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  • To ascertain that the slower

    2021-06-21

    To ascertain that the slower migrating bands represented phosphorylated CDK5, we performed a phosphatase assay. Lysates of A-366 co-expressing p35 and either WT or D144N CDK5 were immunoprecipitated using an anti-p35 antibody. The immunoprecipitates were then dephosphorylated using SAP along with appropriate controls. As shown in Fig. 1B (Top), D144N CDK5 samples showed a single band in all three conditions (lanes 1–3) suggesting the absence of any post-translational modification. The WT CDK5 on the other hand, showed the slower migrating bands in the control lane without SAP (lane 4). Upon treatment with SAP, the slower migrating bands disappeared and instead showed a single band (lane 5) comparable in mobility to that of D144N CDK5 suggesting that the mobility shift seen in lane 4 was due to phosphorylation of CDK5. As a negative control, when SAP was added along with EDTA (SAP inhibitor), the slower migrating phosphorylated bands of CDK5 were restored (lane 6). As expected, we also observed the same pattern for p35 i.e. modified p35 bands in lanes 4 and 6 but not in lanes 1–3 and 5. Together, these results confirmed that the bands showing retarded electrophoretic mobility represent phosphorylated CDK5/p35 and that the phosphorylation of CDK5 is dependent on its catalytic activity. Identification of the phosphosites on CDK5: Next we wished to identify the phosphosites on CDK5. To do so, we performed an in vitro kinase assay on the commercially available recombinant His6-CDK5 and GST-p35 complex (Fig. S1A). A total of seven residues on CDK5 were identified as putative phosphosites using mass spectrometry (Fig. S1B). Next, we mapped these sites on the CDK5-p25 crystal structure (PDB ID-3O0G; Fig. 2A) [18,19]. Out of all the sites, S47 seemed the most interesting as it is part of the cyclin binding element and is within favorable distance to make a H-bond with the backbone of I241 in p25 (Fig. 2A, Inset). If phosphorylated, the phosphate group at S47 may perturb the H-bond and possibly may also pose a steric clash with the p25-binding interface. When we aligned the partial sequence encompassing S47 in CDK5 from different species, it appeared to be evolutionarily conserved with a serine at this position in human, bovine, rat, mouse and drosophila CDK5 and a substitution with threonine in yeast (Fig. 2B). Phosphomimetic mutation at S47 abolishes CDK5-p35 binding: In order to directly test the effect of phosphorylation on S47, we generated phosphomimetic (S47D) and non-phosphorylatable (S47A) mutations on CDK5. The CDK5 constructs were co-transfected with p35 in Cos7 cells and the cell lysates were analyzed by western blotting. As expected, the modified bands were absent in the D144N mutant (Fig. 2C; lane 1). Both WT and S47A CDK5, on the other hand, showed the slower migrating bands suggesting that the S47A mutant retains its kinase activity (Fig. 2C; lanes 2,3). Interestingly, the S47D mutant lacked the phospho-modified bands (lane 4) suggesting that this mutation may render CDK5 inactive. We next performed a co-immunoprecipitation assay to test if the inactivity of the S47D mutant is due to the loss of binding to p35. Lysates of cells co-expressing p35 and CDK5 constructs (D144N, WT, S47A and S47D) were immunoprecipitated using the p35 antibody and analyzed by western blotting. The D144N mutant bound to p35 as expected (Fig. 2D; lane 2) because the D144N mutation inactivates the kinase due to its inability to coordinate Mg2+ ion but does not affect the CDK5-p35 interaction [2,20]. The WT and S47A CDK5 also co-immunoprecipitated with p35 (lanes 3,4). Notably, in both WT and S47A CDK5 expressing lysates, p35 preferentially bound to the phosphorylated form of CDK5 represented by the slower migrating band suggesting that one or more of the phosphosites on active CDK5 enhance its interaction with p35. In contrast, the S47D mutant failed to bind to p35 (Fig. 2D; lane 5). Based on these results, it appeared that while the H-bond made by S47 may be dispensable (p35:S47A CDK5 binding was comparable to p35:WT CDK5 binding), phosphorylation at S47 may sterically hinder CDK5-p35 interaction resulting in loss of its kinase activity.