Adaptor protein GRB2 promotes Src tyrosine kinase activation and podosomal organization by protein-tyrosine phosphatase ϵ in osteoclasts.
The non-receptor isoform of protein-tyrosine phosphatase ϵ (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 Tyr-638 by partially active Src. To understand how cyt-PTPe activates Src, we screened 73 Src homology 2 (SH2) domains for binding to Tyr(P)-638 of cyt-PTPe. The SH2 domain of GRB2 bound Tyr(P)-638 of cyt-PTPe most prominently, whereas 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 (OCLs) in culture. Overexpression of GRB2 in OCLs increased activating phosphorylation of Src at Tyr-416 and of cyt-PTPe at Tyr-638; opposite results were obtained when GRB2 expression was reduced by shRNA or by gene inactivation. Phosphorylation of cyt-PTPe at Tyr-683 and its association with GRB2 are integrin-driven processes in OCLs, and cyt-PTPe undergoes autodephosphorylation at Tyr-683, thus limiting Src activation by integrins. Reduced GRB2 expression also reduced the ability of bone marrow precursors to differentiate into OCLs and reduced 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.
From the Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot 76100, Israel.
Einat
E
Levy-Apter
From the Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot 76100, Israel.
Eynat
E
Finkelshtein
Department of Epigenetics and Molecular Carcinogenesis, The University of Texas M. D. Anderson Cancer Center, Smithville, Texas 78957, and.
Vidyasiri
V
Vemulapalli
Department of Biochemistry and the Siebens-Drake Medical Research Institute, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario N6A 5C1, Canada.
Shawn S-C
SS
Li
Department of Epigenetics and Molecular Carcinogenesis, The University of Texas M. D. Anderson Cancer Center, Smithville, Texas 78957, and.
Mark T
MT
Bedford
From the Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot 76100, Israel, ari.elson@weizmann.ac.il.
Ari
A
Elson
D000595:Amino Acid Sequence; D000818:Animals; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002454:Cell Differentiation; D051380:GRB2 Adaptor Protein / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D057507:Mice, 129 Strain; D018345:Mice, Knockout; D010010:Osteoclasts / Q000201:enzymology*; D010766:Phosphorylation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011499:Protein Processing, Post-Translational; D054630:Receptor-Like Protein Tyrosine Phosphatases, Class 4 / Q000502:physiology*; D019061:src-Family Kinases / Q000378:metabolism*
D015815:Cell Adhesion Molecules; D051380:GRB2 Adaptor Protein; C495949:Grb2 protein, mouse; D019061:src-Family Kinases; C098067:Ptpre protein, mouse; D054630:Receptor-Like Protein Tyrosine Phosphatases, Class 4
Cell Adhesion; Cell Signaling; Growth Factor Receptor-bound Protein 2 (GRB2); Osteoclast; Podosome; Protein Phosphorylation; Src; Src Homology 2 Domain (SH2 Domain); Tyrosine Protein Phosphatase (Tyrosine Phosphatase)