically relevant substrates. Here, we show that serum treatment causes the translocation of two human MAP kinase isoforms, p40mapk and p41mapk, from the cytosol into the nucleus. In addition, we report that p41mapk (but not p40mapk) is localized at the cell surface ruffling membrane in serum-treated cells. To investigate whether the protein kinase activity of MAP kinase is required for serum-induced redistribution within the cell, we constructed mutated kinase-negative forms of p40mapk and p41mapk. The kinase-negative MAP kinases were not observed to localize to the cell surface ruffling membrane. In contrast, the kinase-negative MAP kinases were observed to be translocated to the nucleus. Intrinsic MAP kinase activity is therefore required only for localization at the cell surface and is not required for transport into the nucleus. Together, these data demonstrate that the pattern of serum-induced redistribution of p40mapk is different from p41mapk. Thus, in addition to common targets of signal transduction, it is possible that these MAP kinase isoforms may differentially regulate targets located in distinct sub-cellular compartments.     

Purification and characterization from bovine brain cytosol of two GTPase-activating proteins specific for smg p21, a GTP-binding protein having the same effector domain as c-ras p21s

The smg-21 GTP-binding protein (smg p21) has the same effector domain as the ras proteins (ras p21s) and is identical with the proteins of the rap1A and Krev-1 genes. In this paper, two proteins stimulating the GTPase activity of smg p21 are partially purified from bovine brain cytosol. These proteins, designated as smg p21 GTPase-activating protein (GAP) 1 and 2, are separated from a c-ras p21 GAP described previously by column chromatographies. smg p21 GAP1 and -2 stimulate the GTPase activity of only smg p21 but not that of c-Ha-ras p21 or the rho and smg-25A GTP-binding proteins. smg p21 GAP1 or -2 does not stimulate the dissociation of guanosine 5'-3-O-(thio)triphosphate or GDP from smg p21. smg p21 GAP1 or -2 themselves do not have GTP/GDP binding or GTPase activity. The Mr values of smg p21 GAP1 and -2 are estimated to be 250-400 x 10(3) and 80-100 x 10(3) by gel filtration and sucrose density gradient ultracentrifugation, respectively. The activity of smg p21 GAP1 and -2 is killed by tryptic digestion or heat boiling. These results indicate that bovine brain contains two smg p21 GAPs in addition to c-ras p21 GAP.     

Tumor promoter phorbol ester reversibly modulates tyrosine dephosphorylatioin/ inactivation of protein kinase FA/GSK-3α in A431 cells

The signal transducrion mechanism of protein kinase F_A/GSK-3α by tyrosine phosphorylation in A431 cells was investigated. Kinase F_A/GSK-3α was found to exist in a highly tyrosine-phosphorylated/activated state in resting cells but could be tyrosine-dephosphorylated and inactivated to ～60% of the control level when cells were acutely treated with 1 μM tumor phorbol ester (TPA) at 37^oC for 30 min, as demonstrated by metabolic ³²P-labeling the cells, followed by immunoprecipitation and two-dimensional phosphoamino acid analysis and by immunodetection in an antikinase F_A/GSK-3α immunoprecipitate kinase assay. Conversely, when cells were chronically treated with 1 μM TPA at 37°C for 24 h and processed under identical condetions, kinase F_A/GSK-3α was found to be rephosphorylated on tyrosine residue and reactivated to ～130% of the original control level. Taken together, the results provide initial evidence that the phosphotyrosine content and cellular activity of kinase F_A/GSK-3α can be modulated in a reversible manner by short-term and long-term exposure of A431 cells to TPA. Since acute exposure of cells to TPA causes up-regulation of cellular protein kinase C (PKC) activity and prolonged exposure to TPA causes down-regulation of PKC, the results further suggest that the TPA-mediated modulation of PKC may play a role in the regulation of tyrosine phosphorylation and concurrent activation of kinase F_A/GSK-3α in cells, representing a new mode of signal transduction pathway for the regulation of this multisubstrate/multifunctional protein kinase in cells.     

Dissociation of mitogen-activated protein kinase activation from p125 focal adhesion kinase tyrosine phosphorylation in Swiss 3T3 cells stimulated by bombesin, lysophosphatidic acid, and platelet-derived growth factor.

The experiments presented here were designed to examine the contribution of p125 focal adhesion kinase (p125FAK) tyrosine phosphorylation to the activation of the mitogen-activated protein kinase cascade induced by bombesin, lysophosphatidic acid (LPA), and platelet-derived growth factor (PDGF) in Swiss 3T3 cells. We found that tyrosine phosphorylation of p125FAK in response to these growth factors is completely abolished in cells treated with cytochalasin D or in cells that were suspended in serum-free medium for 30 min. In marked contrast, the activation of p42mapk by these factors was independent of the integrity of the actin cytoskeleton and of the interaction of the cells with the extracellular matrix. The protein kinase C inhibitor GF 109203X and down-regulation of protein kinase C by prolonged pretreatment of cells with phorbol esters blocked bombesin-stimulated activation of p42mapk, p90rsk, and MAPK kinase-1 but did not prevent bombesin-induced tyrosine phosphorylation of p125FAK. Furthermore, LPA-induced p42mapk activation involved a pertussis toxin-sensitive guanylate nucleotide-binding protein, whereas tyrosine phosphorylation of p125FAK in response to LPA was not prevented by pretreatment with pertussis toxin. Finally, PDGF induced maximum p42mapk activation at concentrations (30 ng/ml) that failed to induce tyrosine phosphorylation of p125FAK. Thus, our results demonstrate that p42mapk activation in response to bombesin, LPA, and PDGF can be dissociated from p125FAK tyrosine phosphorylation in Swiss 3T3 cells.     

Agonist-induced association of the p21ras GTPase-activating protein with phosphatidylinositol 3-kinase.

The signal transduction properties of the 21-kDa GTP-binding proteins, encoded by the ras genes, are only partly known. In a recent report, we demonstrated that the signaling pathway of p21ras, like that of several growth factors, is closely associated with phosphatidylinositol 3-kinase (PtdIns 3-kinase) activity. We showed that insulin-like growth factor-1 (IGF-1) and insulin increased the phosphatidylinositol 3-kinase activity in immunoprecipitates obtained with anti-phosphotyrosine and anti-ras antibodies in Ha-ras-transformed epithelial cells. Several findings in this previous study suggested that an additional protein was likely to be associated with the PtdIns 3-kinase. The suggestion that p21ras GTPase-activating protein (GAP) acts not only as a regulator of p21ras activity but also as a direct downstream target in the signaling pathway of p21ras led us to investigate the possible association of PtdIns 3-kinase with GAP. The stimulation of Ha-ras-transformed epithelial cells with IGF-1 caused an increased association of PtdIns 3-kinase activity with GAP, as seen by immunoprecipitation with anti-p21ras and anti-GAP antibodies. The 85-kDa regulatory subunit of PtdIns 3-kinase was present in immunoprecipitates obtained with antibodies against GAP and p21ras of IGF-1 stimulated cells. These data suggest that GAP acts as a downstream target for p21ras via its association with PtdIns 3-kinase.     

Cytosolic and Nuclear Mitogen-Activated Protein Kinases Are Regulated by Distinct Mechanisms

We have investigated the regulation and localization of mitogen-activated protein kinase (MAPK) and mitogen-activated protein kinase kinase (MAPKK) in both cytosolic and nuclear fractions of glomerular mesangial cells. p42 MAPK was localized by both immunoblot and kinase activity in both cytosol and nucleus and was rapidly activated, in both fractions, by fetal bovine serum and TPA. Downregulation of protein kinase C (PKC) by TPA inhibited stimulation of cytosolic p42 MAPK, but unexpectedly had no effect on stimulated p42 MAPK in the nucleus. Next we studied the upstream kinase p45 MAPKK by indirect immunofluorescence microscopy, Western blot analysis, and kinase specific activity. Unlike MAPK, p45 MAPKK is almost exclusively cytosolic in resting cells and kinase activity stimulated by TPA is restricted to the cytosol. Interestingly, PKC downregulation for 24 h with TPA dramatically enhanced nuclear MAPKK as assessed by all three techniques. Cytosolic stimulated MAPKK was attenuated in PKC downregulation. Collectively these results show that in mesangial cells: (i) p42 MAPK and p45 MAPKK localize in both the cytosol and the nucleus, and (ii) PKC exerts a negative effect on nuclear MAPKK activity as documented by PKC downregulation, which augments p45 MAPKK nuclear mass and activity. These results indicate that the dual regulation of these two kinases is under differential control in the cytosol and the nucleus.     

Activation of Ras by insulin in 3T3 L1 cells does not involve GTPase-activating protein phosphorylation.

Insulin-induced differentiation of 3T3 L1 cells to adipocytes can be mimicked by the expression of transfected ras oncogenes but not of the tyrosine-kinase oncogenes src and trk. Expression of two different transfected, dominant inhibitory ras mutants resulted in significant inhibition of insulin-induced differentiation, suggesting that endogenous Ras proteins are mediators of insulin signaling in these cells. Exposure of untransfected 3T3 L1 cells to insulin resulted in significant formation of the active Ras.GTP complex, at levels comparable with those resulting from exposure to platelet-derived growth factor. However, whereas exposure of the same cells to platelet-derived growth factor resulted in significant tyrosine phosphorylation of the p21ras GTPase-activating protein (GAP), insulin-treated cells did not show any detectable levels of de novo GAP tyrosine phosphorylation. Interestingly, insulin caused tyrosine phosphorylation of the p62 polypeptide coprecipitated with GAP by anti-GAP antibodies. Insulin-induced activation of cytosolic MAP kinase activity in untransfected 3T3 L1 cells was also mimicked by Ras expression (in the absence of insulin) in the same cells transfected with an inducible ras construct. These results confirm that Ras proteins participate in insulin signaling pathways in these mammalian cells and indicate that activation of cytosolic MAP kinases is an early event occurring downstream from Ras activation. However, tyrosine phosphorylation of GAP appears not to be a significant upstream regulatory event in the activation of Ras by insulin.