Identification of Novel SHPS-1-associated Proteins and Their Roles in Regulation of Insulin-like Growth Factor-dependent Responses in Vascular Smooth Muscle Cells*
- From the Department of Medicine, University of North Carolina, School of Medicine, Chapel Hill, North Carolina 27599
- ‡ To whom correspondence should be addressed: Div. of Endocrinology, University of North Carolina at Chapel Hill, CB No. 7170, 8024 Burnett-Womack, Chapel Hill, NC 27599-7170. Tel.: 919-966-4735 Fax: 1-919-966-6025; E-mail: david_clemmons{at}med.unc.edu
Abstract
Tyrosine phosphatase non-receptor type substrate-1 (SHPS-1), a transmembrane protein, plays a vital role in cell migration and proliferation. Our previous studies have shown that insulin-like growth factor-I (IGF-I) stimulates SHPS-1 phosphorylation, leading to recruitment of SHP-2, c-Src, Shc, and Grb2·p85 to phosphorylated SHPS-1. Assembly of this signaling complex is required for optimal stimulation of both mitogen-activated protein and phosphatidylinositol 3-kinase pathways. The main aim of the present study was to identify novel proteins that interacted with the cytoplasmic domain of SHPS-1 (SHPS-1/CD) in response to IGF-I stimulation and define the role of these interactions in mediating specific biological functions. We performed a functional proteomic screening to identify SHPS-1 binding partners using combination of mRNA display and the tandem affinity purification-tag methods. Screening identified a number of proteins not previously known to interact with phosphorylated SHPS-1/CD. These novel SHPS-1 binding partners represent several functional categories including heat shock proteins, protein kinases and phosphatases, and proteins that regulate transcription or translation. In Vivo and in vitro studies suggested that most of the proteins bound to SHPS-1 via binding to one of the four SH2 domain containing proteins, SHP-2, CTK, SUPT6H, and STAT1, that directly bound to SHPS-1. Although the binding of most of these proteins to SHPS-1 was positively regulated by IGF-I, a few were negatively regulated, suggesting differential regulation of protein complexes assembled on SHPS-1/CD in response to IGF-I. Further studies showed that truncation of SHPS-1/CD significantly impaired IGF-I-dependent AKT signal transduction and subsequent biological functions including cell survival, protein synthesis, protein aggregation, and prevention of apoptosis. The results emphasize the importance of formation of SHPS-1 signaling complex induced by IGF-I and provide novel insights into our knowledge of the role of this molecular scaffold in regulation of IGF-I-stimulated signal transduction and biological actions.
Footnotes
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↵* This work was supported, in whole or in part, by National Institutes of Health Grant HL56850.
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The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.
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↵1 The abbreviations used are:
- IGF-I
- insulin-like growth factor-I
- VSMC
- vascular smooth muscle cell
- MAPK
- mitogen-activated protein kinase
- TAP
- tandem affinity purification
- CD
- cytoplasmic domain
- DMEM
- Dulbecco's modified Eagle's medium
- RIPA
- radioimmune precipitation assay buffer
- TNT
- coupled in vitro transcription and translation
- WT
- wild type
- PI3K
- phosphatidylinositol 3-kinase
- HSP
- heat shock protein
- CALR
- calreticulin
- HA
- heamagglutinin
- TEV
- tobacco etch virus
- MT
- mutant type
- CTK
- Csk-type protein tyrosine kinase
- GTP
- guanosine triphosphate
- GRP
- glucose-regulated protein
- ATP
- adenosine triphosphate.
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- Received November 25, 2008.
- Accepted March 16, 2009.
- © 2009 The American Society for Biochemistry and Molecular Biology, Inc.
