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Blood, 1 May 2004, Vol. 103, No. 9, pp. 3305-3312.
Prepublished online as a Blood First Edition Paper on December 4, 2003; DOI 10.1182/blood-2003-06-1861.
Submitted June 10, 2003
Accepted October 24, 2003
G-CSF-induced tyrosine phosphorylation of Gab2 is Lyn kinase dependent and associated with enhanced Akt and differentiative, not proliferative, responses
Quan-Sheng Zhu, Lisa J Robinson, Vera Roginskaya, and Seth J Corey*
Division of Pediatrics, University of Texas M. D. Anderson Cancer Center, Houston, TX, USA
Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
* Corresponding author; email: sjcorey{at}mdanderson.org.
The G-CSF Receptor (G-CSFR) transduces intracellular signals for myeloid cell proliferation, survival, and differentiation through the recruitment of non-receptor protein tyrosine kinases Lyn and Jak2. This results in the tyrosine phosphorylation of a small set of positive and negative adapters and effectors. Gab2 is a newly described adapter molecule, preferentially expressed in hematopoietic cells and associated with PI 3-Kinase. Studies suggest that Gab2 plays both positive and negative roles in cytokine receptor signaling. To investigate what 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 PI 3-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-ERK were greater in proximal truncated than in wild-type G-CSFR cells, suggesting that Gab2 is dissociated from PI 3-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. Altogether, 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.
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