Fyn kinase regulates type II PtdIns 4-kinases in RBL 2H3 cells

Abstract

Type II phosphatidylinositol 4-kinases are implicated in FcεRI-mediated signaling cascades leading to release of inflammatory molecules. Cross-linking of FcεRI on RBL 2H3 cells results in protein tyrosine phosphorylation and activation of type II PtdIns 4-kinase activity. Protein tyrosine kinase(s) that phosphorylate type II PtdIns 4-kinase(s) in RBL 2H3 cells remains elusive and is being addressed in this manuscript. Anti-Fyn kinase antibodies co-immunoprecipitated type II PtdIns 4-kinase activity from FcεRI cross-linked RBL 2H3 cells. In reciprocal assays, His-tagged types II PtdIns 4-kinases were shown to pull down Fyn kinase. Further, anti-Fyn immunoprecipitates were shown to phosphorylate type II PtdIns 4-kinase α and β in in vitro assays. Pull down studies with GST-Fyn-SH2 and GST-Fyn-SH3 domains showed that type II PtdIns 4-kinases associate with Fyn-SH2 domain. Knockdown of Fyn kinase in RBL 2H3 cells abrogated activation of type II PtdIns 4-kinase activity in response to FcεRI cross-linking and type II PtdIns 4-kinase activity in anti-phosphotyrosine immunoprecipitates. Knockdown of Fyn kinase was also strongly correlated with a reduction in β-hexosaminidase release in response to FcεRI cross-linking. These results suggest that type II PtdIns 4-kinases act downstream of Fyn kinase in FcεRI signaling cascades and are regulated by Fyn kinase.