Calmodulin-dependent protein kinase IV (CaM-kinase IV) phosphorylated calmodulin (CaM), which is its own activator, in a poly--Lys [poly(Lys)]-dependent manner. Although CaM-kinase II weakly phosphorylated CaM under the same conditions, CaM-kinase I, CaM-kinase kinase α, 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 μM CaM was about 10 μg/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--Arg could partially substitute for poly(Lys), but protamine, spermine, and poly--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 ^<32>P-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 Thr^<44>. 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 Ca^<2+>/CaM can bind and phosphorylate another CaM with the aid of poly(Lys), leading to a decrease in the activity of CaM.
注記
Elsevier, Ishida, A. ; Kameshita, I. ; Okuno, S. ; Kitani, T. ; Fujisawa, H., ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 407(1), 2002, 72-82
