Related Adhesion Focal Tyrosine Kinase and the Epidermal Growth Factor Receptor Mediate the Stimulation of Mitogen-activated Protein Kinase by the G-protein-coupled P2Y2 Receptor
1998; Elsevier BV; Volume: 273; Issue: 36 Linguagem: Inglês
10.1074/jbc.273.36.23110
ISSN1083-351X
Autores Tópico(s)Receptor Mechanisms and Signaling
ResumoThe activation of growth factor receptors and receptors coupled to heterotrimeric guanine nucleotide-binding proteins (G-proteins) can increase mitogen-activated protein (MAP) kinase activity in many cells. Previously, we demonstrated that the activation of G-protein-coupled P2Y2 receptors by extracellular ATP and UTP stimulated MAP (p42 ERK2) kinase by a mechanism that was dependent on the elevation of [Ca2+]i and the activation of related adhesion focal tyrosine kinase (RAFTK) (also called PYK2, CAKβ, and CADTK) and protein kinase C (PKC). Here, we examine further the signaling cascade between the P2Y2 receptor and MAP kinase. MAP kinase was transiently activated by exposure of PC12 cells to UTP. UTP, ionomycin, and phorbol ester (phorbol 12-myristate 13-acetate) increased MAP kinase activity and also promoted the tyrosine phosphorylation of RAFTK, the epidermal growth factor (EGF) receptor, SHC, and p120cbl. Down-regulation of PKC and inhibition of the elevation of [Ca2+]i, conditions that block the activation of MAP kinase, also blocked the increases in the tyrosine phosphorylation of RAFTK and the EGF receptor. AG1478, a tyrphostin selective for the EGF receptor, reduced the activation of MAP kinase, the tyrosine phosphorylation of SHC, the association of Grb2 with SHC, and the tyrosine phosphorylation of the EGF receptor and p120cbl but did not block the tyrosine phosphorylation of RAFTK. The similar effects of UTP, ionomycin, and phorbol 12-myristate 13-acetate (PMA) on these signaling proteins demonstrate that the two signaling molecules from phosphatidylinositol 4,5-bisphosphate hydrolysis ([Ca2+]i, from inositol 1,4,5-trisphosphate production, and diacylglycerol) can individually initiate the activation of MAP kinase in an EGF receptor-dependent manner. These results demonstrate that the P2Y2 receptor-mediated transactivation of the EGF receptor occurs at a point downstream of RAFTK and indicate that the EGF receptor is required for P2Y2 receptor-mediated MAP kinase activation. Although P2Y2 and EGF receptors may both activate a similar multiprotein signaling cascade immediately upstream of MAP kinase, the P2Y2 receptor appears to uniquely utilize [Ca2+]i, PKC, and, subsequently, RAFTK. The activation of growth factor receptors and receptors coupled to heterotrimeric guanine nucleotide-binding proteins (G-proteins) can increase mitogen-activated protein (MAP) kinase activity in many cells. Previously, we demonstrated that the activation of G-protein-coupled P2Y2 receptors by extracellular ATP and UTP stimulated MAP (p42 ERK2) kinase by a mechanism that was dependent on the elevation of [Ca2+]i and the activation of related adhesion focal tyrosine kinase (RAFTK) (also called PYK2, CAKβ, and CADTK) and protein kinase C (PKC). Here, we examine further the signaling cascade between the P2Y2 receptor and MAP kinase. MAP kinase was transiently activated by exposure of PC12 cells to UTP. UTP, ionomycin, and phorbol ester (phorbol 12-myristate 13-acetate) increased MAP kinase activity and also promoted the tyrosine phosphorylation of RAFTK, the epidermal growth factor (EGF) receptor, SHC, and p120cbl. Down-regulation of PKC and inhibition of the elevation of [Ca2+]i, conditions that block the activation of MAP kinase, also blocked the increases in the tyrosine phosphorylation of RAFTK and the EGF receptor. AG1478, a tyrphostin selective for the EGF receptor, reduced the activation of MAP kinase, the tyrosine phosphorylation of SHC, the association of Grb2 with SHC, and the tyrosine phosphorylation of the EGF receptor and p120cbl but did not block the tyrosine phosphorylation of RAFTK. The similar effects of UTP, ionomycin, and phorbol 12-myristate 13-acetate (PMA) on these signaling proteins demonstrate that the two signaling molecules from phosphatidylinositol 4,5-bisphosphate hydrolysis ([Ca2+]i, from inositol 1,4,5-trisphosphate production, and diacylglycerol) can individually initiate the activation of MAP kinase in an EGF receptor-dependent manner. These results demonstrate that the P2Y2 receptor-mediated transactivation of the EGF receptor occurs at a point downstream of RAFTK and indicate that the EGF receptor is required for P2Y2 receptor-mediated MAP kinase activation. Although P2Y2 and EGF receptors may both activate a similar multiprotein signaling cascade immediately upstream of MAP kinase, the P2Y2 receptor appears to uniquely utilize [Ca2+]i, PKC, and, subsequently, RAFTK. G-protein-coupled receptor phorbol 12-myristate 13-acetate protein kinase C diacylglycerol epidermal growth factor extracellular signal-regulated protein kinase nerve growth factor related adhesion focal tyrosine kinase 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, acetoxymethyl ester mitogen-activated protein platelet-derived growth factor. Recent studies have demonstrated that growth factor receptors and G-protein-coupled receptors (GPCRs)1 may both activate the same signal transduction molecules and utilize the same signaling cascades in cells. One of the most common signaling events mediated by both types of receptors is the activation of mitogen-activated protein (MAP) kinase, although portions of the signaling cascade between the receptors and MAP kinase can be different for growth factor receptors and GPCRs, particularly pertaining to the involvement of members of the PKC family of proteins. Previously, we examined the effects of extracellular nucleotides on PC12 cells (1Soltoff S.P. Avraham H. Avraham S. Cantley L.C. J. Biol. Chem. 1998; 273: 2653-2660Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Extracellular nucleotides can bind to P2-type purinoceptors, which constitute a large family of receptors that are either ion channels (P2Xsubtypes) or else coupled to G-proteins (P2Y subtypes) and which vary in their tissue distribution (2Kennedy C. Leff P. Trends Pharmacol. Sci. 1995; 16: 168-174Abstract Full Text PDF PubMed Scopus (165) Google Scholar, 3Burnstock G. Ciba Found. Symp. 1996; 198: 1-28PubMed Google Scholar). Previously, we observed that both extracellular ATP and UTP increased MAP kinase activity in a nucleotide concentration-dependent manner in PC12 cells, which also respond to EGF and NGF with increases in MAP kinase activity. For both ATP and UTP, the EC50 value was ∼25 μm, and 100 μm promoted nearly a maximal effect (1Soltoff S.P. Avraham H. Avraham S. Cantley L.C. J. Biol. Chem. 1998; 273: 2653-2660Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). These results were consistent with the nucleotide-dependent activation of the P2Y2receptor. This purinoceptor was previously designated P2U, because it does not discriminate between ATP and UTP on the basis of potency. The P2Y2 receptor-mediated activation of MAP kinase involved the elevation of [Ca2+]i, the activation of PKC, and the tyrosine phosphorylation and activation of RAFTK (1Soltoff S.P. Avraham H. Avraham S. Cantley L.C. J. Biol. Chem. 1998; 273: 2653-2660Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). In contrast, these signaling events did not appear to play a major role in the mechanism of EGF-initiated increase in MAP kinase activity.The effects of a number of GPCRs have been reported to involve increases in tyrosine phosphorylation. Signaling proteins such as SHC (4Linseman D.A. Benjamin C.W. Jones D.A. J. Biol. Chem. 1995; 270: 12563-12568Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 5Luttrell 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) and members of the Src kinase family may contribute to GPCR-mediated activation of signaling pathways, including MAP kinase (4Linseman D.A. Benjamin C.W. Jones D.A. J. Biol. Chem. 1995; 270: 12563-12568Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 5Luttrell 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, 6Wan Y. Kurosaki T. Huang X.-Y. Nature. 1996; 380: 541-544Crossref PubMed Scopus (256) Google Scholar, 7Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (876) Google Scholar, 8Della Rocca G.J. van Biesen T. Daaka Y. Luttrell D.K. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 19125-19132Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 9Daub H. Weiss F.U. Wallasch C. Ullrich A. Nature. 1996; 379: 557-560Crossref PubMed Scopus (1314) Google Scholar, 10Daub H. Wallasch C. Lankenau A. Herrlich A. Ullrich A. EMBO J. 1997; 16: 7032-7044Crossref PubMed Scopus (586) Google Scholar, 11Eguchi S. Numaguchi K. Iwasaki H. Matsumoto T. Yamakawa T. Utsunomiya H. Motley E.D. Kawakatsu H. Owada K.M. Hirata Y. Marumo F. Inagami T. J. Biol. Chem. 1998; 273: 8890-8896Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar). In addition, the Gβγ subunit of heterotrimeric G-proteins can mediate MAP kinase activation (5Luttrell 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, 12van Biesen T. Hawes B.E. Luttrell D.K. Krueger K.M. Touhara K. Porfiri E. Sakaue M. Luttrell L.M. Lefkowitz R.J. Nature. 1995; 376: 781-784Crossref PubMed Scopus (525) Google Scholar, 13Crespo P. Xu N. Simonds W.F. Gutkind J.S. Nature. 1994; 369: 418-420Crossref PubMed Scopus (758) Google Scholar). GPCRs also can transactivate growth factor receptors, including the EGF receptor and the PDGF receptor (4Linseman D.A. Benjamin C.W. Jones D.A. J. Biol. Chem. 1995; 270: 12563-12568Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 9Daub H. Weiss F.U. Wallasch C. Ullrich A. Nature. 1996; 379: 557-560Crossref PubMed Scopus (1314) Google Scholar, 10Daub H. Wallasch C. Lankenau A. Herrlich A. Ullrich A. EMBO J. 1997; 16: 7032-7044Crossref PubMed Scopus (586) Google Scholar, 11Eguchi S. Numaguchi K. Iwasaki H. Matsumoto T. Yamakawa T. Utsunomiya H. Motley E.D. Kawakatsu H. Owada K.M. Hirata Y. Marumo F. Inagami T. J. Biol. Chem. 1998; 273: 8890-8896Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar). Several reports suggest that the EGF receptor and other proteins may serve as a scaffolding structure or as an adaptor protein to which other signaling proteins may be recruited in response to GPCR signaling (5Luttrell 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, 14Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1887) Google Scholar). Proteins serving in this capacity can localize proteins to a particular region of the cell or a microdomain of a membrane. This protein localization may allow for their biochemical function, which may include substrate phosphorylation or product generation, to be performed at a physiologically relevant site.In the present report, we continued our examination of the activation of MAP kinase by UTP and other stimuli and focused particular attention on the involvement of RAFTK and the potential involvement of the EGF receptor. RAFTK (also called PYK2, CAKβ, and CADTK) is a protein-tyrosine kinase that is activated by elevations of [Ca2+]i or by phorbol esters, and it is involved in the activation of MAP kinase by some stimuli (7Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (876) Google Scholar, 15Lev S. Moreno H. Martinez R. Canoll P. Peles E. Mussachio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1246) Google Scholar). Consistent with our previous results, our new studies suggest a role for RAFTK in the activation of MAP kinase by UTP. In addition, the EGF receptor appears to be directly involved in the signaling cascade that leads to MAP kinase activation by UTP, ionomycin, and PMA. A tyrphostin (AG1478) that blocks the EGF receptor tyrosine kinase also blocks UTP-, PMA-, and ionomycin-promoted MAP kinase activity, SHC tyrosine phosphorylation, and Grb2 association with SHC. AG1478 also blocks the UTP- and ionomycin-initiated tyrosine phosphorylation of other signaling proteins downstream of the EGF receptor, such as p120cbl, but does not block RAFTK tyrosine phosphorylation. The tyrosine phosphorylation of RAFTK is increased by UTP and ionomycin more than by EGF. The results suggest that RAFTK activation is upstream of the EGF receptor and that both proteins are central to the activation of MAP kinase by the G-protein-coupled P2Y2receptor. Although there are similarities in the signaling cascades involved in MAP kinase activation initiated by EGF and UTP, notably EGF receptor and SHC tyrosine phosphorylation, RAFTK and PKC are uniquely involved in the P2Y2 receptor-mediated cascade.DISCUSSIONThe results of these studies demonstrate that activation of the P2Y2 receptor by UTP stimulates MAP kinase in PC12 cells via a cascade of signaling proteins that include RAFTK and the EGF receptor. UTP, which increases [Ca2+]i in these cells (23Barry V.A. Cheek T.R. J. Cell Sci. 1994; 107: 451-462PubMed Google Scholar, 24Murrin R.J.A. Boarder M.R. Mol. Pharmacol. 1992; 41: 561-568PubMed Google Scholar), 2S. P. Soltoff, unpublished results. promotes the tyrosine phosphorylation of RAFTK (Fig. 2 A). This is reduced by down-regulating PKC (Fig. 2 B) as well as by blocking the elevation of [Ca2+]i (1Soltoff S.P. Avraham H. Avraham S. Cantley L.C. J. Biol. Chem. 1998; 273: 2653-2660Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), and both of these alterations reduce the UTP-promoted activation of MAP kinase (1Soltoff S.P. Avraham H. Avraham S. Cantley L.C. J. Biol. Chem. 1998; 273: 2653-2660Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). UTP, ionomycin, and PMA promote the tyrosine phosphorylation of the EGF receptor (Fig. 3). The effects of these stimuli on MAP kinase activity (Fig. 4), the tyrosine phosphorylation of SHC (Fig. 6), and the association of Grb2 with SHC (Fig. 6) were reduced by blocking the EGF receptor tyrosine kinase with the tyrphostin AG1478.A model for the signaling cascade between the P2Y2 receptor and MAP kinase is shown in Fig. 7. In this model, RAFTK is upstream of the EGF receptor and downstream of the elevation of [Ca2+]i and PKC, and the activation of both the EGF receptor and RAFTK is critical to the P2Y2receptor-dependent stimulation of MAP kinase. The results are also consistent with the critical involvement of RAFTK and the EGF receptor in the effects of both ionomycin and PMA on MAP kinase activation in PC12 cells. The EGF receptor may act as a scaffolding or adaptor protein to help to coordinate the signaling molecules involved in the MAP kinase activation scheme outlined in the model (Fig. 7). In this way, the EGF receptor and other proteins (reviewed in Ref. 14Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1887) Google Scholar) may provide an organizational role beyond their intrinsic biochemical function. In this model, the active tyrosine kinase activity of the EGF receptor is also required, since AG1478 blocks the stimulation of MAP kinase and SHC tyrosine phosphorylation.An increasing number of studies have demonstrated the involvement of the EGF receptor and various signaling proteins, including SHC and Src, in the activation of MAP (ERK) kinase by GPCRs. The EGF receptor can be activated by signaling events initiated by Gβγ subunit (5Luttrell 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), ionomycin (19Zwick E. Daub H. Aoki N. Yamaguchi-Aoki Y. Tinhofer I. Maly K. Ullrich A. J. Biol. Chem. 1997; 272: 24767-24770Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 25Rosen L.B. Ginty D.D. Weber M.J. Greenberg M.E. Neuron. 1994; 12: 1207-1221Abstract Full Text PDF PubMed Scopus (595) Google Scholar), and agonists for lysophosphatidic acid, thrombin, endothelin 1, and angiotensin II receptors (5Luttrell 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, 9Daub H. Weiss F.U. Wallasch C. Ullrich A. Nature. 1996; 379: 557-560Crossref PubMed Scopus (1314) Google Scholar, 10Daub H. Wallasch C. Lankenau A. Herrlich A. Ullrich A. EMBO J. 1997; 16: 7032-7044Crossref PubMed Scopus (586) Google Scholar, 11Eguchi S. Numaguchi K. Iwasaki H. Matsumoto T. Yamakawa T. Utsunomiya H. Motley E.D. Kawakatsu H. Owada K.M. Hirata Y. Marumo F. Inagami T. J. Biol. Chem. 1998; 273: 8890-8896Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar). In neuronal cell types, including PC12 cells, [Ca2+]i increases produced by GPCR ligands, calcium ionophores, or membrane depolarization, have been shown to promote neurite outgrowth and activate the EGF receptor, MAP kinase, and other signaling proteins (19Zwick E. Daub H. Aoki N. Yamaguchi-Aoki Y. Tinhofer I. Maly K. Ullrich A. J. Biol. Chem. 1997; 272: 24767-24770Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 20Rosen L.B. Greenberg M.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1113-1118Crossref PubMed Scopus (172) Google Scholar, 25Rosen L.B. Ginty D.D. Weber M.J. Greenberg M.E. Neuron. 1994; 12: 1207-1221Abstract Full Text PDF PubMed Scopus (595) Google Scholar, 26Rusanescu G. Qi H. Thomas S.M. Brugge J.S. Halegoua S. Neuron. 1995; 15: 1415-1425Abstract Full Text PDF PubMed Scopus (233) Google Scholar). Similar to results presented here, the tyrphostin AG1478 blocked MAP kinase activation and the tyrosine phosphorylation of SHC and other signaling proteins promoted by ligands to GPCRs in other studies (9Daub H. Weiss F.U. Wallasch C. Ullrich A. Nature. 1996; 379: 557-560Crossref PubMed Scopus (1314) Google Scholar, 10Daub H. Wallasch C. Lankenau A. Herrlich A. Ullrich A. EMBO J. 1997; 16: 7032-7044Crossref PubMed Scopus (586) Google Scholar, 11Eguchi S. Numaguchi K. Iwasaki H. Matsumoto T. Yamakawa T. Utsunomiya H. Motley E.D. Kawakatsu H. Owada K.M. Hirata Y. Marumo F. Inagami T. J. Biol. Chem. 1998; 273: 8890-8896Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar, 19Zwick E. Daub H. Aoki N. Yamaguchi-Aoki Y. Tinhofer I. Maly K. Ullrich A. J. Biol. Chem. 1997; 272: 24767-24770Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Although there are a number of similarities in the signaling cascades downstream of the GPCRs in different cellular systems, there also are differences. GPCR- and Gβγ-mediated tyrosine phosphorylation of the EGF receptor and SHC were also found to be dependent on Src kinase, and these events were upstream of MAP kinase activation (5Luttrell 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). Since an autophosphorylation-specific antibody did not detect an increase in phosphorylation, these increases in MAP kinase activity did not appear to require the intrinsic kinase activity of the EGF receptor (5Luttrell 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). This conclusion is different from that reached in the present study and one that used both AG1478 and the expression of dominant negative EGF receptor (10Daub H. Wallasch C. Lankenau A. Herrlich A. Ullrich A. EMBO J. 1997; 16: 7032-7044Crossref PubMed Scopus (586) Google Scholar). It is therefore of interest that growth hormone, which binds to a receptor that is a member of the cytokine receptor family, promoted an increase in MAP kinase that was dependent on an increase in EGF receptor tyrosine phosphorylation, but the intrinsic tyrosine kinase activity of the EGF receptor was not required for the activation of MAP kinase (27Yamauchi T. Ueki K. Tobe K. Tamemoto H. Sekine N. Wada M. Honjo M. Takahashi M. Takahashi T. Hirai H. Tushima T. Akanuma Y. Fujita T. Komuro I. Yazaki Y. Kadowaki T. Nature. 1997; 390: 91-96Crossref PubMed Scopus (256) Google Scholar). Thus, the EGF receptor plays an important role in MAP kinase activity in different cell types, but in some cases its kinase activity may not be required.A number of studies indicate that Src is involved in mediating the activation of MAP kinase by GPCRs. Src can co-immunoprecipitate with the EGF receptor in cells exposed to ligands to GPCRs, including lysophosphatidic acid (5Luttrell 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) and angiotensin II (11Eguchi S. Numaguchi K. Iwasaki H. Matsumoto T. Yamakawa T. Utsunomiya H. Motley E.D. Kawakatsu H. Owada K.M. Hirata Y. Marumo F. Inagami T. J. Biol. Chem. 1998; 273: 8890-8896Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar). In PC12 cells, the activation of GPCRs promoted the association of PYK2 (RAFTK) and activated Src, and this coupling was involved in the activation of MAP kinase (7Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (876) Google Scholar). Through the use of dominant negative PYK2 and Src proteins, it was demonstrated that PYK2 and Src were involved in mediating the activation of MAP kinase by the Gi-coupled α2A-adrenergic receptor and the Gq-coupled α1B-adrenergic receptor at a point upstream of SHC in nonneuronal cells (8Della Rocca G.J. van Biesen T. Daaka Y. Luttrell D.K. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 19125-19132Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). An inhibitor (PP1) of Src-like kinases blocked the effects of both GPCR- and EGF receptor-promoted activation of MAP kinase and tyrosine phosphorylation of signaling proteins (including SHC) downstream of EGF receptor activation but produced only a modest reduction in the lysophosphatidic acid-promoted tyrosine phosphorylation of the EGF receptor (10Daub H. Wallasch C. Lankenau A. Herrlich A. Ullrich A. EMBO J. 1997; 16: 7032-7044Crossref PubMed Scopus (586) Google Scholar). This suggests that Src or other kinases may play a regulatory role immediately distal to GPCR-mediated EGF receptor tyrosine kinase activation. A similar conclusion was reached concerning the involvement of Src and the activation of the PDGFα receptor by PDGF. Src was required for efficient PDGF-dependent tyrosine phosphorylation of SHC but not for tyrosine phosphorylation of the receptor itself or for other signaling proteins (phospholipase C-γ1, SHP2) that are recruited to this receptor (28Gelderloos J.A. Rosenkranz S. Bazenet C. Kazlauskas A. J. Biol. Chem. 1998; 273: 5908-5915Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar).The model for the P2Y2 receptor-mediated activation of MAP kinase is generally compatible with many of the studies cited above. Src and/or Src-like kinases may play regulatory roles at multiple points in the signaling cascade between growth factor/G-protein-coupled receptors and MAP kinase, and this may vary with different kinds of cells or GPCRs. In addition, although it is not shown explicitly in the model, the UTP- and PMA-dependent activation of MAP kinase may also involve PKC-mediated effects that are independent of RAFTK and the EGF receptor, since PKC can increase MAP kinase by directly phosphorylating Raf-1 (29Kolch W. Heidecker G. Kochs G. Hummel R. Vahidi H. Mischak H. Finkenzeller G. Marme D. Rapp U.R. Nature. 1993; 364: 249-252Crossref PubMed Scopus (1152) Google Scholar). Based on the degree of inhibition of MAP kinase activity by AG1478, the major P2Y2-mediated pathway is that outlined in Fig. 7.Several observations suggest that the acute activation of PKC by PMA can produce a positive effect on the EGF receptor, including (a) the PMA-promoted increase in the tyrosine phosphorylation of the EGF receptor (Figs. 3 B and 6) and (b) the inhibition by AG1478 of the PMA-promoted MAP kinase activation (Fig. 4) and SHC tyrosine phosphorylation (Fig. 6). In other reports, topical treatment of mice with PMA increased the tyrosine phosphorylation of the EGF receptor in epidermal tissue (30Xian W. Kiguchi K. Imamoto A. Rupp T. Zilberstein A. DiGiovanni J. Cell Growth Differ. 1995; 6: 1447-1455PubMed Google Scholar). Of related interest, PMA and PKC activation also promoted the tyrosine phosphorylation and activation of ErbB2 and ErbB3, other members of the EGF receptor family, in a rat hepatoma cell line (31Emkey R. Kahn C.R. J. Biol. 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Rosner M.R. J. Biol. Chem. 1993; 268: 15536-15543Abstract Full Text PDF PubMed Google Scholar). Thus, it appears that the EGF receptor tyrosine kinase can be either activated (as in our studies) or inhibited by ligands to GPCRs and other stimuli.One may speculate that Src and/or RAFTK, which can be activated by either an elevation of [Ca2+]i or PMA, may contribute to whether there is either GPCR-mediated activation or inhibition of the EGF receptor, but this will require a direct examination. Interestingly, ATP also produced an increase in MAP kinase activity in another PC12 cell line via the activation of an ionotropic P2X-type receptor (which is not coupled to a G-protein) and not via a metabotropic P2Y-type receptor (37Swanson K.D. Reigh C. Landreth G.E. J. Biol. Chem. 1998; 273: 19965-19971Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Although K+-stimulated depolarization and ATP both produced extracellular Ca2+-dependent increases in RAFTK (PYK2) tyrosine phosphorylation and MAP kinase activity in these cells, neither stimulus was found to increase the tyrosine phosphorylation of the EGF receptor.NGF produced a small increase in the tyrosine phosphorylation of the EGF receptor in anti-EGF immunoprecipitates in some experiments (not shown) and in a protein (presumably the EGF receptor) that co-migrated with the EGF receptor in anti-SHC immunoprecipitates (Fig. 6). This is likely to reflect changes in the tyrosine phosphorylation of the EGF receptor by the elevation in [Ca2+]i or the production of DAG in NGF-treated cells. The lack of effect of a significant inhibitory effect of AG1478 on the NGF-promoted tyrosine phosphorylation of SHC (Fig. 6), association of Grb2 with SHC (Fig. 6), and the activation of MAP kinase (not shown) are consistent with the p140trk-mediated activation of MAP kinase by NGF (38Ohmichi M. Pang L. Decker S.J. Saltiel A.R. J. Biol. Chem. 1992; 267: 14604-14610Abstract Full Text PDF PubMed Google Scholar). However, the effects of NGF on the EGF receptor tyrosine phosphorylation suggest that there is cross-talk between these two growth factor receptors as well as between GPCRs and growth factor receptors. Transmodulation of the EGF receptor by the PDGF receptor has been reported (reviewed in Ref. 39Yarden, Y., and Ullrich, A. (eds) (1988) Annu. Rev. Biochem.57, 443–478Google Scholar). In addition, recent studies indicate that the EGF receptor and the PDGFβ receptor can interact directly with each other, perhaps by heterodimerization or oligomerization (40Habib A.A. Hognason T. Ren J. Stefansson K. Ratan R.R. J. Biol. Chem. 1998; 273: 6885-6891Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar).P2Y2 receptors are present on many different types of tissues and cells in culture (reviewed in Refs. 2Kennedy C. Leff P. Trends Pharmacol. 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