Identification of Novel SHPS-1-associated Proteins and Their Roles in Regulation of Insulin-like Growth Factor-dependent Responses in Vascular Smooth Muscle Cells

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 Grb2⅐p85 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

Recruitment of Nox4 to a Plasma Membrane Scaffold Is Required for Localized Reactive Oxygen Species Generation and Sustained Src Activation in Response to Insulin-like Growth Factor-I

“…293FT cells (Invitrogen) were prepared for generation of virus stocks, and VSMC expressing Nox4, Nox4 Y491F, and mitochondria-localized catalase were established using procedures that were described previously (18). VSMC expressing wild type Pyk2, Pyk2 Y881F, wild type SHPS-1, and cytoplasmic domain-deleted SHPS-1, were prepared as described previously (14,19).…”

“…1A). Because our previous studies showed that hyperglycemia induced the formation of a signaling complex on the cytoplasmic domain (CD) of the scaffolding protein SHPS-1 and that IGF-I stimulated Src recruitment to that complex (13,14), we determined whether Src recruitment to SHPS-1 resulted in sustained activation. The results showed that SHPS-1-associated Src activation was also sustained for at least 6 h (Fig.…”

“…This complex includes Src, and SHPS-1-localized Src plays an essential role in mediating IGF-I actions (15). Using unbiased mRNA display, we showed that Nox4 associated with SHPS-1 in response to IGF-I stimulation (14). However, the biological consequences and underlying mechanism of Nox4 recruitment to SHPS-1 have not been investigated.…”

“…Our previous studies have shown that SHPS-1, a plasma membrane protein, functions as a localized scaffold for the formation of a signaling complex, which is required for mediating IGF-I signaling and biological functions in VSMC in responseto hyperglycemia (13,14). This complex includes Src, and SHPS-1-localized Src plays an essential role in mediating IGF-I actions (15).…”

Insulin-like Growth Factor-I-stimulated Insulin Receptor Substrate-1 Negatively Regulates Src Homology 2 Domain-containing Protein-tyrosine Phosphatase Substrate-1 Function in Vascular Smooth Muscle Cells

“…The SHPS-1 cytoplasmic domain containing amino acids 394 -503 was PCR-amplified with the SP6 promotor sequence, following which reverse transcription was performed to obtain mRNA (33). Using this template, SHPS-1/CD was generated, employing in vitro translation using wheat germ extract as per the manufacturer's recommendation (Promega).…”