Src kinase regulates the RACK1/ZBP1 complex and the Y246 of RACK1 is critical for the binding with ZBP1.

<p><b>A</b>, Endogenous RACK1 interacts with Flag-ZBP1 in an anti-Flag immunoprecipitation assay from total lysate of neuroblastoma Flag-ZBP1-transfected cells. Western blotting for endogenous RACK1 and Flag-ZBP1 on proteins eluted from anti-Flag immunoprecipitation assay. Flag transfected cells were used as negative control. Input represents 5% of total lysate. <b>B</b> and <b>C</b>, Src activity regulates the RACK1-ZBP1 complex formation. <b>B</b>, db-cAMP or PACAP treatments of Flag-ZBP1 transfected cells reduced the binding between RACK1 and Flag-ZBP1, whereas PP2 restored the binding, as in untreated cells. <b>C</b> BDNF treatments increased the binding of Flag-ZBP1 to RACK1 in Flag-ZBP1 transfected cells. Src inhibition by Src inhibitor PP2 reduced the RACK1-ZBP1 complex stimulated by BDNF. The density value of immnoprecipitated RACK1 is normalized to that of immunoprecipitated Flag-ZBP1 and summarized in both graphics. Data are graphed as means plus S.D. <b>D</b>, The Src binding and phosphorylation site (Y246) of RACK1 is critical for complex formation. Flag-ZBP1 protein co-immunoprecipitated with GFP-RACK1_wt and GFP-RACK1_Y246F in immunopreciptation assays using anti-GFP antibody, but in the presence of GFP-RACK1_Y246F the binding was reduced. The figures are representative of three independent experiments. The density value of co-immnoprecipitated Flag-ZBP1 is normalized to that of immnoprecipitated GFP-RACK1_wt or GFP-RACK1_Y246F and summarized in both graphic. Data are graphed as means plus S.D.