Platelet-derived growth factor stimulation of GTPase-activating protein tyrosine phosphorylation in control and c-H-ras-expressing NIH 3T3 cells correlates with p21ras activation.

Platelet-derived growth factor (PDGF) stimulation of NIH 3T3 cells leads to the rapid tyrosine phosphorylation of the GTPase-activating protein (GAP) and an associated 64- to 62-kDa tyrosine-phosphorylated protein (p64/62). To assess the functions of these proteins, we evaluated their phosphorylation state in normal NIH 3T3 cells as well as in cells transformed by oncogenically activated v-H-ras or overexpression of c-H-ras genes. No significant GAP tyrosine phosphorylation was observed in unstimulated cultures, while PDGF-BB induced rapid tyrosine phosphorylation of GAP in all cell lines analyzed. In NIH 3T3 cells, we found that PDGF stimulation led to the recovery of between 37 and 52% of GAP molecules by immunoprecipitation with monoclonal antiphosphotyrosine antibodies. Furthermore, PDGF exposure led to a rapid and sustained increase in the levels of p21ras bound to GTP, with kinetics similar to those observed for GAP tyrosine phosphorylation. The PDGF-induced increases in GTP-bound p21ras in NIH 3T3 cells were comparable to the steady-state level observed in serum-starved c-H-ras-overexpressing transformants, conditions in which these cells maintained high rates of DNA synthesis. These results imply that the level of p21ras activation following PDGF stimulation of NIH 3T3 cells is sufficient to support mitogenic stimulation. Addition of PDGF to c-H-ras-overexpressing cells also resulted in a rapid and sustained increase in GTP-bound p21ras. In these cells GAP, but not p64/62, showed increased tyrosine phosphorylation, with kinetics similar to those observed for increased GTP-bound p21ras. All of these findings support a role for GAP tyrosine phosphorylation in p21ras activation and mitogenic signaling.     

Protein phosphorylation in a tetradecanoyl phorbol acetate-nonproliferative variant of 3T3 cells.

The 3T3-TNR9 cell line is a variant of Swiss 3T3 cells which does not respond mitogenically to tumor promoters, but does respond mitogenically to epidermal growth factor, fibroblast growth factor, and serum. To elucidate differences between tumor promoters and polypeptide mitogens in the pathway(s) of mitogenesis which might be responsible for the nonresponsiveness of the 3T3-TNR9 cells, we have examined in these cells the early protein phosphorylation events known to be associated with mitogenesis in the parental 3T3 cells. We find that the 3T3-TNR9 cells display levels of tetradecanoyl phorbol acetate binding and of a calcium- and phospholipid-dependent protein kinase activity which are at least the equal of those seen in the parental 3T3 cells, implicating some postreceptor event in the nonmitogenic phenotype. In addition, we find that phosphorylation of the epidermal growth factor receptor and of 80-kDa and 22-kDa proteins, as well as the tyrosine phosphorylation of a 42-kDa protein, all proceed normally in the nonmitogenic variant, even though these phosphorylations must depend on the activation of different kinases. Thus, all these early phosphorylation reactions are intact in the 3T3-TNR9 cells. Although these phosphorylations may be necessary, they clearly are insufficient to trigger mitogenesis.     

Regulation of dendritic spine morphology by an NMDA receptor-associated Rho GTPase-activating protein, p250GAP

The NMDA receptor regulates spine morphological plasticity by modulating Rho GTPases. However, the molecular mechanisms for NMDA receptor-mediated regulation of Rho GTPases remain elusive. In this study, we show that p250GAP, an NMDA receptor-associated RhoGAP, regulates spine morphogenesis by modulating RhoA activity. Knock-down of p250GAP increased spine width and elevated the endogenous RhoA activity in primary hippocampal neurons. The increased spine width by p250GAP knock-down was suppressed by the expression of a dominant-negative form of RhoA. Furthermore, p250GAP is involved in NMDA receptor-mediated RhoA activation. In response to NMDA receptor activation, exogenously expressed green fluorescent protein (GFP)-tagged p250GAP was redistributed. Thus, these data suggest that p250GAP plays an important role in NMDA receptor-mediated regulation of RhoA activity leading to spine morphological plasticity.     

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.     

Inhibition of DNA synthesis by protein kinase C-activating phorbol esters in NIH/3T3 cells

Fibroblast growth factor (FGF) plus insulin induced DNA synthesis in and proliferation of NIH/3T3 cells. The protein kinase C-activating phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), inhibited both the DNA synthesis and cell proliferation induced by FGF plus insulin. The concentration of TPA required for 50% inhibition of the DNA synthesis was about 5 nM. Phorbol-12,13-dibutyrate, another protein kinase C-activating phorbol ester, also inhibited the DNA synthesis but 4 alpha-phorbol-12,13-didecanoate, known to be inactive for this enzyme, was ineffective. DNA synthesis started at about 12 h after the addition of FGF plus insulin. The inhibitory action of TPA on the DNA synthesis was observed when it was added within 12 h after the addition of FGF plus insulin. These results suggest that phorbol esters exhibit an antiproliferative action through protein kinase C activation in NIH/3T3 cells, and that this action of phorbol esters is due to inhibition of the progression from the late G1 to the S phase of the cell cycle.     

Serum stimulation of NIH 3T3 cells induces the production of lipids able to inhibit GTPase-activating protein activity.

Quiescent NIH 3T3 cells were stimulated with serum prior to the extraction of total cellular lipids. These lipids were fractionated on thin-layer chromatography plates, and individual fractions were tested for the ability to inhibit GTPase-activating protein (GAP) activity. Two separate GAP inhibitory lipids were produced. One behaved similarly to arachidonic acid during silica gel chromatography, whereas the other was related to a phosphoinositide. Further study of the arachidonic acid-related material indicated that it was produced between 1 and 5 min after serum addition but was never observed in high-density, contact-inhibited cultures. The identity of these lipids is under investigation. The possibility raised by these results, that a metabolite of arachidonic acid is involved in mitogenic signaling, was supported by the finding that several lipoxygenase products of arachidonic acid efficiently inhibited GAP activity. These results provide further support for the hypothesis that lipids, GAP, and ras activity function together in the control of cellular proliferation.     

Constitutive c-myb expression in K562 cells inhibits induced erythroid differentiation but not tetradecanoyl phorbol acetate-induced megakaryocytic differentiation.

K562 cells were stably transfected with a plasmid vector constitutively expressing a full-length human c-myb gene. Parental cells possess the dual potential of inducibility of cellular differentiation along two lineages, i.e., erythroid and megakaryocytic. The resulting lineage is dependent on the inducing agent, with a number of compounds being competent to various degrees for inducing erythroid differentiation, while the tumor promoter tetradecanoyl phorbol acetate (TPA) induces a macrophage-like morphology with enhanced expression of proteins associated with megakaryocytes. Exogeneous expression of c-myb in transfected cell lines abrogated erythroid differentiation induced by cadaverine or cytosine arabinoside as assessed by hemoglobin production. However, TPA-induced megakaryocytic differentiation was left intact, as assessed by cell morphology, cytochemical staining, and the expression of the megakaryocytic antigens. These results indicate that c-Myb and protein kinase C play important roles in cellular differentiation of K562 cells and suggest that agents which directly modulate protein kinase C can induce differentiation in spite of constitutively high levels of c-Myb.     

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.     

Type 3 protein kinase C localization to the nuclear envelope of phorbol ester-treated NIH 3T3 cells

We have examined the immunocytochemical localization of protein kinase C (PKC) in NIH 3T3 cells using mAbs that recognize Type 3 PKC. In control cells, the immunofluorescent staining was similar with mAbs directed to either the catalytic or the regulatory domain of PKC. Type 3 PKC localized in a diffuse cytoplasmic pattern, while the nuclei were apparently unstained. Cytoskeletal components also were Treatment of the cells with phorbol 12-myristate 13-acetate (PMA) resulted in a redistribution of PKC with a specific increase in nuclear PKC. Compared to control cells, the staining with the anticatalytic domain mAbs changed markedly, covering the entire cell surface. In contrast, the staining by the antiregulatory domain mAb did not cover the cell surface and the nuclei remained unstained; these results suggest that PKC activation leads to a conformational change of the regulatory domain such that the epitope recognized by the antiregulatory domain mAb is not readily accessible. We have demonstrated by three criteria that PMA treatment specifically increased PKC in the nucleus: (a) immunofluorescent staining in isolated nuclei increased; (b) Western blots showed that our mAbs detected only one protein, the 82-kD PKC, whose level increased in nuclear lysates from PMA-treated cells; and (c) PKC activity increased in nuclear lysates. In fractionation studies we demonstrated that PKC specifically localized to the nuclear envelope fraction. These results demonstrate that PMA activation leads to a rapid redistribution of Type 3 PKC to the nuclear envelope, and suggests that this isozyme may play a role in mediating PKC-induced changes in gene expression.     

pp39mos is associated with p34cdc2 kinase in c-mosxe-transformed NIH 3T3 cells.

We investigated the possible interactions between pp39mos and p34cdc2 kinase in NIH 3T3 cells transformed by c-mosxe. pp39mos is coprecipitated with p34cdc2 when using either anti-PSTAIR antibody or p13suc1-Sepharose beads. Likewise, p34cdc2 is coprecipitated with pp39mos when using anti-mos antibody. However, pp39mos was not present in histone H1 kinase-active p34cdc2 complexes precipitated with anti-p34cdc2 C-terminal peptide antibody even during metaphase of the cell cycle. The molar ratio of p34 to pp39mos in the p13suc1 complex is approximately 2:1. Consistent with the tight association between pp39mos and tubulin, tubulin was also present in equivalent amounts with pp39mos and p34 in the p13suc1 complex. This pp39mos-p34cdc2-tubulin complex may be important in transformation by the mos oncogene.     

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.     

Calcium oscillation associated with reduced protein kinase C activities in ras-transformed NIH3T3 cells

We show here novel intracellular Ca2+ oscillation in v-K-ras-transformed NIH3T3 cells induced by mitogenic peptide hormones, bradykinin and bombesin, as well as fetal calf serum. Induction of the Ca2+ oscillation is strongly correlated with the malignant properties and inversely with PKC activities in vitro and in vivo. These results suggest that the mitogen-induced Ca2+ oscillation is negatively regulated by PKC, which modulates Ca2+ influx in v-K-ras-transformed NIH3T3 cells.     

The Ras/p120 GTPase-activating protein (GAP) interaction is regulated by the p120 GAP pleckstrin homology domain

Pleckstrin homology domains are structurally conserved functional domains that can undergo both protein/protein and protein/lipid interactions. Pleckstrin homology domains can mediate inter- and intra-molecular binding events to regulate enzyme activity. They occur in numerous proteins including many that interact with Ras superfamily members, such as p120 GAP. The pleckstrin homology domain of p120 GAP is located in the NH(2)-terminal, noncatalytic region of p120 GAP. Overexpression of the noncatalytic domains of p120 GAP may modulate Ras signal transduction pathways. Here, we demonstrate that expression of the isolated pleckstrin homology domain of p120 GAP specifically inhibits Ras-mediated signaling and transformation but not normal cellular growth. Furthermore, we show that the pleckstrin homology domain binds the catalytic domain of p120 GAP and interferes with the Ras/GAP interaction. Thus, we suggest that the pleckstrin homology domain of p120 GAP may specifically regulate the interaction of Ras with p120 GAP via competitive intra-molecular binding.     

Defective regulation of mitogen-activated protein kinase activity in a 3T3 cell variant mitogenically nonresponsive to tetradecanoyl phorbol acetate.

Mitogen-activated protein (MAP) kinase is a serine/threonine-specific protein kinase which is activated in response to various mitogenic agonists (e.g., epidermal growth factor, insulin, and the tumor promoter tetradecanoyl phorbol acetate [TPA]) and requires both threonine and tyrosine phosphorylation for activity. This enzyme has recently been shown to be identical or closely related to pp42, a protein which becomes tyrosine phosphorylated in response to mitogenic stimulation. Neither the kinases which regulate MAP kinase/pp42 nor the in vivo substrates for this enzyme are known. Because MAP MAP kinase is activated and phosphorylated in response both to agents which stimulate tyrosine kinase receptors and to agents which stimulate protein kinase C, a serine/threonine kinase, we have examined the regulation and phosphorylation of this enzyme in 3T3-TNR9 cells, a variant cell line partially defective in protein kinase C-mediated signalling. In this communication, we show that in the 3T3-TNR9 variant cell line, TPA does not cause the characteristically rapid phosphorylation of pp42 or the activation and phosphorylation of MAP kinase. This defective response is not due to the absence of the MAP kinase/pp42 protein itself because both tyrosine phosphorylation of MAP kinase/pp42 and its enzymatic activation could be induced by platelet-derived growth factor in the 3T3-TNR9 cells. Thus, the defect in these variant cells apparently resides in some aspect of the regulation of MAP kinase phosphorylation. Since the 3T3-TNR9 cells are also defective with respect to the TPA-induced increase in ribosomal protein S6 kinase, these in vivo results reinforce the earlier in vitro finding that MAP kinase can regulate S6 kinase activity. These findings suggest a key role for MAP kinase in a kinase cascade cascade involved in the control of cell proliferation.     

Modulation of guanine nucleotides bound to Ras in NIH3T3 cells by oncogenes, growth factors, and the GTPase activating protein (GAP)

The mitogenic activity of membrane-associated tyrosine kinases such as Src and the PDGF receptor appear to depend on Ras function. Ras biochemical activity involves regulation of a GTP/GDP cycle and the GTPase activating protein (GAP). Recently, PDGF and v-Src have been shown to stimulate tyrosine phosphorylation of GAP, linking these pathways at the biochemical level. To test whether PDGF and v-Src affect the Ras GTP/GDP cycle, we have measured the guanine nucleotides complexed to Ras in NIH3T3 cells and compared the ratio of GTP to total GTP + GDP detected (percent GTP). In normal quiescent NIH3T3 cells, PDGF stimulated the basal amount of GTP complexed to Ras (7%) by 2.1-fold to 15%. The effect was dependent on PDGF concentration and was observed maximally within 10 min following PDGF challenge. Ras was complexed to 22% GTP in NIH3T3 cells transformed by v-src or v-abl. Overexpression of GAP by 110-fold in NIH3T3 cells reduced the basal level of GTP complexed to Ras to 2.4%; upon challenge with PDGF, Ras was complexed to 6.6% GTP. These results indicate that PDGF receptor activation and tyrosine kinase-encoding oncogene products can stimulate Ras into the GTP complex and that GAP in intact mammalian cells can decrease the amount of GTP complexed to Ras.     

Ras p21 proteins with high or low GTPase activity can efficiently transform NIH/3T3 cells

We sought to determine whether decreased in vitro GTPase activity is uniformly associated with ras p21 mutants possessing efficient transforming properties. Normal H-ras p21-[Gly12-Ala59] as well as an H-ras p21-[Gly12-Thr59] mutant exhibited in vitro GTPase activities at least fivefold higher than either H-ras p21-[Lys12-Ala59] or H-ras p21-[Arg12-Thr59] mutants. Microinjection of as much as 6 X 10(6) molecules/cell of bacterially expressed normal H-ras p21 induced no detectable alterations of NIH/3T3 cells. In contrast, inoculation of 4-5 X 10(5) molecules/cell of each p21 mutant induced morphologic alterations and stimulated DNA synthesis. Moreover, the transforming activity of each mutant expressed in a eukaryotic vector was similar and at least 100-fold greater than that of the normal H-ras gene. These findings establish that activation of efficient transforming properties by ras p21 proteins can occur by mechanisms not involving reduced in vitro GTPase activity.     

Regulation of osteoclastogenesis by three human RANKL isoforms expressed in NIH3T3 cells

Receptor activator of nuclear factor-kappaB ligand (RANKL) induces osteoclastogenesis by binding with the receptor, receptor activator of nuclear factor-kappaB in the presence of macrophage colony-stimulating factor. Three human RANKL isoforms, hRANKL1, hRANKL2, and hRANKL3, were identified. hRANKL1 was identical to previously reported RANKL and possessed intracellular, transmembrane, and extracellular domains, hRANKL2 did not have the intracellular domain, and hRANKL3 did not have the intracellular and transmembrane domains. When bone marrow macrophages were cultured with NIH3T3 cells expressing hRANKL1, osteoclasts were formed, but when cultured with NIH3T3 cells expressing hRANKL2 or hRANKL3, no tartrate resistant acid phosphatase-positive cell was observed. In the coculture system, coexpression of hRANKL3 with hRANKL1 significantly inhibited the formation of osteoclasts by hRANKL1, but coexpression of hRANKL2 with hRANKL1 did not affect the osteoclastogenesis by hRANKL1 significantly. These results suggest that the activity of osteoclastogenesis by hRANKL1 is regulated by the attenuator, hRANKL3.