Figure 5.

Phosphorylation of Abl on Y488 by Src-Kinases Regulates Mig6-Induced Activation of Abl

(A) Coimmunopurification of ectopically expressed Mig6 with wild-type (WT), Y488E, or Y488F forms of c-Abl in HEK239T cells followed by western analysis as indicated. Note that the phosphomimic Y488E and partially the Y488F mutation disrupts the ability of Mig6 to activate c-Abl.

(B) In vitro kinase assay with recombinant active c-Src or EGFR with wild-type (WT), Y488E or Y488F mutant forms of the inactive catalytic domain of c-Abl immunopurified from overexpressing HEK293T cells.

(C) Immunoprecipitation of WT or Y488F mutant c-Abl coexpressed with Mig6 and active c-Src, followed by western analysis as indicated.

(D) Western analysis of endogenous phosphorylation of Abl on Y488 in pMECs cultured in the presence or absence of EGF for 2 hr prior to cell harvest. Values denote relative intensities of p488 bands, normalized against total c-Abl levels.

(E) Western analysis of endogenous phosphorylation of Abl on Y488 in control wild-type MEFs or MEFs lacking Src-family members. Values denote relative intensities of p488 bands, normalized against total c-Abl levels.

(F) Immunopurification of c-Abl coexpressed with Mig6 and EGFR with or without 0.8 μM of the Src inhibitor PP2 and western analysis as indicated. Note the Src-dependent inhibition of Mig6-induced Abl activation by EGFR overexpression.

(G) Western analysis of immunoprecipitated endogenous c-Abl for Y phosphorylation in pMEFs cultured in the presence of EGF, treated with 0.4 or 0.8 μM PP2 for 2 hr. Values denote relative intensities of pY bands, normalized against total immunoprecipitated c-Abl levels, as determined by densitometry.

(H) Induction of cell death by Mig6/Abl but not Src/Abl. Ectopic expression of Abl with or without Mig6 or Src in pMEFs for 16 hr, followed by western analysis as indicated. Graph shows cell death determined as % of cells taking up the viability dye trypan blue (n = 3).

(I) siRNA-mediated silencing of p73 followed by ectopic coexpression of Abl and Mig6 in pMEFs for 16 hr, and western analysis as indicated. Graph shows cell death determined as % of cells taking up the viability dye trypan blue (n = 3).

(J) C-Abl immunofluorescence in WT or Errfi1 KO pMECs cultured in absence of EGF for 2 hr. Note the reduced nuclear staining of c-Abl in the absence of Mig6. Images are representative of three independent experiments.

Indicated p values were determined by two-tailed unpaired Student's t test, while the error bars represent SEM.