V-1 is a 12-kDa protein consisting of three consecutive ANK repeats, which are believed to serve as the surface for protein-protein interactions. It is thought to have a role in neural development for its temporal profile of expression during murine cerebellar development, but its precise role remains unknown. Here we applied the proteomic approach to search for protein targets that interact with V-1. The V-1 cDNA attached with a tandem affinity purification tag was expressed in the cultured 293T cells, and the protein complex formed within the cells were captured and characterized by mass spectrometry. We detected two polypeptides specifically associated with V-1, which were identified as the α and β subunits of the capping protein (CP, alternatively called CapZ or β-actinin). CP regulates actin polymerization by capping the barbed end of the actin filament. The V-1·CP complex was detected not only in cultured cells transfected with the V-1 cDNA but also endogenously in cells as well as in murine cerebellar extracts. An analysis of the V-1/CP interaction by surface plasmon resonance spectroscopy showed that V-1 formed a stable complex with the CP heterodimer with a dissociation constant of 1.2 × 10−7m and a molecular stoichiometry of ∼1:1. In addition, V-1 inhibited the CP-regulated actin polymerization in vitro in a dose-dependent manner. Thus, our results suggest that V-1 is a novel component that regulates the dynamics of actin polymerization by interacting with CP and thereby participates in a variety of cellular processes such as actin-driven cell movements and motility during neuronal development. V-1 is a 12-kDa protein consisting of three consecutive ANK repeats, which are believed to serve as the surface for protein-protein interactions. It is thought to have a role in neural development for its temporal profile of expression during murine cerebellar development, but its precise role remains unknown. Here we applied the proteomic approach to search for protein targets that interact with V-1. The V-1 cDNA attached with a tandem affinity purification tag was expressed in the cultured 293T cells, and the protein complex formed within the cells were captured and characterized by mass spectrometry. We detected two polypeptides specifically associated with V-1, which were identified as the α and β subunits of the capping protein (CP, alternatively called CapZ or β-actinin). CP regulates actin polymerization by capping the barbed end of the actin filament. The V-1·CP complex was detected not only in cultured cells transfected with the V-1 cDNA but also endogenously in cells as well as in murine cerebellar extracts. An analysis of the V-1/CP interaction by surface plasmon resonance spectroscopy showed that V-1 formed a stable complex with the CP heterodimer with a dissociation constant of 1.2 × 10−7m and a molecular stoichiometry of ∼1:1. In addition, V-1 inhibited the CP-regulated actin polymerization in vitro in a dose-dependent manner. Thus, our results suggest that V-1 is a novel component that regulates the dynamics of actin polymerization by interacting with CP and thereby participates in a variety of cellular processes such as actin-driven cell movements and motility during neuronal development. The V-1 protein was originally identified in the murine cerebellum as one of the proteins expressed significantly at the initial stage of postnatal development (1Taoka M. Yamakuni T. Song S.Y. Yamakawa Y. Seta K. Okuyama T. Isobe T. Eur. J. Biochem. 1992; 207: 615-620Google Scholar), particularly in the regions where synaptic formation and neuronal migration occur actively during neurogenesis (2Taoka M. Isobe T. Okuyama T. Watanabe M. Kondo H. Yamakawa Y. Ozawa F. Hishinuma F. Kubota M. Minegishi A. Song S.Y. Yamakuni T. J. Biol. Chem. 1994; 269: 9946-9951Google Scholar,3Fujigasaki H. Song S.Y. Kobayashi T. Yamakuni T. Mol. Brain Res. 1996; 40: 203-213Google Scholar). V-1 consists of 117 amino acids containing three contiguous repeats of the ANK motif, alternatively called the cdc10/SWI6 motif (1Taoka M. Yamakuni T. Song S.Y. Yamakawa Y. Seta K. Okuyama T. Isobe T. Eur. J. Biochem. 1992; 207: 615-620Google Scholar, 2Taoka M. Isobe T. Okuyama T. Watanabe M. Kondo H. Yamakawa Y. Ozawa F. Hishinuma F. Kubota M. Minegishi A. Song S.Y. Yamakuni T. J. Biol. Chem. 1994; 269: 9946-9951Google Scholar), which is crucial for a large number of protein-protein interactions (4Sedgwick S.G. Smerdon S.J. Trends Biochem. Sci. 1999; 24: 311-316Google Scholar). Previous studies suggested the potential role of V-1 in the signal transduction pathways leading to catecholamine synthesis or to cardiac hypertrophy. For example, Yamakuni et al. (5Yamakuni T. Yamamoto T. Hoshino M. Song S.Y. Yamamoto H. Kunikata-Sumitomo M. Minegishi A. Kubota M. Ito M. Konishi S. J. Biol. Chem. 1998; 273: 27051-27054Google Scholar, 6Suzuki T. Inagaki H. Yamakuni T. Nagatsu T. Ichinose H. Biochem. Biophys. Res. Commun. 2002; 293: 962-968Google Scholar, 7Suzuki T. Yamakuni T. Hagiwara M. Ichinose H. J. Biol. Chem. 2002; 277: 40768-40774Google Scholar) demonstrate that the overexpression of V-1 caused a significant increase in the catecholamine level in PC12 cells, presumably through the transcriptional activation of the genes for catecholamine synthesis. In other reports (8Gupta S. Sen S. Biochim. Biophys. Acta. 2002; 1589: 247-260Google Scholar, 9Knuefermann P. Chen P. Misra A. Shi S.P. Abdellatif M. Sivasubramanian N. J. Biol. Chem. 2002; 277: 23888-23897Google Scholar, 10Sivasubramanian N. Adhikary G. Sil P.C. Sen S. J. Biol. Chem. 1996; 271: 2812-2816Google Scholar, 11Sil P. Kandaswamy V. Sen S. Circ. Res. 1998; 82: 1173-1188Google Scholar), V-1 was designated as “myotrophin” and was shown to participate in the cell signaling pathways for the NFkB-mediated activation of protein synthesis in the myocytes. Thus, both of these studies (5Yamakuni T. Yamamoto T. Hoshino M. Song S.Y. Yamamoto H. Kunikata-Sumitomo M. Minegishi A. Kubota M. Ito M. Konishi S. J. Biol. Chem. 1998; 273: 27051-27054Google Scholar, 9Knuefermann P. Chen P. Misra A. Shi S.P. Abdellatif M. Sivasubramanian N. J. Biol. Chem. 2002; 277: 23888-23897Google Scholar) suggested the roles of V-1 in the biological events taking place in the nucleus. However, no biological function has been attributed to V-1 in the cytoplasm in which this molecule predominantly resides within the cells and tissues (5Yamakuni T. Yamamoto T. Hoshino M. Song S.Y. Yamamoto H. Kunikata-Sumitomo M. Minegishi A. Kubota M. Ito M. Konishi S. J. Biol. Chem. 1998; 273: 27051-27054Google Scholar, 9Knuefermann P. Chen P. Misra A. Shi S.P. Abdellatif M. Sivasubramanian N. J. Biol. Chem. 2002; 277: 23888-23897Google Scholar).In this study, we screened for V-1-binding proteins by a novel proteomic approach that combined the tandem affinity purification (TAP) 1The abbreviations used are: TAP, tandem affinity purification; CP, capping protein; GST, glutathioneS-transferase; MS/MS, tandem mass spectrometry; SPR, surface plasmon resonance; CBB, Coomassie Brilliant Blue; 293T, human embryonic kidney 293 T cells 1The abbreviations used are: TAP, tandem affinity purification; CP, capping protein; GST, glutathioneS-transferase; MS/MS, tandem mass spectrometry; SPR, surface plasmon resonance; CBB, Coomassie Brilliant Blue; 293T, human embryonic kidney 293 T cells procedure (12Rigaut G. Shevchenko A. Rutz B. Wilm M. Mann M. Seraphin B. Nat. Biotechnol. 1999; 17: 1030-1032Google Scholar) and mass spectrometry (MS). Following this strategy, we identified capping protein (CP) as a V-1-binding protein. We confirmed the existence of the V-1·CP complex not only in cultured cells transfected with the TAP-tagged V-1 but also endogenously in cells and rat cerebellar extracts. Furthermore, we found that V-1 inhibited the CP-regulated actin polymerization. On the basis of these results, the possible role of V-1 in neuronal development is discussed.DISCUSSIONCP is one of the F-actin-binding proteins that caps the barbed end of actin filaments and nucleates the actin polymerization in a Ca2+-independent (22Maruyama K. Kimura S. T. M. K. S. J. Biochem. Scholar, S.G. V. Scholar, K. H. M. S. Yamamoto H. Ito M. J. Biol. Chem. Scholar, S. J. Biol. Chem. Scholar) and a S.G. Scholar, H. P. M. Biochem. Biophys. Res. Commun. Scholar, K. J. Biol. 1996; Scholar, S. Biochim. Biophys. Acta. Scholar). is thought to the of is to the expression level of CP P.C. Scholar) and CP is for the in vitro of the cell 1999; Scholar, P. Nat. Biol. Scholar). In this study, we have shown that the V-1 protein a stable complex with CP in vitro as well as in and and the of actin polymerization. we that V-1 participates in the of actin dynamics in the cells the interaction with to the was the tandem affinity purification of the V-1 complex with a tag by protein by mass spectrometry. The was originally to interactions proteins (12Rigaut G. Shevchenko A. Rutz B. Wilm M. Mann M. Seraphin B. Nat. Biotechnol. 1999; 17: 1030-1032Google Scholar). We a expression for the and applied to the 293T cell the V-1 protein is a cellular component and a expression was used for the the to a of the V-1·CP complex for by tandem mass spectrometry with mass spectrometry is to novel protein interactions not only in but also in V-1 protein consists of three consecutive ANK repeats with of (1Taoka M. Yamakuni T. Song S.Y. Yamakawa Y. Seta K. Okuyama T. Isobe T. Eur. J. Biochem. 1992; 207: 615-620Google Scholar, 2Taoka M. Isobe T. Okuyama T. Watanabe M. Kondo H. Yamakawa Y. Ozawa F. Hishinuma F. Kubota M. Minegishi A. Song S.Y. Yamakuni T. J. Biol. Chem. 1994; 269: 9946-9951Google Scholar, Y. S. Sen S. J. 1998; Scholar). The ANK is a motif found in proteins S. Misra S. M. T. P. A. A. F. P. J. S.J. B. J. S. Scholar) and interactions with a of protein targets (4Sedgwick S.G. Smerdon S.J. Trends Biochem. Sci. 1999; 24: 311-316Google Scholar). In the of V-1 by spectroscopy Y. S. Sen S. J. 1998; Scholar), the ANK repeats the where the α one a and the the other of the The and the surface of the α a which is believed to for the with the identified CP as a potential of V-1. V-1 to the CP heterodimer consisting of the α and β subunits but not of the two is with that of the CP subunits was and not actin in vitro and in J. Biol. 1992; Scholar, J. Biol. 1992; Scholar, J. Biol. 1999; Scholar). Thus, that V-1 the of the CP heterodimer by its ANK repeats and the surface in the of the J. Biol. 1992; Scholar). However, this is the molecular by which V-1 the interaction of CP and G. K. J. Biol. Scholar) and S. P. J. Biol. Scholar), are to is a protein containing the the the found in actin and the that actin G. K. J. Biol. Scholar). The of CP, but its with CP is unknown. is a actin protein of two by a and has a role in the of actin S. M. P. J. Sci. 2002; Scholar). also a stable complex with CP but not its V-1 is a protein and to or Thus, V-1 to a novel protein was as which actin by with the barbed of actin and CP M. 2002; Scholar). The actin in the cell filaments in the V-1 in the of CP-regulated actin but a attributed to the cell is studies suggested the potential roles of V-1 in events such as the transcriptional activation of a of in catecholamine synthesis (5Yamakuni T. Yamamoto T. Hoshino M. Song S.Y. Yamamoto H. Kunikata-Sumitomo M. Minegishi A. Kubota M. Ito M. Konishi S. J. Biol. Chem. 1998; 273: 27051-27054Google Scholar, 6Suzuki T. Inagaki H. Yamakuni T. Nagatsu T. Ichinose H. Biochem. Biophys. Res. Commun. 2002; 293: 962-968Google Scholar, 7Suzuki T. Yamakuni T. Hagiwara M. Ichinose H. J. Biol. Chem. 2002; 277: 40768-40774Google Scholar) or the of synthesis (8Gupta S. Sen S. Biochim. Biophys. Acta. 2002; 1589: 247-260Google Scholar, 9Knuefermann P. Chen P. Misra A. Shi S.P. Abdellatif M. Sivasubramanian N. J. Biol. Chem. 2002; 277: 23888-23897Google Scholar, 10Sivasubramanian N. Adhikary G. Sil P.C. Sen S. J. Biol. Chem. 1996; 271: 2812-2816Google Scholar, A. A. S. M. J. Scholar). for the that V-1 in the molecular events taking place in the cytoplasm in which the of V-1 resides within the cells as by studies (5Yamakuni T. Yamamoto T. Hoshino M. Song S.Y. Yamamoto H. Kunikata-Sumitomo M. Minegishi A. Kubota M. Ito M. Konishi S. J. Biol. Chem. 1998; 273: 27051-27054Google Scholar, 9Knuefermann P. Chen P. Misra A. Shi S.P. Abdellatif M. Sivasubramanian N. J. Biol. Chem. 2002; 277: 23888-23897Google Scholar). V-1 also to a protein in of amino with no studies the temporal profile of V-1 expression in the murine cerebellum at postnatal (2Taoka M. Isobe T. Okuyama T. Watanabe M. Kondo H. Yamakawa Y. Ozawa F. Hishinuma F. Kubota M. Minegishi A. Song S.Y. Yamakuni T. J. Biol. Chem. 1994; 269: 9946-9951Google Scholar). V-1 expression is particularly significant during the migration of cells the to to synaptic with the the dynamics of actin polymerization a role in the of cells, the of the barbed of actin filaments with the of the J. Sci. Scholar) and the migration of cells J. Scholar). with the results suggest that V-1 have a role in the actin-driven cell movements and motility such as cell migration and The V-1 protein was originally identified in the murine cerebellum as one of the proteins expressed significantly at the initial stage of postnatal development (1Taoka M. Yamakuni T. Song S.Y. Yamakawa Y. Seta K. Okuyama T. Isobe T. Eur. J. Biochem. 1992; 207: 615-620Google Scholar), particularly in the regions where synaptic formation and neuronal migration occur actively during neurogenesis (2Taoka M. Isobe T. Okuyama T. Watanabe M. Kondo H. Yamakawa Y. Ozawa F. Hishinuma F. Kubota M. Minegishi A. Song S.Y. Yamakuni T. J. Biol. Chem. 1994; 269: 9946-9951Google Scholar,3Fujigasaki H. Song S.Y. Kobayashi T. Yamakuni T. Mol. Brain Res. 1996; 40: 203-213Google Scholar). V-1 consists of 117 amino acids containing three contiguous repeats of the ANK motif, alternatively called the cdc10/SWI6 motif (1Taoka M. Yamakuni T. Song S.Y. Yamakawa Y. Seta K. Okuyama T. Isobe T. Eur. J. Biochem. 1992; 207: 615-620Google Scholar, 2Taoka M. Isobe T. Okuyama T. Watanabe M. Kondo H. Yamakawa Y. Ozawa F. Hishinuma F. Kubota M. Minegishi A. Song S.Y. Yamakuni T. J. Biol. Chem. 1994; 269: 9946-9951Google Scholar), which is crucial for a large number of protein-protein interactions (4Sedgwick S.G. Smerdon S.J. Trends Biochem. Sci. 1999; 24: 311-316Google Scholar). Previous studies suggested the potential role of V-1 in the signal transduction pathways leading to catecholamine synthesis or to cardiac hypertrophy. For example, Yamakuni et al. (5Yamakuni T. Yamamoto T. Hoshino M. Song S.Y. Yamamoto H. Kunikata-Sumitomo M. Minegishi A. Kubota M. Ito M. Konishi S. J. Biol. Chem. 1998; 273: 27051-27054Google Scholar, 6Suzuki T. Inagaki H. Yamakuni T. Nagatsu T. Ichinose H. Biochem. Biophys. Res. Commun. 2002; 293: 962-968Google Scholar, 7Suzuki T. Yamakuni T. Hagiwara M. Ichinose H. J. Biol. Chem. 2002; 277: 40768-40774Google Scholar) demonstrate that the overexpression of V-1 caused a significant increase in the catecholamine level in PC12 cells, presumably through the transcriptional activation of the genes for catecholamine synthesis. In other reports (8Gupta S. Sen S. Biochim. Biophys. Acta. 2002; 1589: 247-260Google Scholar, 9Knuefermann P. Chen P. Misra A. Shi S.P. Abdellatif M. Sivasubramanian N. J. Biol. Chem. 2002; 277: 23888-23897Google Scholar, 10Sivasubramanian N. Adhikary G. Sil P.C. Sen S. J. Biol. Chem. 1996; 271: 2812-2816Google Scholar, 11Sil P. Kandaswamy V. Sen S. Circ. Res. 1998; 82: 1173-1188Google Scholar), V-1 was designated as “myotrophin” and was shown to participate in the cell signaling pathways for the NFkB-mediated activation of protein synthesis in the myocytes. Thus, both of these studies (5Yamakuni T. Yamamoto T. Hoshino M. Song S.Y. Yamamoto H. Kunikata-Sumitomo M. Minegishi A. Kubota M. Ito M. Konishi S. J. Biol. Chem. 1998; 273: 27051-27054Google Scholar, 9Knuefermann P. Chen P. Misra A. Shi S.P. Abdellatif M. Sivasubramanian N. J. Biol. Chem. 2002; 277: 23888-23897Google Scholar) suggested the roles of V-1 in the biological events taking place in the nucleus. However, no biological function has been attributed to V-1 in the cytoplasm in which this molecule predominantly resides within the cells and tissues (5Yamakuni T. Yamamoto T. Hoshino M. Song S.Y. Yamamoto H. Kunikata-Sumitomo M. Minegishi A. Kubota M. Ito M. Konishi S. J. Biol. Chem. 1998; 273: 27051-27054Google Scholar, 9Knuefermann P. Chen P. Misra A. Shi S.P. Abdellatif M. Sivasubramanian N. J. Biol. Chem. 2002; 277: 23888-23897Google Scholar). In this study, we screened for V-1-binding proteins by a novel proteomic approach that combined the tandem affinity purification (TAP) 1The abbreviations used are: TAP, tandem affinity purification; CP, capping protein; GST, glutathioneS-transferase; MS/MS, tandem mass spectrometry; SPR, surface plasmon resonance; CBB, Coomassie Brilliant Blue; 293T, human embryonic kidney 293 T cells 1The abbreviations used are: TAP, tandem affinity purification; CP, capping protein; GST, glutathioneS-transferase; MS/MS, tandem mass spectrometry; SPR, surface plasmon resonance; CBB, Coomassie Brilliant Blue; 293T, human embryonic kidney 293 T cells procedure (12Rigaut G. Shevchenko A. Rutz B. Wilm M. Mann M. Seraphin B. Nat. Biotechnol. 1999; 17: 1030-1032Google Scholar) and mass spectrometry (MS). Following this strategy, we identified capping protein (CP) as a V-1-binding protein. We confirmed the existence of the V-1·CP complex not only in cultured cells transfected with the TAP-tagged V-1 but also endogenously in cells and rat cerebellar extracts. Furthermore, we found that V-1 inhibited the CP-regulated actin polymerization. On the basis of these results, the possible role of V-1 in neuronal development is is one of the F-actin-binding proteins that caps the barbed end of actin filaments and nucleates the actin polymerization in a Ca2+-independent (22Maruyama K. Kimura S. T. M. K. S. J. Biochem. Scholar, S.G. V. Scholar, K. H. M. S. Yamamoto H. Ito M. J. Biol. Chem. Scholar, S. J. Biol. Chem. Scholar) and a S.G. Scholar, H. P. M. Biochem. Biophys. Res. Commun. Scholar, K. J. Biol. 1996; Scholar, S. Biochim. Biophys. Acta. Scholar). is thought to the of is to the expression level of CP P.C. Scholar) and CP is for the in vitro of the cell 1999; Scholar, P. Nat. Biol. Scholar). In this study, we have shown that the V-1 protein a stable complex with CP in vitro as well as in and and the of actin polymerization. we that V-1 participates in the of actin dynamics in the cells the interaction with to the was the tandem affinity purification of the V-1 complex with a tag by protein by mass spectrometry. The was originally to interactions proteins (12Rigaut G. Shevchenko A. Rutz B. Wilm M. Mann M. Seraphin B. Nat. Biotechnol. 1999; 17: 1030-1032Google Scholar). We a expression for the and applied to the 293T cell the V-1 protein is a cellular component and a expression was used for the the to a of the V-1·CP complex for by tandem mass spectrometry with mass spectrometry is to novel protein interactions not only in but also in V-1 protein consists of three consecutive ANK repeats with of (1Taoka M. Yamakuni T. Song S.Y. Yamakawa Y. Seta K. Okuyama T. Isobe T. Eur. J. Biochem. 1992; 207: 615-620Google Scholar, 2Taoka M. Isobe T. Okuyama T. Watanabe M. Kondo H. Yamakawa Y. Ozawa F. Hishinuma F. Kubota M. Minegishi A. Song S.Y. Yamakuni T. J. Biol. Chem. 1994; 269: 9946-9951Google Scholar, Y. S. Sen S. J. 1998; Scholar). The ANK is a motif found in proteins S. Misra S. M. T. P. A. A. F. P. J. S.J. B. J. S. Scholar) and interactions with a of protein targets (4Sedgwick S.G. Smerdon S.J. Trends Biochem. Sci. 1999; 24: 311-316Google Scholar). In the of V-1 by spectroscopy Y. S. Sen S. J. 1998; Scholar), the ANK repeats the where the α one a and the the other of the The and the surface of the α a which is believed to for the with the identified CP as a potential of V-1. V-1 to the CP heterodimer consisting of the α and β subunits but not of the two is with that of the CP subunits was and not actin in vitro and in J. Biol. 1992; Scholar, J. Biol. 1992; Scholar, J. Biol. 1999; Scholar). Thus, that V-1 the of the CP heterodimer by its ANK repeats and the surface in the of the J. Biol. 1992; Scholar). However, this is the molecular by which V-1 the interaction of CP and G. K. J. Biol. Scholar) and S. P. J. Biol. Scholar), are to is a protein containing the the the found in actin and the that actin G. K. J. Biol. Scholar). The of CP, but its with CP is unknown. is a actin protein of two by a and has a role in the of actin S. M. P. J. Sci. 2002; Scholar). also a stable complex with CP but not its V-1 is a protein and to or Thus, V-1 to a novel protein was as which actin by with the barbed of actin and CP M. 2002; Scholar). The actin in the cell filaments in the V-1 in the of CP-regulated actin but a attributed to the cell is studies suggested the potential roles of V-1 in events such as the transcriptional activation of a of in catecholamine synthesis (5Yamakuni T. Yamamoto T. Hoshino M. Song S.Y. Yamamoto H. Kunikata-Sumitomo M. Minegishi A. Kubota M. Ito M. Konishi S. J. Biol. Chem. 1998; 273: 27051-27054Google Scholar, 6Suzuki T. Inagaki H. Yamakuni T. Nagatsu T. Ichinose H. Biochem. Biophys. Res. Commun. 2002; 293: 962-968Google Scholar, 7Suzuki T. Yamakuni T. Hagiwara M. Ichinose H. J. Biol. Chem. 2002; 277: 40768-40774Google Scholar) or the of synthesis (8Gupta S. Sen S. Biochim. Biophys. Acta. 2002; 1589: 247-260Google Scholar, 9Knuefermann P. Chen P. Misra A. Shi S.P. Abdellatif M. Sivasubramanian N. J. Biol. Chem. 2002; 277: 23888-23897Google Scholar, 10Sivasubramanian N. Adhikary G. Sil P.C. Sen S. J. Biol. Chem. 1996; 271: 2812-2816Google Scholar, A. A. S. M. J. Scholar). for the that V-1 in the molecular events taking place in the cytoplasm in which the of V-1 resides within the cells as by studies (5Yamakuni T. Yamamoto T. Hoshino M. Song S.Y. Yamamoto H. Kunikata-Sumitomo M. Minegishi A. Kubota M. Ito M. Konishi S. J. Biol. Chem. 1998; 273: 27051-27054Google Scholar, 9Knuefermann P. Chen P. Misra A. Shi S.P. Abdellatif M. Sivasubramanian N. J. Biol. Chem. 2002; 277: 23888-23897Google Scholar). V-1 also to a protein in of amino with no studies the temporal profile of V-1 expression in the murine cerebellum at postnatal (2Taoka M. Isobe T. Okuyama T. Watanabe M. Kondo H. Yamakawa Y. Ozawa F. Hishinuma F. Kubota M. Minegishi A. Song S.Y. Yamakuni T. J. Biol. Chem. 1994; 269: 9946-9951Google Scholar). V-1 expression is particularly significant during the migration of cells the to to synaptic with the the dynamics of actin polymerization a role in the of cells, the of the barbed of actin filaments with the of the J. Sci. Scholar) and the migration of cells J. Scholar). with the results suggest that V-1 have a role in the actin-driven cell movements and motility such as cell migration and CP is one of the F-actin-binding proteins that caps the barbed end of actin filaments and nucleates the actin polymerization in a Ca2+-independent (22Maruyama K. Kimura S. T. M. K. S. J. Biochem. Scholar, S.G. V. Scholar, K. H. M. S. Yamamoto H. Ito M. J. Biol. Chem. Scholar, S. J. Biol. Chem. Scholar) and a S.G. Scholar, H. P. M. Biochem. Biophys. Res. Commun. Scholar, K. J. Biol. 1996; Scholar, S. Biochim. Biophys. Acta. Scholar). is thought to the of is to the expression level of CP P.C. Scholar) and CP is for the in vitro of the cell 1999; Scholar, P. Nat. Biol. Scholar). In this study, we have shown that the V-1 protein a stable complex with CP in vitro as well as in and and the of actin polymerization. we that V-1 participates in the of actin dynamics in the cells the interaction with to the was the tandem affinity purification of the V-1 complex with a tag by protein by mass spectrometry. The was originally to interactions proteins (12Rigaut G. Shevchenko A. Rutz B. Wilm M. Mann M. Seraphin B. Nat. Biotechnol. 1999; 17: 1030-1032Google Scholar). We a expression for the and applied to the 293T cell the V-1 protein is a cellular component and a expression was used for the the to a of the V-1·CP complex for by tandem mass spectrometry with mass spectrometry is to novel protein interactions not only in but also in The V-1 protein consists of three consecutive ANK repeats with of (1Taoka M. Yamakuni T. Song S.Y. Yamakawa Y. Seta K. Okuyama T. Isobe T. Eur. J. Biochem. 1992; 207: 615-620Google Scholar, 2Taoka M. Isobe T. Okuyama T. Watanabe M. Kondo H. Yamakawa Y. Ozawa F. Hishinuma F. Kubota M. Minegishi A. Song S.Y. Yamakuni T. J. Biol. Chem. 1994; 269: 9946-9951Google Scholar, Y. S. Sen S. J. 1998; Scholar). The ANK is a motif found in proteins S. Misra S. M. T. P. A. A. F. P. J. S.J. B. J. S. Scholar) and interactions with a of protein targets (4Sedgwick S.G. Smerdon S.J. Trends Biochem. Sci. 1999; 24: 311-316Google Scholar). In the of V-1 by spectroscopy Y. S. Sen S. J. 1998; Scholar), the ANK repeats the where the α one a and the the other of the The and the surface of the α a which is believed to for the with the identified CP as a potential of V-1. V-1 to the CP heterodimer consisting of the α and β subunits but not of the two is with that of the CP subunits was and not actin in vitro and in J. Biol. 1992; Scholar, J. Biol. 1992; Scholar, J. Biol. 1999; Scholar). Thus, that V-1 the of the CP heterodimer by its ANK repeats and the surface in the of the J. Biol. 1992; Scholar). However, this is the molecular by which V-1 the interaction of CP and G. K. J. Biol. Scholar) and S. P. J. Biol. Scholar), are to is a protein containing the the the found in actin and the that actin G. K. J. Biol. Scholar). The of CP, but its with CP is unknown. is a actin protein of two by a and has a role in the of actin S. M. P. J. Sci. 2002; Scholar). also a stable complex with CP but not its V-1 is a protein and to or Thus, V-1 to a novel protein was as which actin by with the barbed of actin and CP M. 2002; Scholar). The actin in the cell filaments in the V-1 in the of CP-regulated actin but a attributed to the cell is unknown. Previous studies suggested the potential roles of V-1 in events such as the transcriptional activation of a of in catecholamine synthesis (5Yamakuni T. Yamamoto T. Hoshino M. Song S.Y. Yamamoto H. Kunikata-Sumitomo M. Minegishi A. Kubota M. Ito M. Konishi S. J. Biol. Chem. 1998; 273: 27051-27054Google Scholar, 6Suzuki T. Inagaki H. Yamakuni T. Nagatsu T. Ichinose H. Biochem. Biophys. Res. Commun. 2002; 293: 962-968Google Scholar, 7Suzuki T. Yamakuni T. Hagiwara M. Ichinose H. J. Biol. Chem. 2002; 277: 40768-40774Google Scholar) or the of synthesis (8Gupta S. Sen S. Biochim. Biophys. Acta. 2002; 1589: 247-260Google Scholar, 9Knuefermann P. Chen P. Misra A. Shi S.P. Abdellatif M. Sivasubramanian N. J. Biol. Chem. 2002; 277: 23888-23897Google Scholar, 10Sivasubramanian N. Adhikary G. Sil P.C. Sen S. J. Biol. Chem. 1996; 271: 2812-2816Google Scholar, A. A. S. M. J. Scholar). for the that V-1 in the molecular events taking place in the cytoplasm in which the of V-1 resides within the cells as by studies (5Yamakuni T. Yamamoto T. Hoshino M. Song S.Y. Yamamoto H. Kunikata-Sumitomo M. Minegishi A. Kubota M. Ito M. Konishi S. J. Biol. Chem. 1998; 273: 27051-27054Google Scholar, 9Knuefermann P. Chen P. Misra A. Shi S.P. Abdellatif M. Sivasubramanian N. J. Biol. Chem. 2002; 277: 23888-23897Google Scholar). V-1 also to a protein in of amino with no studies the temporal profile of V-1 expression in the murine cerebellum at postnatal (2Taoka M. Isobe T. Okuyama T. Watanabe M. Kondo H. Yamakawa Y. Ozawa F. Hishinuma F. Kubota M. Minegishi A. Song S.Y. Yamakuni T. J. Biol. Chem. 1994; 269: 9946-9951Google Scholar). V-1 expression is particularly significant during the migration of cells the to to synaptic with the the dynamics of actin polymerization a role in the of cells, the of the barbed of actin filaments with the of the J. Sci. Scholar) and the migration of cells J. Scholar). with the results suggest that V-1 have a role in the actin-driven cell movements and motility such as cell migration and
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