TY - JOUR
T1 - Activation of type II calcium/calmodulin-dependent protein kinase by Ca2+/calmodulin is inhibited by autophosphorylation of threonine within the calmodulin-binding domain
AU - Patton, B. L.
AU - Miller, S. G.
AU - Kennedy, M. B.
PY - 1990
Y1 - 1990
N2 - It is now well established that autophosphorylation of a threonine residue located next to each calmodulin-binding domain in the subunits of type II Ca2+/calmodulin-dependent protein kinase causes the kinase to remain active, although at a reduced rate, after Ca2+ is removed from the reaction. This autophosphorylated form of the kinase is still sensitive to Ca2+/calmodulin, which is required for a maximum catalytic rate. After removal of Ca2+, new sites are autophosphorylated by the partially active kinase. Autophosphorylation of these sites abolishes sensitivity of the kinase to Ca2+/calmodulin (Hashimoto, Y., Schworer, C.M., Colbran, R.J., and Soderling, T.R. (1987) J. Biol. Chem. 262, 8051-8055). We have identified two pairs of homologous residues, Thr305 and Ser314 in the α subunit and Thr306 and Ser315 in the β subunit, that are autophosphorylated only after removal of Ca2+ from an autophosphorylation reaction. The sites were identified by direct sequencing of labeled tryptic phosphopeptides isolated by reverse-phase high pressure liquid chromatography. Thr305-306 is rapidly dephosphorylated by purified protein phosphatases 1 and 2A, whereas Ser314-315 is resistant to dephosphorylation. We have shown by selective dephosphorylation that the presence of phosphate on Thr305-306 blocks sensitivity of the kinase to Ca2+/calmodulin. In contrast, the presence of phosphate on Ser314-315 is associated with an increase in the K(act) for Ca2+/calmodulin of only about 2-fold, producing a relatively small decrease in sensitivity to Ca2+/calmodulin.
AB - It is now well established that autophosphorylation of a threonine residue located next to each calmodulin-binding domain in the subunits of type II Ca2+/calmodulin-dependent protein kinase causes the kinase to remain active, although at a reduced rate, after Ca2+ is removed from the reaction. This autophosphorylated form of the kinase is still sensitive to Ca2+/calmodulin, which is required for a maximum catalytic rate. After removal of Ca2+, new sites are autophosphorylated by the partially active kinase. Autophosphorylation of these sites abolishes sensitivity of the kinase to Ca2+/calmodulin (Hashimoto, Y., Schworer, C.M., Colbran, R.J., and Soderling, T.R. (1987) J. Biol. Chem. 262, 8051-8055). We have identified two pairs of homologous residues, Thr305 and Ser314 in the α subunit and Thr306 and Ser315 in the β subunit, that are autophosphorylated only after removal of Ca2+ from an autophosphorylation reaction. The sites were identified by direct sequencing of labeled tryptic phosphopeptides isolated by reverse-phase high pressure liquid chromatography. Thr305-306 is rapidly dephosphorylated by purified protein phosphatases 1 and 2A, whereas Ser314-315 is resistant to dephosphorylation. We have shown by selective dephosphorylation that the presence of phosphate on Thr305-306 blocks sensitivity of the kinase to Ca2+/calmodulin. In contrast, the presence of phosphate on Ser314-315 is associated with an increase in the K(act) for Ca2+/calmodulin of only about 2-fold, producing a relatively small decrease in sensitivity to Ca2+/calmodulin.
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M3 - Article
C2 - 2162838
AN - SCOPUS:0025294106
SN - 0021-9258
VL - 265
SP - 11204
EP - 11212
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 19
ER -