Figure 5.

The PTPN21 phosphatase physically associates with the C-terminal SH3 domain of GRB2. (A) GST fusion proteins of the N-terminal, C-terminal SH3 domain as well as the SH2 domain of GRB2 were incubated with a protein extract of HeLa cells transiently transfected with Flag-PTPN21 in presence or in absence of constitutively activated Y527F SRC kinase. Affinity-purified SH2 and SH3 domains ligands were separated by SDS-PAGE and transferred onto cellulose membranes. The blots were probed with anti-Flag (WB: α-4G10) and anti-phosphotyrosine (WB: α-4G10) antibodies. The cell lysate (input) and the sample affinity-purified with the GST protein (GST) were used as controls. (B) A schematic representation of the modular domain structure of GRB2 and PTPN21 proteins is represented. According to our data, the SH3 binding motif (⁵⁶⁴RPPPPYPPPRP⁵⁷⁴) of PTPN21 binds the GRB2 C-terminal SH3 domain. (C) HeLa cells were transiently co-transfected with Flag-PTPN21 and with constitutively activated Y527F SRC kinase expression plasmids. After cell lysis, whole protein extracts were immunoprecipitated with anti-GRB2 antibody. The membrane was probed with anti-GRB2 (WB: α-GRB2), anti-Flag (WB: α-Flag) and anti-phospho tyrosine (WB: α-4G10) antibodies.