title: A 60 kDa polypeptide of skeletal-muscle sarcoplasmic reticulum is a calmodulin-dependent protein kinase that associates with and phosphorylates several membrane proteins.
abstract: Activation of a calmodulin (CaM)-dependent protein kinase associated with rabbit skeletal-muscle sarcoplasmic reticulum (SR) results in the phosphorylation of polypeptides of 450, 360, 165, 105, 89, 60, 34 and 20 kDa. Radioligand-binding studies indicated that a membrane-bound 60 kDa polypeptide contained both CaM- and ATP-binding domains. Under renaturing conditions on nitrocellulose blots, the 60 kDa polypeptide of the membrane exhibited CaM-dependent autophosphorylation activity, suggesting that it was the CaM-dependent protein kinase of SR. Ca2+/CaM-independent autophosphorylation of polypeptides of 62 and 45 kDa was found to occur in the light SR, whereas the Ca2+/CaM-dependent autophosphorylation activity was enriched in the heavy SR. Both these kinase activities were absent from transverse tubules, although these membranes were enriched in CaM-binding polypeptides of 160, 100 and 80 kDa. In the absence of Ca2+, CaM bound to a 33 kDa polypeptide of the membrane. The purified ryanodine receptor was not phosphorylated by the purified CaM kinase, although it was a substrate for protein kinase C. Affinity-purified antibodies to brain CaM kinase II cross-reacted with the 60 kDa polypeptide in Western blots and immunoprecipitated the 60 kDa polypeptide, along with the 360, 105, 89, 34 and 20 kDa phosphoproteins, from Nonidet-P-40-solubilized SR membranes. Antibodies raised against the 60 kDa kinase polypeptide did not cross-react with the other phosphoproteins, suggesting that these polypeptides were distinct and unrelated. Subcellular distribution of the 60 kDa kinase indicated the specific association of the polypeptide with the junctional-face membrane of SR. The CaM-dependent incorporation of 32P into various membrane proteins was inhibited by the CaM kinase II fragment (290-309), with an IC50 value of 2 nM for the inhibition of incorporation into the 60 kDa kinase polypeptide. Recent studies [Wang and Best (1992) Nature (London) 359, 739-741] have shown that a CaM kinase activity intrinsic to the membrane can inactivate the Ca(2+)-release channel of skeletal muscle SR. Since our results demonstrate that the 60 kDa polypeptide of SR is a CaM-dependent protein kinase, we suggest that this kinase, through its associations, may be responsible for gating the Ca(2+)-release channel.
authors.affiliation: Department of Pharmacology, University of Ottawa, Ontario, Canada.
authors.forename: J J
authors.initials: JJ
authors.lastname: Leddy
authors.forename: B J
authors.initials: BJ
authors.lastname: Murphy
authors.lastname: Qu-Yi
authors.forename: J P
authors.initials: JP
authors.lastname: Doucet
authors.forename: C
authors.initials: C
authors.lastname: Pratt
authors.forename: B S
authors.initials: BS
authors.lastname: Tuana
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D015220:Calcium Channels / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D007163:Immunosorbent Techniques; D008565:Membrane Proteins / Q000378:metabolism*; D008970:Molecular Weight; D009124:Muscle Proteins / Q000378:metabolism; D009132:Muscles / Q000201:enzymology*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
chemical_list: D015220:Calcium Channels; D002147:Calmodulin; D008565:Membrane Proteins; D009124:Muscle Proteins; D010750:Phosphoproteins; D019837:Ryanodine Receptor Calcium Release Channel; D000255:Adenosine Triphosphate; D017871:Calcium-Calmodulin-Dependent Protein Kinases