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• W2029304025 abstract "The human sst4 receptor, recombinantly expressed in Chinese hamster ovary cells, mediates proliferative activity of the peptide hormone somatostatin. This effect was shown to involve activation of pertussis toxin-sensitive G proteins and was inhibited by overexpression of the βγ-sequestrant, transducin. Somatostatin-induced proliferation was abolished by the MEK1 inhibitor, PD 98059, whereas the Src inhibitor, PP1, had no effect. A marked increase was observed in the phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1 and ERK2) 10 min after sst4 receptor activation, which was blocked by pertussis toxin, decreased by PP1 and the βγ-sequestrant, but unaffected by PD 98059. In contrast, the somatostatin-induced phosphorylation of ERK obtained at 4 h, although sensitive to both pertussis toxin and transducin, was unaffected by PP1 but ablated by PD 98059. Protein kinase C inhibition also abolished this somatostatin-induced sustained phosphorylation of ERK, together with the associated increase in cell proliferation. Expression of dominant negative Ras (N17) failed to significantly reduce the proliferative effect mediated by the sst4 receptor but markedly attenuated the acute phase of the somatostatin-induced phosphorylation of ERK obtained at 10 min. In contrast, the phosphorylation induced at 4 h was unaffected. We conclude that ERK activation by Gi/o-coupled sst4receptors involves a Src and Ras-dependent acute phase, but the proliferative response is dependent upon the prolonged ERK-induced activity, mediated by protein kinase C. The human sst4 receptor, recombinantly expressed in Chinese hamster ovary cells, mediates proliferative activity of the peptide hormone somatostatin. This effect was shown to involve activation of pertussis toxin-sensitive G proteins and was inhibited by overexpression of the βγ-sequestrant, transducin. Somatostatin-induced proliferation was abolished by the MEK1 inhibitor, PD 98059, whereas the Src inhibitor, PP1, had no effect. A marked increase was observed in the phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1 and ERK2) 10 min after sst4 receptor activation, which was blocked by pertussis toxin, decreased by PP1 and the βγ-sequestrant, but unaffected by PD 98059. In contrast, the somatostatin-induced phosphorylation of ERK obtained at 4 h, although sensitive to both pertussis toxin and transducin, was unaffected by PP1 but ablated by PD 98059. Protein kinase C inhibition also abolished this somatostatin-induced sustained phosphorylation of ERK, together with the associated increase in cell proliferation. Expression of dominant negative Ras (N17) failed to significantly reduce the proliferative effect mediated by the sst4 receptor but markedly attenuated the acute phase of the somatostatin-induced phosphorylation of ERK obtained at 10 min. In contrast, the phosphorylation induced at 4 h was unaffected. We conclude that ERK activation by Gi/o-coupled sst4receptors involves a Src and Ras-dependent acute phase, but the proliferative response is dependent upon the prolonged ERK-induced activity, mediated by protein kinase C. extracellular signal-regulated kinase mitogen-activated protein basic fibroblast growth factor Chinese hamster ovary platelet-derived growth factor Tris-buffered saline MAP kinase kinase The peptide hormone somatostatin induces numerous biological actions, most of which are inhibitory, by interacting with cell membrane receptors of which five types, named sst1–5, have been heterologously expressed in different cells within the last few years (1Hoyer D. Bell G.I. Berelowitz M. Epelbaum J. Feniuk W. Humphrey P.P.A. O'Carroll A-M. Patel Y.C. Schonbrunn A. Taylor J.E. Reisine T. Trends Pharmacol. Sci. 1995; 16: 86-88Abstract Full Text PDF PubMed Scopus (537) Google Scholar). The growth inhibitory effects of somatostatin are well documented as it is considered to be the physiological regulator of growth hormone release (2Lamberts S.W.J. Oosterom R. Neufeld M. Del Pozo E. J. Clin. Endocrinol. Metab. 1985; 60: 1161-1165Crossref PubMed Scopus (134) Google Scholar). As well as reducing the circulatory levels of several other potential mitogenic hormones and growth factors (3Gerich J.E. Am. J. Med. 1981; 70: 619-626Abstract Full Text PDF PubMed Scopus (33) Google Scholar), somatostatin has also been shown to have a direct action on cellular proliferation and tissue development, with therapeutic potential in retarding the growth of tumor (4Hofland L.J. Lamberts S.W.J. Baillière's Clin. Endocrinol. Metab. 1996; 10: 163-176Abstract Full Text PDF PubMed Scopus (63) Google Scholar) and vascular smooth muscle cells (5Eriksen U.H. Amtorp O. Bagger J.P. Emanuelsson H. Foegh M. Henningsen P. Saunamäki K. Schaeffer M. Thayssen P. Ørskov H. Kuntz R.E. Popma J.J. Am. Heart J. 1995; 130: 1-8Crossref PubMed Scopus (70) Google Scholar).Numerous reports have demonstrated the expression of a high density of somatostatin receptors on a variety of human cancer cells, including most tumors of neuroendocrine origin (including gastroenteropancreatic tumors), small cell lung carcinomas, brain tumors (glial tumors and meningiomas), lymphomas, and melanomas as well as colorectal, pituitary, kidandney, and breast tumors (6Reubi J.C. Schaer J.C. Waser B. Mengod G. Cancer Res. 1994; 54: 3455-3459PubMed Google Scholar, 7Evers B.M. Parekh D. Townsend C.M. Thompson J.C. Ann. Surg. 1991; 213: 190-198Crossref PubMed Scopus (79) Google Scholar). The antiproliferative action of either somatostatin or its analogue octreotide, however, does not correlate with this expression, having inhibitory actions on pancreatic (8Liebow C. Reilly C. Serrano M. Schally A.V. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2003-2007Crossref PubMed Scopus (308) Google Scholar) and breast tumors (9Pagliacci M.C. Tognellini R. Grignani F. Nicoletti I. Endocrinology. 1991; 129: 2555-2562Crossref PubMed Scopus (86) Google Scholar), although eliciting no effect on the growth of small cell lung (10Macaulay V.M. Smith I.E. Everard M.J. Teale J.D. Reubi J.C. Millar J.L. Br. J. Cancer. 1991; 64: 451-456Crossref PubMed Scopus (71) Google Scholar) and colon tumors (11Dy D.Y. Whitehead R.H. Morris D.L. Cancer Res. 1992; 52: 917-923PubMed Google Scholar). Growth-promoting effects of somatostatin have also been described in vitro on human pancreatic carcinoid (12Ishizuka J. Beauchamp R.D. Evers B.M. Townsend C.M. Thompson J.C. Biochem. Biophys. Res. Commun. 1992; 185: 577-581Crossref PubMed Scopus (21) Google Scholar) and epidermoid carcinoma cells (13Kamiya Y. Ohmura E. Arai M. Fuji T. Hayakawa F. Ito J. Kawaguchi M. Tsushima T. Sakuma N. Biochem. Biophys. Res. Commun. 1993; 191: 302-307Crossref PubMed Scopus (13) Google Scholar), whereas in both rat mesangial cells (14Ruiz-Torres P. Lucio F.J. González-Rubio M. Rodríguez-Puyol M. Rodríguez-Puyol D. Biochem. Biophys. Res. Commun. 1993; 195: 1057-1062Crossref PubMed Scopus (23) Google Scholar) and human pancreatic MIA-Pa-Ca-2 cells (15Douziech N. Calvo E. Coulombe Z. Muradia G. Bastien J. Aubin R.A. Lajas A. Morisset J. Endocrinology. 1999; 140: 765-777Crossref PubMed Scopus (53) Google Scholar), somatostatin stimulates proliferation in the absence of serum but inhibits the growth of proliferating cells.Little is known as to the identity of the receptor types mediating the proliferative or antiproliferative responses of somatostatin in tissues, and information has been restricted to studies involving partially selective receptor analogues (16Lauder H. Sellers L.A. Fan T-P.D. Feniuk W. Humphrey P.P.A. Br. J. Pharmacol. 1997; 122: 663-670Crossref PubMed Scopus (21) Google Scholar). Activation of either mouse recombinant sst2 or sst5 receptors, however, has been shown to inhibit serum-induced proliferation (17Buscail L. Estève J-P. Saint-Laurent N. Bertrand V. Reisine T. O'Carroll A-M. Bell G.I. Schally A.V. Vaysse N. Susini C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1580-1584Crossref PubMed Scopus (297) Google Scholar), whereas stimulation of the human recombinant sst4 receptor type induces proliferation in the absence of other mitogenic agents (18Sellers L.A. Feniuk W. Humphrey P.P.A. Lauder H. J. Biol. Chem. 1999; 274: 16423-16430Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Interestingly, the recently cloned rat sst2(b) receptor splice variant has also been shown to induce a proliferative response, in marked contrast to the antiproliferative property mediated by the rat sst2(a) receptor following recombinant expression in the same host cell line (19Alderton F. Fan T-P.D. Schindler M. Humphrey P.P.A. Br. J. Pharmacol. 1998; 125: 1630-1633Crossref PubMed Scopus (24) Google Scholar).The molecular determinants that mediate the proliferative outcome of somatostatin receptors have not yet been fully clarified. All five human receptor types are functionally coupled to inhibition of adenylate cyclase via pertussis toxin-sensitive G proteins (20Patel Y.C. Greenwood M.T. Warszynska A. Panetta R. Srikant C.B. Biochem. Biophys. Res. Commun. 1994; 198: 605-612Crossref PubMed Scopus (216) Google Scholar) and can mediate phospholipase C activation with subsequent calcium mobilization (21Akbar M. Okajima F. Tomura H. Majid M.A. Yamada Y. Seino S. Kondo Y. FEBS Lett. 1994; 348: 192-196Crossref PubMed Scopus (92) Google Scholar). Stimulation of sst1 and sst2 receptors has been shown to activate a protein-tyrosine phosphatase activity (22Buscail L. Delesque N. Estève J-P. Saint-Laurent N. Prats H. Clerc P. Robberecht P. Bell G.I. Liebow C. Schally A.V. Vaysse N. Susini C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2315-2319Crossref PubMed Scopus (321) Google Scholar), and it has been suggested that such an activity may counteract the growth-promoting properties of receptors containing an intrinsic tyrosine kinase domain (23Pan M.G. Florio T. Stork P.J.S. Science. 1992; 256: 1215-1217Crossref PubMed Scopus (195) Google Scholar). The inhibition of basic fibroblast growth factor-stimulated proliferation by activation of human sst1receptors has also been proposed to be due to the induction of the cell cycle inhibitor, p21cip1/WAF1, shown in a recombinant system following the synergistic activation of extracellular signal-regulated kinase (ERK)1 by the growth factor in the presence of somatostatin (24Florio T. Yao H. Carey K.D. Dillon T.J. Stork P.J.S. Mol. Endocrinol. 1999; 13: 24-37Crossref PubMed Scopus (116) Google Scholar).We have previously demonstrated that stimulation of recombinantly expressed sst4 receptors by somatostatin gives rise to both a marked transient increase as well as a sustained period of ERK1 and ERK2 phosphorylation (18Sellers L.A. Feniuk W. Humphrey P.P.A. Lauder H. J. Biol. Chem. 1999; 274: 16423-16430Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). ERK phoshorylation at threonine 183 and tyrosine 185 are widely used indices of mitogen-activated protein (MAP) kinase activation by the ERK kinase, MEK1. We have also provided evidence using a somatostatin analogue that induces only transient phosphorylation of ERK following sst4 receptor activation that it is the sustained component of MAP kinase activity that is critical for the induced proliferative response. Other Giprotein-coupled receptors (25van Biesen T. Luttrell L.M. Hawes B.E. Lefkowitz R.J. Endocr. Rev. 1996; 17: 698-714Crossref PubMed Scopus (390) Google Scholar) have recently been shown to utilize the MAP kinase cascade through a Src-dependent mechanism following release of βγ subunits. In this study therefore, we have examined the ability of the human sst4 receptor to activate intracellular signaling components that converge on the MAP kinase cascade and in particular to see if a differential requirement can be demonstrated for their involvement in mediating the acute or prolonged phases of ERK phosphorylation. In addition, the resultant effect on cell proliferation of any change detected in the somatostatin-induced ERK phosphorylation following effector inhibition was evaluated by direct cell counting using a model to determine the re-population of denuded areas in a previously confluent monolayer (16Lauder H. Sellers L.A. Fan T-P.D. Feniuk W. Humphrey P.P.A. Br. J. Pharmacol. 1997; 122: 663-670Crossref PubMed Scopus (21) Google Scholar). The effect of somatostatin on basal proliferation in the absence of exogenously added mitogenic agents was examined, and responses induced by basic fibroblast growth factor (bFGF) were used as a comparison.DISCUSSIONActivation of the MAP kinase cascade, which in most systems requires Ras and Raf, is a universal downstream response to the stimulation of most receptor protein-tyrosine kinases and has been demonstrated following activation of the G protein-coupled somatostatin sst4 receptor (18Sellers L.A. Feniuk W. Humphrey P.P.A. Lauder H. J. Biol. Chem. 1999; 274: 16423-16430Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 28Bito H. Mori M. Sakanaka C. Takano T. Honda Z. Gotoh Y. Nishida E. Shimizu T. J. Biol. Chem. 1994; 269: 12722-12730Abstract Full Text PDF PubMed Google Scholar). Using a well characterized model to determine the re-population of denuded areas in an otherwise confluent monolayer, we have previously demonstrated that the somatostatin-induced proliferative response of this receptor type depends on the sustained activation of ERK1 and ERK2 and independent of a robust transient phase (18Sellers L.A. Feniuk W. Humphrey P.P.A. Lauder H. J. Biol. Chem. 1999; 274: 16423-16430Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). One of the advantages of this model is that cells are synchronized in G0 or early G1of the cell cycle at the onset of the investigative period, and to substantiate that effects are on a proliferative rather than a motogenic process, the total number of cells forming the regenerating monolayer after 24 h in the presence of test agents was determined in the current study. The aim of this investigation was to attempt to identify the transduction effectors involved in mediating the sustained activation of ERK1 and ERK2 by somatostatin sst4 receptors and, hence, those responsible for inducing the proliferative response.The protein-tyrosine kinase inhibitors, genistein and lavendustin A, had no effect on basal cell numbers at concentrations that abolished the increased proliferation induced by bFGF. The signaling cascades activated by bFGF receptors would thus seem dependent on this type of phosphate transfer process for transduction of the proliferative function and is compatible with the well characterized mechanism through which this family of receptors mediate their mitogenic effects (29Heldin C-H. Cell. 1995; 80: 213-223Abstract Full Text PDF PubMed Scopus (1427) Google Scholar). It would also appear that transmission of the growth-promoting activity of sst4 receptors is similarly dependent on a protein-tyrosine kinase activity. However, in contrast to the growth factor receptor, which contains an intrinsic tyrosine kinase domain within the COOH terminus of each subunit forming the active dimer, the site of intervention of these kinase blockers in the transduction process for sst4 receptor-mediated growth must be localized to secondary effectors. The lack of effect of genistein on the somatostatin-induced ERK phosphorylation determined at time points representative of the acute and sustained phases of MAP kinase activation suggests that either a parallel cooperative pathway utilizing a tyrosine kinase is essential for growth or, alternatively, that the kinase lies exclusively downstream from MAP kinase. Evidence for a pertussis toxin-insensitive pathway mediating tyrosine phosphorylation of the transcription factor STAT3 has recently been provided for the sst4 receptor (18Sellers L.A. Feniuk W. Humphrey P.P.A. Lauder H. J. Biol. Chem. 1999; 274: 16423-16430Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), and it may be that the genistein-sensitive effector is situated within this particular cascade (see Fig. 5). Interestingly, it has also been shown in this previous study that only after the additional phosphorylation on serine residues of this same transcription factor as a consequence of prolonged MAP kinase activation, could a proliferative response be induced by somatostatin.The growth-promoting effect of somatostatin is additionally dependent on a pertussis toxin-sensitive pathway that distinguishes its proliferative mechanism from that of bFGF. Other Giprotein-coupled receptors have been shown to mediate rapid tyrosine phosphorylation of several proteins that participate in mitogenic signal transduction such as the adapter protein Shc (30Cazaubon S.M. Ramos-Morales F. Fischer S. Schweighoffer F. Strosberg A.D. Couraud P.O. J. Biol. Chem. 1994; 269: 24805-24809Abstract Full Text PDF PubMed Google Scholar), which is a major substrate for Src kinase. The mechanism whereby these receptors stimulate tyrosine phosphorylation is poorly understood, although activation of the Src-family kinases by several G protein-coupled receptors has been reported (31Chen Y.H. Pouyssegur J. Courtneidge S.A. van Obberghen Schilling E. J. Biol. Chem. 1994; 269: 27372-27377Abstract Full Text PDF PubMed Google Scholar). In addition, activation of Src seems to account for the Gi-mediated tyrosine phosphorylation events that direct recruitment of the Shc and Grb2 adaptor proteins to the membrane (32Luttrell L.M. Della Rocca G.J. van Biesen T. Luttrell D.K. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 4637-4644Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar), thus providing a route into the Ras-ERK cascade (Fig. 5).To determine whether activation of the MAP kinase cascade was a prerequisite for processing the growth effects induced by somatostatin, the selective MEK1 inhibitor, PD 98059 (33Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3247) Google Scholar), was used in the proliferation model. It is well documented that the dual-specific kinase MEK stimulates ERK by phosphorylation on threonine (Thr-183) and tyrosine (Tyr-185) residues, which following subsequent translocation into the nucleus, activates transcription factors, resulting in enhanced cell growth (34Hill C.S. Treisman R. Cell. 1995; 80: 199-211Abstract Full Text PDF PubMed Scopus (1195) Google Scholar). In this study, the proliferative effect of somatostatin was abolished by PD 98059, confirming that the MAP kinase cascade is critical for the growth-promoting effect of somatostatin by the sst4 receptor. In contrast, bFGF-stimulated proliferation in the same host cell was only partially inhibited on co-application with PD 98059, which is consistent with the ability of this receptor type to recruit a multitude of secondary effectors and initiate a number of distinct, yet parallel signaling pathways. An involvement of MEK1 in the sst4 receptor-mediated proliferative response was further supported by the demonstration of increased phosphorylation of ERK1 and ERK2 following somatostatin treatment. However, although both the acute and sustained phases of MAP kinase activation were abolished by pertussis toxin, a differential effect on the temporally distinct activities was observed following MEK1 inhibition. Abolition of the sustained phase with PD 98059, although having no observable effect on the transient activity, is supportive evidence for the requirement of the prolonged activation of MAP kinase in mediating cell growth. The lack of effect of the MEK1 inhibitor on the marked transient phosphorylation of ERK is possibly due to the ineffectiveness of the concentration of PD 98059 administered with somatostatin and is in keeping with other reports showing that high levels of MAP kinase activity are PD 98059-insensitive (33Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3247) Google Scholar).Several Gi-coupled receptors have been shown to mediate MAP kinase activation through the βγ-component of the G protein possibly through the activation of the Src-family of tyrosine kinases (35Luttrell L.M. Hawes B.E. van Biesen T. Luttrell D.K. Lansing T.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 19443-19450Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar). The proliferative response induced by somatostatin was inhibited following overexpression of the βγ-sequestering protein, transducin, in contrast to the lack of effect on bFGF-induced growth. However, the Src-family inhibitor, PP1 (36Hanke J.H. Gardner J.P. Dow R.L. Changelian P.S. Brissette W.H. Weringer E.J. Pollok B.A. Connelly P.A. J. Biol. Chem. 1996; 271: 695-701Abstract Full Text Full Text PDF PubMed Scopus (1781) Google Scholar), failed to reduce the proliferative effect induced by either mitogen in this cell line. The bFGF results were somewhat unexpected since it is well known that Src is a co-transducer of mitogenic signals arising from a number of tyrosine kinase growth factor receptors, such as platelet-derived growth factor or epidermal growth factor receptors (37Broome M.A. Hunter T. J. Biol. Chem. 1996; 271: 16798-16806Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). However, the association of Src with bFGF receptors appears to be cell-specific (38Roche S. Koegl M. Barone M.V. Roussel M.F. Courtneidge S.A. Mol. Cell. Biol. 1995; 15: 1102-1109Crossref PubMed Google Scholar), and in this respect, bFGF-induced proliferation in vascular smooth muscle cells has been shown to be partially inhibited by the Src inhibitor PP1, as determined by the same model system employed in this current study. 2L. A. Sellers, unpublished data. PP1 in CHO K1 cells transfected with the somatostatin sst4 receptor was shown to reduce platelet-derived growth factor-stimulated growth.The inability of PP1 to inhibit somatostatin-induced proliferation suggests Src is not involved in this response mechanism, and therefore the process through which MEK is activated appears to be very different to that employed by other Gi protein-coupled receptors (35Luttrell L.M. Hawes B.E. van Biesen T. Luttrell D.K. Lansing T.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 19443-19450Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar). However, further examination of the somatostatin-induced ERK phosphorylation showed that the transient phase was sensitive to the Src inhibitor, in contrast to the PP1-independent prolonged phosphorylation. The attenuation of the transient phosphorylation of ERK by the Src inhibitor without any resultant effect on the proliferative response again suggests that it is the sustained activation of MAP kinase that is critical for proliferation. In addition, the sensitivity of the transient and not the prolonged phase of ERK phosphorylation to Src-inhibition provides evidence that different transduction events are involved in mediating the temporally distinct MAP kinase activities. The involvement of Src in mediating the acute phase of ERK phosphorylation and the insensitivity of this component to genistein seem incompatible. However, genistein is a nonselective tyrosine kinase inhibitor, and it may be that in this system, Src activity is unaffected by the concentration of genistein used.The mechanisms by which Gi and Gq-coupled receptors typically activate MAP kinase are through Ras-dependent or protein kinase C-dependent pathways, respectively. However, a few exceptions to this rule have been recently reported for Gq-coupled receptors in that MAP kinase can be activated through a pertussis toxin-insensitive but protein kinase C-independent pathway (39Charlesworth A. Rozengurt E. Oncogene. 1997; 14: 2323-2329Crossref PubMed Scopus (34) Google Scholar). In this study, we have demonstrated that the transient phosphorylation of ERK by sst4 receptors is sensitive to both transducin and dominant negative Ras (N17) but unaffected following protein kinase C inhibition. These results are also in accord with the βγ-mediated Src stimulation utilized by other Gi-coupled receptors to activate MAP kinase through a Ras-dependent mechanism (Fig.5). In addition, these data are also consistent with the acute phase of MAP kinase activity not being involved in mediating a growth response, as expression of N17Ras had no effect on somatostatin-induced proliferation or the prolonged activation of MAP kinase.Since it appears that the sustained activation of MAP kinase, required for the somatostatin-induced proliferative effect, utilizes a distinct but convergent pathway to that mediating the transient Ras-dependent ERK phosphorylation, we examined the involvement of protein kinase C, which can activate the Ras-ERK cascade at the point of Raf (40Kolch W. Heidecker G. Kochs G. Hummel R. Vahidi H. Mischak H. Finkenzeller G. Marme D. Rapp U.R. Nature. 1993; 363: 249-252Crossref Scopus (1153) Google Scholar) (Fig. 5). Both the proliferation and sustained phosphorylation of ERK1 and ERK2 induced by somatostatin were abolished following protein kinase C blockade. This suggests that protein kinase C involvement is critical for the growth response and is placed upstream to ERK activation, consistent with other reports investigating MAP kinase stimulation through Gq-coupled receptors (39Charlesworth A. Rozengurt E. Oncogene. 1997; 14: 2323-2329Crossref PubMed Scopus (34) Google Scholar). Receptor tyrosine kinase-mediated activation of Raf-1 is coupled to Ras, and bFGF-induced proliferation of CHO K1 cells used in this study was N17Ras-sensitive together with both the acute and sustained phases of ERK phosphorylation. By contrast, protein kinase C-mediated activation of Raf-1 is thought to be Ras-independent and is in keeping with the lack of effect on the prolonged MAP kinase phosphorylation and the induced proliferative response observed in this study following application of somatostatin to cells overexpressing N17Ras. Activation of Raf-1 by protein kinase C has been shown to be insensitive to dominant negative Ras (41Marais R. Light Y. Mason C. Paterson H. Olson M.F. Marshall C.J. Science. 1998; 280: 109-112Crossref PubMed Scopus (398) Google Scholar), indicating that protein kinase C activates Raf by a mechanism distinct from that initiated by activation of receptor tyrosine kinases. Although in this study the acute phase of ERK phosphorylation induced by bFGF was unaffected by a protein kinase C inhibitor, the sustained component was reduced, and the proliferative effect was abolished. It thus appears that it is the sustained ERK activity that is also critical for bFGF to induce cell proliferation. However, in contrast to that following sst4 receptor activation, the sustained phase of ERK phosphorylation induced by bFGF appears to involve both Ras- and protein kinase C-dependent mechanisms, and both seem to be required for the proliferative effect.The protein kinase C family has at least 11 members, 6 of which (δ, ε, γ, ι, μ, ζ) have been shown to be expressed in the CHO K1 cells used in this study.2 Both the typical and atypical protein kinase C isozymes are activated by diacylglycerol, which is produced by the metabolism of phosphatidylinositols. Although sst4 receptors have been shown to mediate inositol 1,4,5,-trisphosphate production (21Akbar M. Okajima F. Tomura H. Majid M.A. Yamada Y. Seino S. Kondo Y. FEBS Lett. 1994; 348: 192-196Crossref PubMed Scopus (92) Google Scholar), it has not been determined here if through βγ release, the subsequent stimulation of phospholipase C-β is mandatory for the sustained ERK phosphorylation induced by somatostatin. Activation of MAP kinase after sst4 receptor stimulation has been shown to be dependent on phosphoinositide 3-OH kinase (42Sakanaka C. Ferby I. Waga I. Bito H. Shimizu T. Biochem. Biophys. Res. Commun. 1994; 205: 18-23Crossref PubMed Scopus (54) Google Scholar). Signaling targets of the lipid products of this kinase activity include the calcium-independent protein kinase C isoforms (43Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1218) Google Scholar), raising the possibility that ERK activation through protein kinase C in this study could be via a calcium-independent pathway. Involvement of phosphoinositide 3-OH kinase in the prolonged activation of ERK would also be in keeping with the Gβγ dependence of both this and the proliferative events observed after somatostatin application, as a phosphoinositide 3-OH kinase responsive to βγ subunits has recently been cloned (44Stephens L.R. Eguinoa A. Erdjument-Bromage H. Lui M. Cooke F. Coadwell J. Smrcka A.S. Thelen M. Cadwallader K. Tempst P. Hawkins P.T. Cell. 1997; 89: 105-114Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar).Very little evidence is currently available as to the identity of the molecular determinants responsible for the sustained activation of MAP kinase. Recently it has been shown that in PC12 cells, Ras must be activated for the initial phase of ERK activation following stimulation of the nerve growth factor receptor (TrkA), but the sustained phase involves another small GTPase, Rap1 (45York R.D. Yao H. Dillon T. Ellig C.L. Eckert S.P. McCleskey E.W. Stork P.J.S. Nature. 1998; 392: 622-626Crossref PubMed Scopus (757) Google Scholar). In this study we have demonstrated that sst4 receptors can stimulate cellular proliferation through transduction mechanisms with a critical requirement for a sustained, protein kinase C-dependent activation of MAP kinase. Stimulation of MAP kinase has been shown to regulate a diverse range of functional responses, sometimes with opposing effects. For example, although we have shown a critical requirement for ERK activity in the sst4 receptor-mediated proliferative effect, this activity also appears to be necessary for the growth inhibitory re" @default.
• W2029304025 created "2016-06-24" @default.
• W2029304025 creator A5089214561 @default.
• W2029304025 date "1999-08-01" @default.
• W2029304025 modified "2023-09-27" @default.
• W2029304025 title "Prolonged Activation of Extracellular Signal-regulated Kinase by a Protein Kinase C-dependent and N17Ras-insensitive Mechanism Mediates the Proliferative Response of Gi/o-coupled Somatostatin sst4 Receptors" @default.
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