Substrate specificity of Ca2+/calmodulin-dependent protein kinase phosphatase: Kinetic studies using synthetic phosphopeptides as model substrates

Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKPase) dephosphory lates and regulates multifunctional Ca2+/calmodulin-dependent protein kinas es, In order to elucidate the mechanism of substrate recognition by CaMKPas e, we chemically synthesized a variety of phosphopeptide analogs and carrie d out kinetic analysis using them as CaMKPase substrates. This is the first report using systematically synthesized phosphopeptides as substrates for kinetic studies on substrate specificities of protein Ser/Thr phosphatases. CaMKPase was shown to be a protein Ser/Thr phosphatase having a strong pre ference for a phospho-Thr residue. A Pro residue adjacent to the dephosphor ylation site on the C-terminal side and acidic clusters around the dephosph orylation site had detrimental effects on dephosphorylation by CaMKPase. De letion analysis of a model substrate peptide revealed that the minimal leng th of the substrate peptide was only 2 to 3 amino acid residues including t he dephosphorylation site. The residues on the C-terminal side of the depho sphorylation site were not essential for dephosphorylation, whereas the res idue adjacent to the dephosphorylation site on the N-terminal side was esse ntial. Ala-scanning analysis suggested that CaMKPase did not recognize a sp ecific motif around the dephosphorylation site. Myosin light chain phosphor ylated by protein kinase C and Erk2 phosphorylated by MEK1 were poor substr ates for CaMKPase, while a synthetic phosphopeptide corresponding to the se quence around the phosphorylation site of the former was not dephosphorylat ed by CaMKPase but that of the latter was fairly good substrate. These data suggest that substrate specificity of CaMKPase is determined by higher-ord er structure of the substrate protein rather than by the primary structure around its dephosphorylation site. Use of phosphopeptide substrates also re vealed that poly-L-lysine, an activator for CaMKPase, activated the enzyme mainly through increase in the V-max values.