Phosphorylation of calmodulin by Ca2+/calmodulin-dependent protein kinase IV.
Calmodulin-dependent protein kinase IV (CaM-kinase IV) phosphorylated calmodulin (CaM), which is its own activator, in a poly-L-Lys [poly(Lys)]-dependent manner. Although CaM-kinase II weakly phosphorylated CaM under the same conditions, CaM-kinase I, CaM-kinase kinase alpha, and cAMP-dependent protein kinase did not phosphorylate CaM. Polycations such as poly(Lys) were required for the phosphorylation. The optimum concentration of poly(Lys) for the phosphorylation of 1 microM CaM was about 10 microg/ml, but poly(Lys) strongly inhibited CaM-kinase IV activity toward syntide-2 at this concentration, suggesting that the phosphorylation of CaM is not due to simple activation of the catalytic activity. Poly-L-Arg could partially substitute for poly(Lys), but protamine, spermine, and poly-L-Glu/Lys/Tyr (6/3/1) could not. When phosphorylation was carried out in the presence of poly(Lys) having various molecular weights, poly(Lys) with a higher molecular weight resulted in a higher degree of phosphorylation. Binding experiments using fluorescence polarization suggested that poly(Lys) mediates interaction between the CaM-kinase IV/CaM complex and another CaM. The 32P-labeled CaM was digested with BrCN and Achromobacter protease I, and the resulting peptides were purified by reversed-phase HPLC. Automated Edman sequence analysis of the peptides, together with phosphoamino acid analysis, indicated that the major phosphorylation site was Thr44. Activation of CaM-kinase II by the phosphorylated CaM was significantly lower than that by the nonphosphorylated CaM. Thus, CaM-kinase IV activated by binding Ca2+/CaM can bind and phosphorylate another CaM with the aid of poly(Lys), leading to a decrease in the activity of CaM.
Ishida
Atsuhiko
A
Department of Biochemistry, Asahikawa Medical College, Asahikawa, Japan. aishida@asahikawa-med.ac.jp
Kameshita
Isamu
I
Okuno
Sachiko
S
Kitani
Takako
T
Fujisawa
Hitoshi
H
D000595:Amino Acid Sequence; D000818:Animals; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011107:Polylysine / Q000378:metabolism; D011479:Protamines / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D013096:Spermine / Q000737:chemistry / Q000378:metabolism
D002147:Calmodulin; D011479:Protamines; D011107:Polylysine; D013096:Spermine; D017868:Cyclic AMP-Dependent Protein Kinases; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase