Granulocyte colony-stimulating factor (G-CSF) stimulation of myeloid cells induced tyrosine-phosphorylation of cellular proteins. One of the tyrosine-phosphorylated proteins was found to be a scaffold protein, Grb2-associated binding protein 2 (Gab2). Another member of Gab family protein, Gab3, was exogenously overexpressed in neutrophil progenitor cells to make the Gab3 protein to compete with the endogenous Gab2 for the G-CSF dependent signaling. In Gab3-overexpressed cells, the level of tyrosine phosphorylation of endogenous Gab2 by G-CSF stimulation was markedly down-regulated, while the phosphorylation of Gab3 was significantly enhanced. The Gab3-overexpressed cells continuously proliferated in the medium containing G-CSF and lost the ability to differentiate to the mature neutrophil, characterized by the lobulated nucleus. The G-CSF stimulationdependent tyrosine phosphorylation of Gab3, the association of SHP2 to Gab3 and the following MAPK activation were prolonged in the Gab3-overexpressed cells, compared to the parental cells, where the binding of SHP2 to Gab2 protein and thereby the activation of MAPK were not sustained after G-CSF stimulation. Inhibition of MAPK by pharmaceutical inhibitor restored the Gab3-overexpressed cells to the ability to differentiate to mature neutrophil. Therefore, G-CSF dependent Gab2 phosphorylation and following its downregulation led the short-term MAPK activation. The down-regulation of MAPK after transient Gab2 phosphorylation was necessary for the consequent neutrophil differentiation induced by G-CSF stimulation.
Gab2 is one of the most heavily tyrosine-phosphorylated protein in granulocyte precursor cell, GM-I62-1, upon G-CSF stimulation. Effects of several pharmacological inhibitors to tyrosine kinases were examined on the tyrosine phosphorylation of Gab2 upon G-CSF stimulation. Specific Src kinase inhibitor, PP1, had marginal effects, if any, on the Gab2 tyrosine phosphorylation. In contrast, Syk specific inhibitors, Piceatannol and GS-9973, had partial but distinct inhibitory effects on the Gab2 tyrosine phosphorylation upon G-CSF stimulation. When the tyrosine residues are phosphorylated, downstream effector molecules, SHP2 and p85 subunit of PI3K, are known to bind to these phosphorylated residues of Gab2 through their SH2 domains. Pretreatments of cells with Syk specific inhibitors reduced the SHP2 binding to Gab2. However, no inhibitory effect of these inhibitors on p85 binding to Gab2 was observed. These observations indicated that Syk phosphorylates tyrosine residues of SHP2 binding sites of Gab2, and that the tyrosine residues of Gab2 to which p85 subunit of PI3K binds upon G-CSF stimulation are phosphorylated by other tyrosine kinase(s).
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