Integrin Activates Receptor-Like Protein Tyrosine Phosphatase α, Src, and Rho to Increase Prolactin Gene Expression through a Final Phosphatidylinositol 3-Kinase/Cytoskeletal Pathway that Is Additive with Insulin

0013-7227/05/$15.00/0 Printed in U.S.A. Endocrinology 146(8):3535–3546 Copyright © 2005 by The Endocrine Society doi: 10.1210/en.2004-1386 Integrin Activates Receptor-Like Protein Tyrosine Phosphatase ␣, Src, and Rho to Increase Prolactin Gene Expression through a Final Phosphatidylinositol 3-Kinase/Cytoskeletal Pathway that Is Additive with Insulin Anthony I. Vulin, Kirsten K. Jacob, and Frederick M. Stanley Department of Pharmacology (A.I.V., K.K.J., F.M.S.) and Kaplan Cancer Center (F.M.S.), New York University School of Medicine, New York, New York 10016 We previously showed that receptor-like protein tyrosine phosphatase (RPTP)-␣ inhibited insulin-increased prolactin gene transcription. Others suggested that RPTP␣ was a key intermediary between integrins and activation of Src. We present evidence that inhibition of insulin-increased prolactin gene transcription was secondary to RPTP␣ activation of Src, reflecting its role as mediator of integrin responses. Src kinase activity was increased in GH4 cells transiently or stably expressing RPTP␣ and cells plated on the integrin-␣5␤1 ligand fibronectin. C-terminal Src kinase inactivated Src and blocked RPTP␣ inhibition of insulin-increased prolactin gene transcription. Expression of dominant-negative Src also prevented the RPTP␣-mediated inhibition of insulin-increased prolactin gene expression. Low levels of a constitutively active Src mutant (SrcY/F) stimulated whereas higher expression levels of Src Y/F inhibited prolactin gene expression. T HE INSULIN-INCREASED prolactin gene transcription seen in GH4 cells, a prolactin-producing pituitary tumor cell line, is an important model for studying gene transcription activated by insulin (1). Autophosphorylation of the insulin receptor upon ligand binding activates its kinase activity (1). The insulin receptor then phosphorylates substrates, e.g. the insulin receptor substrates (IRSs) and Shc that activate downstream signaling pathways to mediate the diverse effects of insulin (2). Insulin-activated prolactin gene transcription depends on activation of phosphatidyl inositol 3-kinase (PI 3-kinase) (2). The subsequent phosphatidyl inositol-3,4,5-trisphosphate-dependent kinase(s) have not yet been identified, but this cascade results in the activation of Elk-1. Elk-1 was identified as the transcription factor that binds the insulin response element of the prolactin promoter First Published Online May 5, 2005 Abbreviations: CAT, Chloramphenicol acetyltransferase; CMV, cytomegalovirus; CSK, C-terminal Src kinase; EGF, epithelial growth factor; FAK, focal adhesion kinase; GFP, green fluorescent protein; HIR, human insulin receptor; IRS, insulin receptor substrate; PI 3-kinase, phosphatidylinositol 3-kinase; Prl, prolactin; PSF, PTB-associated splicing factor; PTP, protein tyrosine phosphatase; RPTP, receptor-like protein tyrosine phosphatase; WT, wild type. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community. Src-increased prolactin gene transcription was inhibited by expression of a blocking Rho-mutant (RhoN19), suggesting that Src acted through or required active Rho. Experiments with an activated Rho-mutant (RhoL63) demonstrated a biphasic activation/repression of prolactin gene transcription that was similar to the effect of Src. The effects of both Src and Rho were phosphatidylinositol 3-kinase dependent. Expression of SrcY/F or RhoL63 altered the actin cytoskeleton and morphology of GH4 cells. Taken together, these data suggest a physiological pathway from the cell matrix to increased prolactin gene transcription mediated by RPTP␣/Src/Rho/ phosphatidylinositol 3-kinase and cytoskeletal change that is additive with effects of insulin. Over activation of this pathway, however, caused extreme alteration of the cytoskeleton that blocked activation of the prolactin gene. (Endocrinology 146: 3535–3546, 2005) and is required for insulin-increased prolactin gene expression (3). RPTP␣ blocks insulin-increased prolactin gene transcription in GH4 cells (2). RPTP␣ is a member of the protein tyrosine phosphatase (PTP)ase family that includes the receptor-like, membrane-spanning PTPases and cytosolic PTPases. Receptor-like PTPases are characterized by an extracellular domain of variable length, a single membranespanning domain, and one or two catalytic domains on the intracellular portion of the molecule (4). RPTP␣ does not dephosphorylate the insulin receptor or its immediate substrates shc and IRS-1 in our experiments (2), but others have found some effects of RPTP␣ on the association of PI 3-kinase with IRS-1, although this did not affect downstream signaling (5). This suggests that its effects on insulin-increased prolactin gene expression are mediated by downstream signaling molecules. Src represents a potential mediator downstream of RPTP␣ because RPTP␣ dephosphorylates and activates c-Src (6) and RPTP␣⫺/⫺ fibroblasts had a phenotype similar to Src⫺/⫺ fibroblasts (6 – 8). Studies with v-Src and activated c-Src demonstrate that the Src family of nonreceptor tyrosine kinases activate numerous signaling pathways, many of which are also activated by receptor tyrosine kinases (9). A triple knockout of Src, Fyn, and Yes, however, causes defective integrin-signaling but does not affect signaling by receptor tyrosine kinases. This 3535 3536 Endocrinology, August 2005, 146(8):3535–3546 indicates that Src might be important for integrin signaling. Src’s importance to integrin signaling is supported by the role that many Src substrates [e.g. cortactin, focal adhesion kinase (FAK), paxillin, p130CAS, and vinculin] play in cytoskeletal organization and cell adhesion (9). In response to integrin activation, perinuclear localized c-Src translocates to the cell membrane in which it is activated by dephosphorylation or protein-protein interaction. A study of integrin signaling in mouse fibroblasts showed the importance of RPTP␣ for force generation by Src family tyrosine kinases (10). Active Src causes a rearrangement in cytoskeletal organization. Thus, Src-related kinases are likely to be crucial mediators of cell-cell/cell-substrate interactions. Insulin/IGF-I and integrin signaling are interdependent (11, 12). Integrins are essential for insulin secretion by isolated pancreatic ␤-cells (13). This may be due to their importance for calcium release from intracellular stores. Insulin receptor directly interacts with integrin-␣V␤3 (11) and FAK and Src directly phosphorylate the insulin receptor (14). Internalization of insulin receptor is also dependent on interaction of cells with the extracellular matrix (15). Ligation of ␣6␤4-integrin in breast cancer cell lines results in phosphorylation of IRS-1 and IRS-2 and PI 3-kinase activation (12, 16). IRS-1 expression is dependent on cell adhesion and FAK (17, 18). Conversely, insulin activates PI 3-kinase and ERK through FAK (19). SHP-2 (...truncated)