p35Nck5a associates with and activates Cdk5 in differentiating HL60 cells.
(A) HL60 cells were transfected with an empty pCMV vector (“Vector”) or with a vector expressing hemagglutinin (HA)-tagged Cdk5, and either left untreated (C) or exposed to 10−7M 1,25D3 (D3) for 48 hours. The cell lysates were immunoprecipitated with an HA antibody and the immunoprecipitate incubated with histone H1 (H1) substrate under kinase assay conditions. The panel shows an autoradiogram for 32P-labeled histone H1 and demonstrates that transfected (HA tagged) Cdk5 becomes activated as a kinase in differentiating (1,25D3-treated) but not in undifferentiated HL60 cells. The lane marked “control IP” represents a control where the beads used for immunoprecipitation were not coated with the antibody. (B) Lysates of untreated HL60 cells. HL60 cells treated with 10−7M 1,25D3 for 48 hours and fresh whole mouse brain cell extract were immunoprecipitated (IP) with antibody to Cdk5. The lysates were incubated with γ32P-ATP in kinase assay buffer without an exogenous substrate then subjected to gel electrophoresis. The upper panel shows the immunoreactive p35 protein demonstrated by immunoblotting (IB), the lower panel the phosphorylation of the p35 protein by autoradiography for 32P. Mouse brain was used as a positive control of the previously reported autophosphorylation of p35Nck5a by neural cells. BP indicates a control in which the immunoprecipitating Cdk5 antibody was blocked with the immunizing peptide. The data illustrate 4 similar experiments.
