Smooth Muscle Archvillin Is an ERK Scaffolding Protein
2009; Elsevier BV; Volume: 284; Issue: 26 Linguagem: Inglês
10.1074/jbc.m109.002386
ISSN1083-351X
AutoresSamudra S. Gangopadhyay, Edouard Kengni, Sarah Appel, Cynthia Gallant, Hak Rim Kim, Paul C. Leavis, Jon P. DeGnore, Kathleen G. Morgan,
Tópico(s)Melanoma and MAPK Pathways
ResumoERK influences a number of pathways in all cells, but how ERK activities are segregated between different pathways has not been entirely clear. Using immunoprecipitation and pulldown experiments with domain-specific recombinant fragments, we show that smooth muscle archvillin (SmAV) binds ERK and members of the ERK signaling cascade in a domain-specific, stimulus-dependent, and pathway-specific manner. MEK binds specifically to the first 445 residues of SmAV. B-Raf, an upstream regulator of MEK, constitutively interacts with residues 1–445 and 446–1250. Both ERK and 14-3-3 bind to both fragments, but in a stimulus-specific manner. Phosphorylated ERK is associated only with residues 1–445. An ERK phosphorylation site was determined by mass spectrometry to reside at Ser132. A phospho-antibody raised to this site shows that the site is phosphorylated during α-agonist-mediated ERK activation in smooth muscle tissue. Phosphorylation of SmAV by ERK decreases the association of phospho-ERK with SmAV. These results, combined with previous observations, indicate that SmAV serves as a new ERK scaffolding protein and provide a mechanism for regulation of ERK binding, activation, and release from the signaling complex. ERK influences a number of pathways in all cells, but how ERK activities are segregated between different pathways has not been entirely clear. Using immunoprecipitation and pulldown experiments with domain-specific recombinant fragments, we show that smooth muscle archvillin (SmAV) binds ERK and members of the ERK signaling cascade in a domain-specific, stimulus-dependent, and pathway-specific manner. MEK binds specifically to the first 445 residues of SmAV. B-Raf, an upstream regulator of MEK, constitutively interacts with residues 1–445 and 446–1250. Both ERK and 14-3-3 bind to both fragments, but in a stimulus-specific manner. Phosphorylated ERK is associated only with residues 1–445. An ERK phosphorylation site was determined by mass spectrometry to reside at Ser132. A phospho-antibody raised to this site shows that the site is phosphorylated during α-agonist-mediated ERK activation in smooth muscle tissue. Phosphorylation of SmAV by ERK decreases the association of phospho-ERK with SmAV. These results, combined with previous observations, indicate that SmAV serves as a new ERK scaffolding protein and provide a mechanism for regulation of ERK binding, activation, and release from the signaling complex. The ERK 3The abbreviations used are: ERKextracellular signal-regulated kinaseSmAVsmooth muscle archvillinN-SmAV1residues 1–445 of SmAVN-SmAV2residues 446–1250 of SmAVMAPKmitogen-activated protein kinasePSSphysiological salt solutionPEphenylephrineMEKMAPK/ERK kinaseDTTdithiothreitolIPimmunoprecipitationTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineLCliquid chromatographyMS/MStandem mass spectrometry. 3The abbreviations used are: ERKextracellular signal-regulated kinaseSmAVsmooth muscle archvillinN-SmAV1residues 1–445 of SmAVN-SmAV2residues 446–1250 of SmAVMAPKmitogen-activated protein kinasePSSphysiological salt solutionPEphenylephrineMEKMAPK/ERK kinaseDTTdithiothreitolIPimmunoprecipitationTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineLCliquid chromatographyMS/MStandem mass spectrometry. cascade has long been known to be central to the activation of cellular processes such as proliferation, differentiation, and oncogenic transformation (1Chuderland D. Seger R. Mol. Biotechnol. 2005; 29: 57-74Crossref PubMed Scopus (87) Google Scholar). More recently, it has become clear that this cascade also plays a major role in the regulation of motility and contractility (2Pullikuth A.K. Catling A.D. Cell Signal. 2007; 19: 1621-1632Crossref PubMed Scopus (123) Google Scholar, 3Morgan K.G. Gangopadhyay S.S. J. Appl. Physiol. 2001; 91: 953-962Crossref PubMed Scopus (212) Google Scholar, 4Taggart M.J. Morgan K.G. Semin. Cell Dev. Biol. 2007; 18: 296-304Crossref PubMed Scopus (21) Google Scholar). The MAPK serine/threonine family of protein kinases, of which ERK is a member, are evolutionarily conserved and are activated by a mechanism that includes protein kinase cascades. Original studies performed with Saccharomyces cerevisiae have demonstrated the importance of scaffold proteins in providing coordination and specificity of the MAPK cascades. The yeast protein Ste5 is a critical regulator of the mating response in yeast because of its ability to act as a scaffold to assemble the MAPK kinase kinase homolog Ste11 and the MEK homolog Ste7 to activate the MAPK, Fus3. No mammalian protein shares significant similarity at the sequence level to Ste5 (2Pullikuth A.K. Catling A.D. Cell Signal. 2007; 19: 1621-1632Crossref PubMed Scopus (123) Google Scholar), but several mammalian proteins have been shown to exert scaffolding functions for parts of the MAPK activation cascades. Of note, with respect to ERK activation, are β-arrestin-1,2, which binds to Raf-1, MEK1, and ERK2; KSR, which associates with Raf, MEK1/2, ERK1/2, and 14-3-3; MEKK1, which associates with Raf-1, MEK1, and ERK2; and MP1, which binds to MEK1, ERK1, and p14 (5Morrison D.K. Davis R.J. Annu. Rev. Cell Dev. Biol. 2003; 19: 91-118Crossref PubMed Scopus (645) Google Scholar, 6Dhillon A.S. Kolch W. Arch. Biochem. Biophys. 2002; 404: 3-9Crossref PubMed Scopus (155) Google Scholar).We have previously reported the identification, in smooth muscle tissue, of a new splice variant of the supervillin family, smooth muscle archvillin (SmAV) (7Gangopadhyay S.S. Takizawa N. Gallant C. Barber A.L. Je H.D. Smith T.C. Luna E.J. Morgan K.G. J. Cell Sci. 2004; 117: 5043-5057Crossref PubMed Scopus (39) Google Scholar) that is co-targeted to the cell cortex with ERK during α-agonist activation. Antisense knockdown of SmAV was shown to inhibit ERK activation, and it was postulated that ERK might form a macromolecular complex with SmAV to regulate its signaling function. In the present study we directly demonstrate the agonist- and pathway-specific formation of a complex containing SmAV and members of the ERK signaling module, MEK, B-Raf, and 14-3-3 both in vitro and in vivo in smooth muscle cells. Thus, SmAV functions as a pathway-specific scaffold to couple ERK activation in a spatially restricted manner to select outcomes.DISCUSSIONThe results presented here, together with past cellular studies from our group (7Gangopadhyay S.S. Takizawa N. Gallant C. Barber A.L. Je H.D. Smith T.C. Luna E.J. Morgan K.G. J. Cell Sci. 2004; 117: 5043-5057Crossref PubMed Scopus (39) Google Scholar), demonstrate that SmAV displays properties that allow it to function as an ERK scaffolding protein. Scaffolds have been defined as proteins, generally with no applicable enzymatic activity "that interact with a signaling pathway to create a functional signaling module and to control the specificity of signal transduction" (5Morrison D.K. Davis R.J. Annu. Rev. Cell Dev. Biol. 2003; 19: 91-118Crossref PubMed Scopus (645) Google Scholar). We have previously shown that SmAV knockdown in vascular smooth muscle tissue inhibits ERK activation and contraction induced by the α-agonist PE (7Gangopadhyay S.S. Takizawa N. Gallant C. Barber A.L. Je H.D. Smith T.C. Luna E.J. Morgan K.G. J. Cell Sci. 2004; 117: 5043-5057Crossref PubMed Scopus (39) Google Scholar). We demonstrate here that N-terminal SmAV sequence (N-SmAV-1) associates with ERK, MEK, Raf, and 14-3-3, all known members of a classical ERK signaling pathway, in an agonist- and pathway-specific manner. Taken together, these results demonstrate an ERK scaffolding function for this recently described smooth muscle protein.Two ERK pathways, targeted to two different ERK substrates have previously been shown to co-exist in the cell type from which SmAV was first identified (17Wier W.G. Morgan K.G. Rev. Physiol. Biochem. Pharmacol. 2003; 150: 91-139Crossref PubMed Scopus (97) Google Scholar). α-Agonist activation has been shown to lead to ERK activation and subsequent phosphorylation of caldesmon at an ERK phosphorylation site. Phosphorylation at this site has been shown to increase actomyosin ATPase activity (18Gerthoffer W.T. Yamboliev I.A. Shearer M. Pohl J. Haynes R. Dang S. Sato K. Sellers J.R. J. Physiol. 1996; 495: 597-609Crossref PubMed Scopus (99) Google Scholar, 19Adam L.P. Franklin M.T. Raff G.J. Hathaway D.R. Circ. Res. 1995; 76: 183-190Crossref PubMed Google Scholar) and, hence, contractility. In contrast, depolarization-mediated activation leads to activation of calcium-calmodulin-kinase 2 and subsequent activation of ERK, but in this case, ERK activation leads to increased myosin phosphorylation and contractility, presumably via ERK-mediated phosphorylation of myosin light chain kinase. In the present study we showed that α-adrenoreceptor activation of tissues with PE causes a greater fold increase in the association of N-SmAV1 in pulldown experiments with ERK, phospho-ERK, MEK, and 14-3-3 than does depolarization of tissues even though both modes of stimulation lead to ERK activation. Previously, PE stimulation has been shown to target ERK to the cell surface (8Khalil R.A. Morgan K.G. Am. J. Physiol. Cell Physiol. 1993; 265: C406-C411Crossref PubMed Google Scholar), but depolarization leads to a homogeneous cellular distribution of ERK (20Marganski W.A. Gangopadhyay S.S. Je H.D. Gallant C. Morgan K.G. Circ. Res. 2005; 97: 541-549Crossref PubMed Scopus (66) Google Scholar). Interestingly, both SmAV and ERK translocate to the membrane upon PE stimulation at the same time point (4 min) in isolated ferret aorta cells, as has been shown previously (7Gangopadhyay S.S. Takizawa N. Gallant C. Barber A.L. Je H.D. Smith T.C. Luna E.J. Morgan K.G. J. Cell Sci. 2004; 117: 5043-5057Crossref PubMed Scopus (39) Google Scholar, 8Khalil R.A. Morgan K.G. Am. J. Physiol. Cell Physiol. 1993; 265: C406-C411Crossref PubMed Google Scholar). The stimulus-specific nature of N-SmAV1 binding to ERK signaling partners could serve to spatially sequester the α-agonist-activated ERK pathway from the depolarization-activated ERK pathway and provide a mechanism whereby ERK in the intracellular environment chooses the signaling pathway to follow in response to a specific agonist.Multiple cellular studies have demonstrated a biphasic targeting of MAPKs, first from the cytosol to the cell cortex/membrane and, subsequently, either to the nucleus in proliferating cells (21Pouysségur J. Lenormand P. Eur. J. Biochem. 2003; 270: 3291-3299Crossref PubMed Scopus (154) Google Scholar, 22Gonzalez F.A. Seth A. Raden D.L. Bowman D.S. Fay F.S. Davis R.J. J. Cell Biol. 1993; 122: 1089-1101Crossref PubMed Scopus (282) Google Scholar) or to the contractile filaments in differentiated smooth muscle (8Khalil R.A. Morgan K.G. Am. J. Physiol. Cell Physiol. 1993; 265: C406-C411Crossref PubMed Google Scholar). A possible mechanism triggering the release from its cortical targeting has been lacking. We have now shown that ERK, after preferentially associating with N-SmAV1 and becoming activated, uses N-SmAV1 as a substrate and phosphorylates (in vitro and in vivo) N-SmAV1 at Ser132. We have also shown that ERK-mediated phosphorylation of N-SmAV1 decreases the association of active ERK from the scaffold. The release of active ERK then allows the subsequent targeting to the final substrate of ERK. The binding of unphosphorylated ERK to N-SmAV2 may feed substrate to activated MEK associated with N-SmAV1.Leinweber et al. (23Leinweber B.D. Leavis P.C. Grabarek Z. Wang C.L. Morgan K.G. Biochem. J. 1999; 344: 117-123Crossref PubMed Scopus (124) Google Scholar) showed that the ERK-binding domain of calponin is the N-terminal calponin homology domain, which Gangopadhyay et al. (7Gangopadhyay S.S. Takizawa N. Gallant C. Barber A.L. Je H.D. Smith T.C. Luna E.J. Morgan K.G. J. Cell Sci. 2004; 117: 5043-5057Crossref PubMed Scopus (39) Google Scholar) showed is the domain of calponin that binds the C-terminal end of SmAV. These past studies can be combined with the work presented here in a model shown in Fig. 10, whereby (a) ERK, bound to the calponin homology domain of calponin, colocalizes with SmAV in the cell cortex (7Gangopadhyay S.S. Takizawa N. Gallant C. Barber A.L. Je H.D. Smith T.C. Luna E.J. Morgan K.G. J. Cell Sci. 2004; 117: 5043-5057Crossref PubMed Scopus (39) Google Scholar), and binding of the calponin homology domain to SmAV 1823–2073 releases ERK. (b) ERK binds the two ERK-binding sites in the N-terminal portion of SmAV. (c) MEK is recruited to SmAV and activated by B-Raf. (d) ERK is phosphorylated and activated by MEK. (e) Activated ERK phosphorylates Ser132 of SmAV, and phosphorylation of SmAV may cause a conformational change in the SmAV molecule or the charge difference may repel ERK from SmAV, releasing phospho-ERK. (f) phospho-ERK associates with and phosphorylates caldesmon. (g) Phosphorylation of caldesmon makes the actin available for the interaction with myosin, which in turn increases contractility. In summary, we have shown that SmAV displays the properties necessary to function as an ERK scaffold, creating a functional signaling module that can control the stimulus specificity of signal transduction. The ERK 3The abbreviations used are: ERKextracellular signal-regulated kinaseSmAVsmooth muscle archvillinN-SmAV1residues 1–445 of SmAVN-SmAV2residues 446–1250 of SmAVMAPKmitogen-activated protein kinasePSSphysiological salt solutionPEphenylephrineMEKMAPK/ERK kinaseDTTdithiothreitolIPimmunoprecipitationTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineLCliquid chromatographyMS/MStandem mass spectrometry. 3The abbreviations used are: ERKextracellular signal-regulated kinaseSmAVsmooth muscle archvillinN-SmAV1residues 1–445 of SmAVN-SmAV2residues 446–1250 of SmAVMAPKmitogen-activated protein kinasePSSphysiological salt solutionPEphenylephrineMEKMAPK/ERK kinaseDTTdithiothreitolIPimmunoprecipitationTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineLCliquid chromatographyMS/MStandem mass spectrometry. cascade has long been known to be central to the activation of cellular processes such as proliferation, differentiation, and oncogenic transformation (1Chuderland D. Seger R. Mol. Biotechnol. 2005; 29: 57-74Crossref PubMed Scopus (87) Google Scholar). More recently, it has become clear that this cascade also plays a major role in the regulation of motility and contractility (2Pullikuth A.K. Catling A.D. Cell Signal. 2007; 19: 1621-1632Crossref PubMed Scopus (123) Google Scholar, 3Morgan K.G. Gangopadhyay S.S. J. Appl. Physiol. 2001; 91: 953-962Crossref PubMed Scopus (212) Google Scholar, 4Taggart M.J. Morgan K.G. Semin. Cell Dev. Biol. 2007; 18: 296-304Crossref PubMed Scopus (21) Google Scholar). The MAPK serine/threonine family of protein kinases, of which ERK is a member, are evolutionarily conserved and are activated by a mechanism that includes protein kinase cascades. Original studies performed with Saccharomyces cerevisiae have demonstrated the importance of scaffold proteins in providing coordination and specificity of the MAPK cascades. The yeast protein Ste5 is a critical regulator of the mating response in yeast because of its ability to act as a scaffold to assemble the MAPK kinase kinase homolog Ste11 and the MEK homolog Ste7 to activate the MAPK, Fus3. No mammalian protein shares significant similarity at the sequence level to Ste5 (2Pullikuth A.K. Catling A.D. Cell Signal. 2007; 19: 1621-1632Crossref PubMed Scopus (123) Google Scholar), but several mammalian proteins have been shown to exert scaffolding functions for parts of the MAPK activation cascades. Of note, with respect to ERK activation, are β-arrestin-1,2, which binds to Raf-1, MEK1, and ERK2; KSR, which associates with Raf, MEK1/2, ERK1/2, and 14-3-3; MEKK1, which associates with Raf-1, MEK1, and ERK2; and MP1, which binds to MEK1, ERK1, and p14 (5Morrison D.K. Davis R.J. Annu. Rev. Cell Dev. Biol. 2003; 19: 91-118Crossref PubMed Scopus (645) Google Scholar, 6Dhillon A.S. Kolch W. Arch. Biochem. Biophys. 2002; 404: 3-9Crossref PubMed Scopus (155) Google Scholar). extracellular signal-regulated kinase smooth muscle archvillin residues 1–445 of SmAV residues 446–1250 of SmAV mitogen-activated protein kinase physiological salt solution phenylephrine MAPK/ERK kinase dithiothreitol immunoprecipitation N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine liquid chromatography tandem mass spectrometry. extracellular signal-regulated kinase smooth muscle archvillin residues 1–445 of SmAV residues 446–1250 of SmAV mitogen-activated protein kinase physiological salt solution phenylephrine MAPK/ERK kinase dithiothreitol immunoprecipitation N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine liquid chromatography tandem mass spectrometry. We have previously reported the identification, in smooth muscle tissue, of a new splice variant of the supervillin family, smooth muscle archvillin (SmAV) (7Gangopadhyay S.S. Takizawa N. Gallant C. Barber A.L. Je H.D. Smith T.C. Luna E.J. Morgan K.G. J. Cell Sci. 2004; 117: 5043-5057Crossref PubMed Scopus (39) Google Scholar) that is co-targeted to the cell cortex with ERK during α-agonist activation. Antisense knockdown of SmAV was shown to inhibit ERK activation, and it was postulated that ERK might form a macromolecular complex with SmAV to regulate its signaling function. In the present study we directly demonstrate the agonist- and pathway-specific formation of a complex containing SmAV and members of the ERK signaling module, MEK, B-Raf, and 14-3-3 both in vitro and in vivo in smooth muscle cells. Thus, SmAV functions as a pathway-specific scaffold to couple ERK activation in a spatially restricted manner to select outcomes. DISCUSSIONThe results presented here, together with past cellular studies from our group (7Gangopadhyay S.S. Takizawa N. Gallant C. Barber A.L. Je H.D. Smith T.C. Luna E.J. Morgan K.G. J. Cell Sci. 2004; 117: 5043-5057Crossref PubMed Scopus (39) Google Scholar), demonstrate that SmAV displays properties that allow it to function as an ERK scaffolding protein. Scaffolds have been defined as proteins, generally with no applicable enzymatic activity "that interact with a signaling pathway to create a functional signaling module and to control the specificity of signal transduction" (5Morrison D.K. Davis R.J. Annu. Rev. Cell Dev. Biol. 2003; 19: 91-118Crossref PubMed Scopus (645) Google Scholar). We have previously shown that SmAV knockdown in vascular smooth muscle tissue inhibits ERK activation and contraction induced by the α-agonist PE (7Gangopadhyay S.S. Takizawa N. Gallant C. Barber A.L. Je H.D. Smith T.C. Luna E.J. Morgan K.G. J. Cell Sci. 2004; 117: 5043-5057Crossref PubMed Scopus (39) Google Scholar). We demonstrate here that N-terminal SmAV sequence (N-SmAV-1) associates with ERK, MEK, Raf, and 14-3-3, all known members of a classical ERK signaling pathway, in an agonist- and pathway-specific manner. Taken together, these results demonstrate an ERK scaffolding function for this recently described smooth muscle protein.Two ERK pathways, targeted to two different ERK substrates have previously been shown to co-exist in the cell type from which SmAV was first identified (17Wier W.G. Morgan K.G. Rev. Physiol. Biochem. Pharmacol. 2003; 150: 91-139Crossref PubMed Scopus (97) Google Scholar). α-Agonist activation has been shown to lead to ERK activation and subsequent phosphorylation of caldesmon at an ERK phosphorylation site. Phosphorylation at this site has been shown to increase actomyosin ATPase activity (18Gerthoffer W.T. Yamboliev I.A. Shearer M. Pohl J. Haynes R. Dang S. Sato K. Sellers J.R. J. Physiol. 1996; 495: 597-609Crossref PubMed Scopus (99) Google Scholar, 19Adam L.P. Franklin M.T. Raff G.J. Hathaway D.R. Circ. Res. 1995; 76: 183-190Crossref PubMed Google Scholar) and, hence, contractility. In contrast, depolarization-mediated activation leads to activation of calcium-calmodulin-kinase 2 and subsequent activation of ERK, but in this case, ERK activation leads to increased myosin phosphorylation and contractility, presumably via ERK-mediated phosphorylation of myosin light chain kinase. In the present study we showed that α-adrenoreceptor activation of tissues with PE causes a greater fold increase in the association of N-SmAV1 in pulldown experiments with ERK, phospho-ERK, MEK, and 14-3-3 than does depolarization of tissues even though both modes of stimulation lead to ERK activation. Previously, PE stimulation has been shown to target ERK to the cell surface (8Khalil R.A. Morgan K.G. Am. J. Physiol. Cell Physiol. 1993; 265: C406-C411Crossref PubMed Google Scholar), but depolarization leads to a homogeneous cellular distribution of ERK (20Marganski W.A. Gangopadhyay S.S. Je H.D. Gallant C. Morgan K.G. Circ. Res. 2005; 97: 541-549Crossref PubMed Scopus (66) Google Scholar). Interestingly, both SmAV and ERK translocate to the membrane upon PE stimulation at the same time point (4 min) in isolated ferret aorta cells, as has been shown previously (7Gangopadhyay S.S. Takizawa N. Gallant C. Barber A.L. Je H.D. Smith T.C. Luna E.J. Morgan K.G. J. Cell Sci. 2004; 117: 5043-5057Crossref PubMed Scopus (39) Google Scholar, 8Khalil R.A. Morgan K.G. Am. J. Physiol. Cell Physiol. 1993; 265: C406-C411Crossref PubMed Google Scholar). The stimulus-specific nature of N-SmAV1 binding to ERK signaling partners could serve to spatially sequester the α-agonist-activated ERK pathway from the depolarization-activated ERK pathway and provide a mechanism whereby ERK in the intracellular environment chooses the signaling pathway to follow in response to a specific agonist.Multiple cellular studies have demonstrated a biphasic targeting of MAPKs, first from the cytosol to the cell cortex/membrane and, subsequently, either to the nucleus in proliferating cells (21Pouysségur J. Lenormand P. Eur. J. Biochem. 2003; 270: 3291-3299Crossref PubMed Scopus (154) Google Scholar, 22Gonzalez F.A. Seth A. Raden D.L. Bowman D.S. Fay F.S. Davis R.J. J. Cell Biol. 1993; 122: 1089-1101Crossref PubMed Scopus (282) Google Scholar) or to the contractile filaments in differentiated smooth muscle (8Khalil R.A. Morgan K.G. Am. J. Physiol. Cell Physiol. 1993; 265: C406-C411Crossref PubMed Google Scholar). A possible mechanism triggering the release from its cortical targeting has been lacking. We have now shown that ERK, after preferentially associating with N-SmAV1 and becoming activated, uses N-SmAV1 as a substrate and phosphorylates (in vitro and in vivo) N-SmAV1 at Ser132. We have also shown that ERK-mediated phosphorylation of N-SmAV1 decreases the association of active ERK from the scaffold. The release of active ERK then allows the subsequent targeting to the final substrate of ERK. The binding of unphosphorylated ERK to N-SmAV2 may feed substrate to activated MEK associated with N-SmAV1.Leinweber et al. (23Leinweber B.D. Leavis P.C. Grabarek Z. Wang C.L. Morgan K.G. Biochem. J. 1999; 344: 117-123Crossref PubMed Scopus (124) Google Scholar) showed that the ERK-binding domain of calponin is the N-terminal calponin homology domain, which Gangopadhyay et al. (7Gangopadhyay S.S. Takizawa N. Gallant C. Barber A.L. Je H.D. Smith T.C. Luna E.J. Morgan K.G. J. Cell Sci. 2004; 117: 5043-5057Crossref PubMed Scopus (39) Google Scholar) showed is the domain of calponin that binds the C-terminal end of SmAV. These past studies can be combined with the work presented here in a model shown in Fig. 10, whereby (a) ERK, bound to the calponin homology domain of calponin, colocalizes with SmAV in the cell cortex (7Gangopadhyay S.S. Takizawa N. Gallant C. Barber A.L. Je H.D. Smith T.C. Luna E.J. Morgan K.G. J. Cell Sci. 2004; 117: 5043-5057Crossref PubMed Scopus (39) Google Scholar), and binding of the calponin homology domain to SmAV 1823–2073 releases ERK. (b) ERK binds the two ERK-binding sites in the N-terminal portion of SmAV. (c) MEK is recruited to SmAV and activated by B-Raf. (d) ERK is phosphorylated and activated by MEK. (e) Activated ERK phosphorylates Ser132 of SmAV, and phosphorylation of SmAV may cause a conformational change in the SmAV molecule or the charge difference may repel ERK from SmAV, releasing phospho-ERK. (f) phospho-ERK associates with and phosphorylates caldesmon. (g) Phosphorylation of caldesmon makes the actin available for the interaction with myosin, which in turn increases contractility. In summary, we have shown that SmAV displays the properties necessary to function as an ERK scaffold, creating a functional signaling module that can control the stimulus specificity of signal transduction. The results presented here, together with past cellular studies from our group (7Gangopadhyay S.S. Takizawa N. Gallant C. Barber A.L. Je H.D. Smith T.C. Luna E.J. Morgan K.G. J. Cell Sci. 2004; 117: 5043-5057Crossref PubMed Scopus (39) Google Scholar), demonstrate that SmAV displays properties that allow it to function as an ERK scaffolding protein. Scaffolds have been defined as proteins, generally with no applicable enzymatic activity "that interact with a signaling pathway to create a functional signaling module and to control the specificity of signal transduction" (5Morrison D.K. Davis R.J. Annu. Rev. Cell Dev. Biol. 2003; 19: 91-118Crossref PubMed Scopus (645) Google Scholar). We have previously shown that SmAV knockdown in vascular smooth muscle tissue inhibits ERK activation and contraction induced by the α-agonist PE (7Gangopadhyay S.S. Takizawa N. Gallant C. Barber A.L. Je H.D. Smith T.C. Luna E.J. Morgan K.G. J. Cell Sci. 2004; 117: 5043-5057Crossref PubMed Scopus (39) Google Scholar). We demonstrate here that N-terminal SmAV sequence (N-SmAV-1) associates with ERK, MEK, Raf, and 14-3-3, all known members of a classical ERK signaling pathway, in an agonist- and pathway-specific manner. Taken together, these results demonstrate an ERK scaffolding function for this recently described smooth muscle protein. Two ERK pathways, targeted to two different ERK substrates have previously been shown to co-exist in the cell type from which SmAV was first identified (17Wier W.G. Morgan K.G. Rev. Physiol. Biochem. Pharmacol. 2003; 150: 91-139Crossref PubMed Scopus (97) Google Scholar). α-Agonist activation has been shown to lead to ERK activation and subsequent phosphorylation of caldesmon at an ERK phosphorylation site. Phosphorylation at this site has been shown to increase actomyosin ATPase activity (18Gerthoffer W.T. Yamboliev I.A. Shearer M. Pohl J. Haynes R. Dang S. Sato K. Sellers J.R. J. Physiol. 1996; 495: 597-609Crossref PubMed Scopus (99) Google Scholar, 19Adam L.P. Franklin M.T. Raff G.J. Hathaway D.R. Circ. Res. 1995; 76: 183-190Crossref PubMed Google Scholar) and, hence, contractility. In contrast, depolarization-mediated activation leads to activation of calcium-calmodulin-kinase 2 and subsequent activation of ERK, but in this case, ERK activation leads to increased myosin phosphorylation and contractility, presumably via ERK-mediated phosphorylation of myosin light chain kinase. In the present study we showed that α-adrenoreceptor activation of tissues with PE causes a greater fold increase in the association of N-SmAV1 in pulldown experiments with ERK, phospho-ERK, MEK, and 14-3-3 than does depolarization of tissues even though both modes of stimulation lead to ERK activation. Previously, PE stimulation has been shown to target ERK to the cell surface (8Khalil R.A. Morgan K.G. Am. J. Physiol. Cell Physiol. 1993; 265: C406-C411Crossref PubMed Google Scholar), but depolarization leads to a homogeneous cellular distribution of ERK (20Marganski W.A. Gangopadhyay S.S. Je H.D. Gallant C. Morgan K.G. Circ. Res. 2005; 97: 541-549Crossref PubMed Scopus (66) Google Scholar). Interestingly, both SmAV and ERK translocate to the membrane upon PE stimulation at the same time point (4 min) in isolated ferret aorta cells, as has been shown previously (7Gangopadhyay S.S. Takizawa N. Gallant C. Barber A.L. Je H.D. Smith T.C. Luna E.J. Morgan K.G. J. Cell Sci. 2004; 117: 5043-5057Crossref PubMed Scopus (39) Google Scholar, 8Khalil R.A. Morgan K.G. Am. J. Physiol. Cell Physiol. 1993; 265: C406-C411Crossref PubMed Google Scholar). The stimulus-specific nature of N-SmAV1 binding to ERK signaling partners could serve to spatially sequester the α-agonist-activated ERK pathway from the depolarization-activated ERK pathway and provide a mechanism whereby ERK in the intracellular environment chooses the signaling pathway to follow in response to a specific agonist. Multiple cellular studies have demonstrated a biphasic targeting of MAPKs, first from the cytosol to the cell cortex/membrane and, subsequently, either to the nucleus in proliferating cells (21Pouysségur J. Lenormand P. Eur. J. Biochem. 2003; 270: 3291-3299Crossref PubMed Scopus (154) Google Scholar, 22Gonzalez F.A. Seth A. Raden D.L. Bowman D.S. Fay F.S. Davis R.J. J. Cell Biol. 1993; 122: 1089-1101Crossref PubMed Scopus (282) Google Scholar) or to the contractile filaments in differentiated smooth muscle (8Khalil R.A. Morgan K.G. Am. J. Physiol. Cell Physiol. 1993; 265: C406-C411Crossref PubMed Google Scholar). A possible mechanism triggering the release from its cortical targeting has been lacking. We have now shown that ERK, after preferentially associating with N-SmAV1 and becoming activated, uses N-SmAV1 as a substrate and phosphorylates (in vitro and in vivo) N-SmAV1 at Ser132. We have also shown that ERK-mediated phosphorylation of N-SmAV1 decreases the association of active ERK from the scaffold. The release of active ERK then allows the subsequent targeting to the final substrate of ERK. The binding of unphosphorylated ERK to N-SmAV2 may feed substrate to activated MEK associated with N-SmAV1. Leinweber et al. (23Leinweber B.D. Leavis P.C. Grabarek Z. Wang C.L. Morgan K.G. Biochem. J. 1999; 344: 117-123Crossref PubMed Scopus (124) Google Scholar) showed that the ERK-binding domain of calponin is the N-terminal calponin homology domain, which Gangopadhyay et al. (7Gangopadhyay S.S. Takizawa N. Gallant C. Barber A.L. Je H.D. Smith T.C. Luna E.J. Morgan K.G. J. Cell Sci. 2004; 117: 5043-5057Crossref PubMed Scopus (39) Google Scholar) showed is the domain of calponin that binds the C-terminal end of SmAV. These past studies can be combined with the work presented here in a model shown in Fig. 10, whereby (a) ERK, bound to the calponin homology domain of calponin, colocalizes with SmAV in the cell cortex (7Gangopadhyay S.S. Takizawa N. Gallant C. Barber A.L. Je H.D. Smith T.C. Luna E.J. Morgan K.G. J. Cell Sci. 2004; 117: 5043-5057Crossref PubMed Scopus (39) Google Scholar), and binding of the calponin homology domain to SmAV 1823–2073 releases ERK. (b) ERK binds the two ERK-binding sites in the N-terminal portion of SmAV. (c) MEK is recruited to SmAV and activated by B-Raf. (d) ERK is phosphorylated and activated by MEK. (e) Activated ERK phosphorylates Ser132 of SmAV, and phosphorylation of SmAV may cause a conformational change in the SmAV molecule or the charge difference may repel ERK from SmAV, releasing phospho-ERK. (f) phospho-ERK associates with and phosphorylates caldesmon. (g) Phosphorylation of caldesmon makes the actin available for the interaction with myosin, which in turn increases contractility. In summary, we have shown that SmAV displays the properties necessary to function as an ERK scaffold, creating a functional signaling module that can control the stimulus specificity of signal transduction. We thank Dr. Melanie H. Cobb (University of Texas Southwestern Medical Center, Dallas, TX) for providing the ERK construct.
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