Regulatory domain of calcium/calmodulin-dependent protein kinase II. Mechanism of inhibition and regulation by phosphorylation.
Regulatory mechanisms of rat brain Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) were probed using a synthetic peptide (CaMK-(281-309] corresponding to residues 281-309 (alpha-subunit) which contained the calmodulin (CaM)-binding and inhibitory domains and also the initial autophosphorylation site (Thr286). Kinetic analyses indicated that inhibition of a completely Ca2+/CaM-independent form of CaM-kinase II by CaMK-(281-309) was noncompetitive with respect to peptide substrate (syntide-2) but was competitive with respect to ATP. Interaction of CaMK-(281-309) with the ATP-binding site was independently confirmed since inactivation of proteolyzed CaM-kinase II by phenylglyoxal (t1/2 = 7 min) was blocked by ATP analog plus Mg2+ or by CaMK-(281-309). In the presence of Ca2+/CaM, CaMK-(281-309) no longer protected against phenylglyoxal inactivation, consistent with our previous observations (Colbran, R.J., Fong, Y.-L., Schworer, C.M., and Soderling, T.R. (1988) J. Biol. Chem. 263, 18145-18151) that binding of Ca2+/CaM to CaMK-(281-309) 1) blocks its inhibitory property, and 2) enhances its phosphorylation at Thr 286. The present study also showed that phosphorylation of CaMK-(281-309) decreased its inhibitory potency at least 10-fold without affecting its Ca2+/CaM-binding ability. Thus, CaM-kinase II is inactive in the absence of Ca2+/CaM because an inhibitory domain within residues 281-309 interacts with the catalytic domain and blocks ATP binding. Autophosphorylation of Thr286 results in a Ca2+/CaM-independent form of the kinase by disrupting the inhibitory interaction with the catalytic domain.
Colbran
R J
RJ
Howard Hughes Medical Institute, Nashville, Tennessee 37232.
Smith
M K
MK
Schworer
C M
CM
Fong
Y L
YL
Soderling
T R
TR
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D006868:Hydrolysis; D007700:Kinetics; D010446:Peptide Fragments / Q000494:pharmacology; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity
D010446:Peptide Fragments; D047428:Protein Kinase Inhibitors; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases