Src Homology 2 Domain Containing Protein 5 (SH2D5) Binds the Breakpoint Cluster Region Protein, BCR, and Regulates Levels of Rac1-GTP

Elizabeth J Gray,Evangelia Petsalaki,D Andrew James,Richard D Bagshaw,Melissa M Stacey,Oliver Rocks,Anne-Claude Gingras,Tony Pawson

doi:10.1074/jbc.m114.615112

Elizabeth J Gray, Evangelia Petsalaki + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m114.615112

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Abstract

SH2D5 is a mammalian-specific, uncharacterized adaptor-like protein that contains an N-terminal phosphotyrosine-binding domain and a C-terminal Src homology 2 (SH2) domain. We show that SH2D5 is highly enriched in adult mouse brain, particularly in Purkinjie cells in the cerebellum and the cornu ammonis of the hippocampus. Despite harboring two potential phosphotyrosine (Tyr(P)) recognition domains, SH2D5 binds minimally to Tyr(P) ligands, consistent with the absence of a conserved Tyr(P)-binding arginine residue in the SH2 domain. Immunoprecipitation coupled to mass spectrometry (IP-MS) from cultured cells revealed a prominent association of SH2D5 with breakpoint cluster region protein, a RacGAP that is also highly expressed in brain. This interaction occurred between the phosphotyrosine-binding domain of SH2D5 and an NxxF motif located within the N-terminal region of the breakpoint cluster region. siRNA-mediated depletion of SH2D5 in a neuroblastoma cell line, B35, induced a cell rounding phenotype correlated with low levels of activated Rac1-GTP, suggesting that SH2D5 affects Rac1-GTP levels. Taken together, our data provide the first characterization of the SH2D5 signaling protein.

Highlights

Src homology 2 domain containing protein 5 (SH2D5) is a previously uncharacterized protein that resembles the Shc proteins in structural organization
Immunoprecipitation coupled to mass spectrometry (IP-MS) from cultured cells revealed a prominent association of SH2D5 with breakpoint cluster region protein, a RacGAP that is highly expressed in brain
We have characterized an interaction between SH2D5 and breakpoint cluster region protein (BCR), a regulator of Rho GTPases (9, 10) displaying GTPase-activating proteins (GAPs) activity toward Rac, Rho, and Cdc42 proteins (18, 23)

Summary

Background

Src homology 2 domain containing protein 5 (SH2D5) is a previously uncharacterized protein that resembles the Shc proteins in structural organization. SH2D5 is a mammalian-specific, uncharacterized adaptorlike protein that contains an N-terminal phosphotyrosine-binding domain and a C-terminal Src homology 2 (SH2) domain. Immunoprecipitation coupled to mass spectrometry (IP-MS) from cultured cells revealed a prominent association of SH2D5 with breakpoint cluster region protein, a RacGAP that is highly expressed in brain. This interaction occurred between the phosphotyrosine-binding domain of SH2D5 and an NxxF motif located within the N-terminal region of the breakpoint cluster region. This domain architecture is shared with the Shc family of adaptor proteins (see Fig. 1, A and B) Despite this conserved domain organization, the SH2 domain of SH2D5 lacks the critical arginine (␤B5) necessary for Tyr(P) binding. SH2D5 Associates with BCR to Regulate Rac1-GTP Levels ronal cells display low levels of Rac1-GTP and a cell-rounding phenotype

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION