Figure 3.
CaMKII activates ASK1. (A) CaMKII phosphorylates ASK1. GST-tagged kinase negative ASK1 (GST-ASK1KN) and/or CaMKII purified from rat forebrain (purchased from Upstate Bio) were incubated in a kinase buffer in the presence of 2 mM CaCl2/2.4 μM calmodulin and [γ-32P]ATP. Samples were subjected to SDS–PAGE followed by autoradiography (top panel) and immunoblotting with anti-GST (middle panel) and anti-phospho-CaMKII (bottom panel) antibodies. (B) CaMKII does not directly phosphorylate Thr 845 of ASK1. GST-ASK1 bound to glutathione sepharose beads was first treated with Lambda Protein Phosphatase (λPPase; purchased from NEB) to reduce the basal phosphorylation of Thr 845. After washing with PBS including PPase inhibitors, the beads were incubated with purified CaMKII in a kinase buffer in the presence of 2 mM CaCl2/2.4 μM calmodulin. Samples were subjected to SDS–PAGE followed by immunoblotting with anti-phospho-ASK1 (top panel), anti-GST (middle panel) and anti-phospho-CaMKII (bottom panel) antibodies. (C) CaMKII binds ASK1 and induces activating phosphorylation of ASK1 in the immune complex. HEK-293 cells were transfected with HA-tagged kinase negative ASK1 (HA-ASK1KN) and/or Flag-CaMKII. Cell lysates were immunoprecipitated with anti-Flag antibody. An immune complex kinase assay was performed in the presence of either 2 mM EGTA (Ca2+/CaM−) or 2 mM CaCl2/2.4 μM calmodulin (Ca2+/CaM+). The activated state and the amount of co-purified ASK1KN with CaMKII were detected by immunoblotting with anti-phospho-ASK1 (P-ASK1) and anti-HA antibodies, respectively (upper two panels). The immunoprecipitated Flag-CaMKII and the expression level of HA-ASK1KN were confirmed by immunoblotting with anti-Flag and anti-HA antibodies (lower two panels). (D) Activating phosphorylation of ASK1 by CaMKII is dependent on the kinase activity of CaMKII. HEK-293 cells were transfected with the indicated combination of HA-ASK1KN, Flag-CaMKII and Flag-tagged kinase negative CaMKII (Flag-CaMKIIKN). Cell lysates were immunoprecipitated with anti-Flag antibody followed by an immune complex kinase assay as described in (C). The activated state and the amount of co-purified ASK1KN with CaMKII were detected by immunoblotting with anti-phospho-ASK1 (P-ASK1) and anti-HA antibodies, respectively (upper two panels). The immunoprecipitated Flag-CaMKII and its activated state were confirmed by immunoblotting with anti-Flag and anti-phospho-CaMKII antibodies (lower two panels).