FIG. 4.

Snk/Plk2 has kinase activity that is regulated by phosphorylation in its kinase domain. (a) Schematic of Snk/Plk2 mutants. Snk/Plk2 contains an N-terminal kinase domain and a C-terminal Polo box. Single-amino-acid residue substitutions in the kinase domain of conserved residues are indicated with an asterisk. Constructs contain a C-terminal V5-six-histidine tag. (b) 293 cells were transfected with the indicated Snk/Plk2 constructs or empty vector. At 24 h, the cells were harvested for protein and analyzed for expression with mouse anti-V5 antibody. Actin was used to confirm that an equivalent amount of protein was loaded in each lane. (c) 293 cells were transfected with the indicated Snk/Plk2 constructs or empty vector for 48 h. Equal amounts of 293 cell lysates (2.5 × 10⁶ cell equivalent) were harvested and immunoprecipitated with mouse monoclonal anti-polyhistidine agarose and subjected to Western blot analysis (lower panel) or an in vitro protein kinase assay (upper panel) using casein as a substrate. Lysates were immunoblotted for exogenously immunoprecipitated Snk/Plk2 using mouse monoclonal anti-V5-HRP.