G-CSF-induced tyrosine phosphorylation of Gab2 is Lyn kinase dependent and associated with enhanced Akt and differentiative, not proliferative, responses

Abstract

The granulocyte colony-stimulating factor receptor (G-CSFR) transduces intracellular signals for myeloid cell proliferation, survival, and differentiation through the recruitment of nonreceptor protein tyrosine kinases Lyn and janus kinase 2 (Jak2). This results in the tyrosine phosphorylation of a small set of positive and negative adapters and effectors. Grb2-associated binder-2 (Gab2) is a newly described adapter molecule, preferentially expressed in hematopoietic cells and associated with phosphatidylinositol 3 (PI3) kinase. Studies suggest that Gab2 plays both positive and negative roles in cytokine receptor signaling. To investigate the role Gab2 plays in G-CSF receptor-mediated signaling, we have analyzed its activation state and correlated that with wild-type and mutant G-CSF receptors stably expressed in the murine factor-dependent Ba/F3 cell lines. G-CSF-induced tyrosine phosphorylation of Gab2 occurred in the wild-type and single Y-to-F mutants (Y704F, Y729F, and Y744F), but not in the ADA and W650R loss-of-function mutants. Cells expressing truncated proximal G-CSFR, the tyrosine-null (Y4F) G-CSFR, or Y764F mutant receptors had decreased phosphorylation of Gab2. Specific inhibitors of Src kinase (PD173 and PP1) but not Jak2 kinase (AG490) blocked Gab2 phosphorylation. Phosphorylation of Gab2 occurred in wild-type, but not Lyn-deficient, G-CSFR-transfected DT40 B cells. These data propose that Lyn, not Jak2, phosphorylates Gab2 and that maximal phosphorylation of Gab2 requires Y764, a Grb2-binding site. Serine phosphorylation of Akt, a marker of PI3-kinase activity, was detected in both wild-type and truncated proximal domain receptors, but not in the ADA and W650R mutants. Levels of phospho-Akt and phospho-extracellular signal-regulated kinase (phospho-ERK) were greater in proximal truncated than in wild-type G-CSFR cells, suggesting that Gab2 is dissociated from PI3 kinase or ERK activities. Overexpression of Gab2 enhanced the phosphorylation state of Akt, but not of ERK. This inhibited the proliferation of wild-type and truncated G-CSFR-transfected Ba/F3 cells and enhanced their myeloid differentiation. All together, these data indicate that G-CSF treatment leads to Lyn-mediated tyrosine phosphorylation of Gab2, which may serve as an important intermediate of enhanced Akt activity and myeloid differentiation, not growth/survival response. (Blood. 2004; 103:3305-3312)