Inactivation of calmodulin-dependent protein kinase IV by autophosphorylation of serine 332 within the putative calmodulin-binding domain.
When brain calmodulin-dependent protein kinase IV is incubated with calmodulin-dependent protein kinase IV kinase under the phosphorylation conditions in the presence of Ca2+/calmodulin, rapid initial incorporation of 1 mol of phosphate into 1 mol of the enzyme by the action of the kinase kinase occurs, resulting in marked activation of the enzyme, and the subsequent incorporation of more than 3 mol of phosphate by autophosphorylation occurs, resulting in no significant change in the activity (Okuno, S., Kitani, T., and Fujisawa, H. (1994) J. Biochem. (Tokyo) 116, 923-930; Okuno, S., Kitani, T., and Fujisawa, H. (1995) J. Biochem. (Tokyo) 117, 686-690). After the maximal phosphorylation, the continued incubation in the presence of excess EGTA resulted in additional autophosphorylation of the enzyme, leading to a complete loss of the Ca2+/calmodulin-dependent activity, while causing no significant change in the Ca2+/calmodulin-independent activity. The amino acid sequence analysis revealed that the autophosphorylation after removal of Ca2+ occurred on Ser332, Ser333, Ser337, and Ser341. Analysis by site-directed mutagenesis clearly showed that the autophosphorylation site responsible for the inactivation is Ser332. Thus, calmodulin-dependent protein kinase IV activated by the kinase kinase may lose its Ca2+/calmodulin-dependent activity by autophosphorylation on Ser332 located within the putative calmodulin-binding domain in the absence of Ca2+.
Watanabe
S
S
Department of Biochemistry, Asahikawa Medical College, Asahikawa 078, Japan.
Okuno
S
S
Kitani
T
T
Fujisawa
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D002645:Chickens; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016376:Oligonucleotides, Antisense; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051381:Rats; D012694:Serine / Q000378:metabolism*
D002147:Calmodulin; D016376:Oligonucleotides, Antisense; D012694:Serine; D017346:Protein Serine-Threonine Kinases; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C517686:Camk4 protein, rat; D002118:Calcium