G-CSF-Stimulated Erk Activation and Cell Proliferation Involves Jak2-Dependent Phosphorylation of Gab2 Tyrosine 643.
Authors: Robinson Lisa J. , Wang Lin , Xue Jia 
Published: April 2020

Abstract
Granulocyte colony stimulating factor (G-CSF) is the major cytokine regulating neutrophil production. G-CSF has complex effects, both stimulating proliferation and promoting differentiation of neutrophil precursors. Many key intermediates in G-CSF signaling have been identified, but their organization in signaling pathways, and the cellular responses that these pathways subserve, remain poorly understood. Jak2 and Erk1/2, for example, are clearly implicated in G-CSF signaling and both have been linked to proliferation, but their interconnections are unclear. We now report that these signaling proteins are linked through the scaffolding protein Gab2, and that the Gab2 pathway is an important route for transduction of G-CSF proliferative signals. To identify Jak2-dependent signaling events, Jak2 expression and activity were selectively inhibited by transfecting G-CSF responsive cell lines with antisense Jak2 or a dominant-negative Jak2 mutant. Without active Jak2, G-CSF stimulated Erk1/2 activation was markedly diminished; G-CSF-stimulated Gab2 phosphorylation was also inhibited. These results raised the possibility of a link between Jak2, Gab2 and Erk in G-CSF signaling. To identify sites of Jak2-dependent Gab2 phosphorylation, site-directed mutagenesis was used to replace single tyrosine residues in Gab2 with phenylalanine. Mutation of tyrosine 643 (Gab2 Y643F), but not other residues tested, markedly reduced G-CSF-stimulated Gab2 phosphorylation. To determine whether this might represent a site for direct Gab2 phosphorylation by Jak2, the analogous tyrosine residue in a Gab2-GST fusion protein was mutated, and in vitro phosphorylation of the fusion protein by purified active Jak2 or Jak2 immunoprecipitated from G-CSF-stimulated cells was evaluated. Phosphorylation of the tyrosine 643 fusion protein mutant was significantly inhibited compared to phosphorylation of the unmutated form or a tyrosine 614 mutant, suggesting that tyrosine 643 was a site for Jak2-dependent Gab2 phosphorylation. The Gab2 tyrosine 643 mutant was then used to examine downstream consequences of inhibiting Jak2-dependent Gab2 phosphorylation. In cells expressing Gab2 Y643F, G-CSF-stimulated Erk1/2 activation was markedly inhibited. In contrast, G-CSF-stimulated Stat3 and Stat5 phosphorylation were unchanged. Mutation of Gab2 tyrosine 643 also inhibited the G-CSF-stimulated association of Gab2 with Shp2, which is known to link Gab2 and Erk1/2 in other signaling systems. G-CSF-dependent cell proliferation was also examined. Cells transduced with wild-type Gab2 showed a marked increase in thymidine incorporation following G-CSF stimulation; this effect was abrogated by the mutation of Gab2 tyrosine 643. Together, these results suggest that G-CSF-activated Jak2 phosphorylates, perhaps directly, Gab2 tyrosine 643, which then binds Shp2, thereby stimulating Erk1/2 activation. Blocking the Jak2-Gab2-Shp2-Erk1/2 pathway by mutation of Gab2 tyrosine 643 appeared to inhibit cell proliferation, although Jak-Stat pathways were intact. Overall, these findings identify a novel G-CSF signaling pathway and provide evidence of its importance in G-CSF stimulated cell proliferation.