A novel protein phosphatase that dephosphorylates and regulates Ca2+/calmodulin-dependent protein kinase II.
A synthetic peptide corresponding to the autophosphorylation site of Ca2+/calmodulin-dependent protein kinase II (CaMKII) (residues 281-289) was conjugated to paramagnetic particles, and phosphorylated by a constitutively active CaMKII fragment. Using this phosphopeptide conjugate as a substrate, a calyculin A-insensitive, Mn(2+)-dependent, and poly-L-lysine-stimulated protein phosphatase activity was detected in the crude extract of rat brain. The protein phosphatase (designated as CaMKII phosphatase) (CaMKIIPase) was purified to near homogeneity from rat brain. CaMKIIPase showed apparent molecular weights of 54,000 and 65,000, on SDS-polyacrylamide gel electrophoresis and gel-filtration analysis, respectively. It was not inhibited by 100 nM calyculin A or 10 microM okadaic acid. Mn2+, but not Mg2+, was absolutely required for activity. CaMKIIPase was potently activated by polycations. Autophosphorylated CaMKII was dephosphorylated by CaMKIIPase, whereas phosphorylase kinase, mixed histones, myelin basic protein, and alpha-casein (which had been phosphorylated by cAMP-dependent protein kinase) and phosphorylase a (phosphorylated by phosphorylase kinase) were not significantly dephosphorylated. No other proteins than CaMKII in rat brain extract which had been phosphorylated by CaMKII were dephosphorylated. The stimulated Ca(2+)-independent activity of autophosphorylated CaMKII was reversed by the action of CaMKIIPase. Thus, CaMKIIPase appears to be a specialized protein phosphatase for the regulation of CaMKII.
Ishida
A
A
Department of Biochemistry, Asahikawa Medical College, Japan.
Kameshita
I
I
Fujisawa
H
H
D000818:Animals; D001921:Brain / Q000201:enzymology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002412:Cations / Q000378:metabolism; D004789:Enzyme Activation; D008970:Molecular Weight; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation; D011073:Polyamines / Q000378:metabolism; D000071228:Polyelectrolytes; D051381:Rats; D013379:Substrate Specificity
D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002412:Cations; D010749:Phosphoprotein Phosphatases; D011073:Polyamines; D000071228:Polyelectrolytes; C110531:calmodulin dependent protein kinase II phosphatase