Substrate specificity of Ca(2+)/calmodulin-dependent protein kinase phosphatase: kinetic studies using synthetic phosphopeptides as model substrates.
Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKPase) dephosphorylates and regulates multifunctional Ca(2+)/calmodulin-dependent protein kinases. In order to elucidate the mechanism of substrate recognition by CaMKPase, we chemically synthesized a variety of phosphopeptide analogs and carried 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 preference for a phospho-Thr residue. A Pro residue adjacent to the dephosphorylation site on the C-terminal side and acidic clusters around the dephosphorylation site had detrimental effects on dephosphorylation by CaMKPase. Deletion analysis of a model substrate peptide revealed that the minimal length of the substrate peptide was only 2 to 3 amino acid residues including the dephosphorylation site. The residues on the C-terminal side of the dephosphorylation site were not essential for dephosphorylation, whereas the residue adjacent to the dephosphorylation site on the N-terminal side was essential. Ala-scanning analysis suggested that CaMKPase did not recognize a specific motif around the dephosphorylation site. Myosin light chain phosphorylated by protein kinase C and Erk2 phosphorylated by MEK1 were poor substrates for CaMKPase, while a synthetic phosphopeptide corresponding to the sequence around the phosphorylation site of the former was not dephosphorylated by CaMKPase but that of the latter was fairly good substrate. These data suggest that substrate specificity of CaMKPase is determined by higher-order structure of the substrate protein rather than by the primary structure around its dephosphorylation site. Use of phosphopeptide substrates also revealed that poly-L-lysine, an activator for CaMKPase, activated the enzyme mainly through increase in the V(max) values.
Ishida
A
A
Department of Biochemistry, Asahikawa Medical College, Asahikawa 078-8510, Japan. fujisawa@asahikawa-med.ac.jp
Shigeri
Y
Y
Tatsu
Y
Y
Endo
Y
Y
Kameshita
I
I
Okuno
S
S
Kitani
T
T
Takeuchi
M
M
Yumoto
N
N
Fujisawa
H
H
D000595:Amino Acid Sequence / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics / Q000502:physiology; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D007700:Kinetics; D010748:Phosphopeptides / Q000138:chemical synthesis / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D017384:Sequence Deletion / Q000235:genetics / Q000502:physiology; D013379:Substrate Specificity / Q000502:physiology
D010748:Phosphopeptides; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C110531:calmodulin dependent protein kinase II phosphatase; D010749:Phosphoprotein Phosphatases