Gab2 Is Constitutively Phosphorylated in Cells Expressing the Polycythemia Vera-Associated Jak2 V614F Mutant and Links Jak2 to Erk1/2 Activation.

Although chronic myeloid leukemia was one of the first hematologic disorders for which a molecular pathogenesis could be defined, the molecular abnormalities responsible for other myeloproliferative disorders have remained largely unknown. Several recent studies, however, have identified an activating mutation in Janus kinase 2 (Jak2 V614F) as a frequent abnormality in polycythemia vera. Substitution of phenylalanine for valine 614 in Jak2 was found to result in constitutive kinase activity. In erythroid precursors expressing this mutant, erythropoietin hypersensitivity was seen, and, in an animal model, the mutation was sufficient to produce an erythrocytosis resembling PV. Jak2 is a key intermediate in erythropoietin signal transduction and its phosphorylation and activation of Stat proteins is well-characterized. Its involvement with other downstream pathways, however, is less well-defined but potentially important to the pathogenesis of polycythemia vera. We therefore investigated signaling events downstream from the activated Jak2 V614F mutant in the human erythroleukemia (HEL) cell line. We discovered that the scaffolding protein Gab2 was constitutively phosphorylated in cells expressing the Jak2 mutant; moreover, its phosphorylation was blocked when cells were treated with the Jak2-specific inhibitor AG490. This suggested that Gab2 might represent an additional Jak2 target and one that could link Jak2 to multiple downstream signaling pathways including PI3kinase-Akt and Shp2-Erk1/2. We investigated whether Jak2 could phosphorylated Gab2 in vitro using a Gab2-GST fusion protein and found that purified, activated Jak2 could phosphorylate site(s) in the C-terminus of Gab2. This region contains two tyrosine residues that lie within a consensus sequence for Jak2 phosphorylation (pY-X-X-I/V/L): Gab2 tyrosines 614 and 643. Mutagenesis of tyrosine 643 resulted in a marked inhibition of Jak2-dependent Gab2 phosphorylation in vitro and in vivo. Mutation of tyrosine 643 also inhibited Shp2 association with Gab2 and activation of Erk1/2. These results suggest that Gab2 may provide a link between the activated Jak2 and the Shp2-Erk2 pathway and thereby play a role in the neoplastic effects of mutant Jak2. Interestingly, Gab2 is also implicated in the pathogenic effects of Bcr-Abl, suggesting that the protein interactions of this molecular scaffold could represent a target for therapeutic intervention that could be of benefit in multiple myeloproliferative disorders.