Adaptor Protein GRB2 Promotes Src Tyrosine Kinase Activation and Podosomal Organization by Protein-tyrosine Phosphatase ϵ in Osteoclasts

Abstract The non-receptor isoform of protein tyrosine phosphatase Epsilon (cyt-PTPe) supports adhesion of bone-resorbing osteoclasts by activating Src downstream of integrins. Loss of cyt-PTPe reduces Src activity in osteoclasts, reduces resorption of mineralized matrix both in vivo and in cell culture, and induces mild osteopetrosis in young female PTPe KO mice. Activation of Src by cyt-PTPe is dependent upon this phosphatase undergoing phosphorylation at its C-terminal Y638 by partially-active Src. In order to understand how cyt-PTPe activates Src we screened 73 SH2 domains for binding to pY638 of cyt-PTPe. The SH2 domain of Grb2 bound pY638 of cyt-PTPe most prominently, while the Src SH2 domain did not bind at all, suggesting that Grb2 may link PTPe with downstream molecules. Further studies indicated that Grb2 is required for activation of Src by cyt-PTPe in osteoclast-like cells in culture (OCLs). Overexpression of Grb2 in OCLs increased activating phosphorylation of Src at Y416 and of cyt-PTPe at Y638; opposite results were obtained when Grb2 expression was reduced by shRNA or by gene inactivation. Phosphorylation of cyt-PTPe at Y683 and its association with Grb2 are integrin-driven processes in OCLs; cyt-PTPe auto-dephosphorylates at Y683, thus limiting Src activation by integrins. Reduced Grb2 expression also reduces the ability of bone marrow precursors to differentiate into OCLs and reduces the fraction of OCLs in which podosomal adhesion structures assume organization typical of active, resorbing cells. We conclude that Grb2 physically links cyt-PTPe with Src and enables cyt-PTPe to activate Src downstream of activated integrins in OCLs.