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.
Highlights
Tyrosine phosphatase non-receptor type substrate-1 (SHPS-1), a transmembrane protein, plays a vital role in cell migration and proliferation
Increasing evidence has shown that SHPS-1 is involved in various biological phenomena, including suppression of anchorage-independent cell growth, negative regulation of immune cell responses, self-recognition of red blood cells, mediation of macrophage multi-nucleation, 1 The abbreviations used are: IGF-I, insulin-like growth factor-I; vascular smooth muscle cells (VSMCs), 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
Interaction of SHPS-1/CD with Its Binding Partners Is Regulated by IGF-I—To determine whether SHPS-1 that was present in intact cells could interact with binding partners of interest, and whether these interactions were regulated by IGF-I, we performed co-immunoprecipitation and pulldown assays using lysates prepared from intact VSMC
Summary
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 Grb21⁄7p85 to phosphorylated SHPS-1 Of this signaling complex is required for optimal stimulation of both mitogen-activated protein and phosphatidylinositol 3-kinase pathways. Src homology 2 domain containing protein tyrosine phosphatase (SHP) substrate-1 (SHPS-1) is a transmembrane protein that plays an important role in mediating various cellular responses, including cell migration and proliferation. Two approaches recently used to identify protein binding partners are mRNA display techniques, such as mRNA-protein fusions [17,18,19,20,21] and in vitro virus, based on cell-free co-translation and affinity selection [22,23,24] and tandem affinity purification (TAP)-mass spectrometry methods These methods are well suited for identifying the components of complexes that form in near physiological conditions. The identification of these proteins provides an opportunity to determine their roles in mediating physiological events that occur in VSMCs in response to hyperglycemic stress and IGF-I stimulation
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