PDGF and FGF induce focal adhesion kinase (FAK) phosphorylation at Ser-910: dissociation from Tyr-397 phosphorylation and requirement for ERK activation

A rapid increase in the tyrosine phosphorylation of focal adhesion kinase (FAK) has been extensively documented in cells stimulated by multiple signaling molecules, but very little is known about the regulation of FAK phosphorylation at serine residues. Stimulation of Swiss 3T3 cells with platelet-derived growth factor (PDGF) promoted a striking increase in the phosphorylation of FAK at Ser-910, as revealed by site-specific antibodies that recognized the phosphorylated state of this residue. FAK phosphorylation at Ser-910 could be distinguished from that at Tyr-397 in terms of dose-response relationships and kinetics. Furthermore, the selective phosphoinositide 3-kinase (PI 3-kinase) inhibitors wortmannin and LY 294002 abrogated FAK phosphorylation at Tyr-397 but did not interfere with PDGF-induced FAK phosphorylation at Ser-910. Conversely, treatment with U0126, a potent inhibitor of MEK-mediated ERK activation, prevented FAK phosphorylation at Ser-910 induced by PDGF but did not interfere with PDGF-induced FAK phosphorylation at Tyr-397. These results were extended using growth factors that either stimulate, fibroblast growth factor (FGF), or do not stimulate (insulin) the ERK pathway activation in Swiss 3T3 cells. FGF but not insulin promoted a striking ERK-dependent phosphorylation of FAK at Ser-910. Our results indicate that FAK phosphorylation at Tyr-397 and FAK phosphorylation at Ser-910 are induced in response to PDGF stimulation through different signaling pathways, namely PI 3-kinase and ERK, respectively.     

Differential FAK phosphorylation at Ser-910, Ser-843 and Tyr-397 induced by angiotensin II, LPA and EGF in intestinal epithelial cells

A rapid increase in the tyrosine phosphorylation of the non-receptor tyrosine kinase FAK is a prominent early event in fibroblasts stimulated by a variety of signaling molecules. However, a variety of epithelial cells, including intestinal epithelial cells, show a high basal level of tyrosine phosphorylated FAK that is only slightly further increased by addition of G protein-coupled receptor (GPCR) agonists or growth factors. In this study, we determined whether these stimuli could elicit FAK phosphorylation at serine residues, including Ser-910 and Ser-843. Our results show that multiple agonists including angiotensin II (ANGII), lysophosphatidic acid (LPA), phorbol esters and EGF induced a striking stimulation of FAK phosphorylation at Ser-910 in rat intestinal epithelial IEC-18 cells via an ERK-dependent pathway. In striking contrast, none of these stimuli promoted a significant further increase in FAK phosphorylation at Tyr-397 in these cells. These results were extended using cultures of polarized human colonic epithelial T84 cells. We found that either carbachol or EGF promoted a striking ERK-dependent phosphorylation of FAK at Ser-910, but these agonists caused only slight stimulation of FAK at Tyr-397 in T84 cells. In addition, we demonstrated that GPCR agonists also induced a dramatic increase of FAK phosphorylation at Ser-843 in either IEC-18 or T84 cells. Our results indicate that Ser-910 and Ser-843, rather than Tyr-397, are prominent sites differentially phosphorylated in response to neurotransmitters, bioactive lipids, tumor promoters and growth factors in intestinal epithelial cells.     

G protein-coupled receptor activation rapidly stimulates focal adhesion kinase phosphorylation at Ser-843. Mediation by Ca2+, calmodulin, and Ca2+/calmodulin-dependent kinase II

A rapid increase in the tyrosine phosphorylation of focal adhesion kinase (FAK) has been extensively documented in cells stimulated by multiple signaling molecules, but little is known about the regulation of FAK phosphorylation at serine residues. Stimulation of Swiss 3T3 cells with the G protein-coupled receptor agonists bombesin, vasopressin, or bradykinin induced an extremely rapid (within 5 s) increase in FAK phosphorylation at Ser-843. The phosphorylation of this residue preceded FAK phosphorylation at Tyr-397, the major autophosphorylation site, and FAK phosphorylation at Ser-910. Treatment of intact cells with ionomycin stimulated a rapid increase in FAK phosphorylation at Ser-843, indicating that an increase in intracellular Ca2+ concentration ([Ca2+]i) is a potential pathway leading to FAK-Ser-843 phosphorylation. Indeed, treatment with agents that prevent an agonist-induced increase in [Ca2+]i (e.g. thapsigargin or BAPTA (1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)), interfere with calmodulin function (e.g. trifluoperazine, W13, and W7), or block Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation (KN93) or expression (small interfering RNA) abrogated the rapid FAK phosphorylation at Ser-843 induced by bombesin, bradykinin, or vasopressin. Furthermore, activated CaMKII directly phosphorylated the recombinant COOH-terminal region of FAK at a residue equivalent to Ser-843. Thus, our results demonstrate that G protein-coupled receptor activation induces rapid FAK phosphorylation at Ser-843 through Ca2+, calmodulin, and CaMKII.     

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.     

Bombesin, vasopressin, and endothelin stimulation of tyrosine phosphorylation in Swiss 3T3 cells. Identification of a novel tyrosine kinase as a major substrate.

Neuropeptide-stimulated tyrosine phosphorylation of specific components in Swiss 3T3 cells was investigated using monoclonal antibodies directed against the src transformation-associated substrates p125 focal adhesion kinase (FAK), a novel type of cytosolic tyrosine kinase, and p130. Treatment of Swiss 3T3 cells with the mitogenic peptides bombesin, vasopressin, and endothelin caused a striking increase in the tyrosine phosphorylation of p125FAK, as judged either by anti-phosphotyrosine (anti-Tyr(P)) Western blots of anti-p125FAK immunoprecipitates, or by anti-p125FAK immunoblots of anti-Tyr(P) immunoprecipitates. Bombesin-stimulated tyrosine phosphorylation of p125FAK was detectable within seconds and concentration-dependent (half-maximum effect of 0.3 nM). Neuropeptides also stimulated the tyrosine phosphorylation of a second component of M(r) 130,000, previously identified as the major p130 phosphotyrosyl protein in src-transformed cells. Bombesin stimulated p130 tyrosine phosphorylation with kinetics and concentration dependence similar to those observed for p125FAK. This is the first report to identify substrates for neuropeptide-stimulated tyrosine phosphorylation; the finding that one of these substrates is a tyrosine kinase suggests the existence of a novel signal transduction pathway in the action of mitogenic neuropeptides.     

Src family kinases are required for integrin-mediated but not for G protein-coupled receptor stimulation of focal adhesion kinase autophosphorylation at Tyr-397

Plating suspended Swiss 3T3 cells onto fibronectin-coated dishes promoted phosphorylation of endogenous focal adhesion kinase (FAK) at Tyr-397, the major autophosphorylation site, and at Tyr-577, located in the activation loop, as revealed by site-specific antibodies that recognize the phosphorylated form of these residues. Treatment with the selective Src family kinase inhibitor pyrazolopyrimidine 2 (PP-2) markedly reduced the phosphorylation of both Tyr-397 and Tyr-577 induced by fibronectin. Furthermore, fibronectin-mediated FAK phosphorylation at Tyr-397 was dramatically reduced in SYF cells (deficient in Src, Yes, and Fyn expression). Stimulation of Swiss 3T3 cells with bombesin also induced a rapid increase in the phosphorylation of endogenous FAK at Tyr-397. In contrast to the results obtained with fibronectin, PP-2 did not prevent FAK Tyr-397 phosphorylation stimulated by bombesin at a concentration (10 micrometer) that suppressed bombesin-induced FAK Tyr-577 phosphorylation. Similarly, PP-2 did not prevent Tyr-397 phosphorylation in Swiss 3T3 cells stimulated with other G protein-coupled receptor agonists including vasopressin, bradykinin, endothelin, and lysophosphatidic acid. Lysophosphatidic acid also induced FAK phosphorylation at Tyr-397 in SYF cells. Our results identify, for first time, the existence of Src-dependent and Src-independent pathways leading to FAK autophosphorylation at Tyr-397 stimulated by adhesion-dependent signals and G protein-coupled receptor agonists in the same cell.     

Bombesin and platelet-derived growth factor induce association of endogenous focal adhesion kinase with Src in intact Swiss 3T3 cells.

Stimulation of quiescent Swiss 3T3 cells with bombesin induces a rapid increase in the formation of complexes between focal adhesion kinase (FAK) and Src family members, which can be extracted with a buffer containing Triton, deoxycholate, and SDS but not with a buffer containing Triton alone. An increase in complex formation between FAK and Src in response to bombesin could be detected within 1 min, reached a maximum after 10 min, and declined toward base-line levels after 60 min of bombesin treatment. Bradykinin, endothelin, and lysophosphatidic acid also stimulated FAK-Src complex formation. Bombesin stimulated FAK/Src association through a Ca(2+) and phosphatidylinositol 3'-kinase-independent pathway that requires the integrity of the actin filament network and is partly dependent on functional protein kinase C. Treatment with the selective Src kinase inhibitor PP-2 inhibited both FAK activation and phosphorylation of FAK at Tyr(577) induced by bombesin in intact cells. Platelet-derived growth factor at low concentrations (1-10 ng/ml) also induced FAK-Src complex formation via a pathway that depended on the integrity of the actin cytoskeleton and phosphatidylinositol 3'-kinase. Thus, G protein-coupled receptor agonists and platelet-derived growth factor promote complex formation between endogenous FAK and Src in attached cells through different signal transduction pathways.     

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

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.     

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.     

Dissociation of focal adhesion kinase and paxillin tyrosine phosphorylation induced by bombesin and lysophosphatidic acid from epidermal growth factor receptor transactivation in Swiss 3T3 cells

Tyrosine phosphorylation of the nonreceptor tyrosine kinase p125 focal adhesion kinase (FAK) and the adapter protein paxillin is rapidly increased by multiple agonists, including bombesin (BOM) and lysophosphatidic acid (LPA), through heptahelical G protein-coupled receptors (GPCRs). The pathways involved remain incompletely understood. The experiments presented here were designed to test the role of epidermal growth factor receptor (EGFR) transactivation in the rapid increase of tyrosine phosphorylation of FAK and paxillin induced by GPCR agonists. Our results show that treatment with the selective EGFR tyrosine kinase inhibitor AG 1478, at concentrations that completely blocked the increase in tyrosine phosphorylation of these proteins induced by EGF, did not affect the stimulation of tyrosine phosphorylation of either FAK or paxillin induced by multiple GPCR agonists including LPA, BOM, vasopressin, bradykinin, and endothelin. Similar results were obtained when Swiss 3T3 cells were treated with another highly specific inhibitor of the EGF receptor kinase activity, PD-158780. Collectively, our results clearly dissociate EGFR transactivation from the tyrosine phosphorylation of FAK and paxillin induced by multiple GPCR agonists.     

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.     

Mechanical strain on osteoblasts activates autophosphorylation of focal adhesion kinase and proline-rich tyrosine kinase 2 tyrosine sites involved in ERK activation

The mechanisms involved in the mechanical loading-induced increase in bone formation remain unclear. In this study, we showed that cyclic strain (CS) (10 min, 1% stretch at 0.25 Hz) stimulated the proliferation of overnight serum-starved ROS 17/2.8 osteoblast-like cells plated on type I collagen-coated silicone membranes. This increase was blocked by MEK inhibitor PD-98059. Signaling events were then assessed 0 min, 30 min, and 4 h after one CS period with Western blotting and coimmunoprecipitation. CS rapidly and time-dependently promoted phosphorylation of both ERK2 at Tyr-187 and focal adhesion kinase (FAK) at Tyr-397 and Tyr-925, leading to the activation of the Ras/Raf/MEK pathway. Cell transfection with FAK mutated at Tyr-397 completely blocked ERK2 Tyr-187 phosphorylation. Quantitative immunofluorescence analysis of phosphotyrosine residues showed an increase in focal adhesion plaque number and size in strained cells. CS also induced both Src-Tyr-418 phosphorylation and Src to FAK association. Treatment with the selective Src family kinase inhibitor pyrazolopyrimidine 2 did not prevent CS-induced FAK-Tyr-397 phosphorylation suggesting a Src-independent activation of FAK. CS also activated proline-rich tyrosine kinase 2 (PYK2), a tyrosine kinase highly homologous to FAK, at the 402 phosphorylation site and promoted its association to FAK in a time-dependent manner. Mutation of PYK2 at the Tyr-402 site prevented the ERK2 phosphorylation only at 4 h. Intra and extracellular calcium chelators prevented PYK2 activation only at 4 h. In summary, our data showed that osteoblast response to mitogenic CS was mediated by MEK pathway activation. The latter was induced by ERK2 phosphorylation under the control of FAK and PYK2 phosphorylation orchestrated in a time-dependent manner.     

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 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.     

Nitric oxide stimulates tyrosine phosphorylation of focal adhesion kinase, Src kinase, and mitogen-activated protein kinases in murine fibroblasts

Nitric oxide (NO) can participate in cellular signaling. In this study, monoclonal antibodies against proteins from the growth factor-mediated signalling pathway were used to identify a set of 126-, 56-, 43-, and 40-kDa proteins phosphorylated on tyrosine at NO stimulation of murine fibroblasts overexpressing the human epidermal growth factor receptor. The band corresponding to the 126-kDa protein was FAK. The 56-kDa protein was Src kinase, and the doublet 43- and 40-kDa protein corresponded to the extracellular-regulated MAP kinases (ERK1/ERK2). The effects of NO on focal adhesion complexes were also investigated. FAK was constitutively associated with the adapter protein Grb2 in HER14 cells. Treatment of the cells with the NO donor, sodium nitroprusside, or with EGF did not change this association. We also detected a basal constitutive association of Src kinase with FAK in HER14 cells. In NO-treated cells, this association was stimulated. The doublet 43/40-kDa protein was identical to the ERK1/ERK2 MAP kinases. NO stimulated an increase in ERK1/ERK2 phosphorylation as assessed by a shift in its eletrophoretic mobility and by increased phosphotyrosine immunoreactivity. Furthermore, NO-dependent activation of ERK1/ERK2 depended on the intracellular redox status. Inhibition of glutathione synthesis was necessary to promote activation of the kinases.     

Bombesin and angiotensin II rapidly stimulate Src phosphorylation at Tyr-418 in fibroblasts and intestinal epithelial cells through a PP2-insensitive pathway

Src is activated in response to a variety of growth factors and hormones that bind G protein-coupled receptors (GPCRs), and its activity is regulated by phosphorylation at key sites, including the autophosphorylation site Tyr-418 and the inhibitory site Tyr-529. To better understand the mechanisms controlling Src activation, we examined Src phosphorylation in Swiss 3T3 fibroblasts stimulated with bombesin and in IEC-18 intestinal epithelial cells stimulated with angiotensin II (Ang II). Phosphorylation at Src Tyr-418, the activation loop site, was rapidly and markedly increased after GPCR agonist addition in both cell types. However, treatment of intact cells with the selective Src family kinase inhibitor PP2, at concentrations which abolished Src-mediated phosphorylation of focal adhesion kinase (FAK) at Tyr-577, unexpectedly led to increased phosphorylation at Src Tyr-418 and diminished phosphorylation at Tyr-529. In Swiss 3T3 cells, PP2 enhanced Tyr-418 phosphorylation after 1 min of bombesin stimulation, while in IEC-18 cells, PP2 increased Ang II-stimulated Tyr-418 phosphorylation at all times tested. These results imply that a distinct, non-Src family kinase may be responsible for phosphorylating Src at Tyr-418 in intact fibroblasts and epithelial cells stimulated by GPCR agonists.     

Bombesin induction of c-fos and c-myc proto-oncogenes in Swiss 3T3 cells: significance for the mitogenic response

Bombesin is a potent mitogen for Swiss 3T3 cells and acts synergistically with insulin and other growth factors. We show here that addition of bombesin to quiescent Swiss 3T3 cells causes a striking increase in the levels of c-fos and c-myc mRNAs. Enhanced expression of c-fos (122 +/- 14-fold) occurred within minutes of peptide addition followed by increased expression of c-myc (82 +/- 16-fold). The concentrations of peptide required for half-maximal increase in the levels of c-fos and c-myc mRNAs were 1.0 and 0.9 nM, respectively. The peptide [D-Arg1, D-Pro2, D-Trp7,9, Leu11] substance P which inhibits the binding of bombesin to its receptor and bombesin-stimulated DNA synthesis in Swiss 3T3 cells blocked the increase in c-fos and c-myc mRNA levels promoted by bombesin. Down-regulation of protein kinase C by long-term exposure to phorbol esters prevented c-fos and c-myc induction by bombesin. This and other results indicate that the induction of these proto-oncogenes by bombesin could be mediated by the coordinated effects of protein kinase C activation and Ca2+ mobilization. The marked synergistic effect between bombesin and insulin was used to assess whether the increase in the induction of c-fos and c-myc is an obligatory event in cell activation. In the presence of insulin, bombesin stimulated DNA synthesis at subnanomolar concentrations but had only a small effect on c-fos and c-myc mRNA levels. This apparent dissociation of mitogenesis from proto-oncogene induction was even more dramatic in 3T3 cells with down-regulated protein kinase C. In these cells bombesin stimulated DNA synthesis in the presence of insulin but failed to enhance c-fos and c-myc mRNA levels at comparable concentrations. Thus, the induction of c-fos and c-myc may be a necessary step in the mitogenic response initiated by ligands that act through activation of protein kinase C but the expression of these proto-oncogenes may not be an obligatory event in the stimulation of mitogenesis in 3T3 cells by mitogens that utilise other signalling pathways.     

Bombesin, diacylglycerols, and phorbol esters rapidly stimulate the phosphorylation of an Mr = 80,000 protein kinase C substrate in permeabilized 3T3 cells. Effect of guanine nucleotides

We have used digitonin permeabilization to study the mechanism of bombesin-induced activation of protein kinase C in Swiss 3T3 cells. Protein kinase C-mediated phosphorylations in permeabilized cells were identified using phorbol esters and diacylglycerols. Addition of phorbol 12,13-dibutyrate (PDBu) in the presence of [gamma-32P]ATP and digitonin caused a marked and rapid time- and dose-dependent increase in the phosphorylation of an Mr 80,000 cellular protein (maximum stimulation = 12.6 +/- 1.6-fold after 1 min, EC50 = 27 nM). 1-oleoyl-2-acetylglycerol substituted for PDBu in stimulating the phosphorylation of Mr 80,000 protein (EC50 = 13 microM). Bombesin also caused a striking increase in the phosphorylation of Mr 80,000 protein with a time course similar to that observed with PDBu. This phosphorylation was mimicked by mammalian bombesin-like peptides and blocked by the bombesin antagonists [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P and [Leu13 psi (CH2NH)Leu14]bombesin. Down-regulation of protein kinase C in intact cells by prolonged exposure to PDBu prevented Mr 80,000 protein phosphorylation upon subsequent bombesin addition in digitonin-permeabilized cells. Comigration on one- and two-dimensional gel electrophoresis and phosphopeptide mapping confirmed that the Mr 80,000 protein phosphorylated in permeabilized cells was indistinguishable from the Mr 80,000 protein which is the major protein kinase C substrate in intact cells. The GDP analogue guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) caused a 70% inhibition of the bombesin-induced phosphorylation of Mr 80,000 protein but had no effect on the phosphorylation induced by PDBu. Bombesin stimulated Mr 80,000 protein phosphorylation in permeabilized cells in a dose-dependent manner (EC50 = 4 nM), and GDP beta S shifted the bombesin dose response curve to higher bombesin concentrations (EC50 = 14 nM). These results demonstrate for the first time a growth factor receptor-mediated activation of protein kinase C in permeabilized cells and provide functional evidence for the involvement of a G protein in the transmembrane signaling pathway that mediates the stimulation of protein kinase C by bombesin in Swiss 3T3 cells.     

Neurotensin and EGF induce synergistic stimulation of DNA synthesis by increasing the duration of ERK signaling in ductal pancreatic cancer cells

Neurotensin (NT) and epidermal growth factor (EGF) induced rapid extracellular-regulated protein kinase (ERK) activation through different signaling pathways in the K-Ras mutated human pancreatic carcinoma cell lines PANC-1 and MIA PaCa-2. NT stimulated ERK activation via a protein kinase C (PKC)-dependent (but EGF receptor-independent) pathway in PANC-1 and MIA PaCa-2 cells, whereas EGF promoted ERK activation through a PKC-independent pathway in these cells. Concomitant stimulation of these cells with NT and EGF induced a striking increase in the duration of ERK pathway activation as compared with that obtained in cells treated with each agonist alone. Stimulation with NT + EGF promoted synergistic stimulation of DNA synthesis and anchorage-independent growth. Addition of the MEK inhibitor U0126, either prior to stimulation with NT + EGF or 2 h after stimulation with NT + EGF prevented the synergistic increase in DNA synthesis and suppressed the sustained phase of ERK activation. Furthermore, treatment with the selective PKC inhibitor GF-1 converted the sustained ERK activation in response to NT and EGF into a transient signal and also abrogated the synergistic increase in DNA synthesis. Collectively, our results suggest that the sustained phase of ERK signaling mediates the synergistic effects of NT and EGF on DNA synthesis in pancreatic cancer cells.     

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.