EGF receptor function is required in late G(1) for cell cycle progression induced by bombesin and bradykinin

We examined therole of epidermal growth factor (EGF) receptor (EGFR) tyrosine kinaseactivation in G protein-coupled receptor (GPCR) agonist-inducedmitogenesis in Swiss 3T3 and Rat-1 cells. Addition of EGFR tyrosinekinase inhibitors (e.g., tyrphostin AG-1478) abrogated bombesin-inducedextracellular signal-regulated kinase (ERK) activation in Rat-1 cellsbut not in Swiss 3T3 cells, indicating the importance of cell contextin determining the role of EGFR in ERK activation. In strikingcontrast, treatment with tyrphostin AG-1478 markedly (~70%)inhibited DNA synthesis induced by bombesin in both Swiss 3T3 and Rat-1cells. Similar inhibition of bombesin-induced DNA synthesis in Swiss3T3 cells was obtained using four structurally different inhibitors ofEGFR tyrosine kinase. Furthermore, kinetic analysis indicates that EGFRfunction is necessary for bombesin-induced mitogenesis in mid-lateG₁ in both Swiss 3T3 and Rat-1 cells. Our results indicatethat EGFR kinase activity is necessary in mid-late G₁ forpromoting the accumulation of cyclins D1 and E and implicate EGFRfunction in the coupling of GPCR signaling to the activation of thecell cycle.

     

Protein kinase D (PKD) activation in intact cells through a protein kinase C-dependent signal transduction pathway.

Protein kinase D (PKD) is a serine/threonine protein kinase that is directly stimulated in vitro by phorbol esters and diacylglycerol in the presence of phospholipids. Here, we examine the regulation of PKD in living cells. Our results demonstrate that tumour-promoting phorbol esters, membrane-permeant diacylglycerol and serum growth factors rapidly induced PKD activation in immortalized cell lines (e.g. Swiss 3T3 and Rat-1 cells), in secondary cultures of mouse embryo fibroblasts and in COS-7 cells transiently transfected with a PKD expression construct. PKD activation was maintained during cell disruption and immunopurification and was associated with an electrophoretic mobility shift and enhanced 32P incorporation into the enzyme, but was reversed by treatment with alkaline phosphatase. PKD was activated, deactivated and reactivated in response to consecutive cycles of addition and removal of PDB. PKD activation was completely abrogated by exposure of the cells to the protein kinase C inhibitors GF I and Ro 31-8220. In contrast, these compounds did not inhibit PKD activity when added directly in vitro. Co-transfection of PKD with constitutively activated mutants of PKCs showed that PKCepsilon and eta but not PKCzeta strongly induced PKD activation in COS-7 cells. Thus, our results indicate that PKD is activated in living cells through a PKC-dependent signal transduction pathway.     

PKD in intestinal epithelial cells: rapid activation by phorbol esters, LPA, and angiotensin through PKC

Protein kinase C (PKC) isimplicated in the regulation of multiple important functions inintestinal epithelial cells, but the downstream signaling targets ofPKCs in these cells remain poorly characterized. Here we report thattreatment of normal rat intestinal cell lines IEC-6 and IEC-18 withphorbol 12,13-dibutyrate (PDBu) led to a rapid and strikingPKC-dependent activation of protein kinase D (PKD; also known asPKCµ). Unlike conventional and novel PKCs, PKD did not undergodownregulation in response to prolonged (24 h) exposure of IEC-6 orIEC-18 cells to PDBu. PKD was also rapidly activated in these cells bylysophosphatidic acid (LPA) or angiotensin in a concentration-dependentfashion via a PKC-dependent pathway. EC₅₀ values were 0.1 µM and 2 nM for LPA and angiotensin II, respectively. LPA-induced PKDactivation was prevented selectively by treatment with pertussis toxin.PKD activation was tightly associated with an increase in PKDautophosphorylation at serine 916. Our results identify PKD as a novelearly point of convergence and integration of G_i andG_q signaling in intestinal epithelial cells.

     

Protein kinase D potentiates DNA synthesis induced by Gq-coupled receptors by increasing the duration of ERK signaling in swiss 3T3 cells

Protein kinase D (PKD) potentiates cellular DNA synthesis in response to G protein-coupled receptor (GPCR) agonists but the mechanism(s) involved has not been elucidated. Here, we examined whether PKD overexpression in Swiss 3T3 cells regulates the activation/inactivation kinetics of the extracellular-regulated protein kinase (ERK) in response to the mitogenic GPCR agonists bombesin and vasopressin. Addition of bombesin or vasopressin to Swiss 3T3 cells overexpressing PKD induced a striking increase in the duration of MEK/ERK/RSK activation as compared with cultures of either control Swiss 3T3 cells or Swiss 3T3 cells expressing a kinase-inactive PKD mutant. In contrast, the duration of ERK activation in response to epidermal growth factor, which acts via protein kinase C/PKD-independent pathways, was not increased. Furthermore, bombesin or vasopressin promoted a striking increase in phosphorylation (at Ser-374) and accumulation of c-Fos (the c-fos proto-oncogene product) in Swiss 3T3 cells overexpressing wild-type (but not kinase-inactive) PKD. Inhibition of the sustained phase of ERK/RSK activation abrogated the increase in c-Fos accumulation and DNA synthesis induced by bombesin or vasopressin in PKD-overexpressing cells. Our results demonstrate that PKD selectively potentiates mitogenesis induced by bombesin or vasopressin in Swiss 3T3 cells by increasing the duration of MEK/ERK/RSK signaling.     

Protein kinase D potentiates DNA synthesis and cell proliferation induced by bombesin, vasopressin, or phorbol esters in Swiss 3T3 cells

We examined whether protein kinase D (PKD) overexpression in Swiss 3T3 cells potentiates the proliferative response to either the G protein-coupled receptor agonists bombesin and vasopressin or the biologically active phorbol ester phorbol 12,13-dibutyrate (PDBu). In order to generate Swiss 3T3 cells stably overexpressing PKD, cultures of these cells were infected with retrovirus encoding murine PKD and green fluorescent protein (GFP) expressed as two separate proteins translated from the same mRNA. GFP was used as a marker for selection of PKD-positive cells. PKD overexpressed in Swiss 3T3 cells was dramatically activated by cell treatment with bombesin or PDBu as judged by in vitro kinase autophosphorylation assays and exogenous substrate phosphorylation. Concomitantly, these stimuli induced PKD phosphorylation at Ser(744), Ser(748), and Ser(916). PKD activation and phosphorylation were prevented by exposure of the cells to protein kinase C-specific inhibitors. Addition of bombesin, vasopressin, or PDBu to cultures of Swiss 3T3 cells overexpressing PKD induced a striking increase in DNA synthesis and cell number compared with cultures of Swiss 3T3-GFP cells. In contrast, stimulation of DNA synthesis in response to epidermal growth factor, which acts via protein kinase C/PKD-independent pathways, was not enhanced. Our results demonstrate that overexpression of PKD selectively potentiates mitogenesis induced by bombesin, vasopressin, or PDBu in Swiss 3T3 cells.     

Activation loop Ser744 and Ser748 in protein kinase D are transphosphorylated in vivo.

The importance of activation loop phosphorylation in the regulation of protein kinase D (PKD/protein kinase C (PKC) mu) activity has become controversial. In order to clarify the mechanism(s) of PKD activation, we developed a novel phosphospecific antibody recognizing phosphorylated Ser(748) in PKD (pS748). Western blot analysis with the pS748 antibody, carried out with a variety of PKD forms and in a variety of cell types including full-length PKD transfected in COS-7 and HEK 293 cells, a green fluorescent protein-PKD fusion protein transfected in either Swiss 3T3 fibroblasts or Madin-Darby canine kidney epithelial cells, and endogenous PKD expressed in A20 lymphocytes and Rat-1 fibroblasts, indicated that Ser(748) phosphorylation was absent from unstimulated cells. In contrast, dramatic increases in Ser(748) phosphorylation were induced by phorbol esters, bombesin, or cross-linking of B lymphocyte antigen receptors or by cotransfection with active PKCepsilon or PKCeta. Western analysis using a second phosphospecific antibody, which primarily recognizes PKD phosphorylated at Ser(744), revealed that Ser(744) phosphorylation accompanies Ser(748) phosphorylation during PKD activation in vivo. Ser(744)/Ser(748) phosphorylation requires PKC but not PKD activity, indicative of transphosphorylation. Our results provide new experimental evidence indicating that activation loop phosphorylation at Ser(744) and Ser(748) occurs during PKD activation in vivo and support the notion of a PKC-PKD phosphorylation cascade.     

Cooperation of Gq,Gi, and G12/13 in protein kinase D activation and phosphorylation induced by lysophosphatidic acid

To examine the contribution of different G-protein pathways to lysophosphatidic acid (LPA)-induced protein kinase D (PKD) activation, we tested the effect of LPA on PKD activity in murine embryonic cell lines deficient in Galpha(q/11) (Galpha(q/11) KO cells) or Galpha(12/13) (Galpha(12/13) KO cells) and used cells lacking rhodopsin kinase (RK cells) as a control. In RK and Galpha(12/13) KO cells, LPA induced PKD activation through a phospholipase C/protein kinase C pathway in a concentration-dependent fashion with maximal stimulation (6-fold for RK cells and 4-fold for Galpha(12/13) KO cells in autophosphorylation activity) achieved at 3 microm. In contrast, LPA did not induce any significant increase in PKD activity in Galpha(q/11) KO cells. However, LPA induced a significantly increased PKD activity when Galpha(q/11) KO cells were transfected with Galpha(q). LPA-induced PKD activation was modestly attenuated by prior exposure of RK cells to pertussis toxin (PTx) but abolished by the combination treatments of PTx and Clostridium difficile toxin B. Surprisingly, PTx alone strikingly inhibited LPA-induced PKD activation in a concentration-dependent fashion in Galpha(12/13) KO cells. Similar results were obtained when activation loop phosphorylation at Ser-744 was determined using an antibody that detects the phosphorylated state of this residue. Our results indicate that G(q) is necessary but not sufficient to mediate LPA-induced PKD activation. In addition to G(q), LPA requires additional G-protein pathways to elicit a maximal response with G(i) playing a critical role in Galpha(12/13) KO cells. We conclude that LPA induces PKD activation through G(q), G(i), and G(12) and propose that PKD activation is a point of convergence in the action of multiple G-protein pathways.     

Oxidative stress induces protein kinase C-mediated activation loop phosphorylation and nuclear redistribution of protein kinase D

Oxidative stress induced by cell treatments with H(2)O(2) activates protein kinase D (PKD) via a protein kinase C (PKC)-dependent signal transduction pathway (Waldron, R. T., and Rozengurt, E. (2000) J. Biol. Chem. 275, 17114-17121). Here we show that oxidative stress induces PKC-dependent activation loop Ser(744) and Ser(748) phosphorylation to mediate dose- and time-dependent activation of PKD, both endogenously expressed in Swiss 3T3 cells and stably overexpressed in Swiss 3T3-GFP.PKD cells. Although oxidative stress induced PKD activation loop phosphorylation and activation with identical kinetics, both were dose-dependently blocked by preincubation of cells with selective inhibitors of PKC (GF109203X and G?6983) or c-Src (PP2). Inhibition of Src tyrosine kinase activity eliminated oxidative stress-induced direct PKD tyrosine phosphorylation, but only partially attenuated activation loop phosphorylation and activation. Mutation of a putative tyrosine phosphorylation site on PKD, Tyr(469) to phenylalanine, had no effect on its activation by oxidative stress in transfected COS-7 cells. Similarly, a mutant with Tyr(469) replaced by aspartic acid had increased basal activity but was also further activated by oxidative stress. Thus, PKD tyrosine phosphorylation at this site neither produced full activation by itself nor was required for oxidative stress-induced activation mediated by activation loop phosphorylation. In addition to PKD activation, activation loop phosphorylation in response to oxidative stress also redistributed activated PKD to cell nuclei, as revealed by PKD indirect immunofluorescence, imaging of a PKD-green fluorescent protein fusion construct (GFP-PKD), and analysis of nuclear pellets. Cell preincubation with G?6983 strongly diminished H(2)O(2)-induced nuclear relocalization of GFP-PKD. Taken together, these results indicate that PKC-mediated PKD Ser(744) and Ser(748) phosphorylation induced by oxidative stress integrates PKD activation with redistribution to the nucleus.     

Protein kinase D2 potentiates MEK/ERK/RSK signaling, c-Fos accumulation and DNA synthesis induced by bombesin in Swiss 3T3 cells

Protein kinase D (PKD) plays an important role in mediating cellular DNA synthesis in response to G protein-coupled receptor (GPCR) agonists but the function of other isoforms of the PKD family has been much less explored. Here, we examined whether PKD2 overexpression in Swiss 3T3 cells facilitates DNA synthesis and the activation of the extracellular regulated protein kinase (ERK) pathway in response to the mitogenic GPCR agonist bombesin. We show that PKD2 overexpression markedly potentiated the ability of this agonist to induce DNA synthesis. Addition of bombesin to Swiss 3T3 cells overexpressing PKD2 also induced a striking increase in the duration of MEK/ERK/RSK activation as compared with cultures of control cells. In contrast, neither DNA synthesis nor the duration of ERK activation in response to epidermal growth factor, which acts via protein kinase C/PKD2-independent pathways, was increased. Furthermore, bombesin promoted a striking accumulation of c-Fos protein in cells overexpressing PKD2. Our study demonstrates that PKD2, like PKD, facilitates mitogenesis and supports the hypothesis that an increase in the duration of the ERK signaling leading to accumulation of immediate gene products is one of the mechanisms by which isoforms of the PKD family enhance re-initiation of DNA synthesis by Gq-coupled receptor activation.     

Cell-type specific phosphorylation of threonines T654 and T669 by PKD defines the signal capacity of the EGF receptor.

In Rat-1 fibroblasts epidermal growth factor (EGF), but not platelet-derived growth factor (PDGF) stimulates the activity of the c-Jun N-terminal kinase (JNK). Moreover, PDGF induced suppression of EGF-mediated JNK activation, apparently through protein kinase C (PKC) activation. Further analysis revealed that PKD was specifically activated by PDGF but not EGF in Rat-1 cells. In SF126 glioblastoma cells, however, EGF and PDGF synergistically activated JNK, while neither PDGF nor EGF stimulated PKD activity. In this cell line, overexpression of PKD blocked EGF- and PDGF-induced JNK activation. Mutational analysis further revealed that the EGFR mutant (T654/669E) was incapable of activating JNK and provided evidence that PKD-mediated dual phosphorylation of these critical threonine residues leads to suppression of EGF-induced JNK activation. Our results establish a novel crosstalk mechanism which allows signal integration and definition in cells with many different RTKs.     

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.     

Protein kinase D2 mediates lysophosphatidic acid-induced interleukin 8 production in nontransformed human colonic epithelial cells through NF-kappaB

The signaling pathways mediating lysophosphatidic acid (LPA)-stimulated PKD₂ activation and the potential contribution of PKD₂ in regulating LPA-induced interleukin 8 (IL-8) secretion in nontransformed, human colonic epithelial NCM460 cells were examined. Treatment of serum-deprived NCM460 cells with LPA led to a rapid and striking activation of PKD₂, as measured by in vitro kinase assay and phosphorylation at the activation loop (Ser706/710) and autophosphorylation site (Ser876). PKD₂ activation induced by LPA was abrogated by preincubation with selective PKC inhibitors GF-I and Ro-31-8220 in a dose-dependent manner. These inhibitors did not have any direct inhibitory effect on PKD₂ activity. LPA induced a striking increase in IL-8 production and stimulated NF-B activation, as measured by NF-B-DNA binding, NF-B-driven luciferase reporter activity, and IB phosphorylation. PKD₂ gene silencing utilizing small interfering RNAs targeting distinct PKD₂ sequences dramatically reduced LPA-stimulated NF-B promoter activity and IL-8 production. PKD₂ activation is a novel early event in the biological action of LPA and mediates LPA-stimulated IL-8 secretion in NCM460 cells through a NF-B-dependent pathway. Our results demonstrate, for the first time, the involvement of a member of the PKD family in the production of IL-8, a potent proinflammatory chemokine, by epithelial cells. NCM460 cells; protein kinase C; CXCL8; phorbol esters     

Protein kinase D protects against oxidative stress-induced intestinal epithelial cell injury via Rho/ROK/PKC-delta pathway activation

Protein kinase D (PKD) is a novel protein serine kinase that has recently been implicated in diverse cellular functions, including apoptosis and cell proliferation. The purpose of our present study was 1) to define the activation of PKD in intestinal epithelial cells treated with H₂O₂, an agent that induces oxidative stress, and 2) to delineate the upstream signaling mechanisms mediating the activation of PKD. We found that the activation of PKD is induced by H₂O₂ in both a dose- and time-dependent fashion. PKD phosphorylation was attenuated by rottlerin, a selective PKC- inhibitor, and by small interfering RNA (siRNA) directed against PKC-, suggesting the regulation of PKD activity by upstream PKC-. Activation of PKD was also blocked by a Rho kinase (ROK)-specific inhibitor, Y-27632, as well as by C3, a Rho protein inhibitor, demonstrating that the Rho/ROK pathway also mediates PKD activity in intestinal cells. In addition, H₂O₂-induced PKC- phosphorylation was inhibited by C3 treatment, further suggesting that PKC- is downstream of Rho/ROK. Interestingly, H₂O₂-induced intestinal cell apoptosis was enhanced by PKD siRNA. Together, these results clearly demonstrate that oxidative stress induces PKD activation in intestinal epithelial cells and that this activation is regulated by upstream PKC- and Rho/ROK pathways. Importantly, our findings suggest that PKD activation protects intestinal epithelial cells from oxidative stress-induced apoptosis. These findings have potential clinical implications for intestinal injury associated with oxidative stress (e.g., necrotizing enterocolitis in infants). Rho kinase; protein kinase C-     

CCK2 (CCK(B)/gastrin) receptor mediates rapid protein kinase D (PKD) activation through a protein kinase C-dependent pathway

Addition of gastrin or cholecystokinin octapeptide (CCK-8) to cultures of Rat-1 cells stably transfected with the CCK2 (CCK(B)/gastrin) receptor induced protein kinase D (PKD) activation that was detectable within 1 min and reached a maximum ( approximately 10-fold) after 2.5 min of hormonal stimulation. Half-maximal PKD activation for both CCK-8 and gastrin was achieved at 10 nM. Treatment with various concentrations of the selective PKC inhibitors Ro 31-8220 or GF-I potently blocked PKD activation induced by subsequent addition of CCK-8 in a concentration-dependent fashion. Our results indicate that PKC-dependent PKD activation is a novel early event in the action of gastrin and CCK-8 via CCK2 receptors.     

Tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin does not require extracellular signal-regulated kinase activation in Swiss 3T3 cells stimulated by bombesin or platelet-derived growth factor

The experiments presented here were designed to examine the contribution of the extracellular signal-regulated mitogen-activated protein kinases (ERKs) to the tyrosine phosphorylation of the focal adhesion proteins p125(Fak), p130(Cas), and paxillin induced by G protein-coupled receptors (GPCRs) and tyrosine kinase receptors in Swiss 3T3 cells. Stimulation of these cells with bombesin, lysophosphatidic acid (LPA), endothelin, and platelet-derived growth factor (PDGF) led to a marked increase in the tyrosine phosphorylation of these focal adhesion proteins and in ERK activation. Exposure of the cells to two structurally unrelated mitogen-activated protein kinase or ERK kinase (MEK) inhibitors, PD98059 and U0126, completely abrogated ERK activation but did not prevent tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin. Furthermore, different dose-response relationships were obtained for tyrosine phosphorylation of focal adhesion proteins and for ERK activation in response to PDGF. Putative upstream events in the activation of focal adhesion proteins including actin cytoskeletal reorganization and myosin light chain (MLC) phosphorylation were also not prevented by inhibition of ERK activation. Thus, our results demonstrate that the activation of the ERK pathway is not necessary for the increase of the tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin induced by either GPCRs or tyrosine kinase receptors in Swiss 3T3 cells.     

TNF-α potentiates lysophosphatidic acid-induced COX-2 expression via PKD in human colonic myofibroblasts

The myofibroblast (MFB) has recently been identified as an important mediator of tumor necrosis factor-α (TNF-α)-associated colitis and cancer, but the mechanism(s) involved remains incompletely understood. Here, we show that treatment of 18Co cells, a model of human colonic MFBs, with TNF-α and lysophosphatidic acid (LPA) induced striking synergistic cyclooxygenase-2 (COX-2) protein expression and production of PGE(2). This effect was prevented by the LPA(1) receptor antagonist Ki16425, the G(iα)-specific inhibitor pertussis toxin, and by the preferential protein kinase (PK) C inhibitors GF109203X and Go6983. As a known downstream target of LPA and PKC, we tested whether PKD, recently implicated in the regulation of COX-2 expression in MFB, was involved in this response. TNF-α, while having no detectable effect on the activation of PKD when added alone, augmented PKD activation stimulated by LPA, as measured by PKD autophosphorylation at Ser(910). LPA-induced PKD activation was also inhibited by Ki16425, pertussis toxin, GF109203X, and Go6983. Transfection of 18Co cells with short interfering RNA targeting PKD completely inhibited the synergistic increase in COX-2 protein, demonstrating a critical role of PKD in this response. Our results imply that cross talk between TNF-α and LPA results in the amplification of COX-2 protein expression via a conserved PKD-dependent signaling pathway that appears to involve the LPA(1) receptor and the G protein G(iα). PKD plays a critical role in the expression of COX-2 in human colonic MFBs and may contribute to an inflammatory microenvironment that promotes tumor growth.     

Protein kinase C-independent activation of protein kinase D is involved in BMP-2-induced activation of stress mitogen-activated protein kinases JNK and p38 and osteoblastic cell differentiation

An important role for JNK* and p38 has recently been discovered in the differentiating effect of bone morphogenetic protein 2 (BMP-2) on osteoblastic cells. In this study, we investigated the molecular mechanism by which BMP-2 activates JNK and p38 in MC3T3-E1 osteoblastic cells. Activation of JNK and p38 induced by BMP-2 was blocked by the protein kinase C/protein kinase D (PKC/PKD) inhibitor Go6976 but not by the related compound, Go6983, a selective inhibitor of conventional PKCs. Associated with this inhibitory effect of Go6976, BMP-2 induced a selective and a dose-dependent Ser916 phosphorylation/activation of PKD, which was also blocked by Go6976. In contrast to the recently described PKC-dependent molecular mechanism involved in activation of PKD by G protein-coupled receptor agonists, BMP-2 did not induce a phosphorylation of PKD on Ser744/748. To further document an implication of PKD in activation of JNK and p38 induced by BMP-2, we constructed MC3T3-E1 cells stably expressing PKD antisense oligonucleotide (AS-PKD). In AS-PKD clones having low PKD levels, activation of JNK and p38 by BMP-2, but not of Smad1/5, was markedly impaired compared with empty vector transfected (V-PKD) cells. Analysis of osteoblastic cell differentiation in AS-PKD compared with V-PKD cells showed that mRNA and protein expressions of alkaline phosphatase and osteocalcin induced by BMP-2 were markedly reduced in AS-PKD. In conclusion, results presented in this study indicate that BMP-2 can induce activation of PKD in osteoblastic cells by a PKC-independent mechanism and that this kinase is involved in activation of JNK and p38 induced by BMP-2. Thus, this pathway, in addition to Smads, appears to be essential for the effect of BMP-2 on osteoblastic cell differentiation.     

Signal transduction pathways regulating Rho-mediated stress fibre formation: requirement for a tyrosine kinase.

Lysophosphatidic acid (LPA) and bombesin rapidly stimulate the formation of focal adhesions and actin stress fibres in serum-starved Swiss 3T3 fibroblasts, a process regulated by the small GTP binding protein Rho. To investigate further the signalling pathways leading to these responses, we have tested the roles of three intracellular signals known to be induced by LPA: activation of protein kinase C (PK-C), Ca2+ mobilization and decreased cAMP levels. Neither PK-C activation nor increased [Ca2+]i, alone or in combination, induced stress fibre formation, and in fact activators of PK-C inhibited this response to LPA and bombesin. The G(i)-mediated decrease in cAMP was not required for the response to LPA, and increased cAMP levels did not prevent stress fibre formation. In contrast, the tyrosine kinase inhibitor genistein inhibited the formation of stress fibres induced by both extracellular factors and microinjected Rho protein. Genistein also inhibited the Rho-dependent clustering of phosphotyrosine-containing proteins at focal adhesions, and the increased tyrosine phosphorylation of several proteins including pp125FAK, induced by LPA and bombesin. This suggests a model where Rho-induced activation of a tyrosine kinase is required for the formation of stress fibres.     

Gi-mediated activation of the Ras/MAP kinase pathway involves a 100 kDa tyrosine-phosphorylated Grb2 SH3 binding protein, but not Src nor Shc.

Mitogenic G protein-coupled receptors, such as those for lysophosphatidic acid (LPA) and thrombin, activate the Ras/MAP kinase pathway via pertussis toxin (PTX)-sensitive Gi, tyrosine kinase activity and recruitment of Grb2, which targets guanine nucleotide exchange activity to Ras. Little is known about the tyrosine phosphorylations involved, although Src activation and Shc phosphorylation are thought to be critical. We find that agonist-induced Src activation in Rat-1 cells is not mediated by Gi and shows no correlation with Ras/MAP kinase activation. Furthermore, LPA-induced tyrosine phosphorylation of Shc is PTX-insensitive and Ca2+-dependent in COS cells, but undetectable in Rat-1 cells. Expression of dominant-negative Src or Shc does not affect MAP kinase activation by LPA. Thus, Gi-mediated Ras/MAP kinase activation in fibroblasts and COS cells involves neither Src nor Shc. Instead, we detect a 100 kDa tyrosine-phosphorylated protein (p100) that binds to the C-terminal SH3 domain of Grb2 in a strictly Gi- and agonist-dependent manner. Tyrosine kinase inhibitors and wortmannin, a phosphatidylinositol (PI) 3-kinase inhibitor, prevent p100-Grb2 complex formation and MAP kinase activation by LPA. Our results suggest that the p100-Grb2 complex, together with an upstream non-Src tyrosine kinase and PI 3-kinase, couples Gi to Ras/MAP kinase activation, while Src and Shc act in a different pathway.