IRS-1 Functions as a Molecular Scaffold to Coordinate IGF-I/IGFBP-2 Signaling During Osteoblast Differentiation.

Insulin like growth factor I (IGF-I) and insulin like growth factor binding protein-2 (IGFBP-2) function coordinately to stimulate AKT and osteoblast differentiation. IGFBP-2 binding to receptor protein tyrosine phosphatase β (RPTPβ) stimulates polymerization and inactivation of phosphatase activity. Because phosphatase and tensin homolog (PTEN) is the primary target of RPTPβ, this leads to enhanced PTEN tyrosine phosphorylation and inactivation. However RPTPβ inactivation also requires IGF-I receptor activation. The current studies were undertaken to determine the mechanism by which IGF-I mediates changes in RPTPβ function in osteoblasts. IGFBP-2/IGF-I stimulated vimentin binding to RPTPβ and this was required for RPTPβ polymerization. Vimentin serine phosphorylation mediated its binding to RPTPβ and PKCζ was identified as the kinase that phosphorylated vimentin. To determine the mechanism underlying IGF-I stimulation of PKCζ-mediated vimentin phosphorylation, we focused on insulin receptor substrate-1 (IRS-1). IGF-I stimulated IRS-1 phosphorylation and recruitment of PKCζ and vimentin to phospho-IRS-1. IRS-1 immunoprecipitates containing PKCζ and vimentin were used to confirm that activated PKCζ directly phosphorylated vimentin. PKCζ does not contain a SH-2 domain that is required to bind to phospho-IRS-1. To determine the mechanism of PKCζ recruitment we analyzed the role of p62 (a PKCζ binding protein) that contains a SH2 domain. Exposure to differentiation medium plus IGF-I stimulated PKCζ/p62 association. Subsequent analysis showed the p62/PKCζ complex was co-recruited to IRS-1. Peptides that disrupted p62/PKCζ or p62/IRS-1 inhibited IGF-I/IGFBP-2 stimulated PKCζ activation, vimentin phosphorylation, PTEN tyrosine phosphorylation, AKT activation, and osteoblast differentiation. The importance of these signaling events for differentiation was confirmed in primary mouse calvarial osteoblasts. These results demonstrate the cooperative interaction between RPTPβ and the IGF-I receptor leading to a coordinated series of signaling events that are required for osteoblast differentiation. Our findings emphasize the important role IRS-1 plays in modulating these signaling events and confirm its essential role in facilitating osteoblast differentiation.