Metformin Restrains Pancreatic Duodenal Homeobox-1 (PDX-1) Function by Inhibiting ERK Signaling in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most potent and perilous diseases known, with a median survival rate of 3-5 months due to the combination of only advanced stage diagnosis and ineffective therapeutic options. Metformin (1,1-Dimethylbiguanide hydrochloride), the leading drug used for type 2 diabetes mellitus, emerges as a potential therapy for PDAC and other human cancers. Metformin exerts its anticancer action via a variety of adenosine monophosphate (AMP)-activated protein kinase (AMPK)- dependent and/or AMPK-independent mechanisms. We present data here showing that metformin downregulated pancreatic transcription factor pancreatic duodenal homeobox-1 (PDX-1), suggesting a potential novel mechanism by which metformin exerts its anticancer action. Metformin inhibited PDX-1 expression at both protein and mRNA levels and PDX-1 transactivity as well in PDAC cells. Extracellular signal-regulated kinase (ERK) was identified as a PDX-1-interacting protein by antibody array screening in GFP-PDX-1 stable HEK293 cells. Co-transfection of ERK1 with PDX-1 resulted in an enhanced PDX-1 expression in HEK293 cells in a dose-dependent manner. Immunoprecipitation/Western blotting analysis confirmed the ERK-PDX-1 interaction in PANC-1 cells stimulated by epidermal growth factor (EGF). EGF induced an enhanced PDX-1 expression in PANC-1 cells and this stimulation was inhibited by MEK inhibitor PD0325901. Metformin inhibited EGF-stimulated PDX-1 expression with an accompanied inhibition of ERK kinase activation in PANC- 1 cells. Taken together, our studies show that PDX-1 is a potential novel target for metformin in PDAC cells and that metformin may exert its anticancer action in PDAC by down-regulating PDX-1 via a mechanism involving inhibition of ERK signaling.

CCK(B)/gastrin receptor mediates synergistic stimulation of DNA synthesis and cyclin D1, D3, and E expression in Swiss 3T3 cells.

In order to develop a model system for identifying signaling pathways and cell cycle events involved in gastrin-mediated mitogenesis, we have used high efficiency retroviral-mediated transfection of cholecystokinin (CCK)(B)/gastrin receptor into Swiss 3T3 cells. The retrovirally-transfected CCK(B)/gastrin receptor binds 125I-CCK-8 with high affinity (Kd = 1.1 nM) and is functionally coupled to intracellular signaling pathways including rapid and transient increase in Ca2+ fluxes, protein kinase C-dependent protein kinase D activation, and MEK-dependent ERK1/2 activation. In the presence of insulin, CCK-8 or gastrin induced a 66.5 +/- 8.8-fold (mean +/- SEM, n = 24 in eight independent experiments) increase in cellular DNA synthesis, reaching a level similar to that achieved by stimulation with a saturating concentration of fresh serum, and much greater than the response to each agonist added alone. CCK-8 also induced a striking increase in the expression of cyclins D1, D3, and E and hyperphosphorylation of Rb acting synergistically with insulin. Similar effects were observed when CCK(B)/gastrin receptor was activated in the presence of EGF or bombesin. Our results demonstrate that activation of CCK(B)/gastrin receptor retrovirally-transfected into Swiss 3T3 induces a potent synergistic effect on DNA synthesis, accumulation of cyclins D1, D3, and E and hyperphosphorylation of Rb in combination with insulin, EGF, or bombesin. Thus, the CCK(B)/gastrin receptor transfected into Swiss 3T3 cells provides a novel model system to elucidate mitogenic signal transduction pathways and cell cycle events activated via this receptor.

Regulated nucleocytoplasmic transport of protein kinase D in response to G protein-coupled receptor activation.

Protein kinase D (PKD)/protein kinase C mu is a serine/threonine protein kinase activated by growth factors, antigen-receptor engagement, and G protein-coupled receptor (GPCR) agonists via a phosphorylation-dependent mechanism that requires protein kinase C (PKC) activity. In order to investigate the dynamic mechanisms associated with GPCR signaling, the intracellular distribution of PKD was analyzed in live cells by imaging fluorescent protein-tagged PKD and in fixed cells by immunocytochemistry. We found that PKD shuttled between the cytoplasm and the nucleus in both fibroblasts and epithelial cells. Cell stimulation with mitogenic GPCR agonists that activate PKD induced a transient nuclear accumulation of PKD that was prevented by inhibiting PKC activity. The nuclear import of PKD requires its cys2 domain in conjunction with a nuclear import receptor, while its nuclear export requires its pleckstrin homology domain and a competent Crm1-dependent nuclear export pathway. This study thus characterizes the regulated nuclear transport of a signaling molecule in response to mitogenic GPCR agonists and positions PKD as a serine kinase whose kinase activity and intracellular localization is coordinated by PKC.

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

We examined the role of epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase activation in G protein-coupled receptor (GPCR) agonist-induced mitogenesis in Swiss 3T3 and Rat-1 cells. Addition of EGFR tyrosine kinase inhibitors (e.g., tyrphostin AG-1478) abrogated bombesin-induced extracellular signal-regulated kinase (ERK) activation in Rat-1 cells but not in Swiss 3T3 cells, indicating the importance of cell context in determining the role of EGFR in ERK activation. In striking contrast, treatment with tyrphostin AG-1478 markedly (~70%) inhibited DNA synthesis induced by bombesin in both Swiss 3T3 and Rat-1 cells. Similar inhibition of bombesin-induced DNA synthesis in Swiss 3T3 cells was obtained using four structurally different inhibitors of EGFR tyrosine kinase. Furthermore, kinetic analysis indicates that EGFR function is necessary for bombesin-induced mitogenesis in mid-late G(1) in both Swiss 3T3 and Rat-1 cells. Our results indicate that EGFR kinase activity is necessary in mid-late G(1) for promoting the accumulation of cyclins D1 and E and implicate EGFR function in the coupling of GPCR signaling to the activation of the cell cycle.

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.

Y-27632, an inhibitor of Rho-associated kinases, prevents tyrosine phosphorylation of focal adhesion kinase and paxillin induced by bombesin: dissociation from tyrosine phosphorylation of p130(CAS).

A rapid increase in tyrosine phosphorylation of focal adhesion kinase (FAK), paxillin, and Crk-associated substrate (CAS) are prominent early events triggered by many G protein-coupled receptors (GPCRs), but the mechanisms involved remain unclear. Here, we examined whether the Rho-associated protein serine/threonine kinase family (ROCK) is a critical Rho effector in the pathway that links GPCR activation to the tyrosine phosphorylation of FAK, CAS, and paxillin. Treatment of Swiss 3T3 cells with Y-27632, a preferential inhibitor of ROCK, dramatically inhibited the formation of actin stress fibers, the assembly of focal contacts, and the increase in tyrosine phosphorylation of FAK and paxillin induced by bombesin in these cells. Surprisingly, we found that treatment with Y-27632 did not produce any detectable effect on bombesin-elicited CAS tyrosine phosphorylation even at the highest concentrations of Y-27632 tested. HA-1077, a preferential inhibitor of ROCK activity structurally unrelated to Y-27632, also attenuated the increase in the tyrosine phosphorylation of FAK and paxillin but did not affect the tyrosine phosphorylation of CAS induced by bombesin in Swiss 3T3 cells. The results demonstrate that ROCK-dependent tyrosine phosphorylation of FAK and paxillin can be dissociated from a ROCK-independent pathway leading to tyrosine phosphorylation of CAS.

[D-Arg(1),D-Trp(5,7,9),Leu(11)]Substance P inhibits bombesin-induced mitogenic signal transduction mediated by both G(q) and G(12) in Swiss 3T3cells.

Substance P (SP) analogues including [d-Arg(1),d-Trp(5,7,9), Leu(11)]SP are broad spectrum neuropeptide antagonists and potential anticancer agents, but their mechanism of action is not fully understood. Here, we examined the mechanism of action of [d-Arg(1), d-Trp(5,7,9),Leu(11)]SP as an inhibitor of G protein-coupled receptor (GPCR)-mediated signal transduction and cellular DNA synthesis in Swiss 3T3 cells. Addition of [d-Arg(1),d-Trp(5,7,9), Leu(11)]SP, at 10 micrometer, caused a striking rightward shift in the dose-response curves of DNA synthesis induced by bombesin, bradykinin, or vasopressin and markedly inhibited the activation of p42(mapk) (ERK-2) and p44(mapk) (ERK-1) induced by these GPCR agonists. In addition, this SP analogue also prevented the protein kinase C-dependent activation of protein kinase D induced by these agonists. [d-Arg(1),d-Trp(5,7,9),Leu(11)]SP, at a concentration (10 micrometer) that inhibited these G(q)-mediated events, also prevented GPCR agonist-induced responses mediated through the G proteins of the G(12) subfamily. These include bombesin-induced assembly of focal adhesions, formation of parallel arrays of actin stress fibers, increase in the tyrosine phosphorylation of focal adhesion kinase (FAK), p130(Cas), and paxillin, and formation of a complex between FAK and Src. We conclude that [d-Arg(1),d-Trp(5,7,9),Leu(11)]SP acts as a mitogenic antagonist of neuropeptide GPCRs blocking signal transduction via both G(q) and G(12).

Lysophosphatidic acid rapidly induces protein kinase D activation through a pertussis toxin-sensitive pathway.

Protein kinase D (PKD) is a serine-threonine protein kinase with distinct structural features and enzymological properties. Herein we demonstrate that lysophosphatidic acid (LPA) induces rapid PKD activation in mouse Swiss 3T3 and Rat-1 cells. LPA induced PKD activation in a concentration-dependent fashion with maximal stimulation (7.6-fold) achieved at 5 microM. Treatment of Swiss 3T3 cells with the protein kinase C (PKC) inhibitors GF-I, Ro-31-8220, and Gö-7874 completely abrogated PKD activation induced by LPA at concentrations that did not inhibit PKD activity when added directly to the in vitro kinase assays. PKD activation induced by LPA was attenuated markedly and selectively by prior exposure of either Swiss 3T3 or Rat-1 cells to pertussis toxin (PTx) in a concentration-dependent manner. In contrast, treatment with the protein tyrosine kinase inhibitor genistein, the MEK inhibitor PD-098059, or the phosphoinositide 3-kinase inhibitor wortmannin did not affect PKD activation in response to LPA. These results provide the first example of PTx-sensitive and PKC-dependent PKD activation and identify a novel G(i)-dependent event in the action of LPA.

Protein kinase D inhibits plasma membrane Na(+)/H(+) exchanger activity.

The regulation of plasma membrane Na(+)/H(+) exchanger (NHE) activity by protein kinase D (PKD), a novel protein kinase C- and phorbol ester-regulated kinase, was investigated. To determine the effect of PKD on NHE activity in vivo, intracellular pH (pH(i)) measurements were made in COS-7 cells by microepifluorescence using the pH indicator cSNARF-1. Cells were transfected with empty vector (control), wild-type PKD, or its kinase-deficient mutant PKD-K618M, together with green fluorescent protein (GFP). NHE activity, as reflected by the rate of acid efflux (J(H)), was determined in single GFP-positive cells following intracellular acidification. Overexpression of wild-type PKD had no significant effect on J(H) (3. 48 +/- 0.25 vs. 3.78 +/- 0.24 mM/min in control at pH(i) 7.0). In contrast, overexpression of PKD-K618M increased J(H) (5.31 +/- 0.57 mM/min at pH(i) 7.0; P < 0.05 vs. control). Transfection with these constructs produced similar effects also in A-10 cells, indicating that native PKD may have an inhibitory effect on NHE in both cell types, which is relieved by a dominant-negative action of PKD-K618M. Exposure of COS-7 cells to phorbol ester significantly increased J(H) in control cells but failed to do so in cells overexpressing either wild-type PKD (due to inhibition by the overexpressed PKD) or PKD-K618M (because basal J(H) was already near maximal). A fusion protein containing the cytosolic regulatory domain (amino acids 637-815) of NHE1 (the ubiquitous NHE isoform) was phosphorylated in vitro by wild-type PKD, but with low stoichiometry. These data suggest that PKD inhibits NHE activity, probably through an indirect mechanism, and represents a novel pathway in the regulation of the exchanger.

Bombesin, vasopressin, endothelin, bradykinin, and platelet-derived growth factor rapidly activate protein kinase D through a protein kinase C-dependent signal transduction pathway.

Protein kinase D (PKD) is a serine/threonine protein kinase that is activated by phorbol esters via protein kinase C in intact cells. To assess the physiological significance of this putative pathway, we examined the regulation of PKD in living cells by mitogenic regulatory peptides and by platelet-derived growth factors (PDGF). Our results demonstrate that bombesin rapidly induces PKD activation in Swiss 3T3 cells, as shown by autophosphorylation and syntide-2 phosphorylation assays. Maximum PKD activation (14-fold above base-line levels) was obtained 90 s after bombesin stimulation. Bombesin also induced PKD activation in Rat-1 cells stably transfected with the bombesin/gastrin releasing peptide (GRP) receptor and in COS-7 cells transiently co-transfected with PKD and bombesin/GRP receptor expression constructs. No inducible kinase activity was demonstrated when COS-7 cells were transfected with a kinase-deficient PKD mutant. Bombesin-mediated PKD activation was prevented by treatment of Swiss 3T3 cells with the protein kinase C inhibitors GF 1092030X and Ro 31-8220. In contrast, these compounds did not inhibit PKD activity when added directly in vitro. Vasopressin, endothelin, and bradykinin also activated PKD in Swiss 3T3 cells through a PKC-dependent pathway. Platelet-derived growth factor-stimulated PKD activation in Swiss 3T3 cells and in porcine aortic endothelial cells stably transfected with PDGF-beta receptors. Treatment with GF 1092030X or Ro 31-8220 inhibited PKD activation induced by PDGF. Thus, our results indicate that PKD is activated by multiple signaling peptides through a protein kinase C-dependent signal transduction pathway in a variety of cell types.

Large induction of c-Myc is not essential for the mitogenic response of Swiss 3T3 fibroblasts.

Quiescent Swiss 3T3 fibroblasts can be stimulated to reenter the cell cycle following stimulation with growth factors. Among these, bombesin is a potent mitogen for Swiss 3T3 cells and can act synergistically with insulin to stimulate DNA synthesis through protein kinase C-independent pathways. One of the earliest nuclear responses of quiescent cells treated with a combination of bombesin and insulin is a dramatic increase in c-Myc expression, and it has been suggested that this proto-oncogene plays a central role in the mitogenic response. In the present study, we have taken two approaches to study the relationship between c-Myc expression and the reinitiation of DNA synthesis. First, low concentrations of bombesin, in the presence of insulin, stimulated DNA synthesis in Swiss 3T3 fibroblasts in the absence of a large increase in c-myc mRNA or protein levels. Second, selective down-regulation of phorbol ester-inducible protein kinase C in Swiss 3T3 cells resulted in a 90% decrease in the induction of c-myc mRNA and an 80% reduction in Myc protein expression but did not affect the mitogenic response to bombesin and insulin. These observations were confirmed in detailed dose-response and time-course experiments. We conclude that the large induction of c-Myc is not an essential event for the entry of Swiss 3T3 fibroblasts into S phase. Quantitation of Myc protein levels using a sensitive ELISA indicated that quiescent cells could enter S phase with only 450 c-Myc molecules per cell. These results indicate that cells in the G0 phase of the cell cycle can be stimulated to reinitiate DNA synthesis with only marginal increases in Myc protein expression.

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.

Protein kinase D (PKD): a novel target for diacylglycerol and phorbol esters.

A novel serine/threonine protein kinase regulated by phorbol esters and diacylglycerol (named PKD) has been identified. PKD contains a cysteine-rich repeat sequence homologous to that seen in the regulatory domain of protein kinase C (PKC). A bacterially expressed NH2-terminal domain of PKD exhibited high affinity phorbol ester binding activity (Kd = 35 nM). Expression of PKD cDNA in COS cells conferred increased phorbol ester binding to intact cells. The catalytic domain of PKD contains all characteristic sequence motifs of serine protein kinases but shows only a low degree of sequence similarity to PKCs. The bacterially expressed catalytic domain of PKD efficiently phosphorylated the exogenous peptide substrate syntide-2 in serine but did not catalyse significant phosphorylation of a variety of other substrates utilised by PKCs and other major second messenger regulated kinases. PKD expressed in COS cells showed syntide-2 kinase activity that was stimulated by phorbol esters in the presence of phospholipids. We propose that PKD may be a novel component in the transduction of diacylglycerol and phorbol ester signals.

Expression and characterization of PKD, a phorbol ester and diacylglycerol-stimulated serine protein kinase.

A novel protein kinase (named PKD) with an NH2-terminal region containing two cysteine-rich motifs has been expressed in COS-7 cells and identified as a receptor for phorbol esters. COS-7 cells transfected with a PKD cDNA construct (pcDNA3-PKD) exhibit a marked (4.8-fold) increase in [3H]phorbol 12,13-dibutyrate binding. An antiserum raised against the COOH-terminal 15 amino acids of PKD specifically recognized a single 110-kDa band in PKD-transfected cells. PKD prepared by elution from immunoprecipitates with the immunizing peptide efficiently phosphorylated the synthetic peptide syntide-2. The enzyme only poorly phosphorylated a variant syntide-2 where arginine 4 has been replaced by an alanine. The addition of [3H]phorbol 12,13-dibutyrate, 1-oleoyl-2-acetylglycerol, or 1,2-dioctanoyl-sn-glycerol in the presence of dioleoylphosphatidylserine stimulated the syntide-2 kinase activity of PKD in a synergistic fashion (4-6-fold). Furthermore, the autophosphorylation of PKD was strikingly stimulated by the same lipid activators (14-24-fold). Similar properties were found with PKD isolated from mouse lung. The substrate specificity of PKD is different from that of previously identified members of the protein kinase C family since it does not efficiently phosphorylate histone III-S, protamine sulfate, or a synthetic peptide based upon the conserved pseudosubstrate region of the protein kinase C family. Taken together, these data unambiguously establish PKD as a phorbol ester receptor and as a novel phospholipid/diacylglycerol-stimulated protein kinase.

Molecular cloning and characterization of protein kinase D: a target for diacylglycerol and phorbol esters with a distinctive catalytic domain.

A serine/threonine protein kinase that binds phorbol esters and diacylglycerol (named protein kinase D, PKD) has been identified. PKD contains membrane localization signals and a cysteine-rich repeat sequence homologous to that seen in the regulatory domain of protein kinase C (PKC). A bacterially expressed N-terminal domain of PKD exhibited high-affinity phorbol ester binding activity (Kd = 35 nM). The diacylglycerol analog 1-oleoyl-2-acetylglycerol inhibited phorbol ester binding in a dose-dependent manner. The catalytic domain of PKD contains all characteristic sequence motifs of serine protein kinases but shows only a low degree of sequence similarity to PKCs. The highest identity is with the catalytic domain of myosin light-chain kinase from Dictyostelium (41%). The bacterially expressed catalytic domain of PKD efficiently phosphorylated the exogenous peptide substrate syntide 2 in serine but did not catalyze significant phosphorylation of a variety of other substrates used by PKCs and other major second messenger regulated kinases. PKD may be an unusual component in the transduction of diacylglycerol and phorbol ester signals.

Bombesin treatment enhances vasopressin receptors in Swiss 3T3 cells.

Cells possess receptors for multiple different peptides that regulate a wide spectrum of biological processes. Although examples of homologous and heterologous downregulation have been reported, relatively little is known about the interaction between different peptides in modulating cellular activities. Here we demonstrate that pretreatment of Swiss 3T3 fibroblasts with 10 nM bombesin for 48 h enhanced the 45Ca2+ efflux acutely stimulated by vasopressin. The effect was not reciprocal, since preincubation with vasopressin did not affect the bombesin-stimulated Ca2+ efflux. Measurement of displaceable [3H] vasopressin binding demonstrated that bombesin pretreatment increases the hormonal binding by 3.8 +/- 0.2-fold (SE; n = 14) measured at 37 degrees C or at 4 degrees C. Scatchard analysis at 4 degrees C indicated that the increased binding reflects an increase in the number of vasopressin receptors without any significant effect on the apparent affinity of binding. Furthermore, addition of cycloheximide completely prevented the increase in [3H] vasopressin binding induced by bombesin. We conclude that long-term bombesin pretreatment induces heterologous enhancement of vasopressin responsiveness by increasing the number of membrane receptors.

Bombesin, vasopressin, and endothelin rapidly stimulate tyrosine phosphorylation of the focal adhesion-associated protein paxillin in Swiss 3T3 cells.

Treatment of Swiss 3T3 cells with bombesin caused a striking increase (21-fold) in the tyrosine phosphorylation of the cytoskeleton-associated protein paxillin, as judged by anti-phosphotyrosine Western blots of anti-paxillin immunoprecipitates. Vasopressin and endothelin also stimulated paxillin tyrosine phosphorylation. Bombesin-stimulated tyrosine phosphorylation of paxillin was detectable within 1 min and was concentration-dependent (half-maximum effect at 0.09 nM). Bombesin stimulation of paxillin tyrosine phosphorylation could be dissociated from both protein kinase C (PKC) activation and the mobilization of Ca2+ from intracellular stores. Activation of PKC in quiescent Swiss 3T3 cells using the tumor promoter phorbol 12,13-dibutyrate (PDB) increased the tyrosine phosphorylation of paxillin in a time-dependent manner but was less effective than bombesin and stimulated detectable phosphorylation only within 5 min, considerably slower than bombesin-induced tyrosine phosphorylation of paxillin. Furthermore, the selective PKC inhibitor, GF109203X, or down-regulation of PKC using prolonged treatment with PDB markedly inhibited the stimulation of paxillin tyrosine phosphorylation by PDB but had little effect on the response to bombesin. In contrast, cytochalasin D, an agent that selectively disrupts the network of actin microfilaments, completely inhibited bombesin- and PDB-induced paxillin tyrosine phosphorylation. This is the first report to identify paxillin as a substrate for neuropeptide-stimulated tyrosine phosphorylation.

Bombesin stimulation of p125 focal adhesion kinase tyrosine phosphorylation. Role of protein kinase C, Ca2+ mobilization, and the actin cytoskeleton.

Activation of protein kinase C (PKC) in quiescent Swiss 3T3 cells using either the tumor promoter phorbol 12,13-dibutyrate (PDB) or diacylglycerols increased the tyrosine phosphorylation of p125 focal adhesion kinase (p125FAK) by 3.8-fold. PDB stimulation of p125FAK tyrosine phosphorylation was detected within 1 min and reached a maximum within 5 min, considerably slower than PDB stimulation of 80K/MARCKS phosphorylation which was maximal within 1 min. In sharp contrast, bombesin-induced tyrosine phosphorylation of p125FAK reached a maximum (8-fold stimulation) within 1 min after addition of the peptide and occurred with a half-maximal effect of 0.08 nM, 6-fold lower than the half-maximal effect of bombesin on 80K/MARCKS phosphorylation. Down-regulation of PKC by prolonged treatment with PDB blocked the effect of PDB on p125FAK tyrosine phosphorylation but had no effect on the response to bombesin. A selective inhibitor of PKC, GF 109203X, markedly inhibited the stimulation of p125FAK tyrosine phosphorylation by PDB but had little effect on the response to bombesin, vasopressin, and endothelin. Bombesin stimulation of tyrosine phosphorylation could also be dissociated from mobilization of Ca2+ from intracellular stores. Depletion of the intracellular Ca2+ pool by treatment with the tumor promoter thapsigargin completely blocked the ability of bombesin to transiently increase the cytosolic Ca2+ concentration but had no effect on bombesin stimulation of p125FAK tyrosine phosphorylation. In contrast, cytochalasin D, an agent which selectively disrupts the network of actin microfilaments, completely inhibited bombesin- and PDB-induced p125FAK tyrosine phosphorylation. Within the same concentration range (0.3-2 microM), the drug had no effect on other early events stimulated by bombesin, including Ca2+ mobilization and activation of PKC. These findings demonstrate that neither the PKC nor Ca2+ pathways are responsible for the rapid stimulation of p125FAK tyrosine phosphorylation by neuropeptide growth factors. Furthermore, the integrity of the actin cytoskeleton is essential for the effects of both PDB and bombesin.