Fig 1: The SH2 domains of SHP2, but not its PTP domain, protect specific GAB1 and GAB2 pY marks.(A) Schematic representation of wild-type, point mutants, and deletion constructs of SHP2 tested. (B, C) Parental-, SHP2 knockout-, or SHP2 knockout U2OS cells stably expressing various SHP2 ‘rescue’ constructs were cultured with or without EGF stimulation (10 nM) for 10 min. Cells were lysed and subjected to Western blotting using anti-pY659-GAB1 and anti-GAB1 antibodies. (D) MDA-MB-468 cells stably expressing wild-type, PTP, or SH2 domains pre-treated with SHP099 were stimulated with EGF and immunoblotted after lysis using the specified antibodies. Western blot results are representative of at least n = 2 independent biological replicates.
Fig 2: Model highlighting coordinated scaffolding and catalytic activities of SHP2.(A) Schematic model showing pY mark protection by stable engagement of a partner protein (e.g. GAB1) with the tandem SH2 domains of SHP2. (B) In the presence of SHP099, pY marks on putative SHP2 interactors are vulnerable to dephosphorylation, and SHP099 is not recruited to sites of RTK signaling. It is also possible that deposition of certain phosphate marks is SHP2 dependent (lower panel).
Fig 3: Effects of SHP099-resistance and pY binding-site mutations on pY abundance at protected sites on GAB1 and GAB2.(A) Schematic illustrating wild-type and SHP099-resistant forms of SHP2. (B) Parental MDA-MB-468 cells or MDA-MB-468 cells stably expressing wild-type or T253M/Q257L SHP2 proteins were pre-treated with SHP099 (10 µM). The cells were then mock treated or stimulated with EGF (10 nM) for 10 min and immunoblotted after lysis using the specified antibodies. (C) Schematic illustrating wild-type and mutated forms of SH2-only tandem fragments of SHP2. (D) Parental MDA-MB-468 cells or MDA-MB-468 cells stably expressing wild-type or mutated tandem SH2 fragments of SHP2 (SH2) as indicated (R32M, R138M, and R32M/R138M, denoted DM here) were pre-treated with SHP099 (10 µM). The cells were then mock treated or stimulated with EGF (10 nM) for 10 min and immunoblotted after lysis using the specified antibodies. Western blot results are representative of at least n = 2 independent biological replicates.
Fig 4: SHP2 inhibition results in reduced pY abundance at its interaction motifs.(A, B) TMT signal-to-noise intensities of GAB1 pY659 (A) and GAB2 pY643 (B) peptides showing dynamic changes in phosphorylation under DMSO- (solid line) and SHP099-treated (dotted line) conditions. (C) MDA-MB-468 cells pre-treated with DMSO carrier or SHP099 (10 µM) for 2 hr were mock treated or stimulated with EGF (10 nM). Total GAB1, total GAB2, GAB1 pY659, and GAB2 pY643 were analyzed by Western blot with both anti-protein and phosphospecific antibodies as a function of time after EGF addition. Western blot results are representative of at least two independent biological replicates.Figure 4—source data 1.Table showing TMT relative abundance values for Y659 of GAB1.Figure 4—source data 2.Table showing TMT relative abundance values for Y643 of GAB2.
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