Phosphorylation of dystrophin and alpha-syntrophin by Ca(2+)-calmodulin dependent protein kinase II.
A Ca(2+)-calmodulin dependent protein kinase activity (DGC-PK) was previously shown to associate with skeletal muscle dystrophin glycoprotein complex (DGC) preparations, and phosphorylate dystrophin and a protein with the same electrophoretic mobility as alpha-syntrophin (R. Madhavan, H.W. Jarrett, Biochemistry 33 (1994) 5797-5804). Here, we show that DGC-PK and Ca(2+)-calmodulin dependent protein kinase II (CaM kinase II) phosphorylate a common site (RSDS(3616)) within the dystrophin C terminal domain that fits the consensus CaM kinase II phosphorylation motif (R/KXXS/T). Furthermore, both kinase activities phosphorylate exactly the same three fusion proteins (dystrophin fusions DysS7 and DysS9, and the syntrophin fusion) out of a panel of eight fusion proteins (representing nearly 100% of syntrophin and 80% of dystrophin protein sequences), demonstrating that DGC-PK and CaM kinase II have the same substrate specificity. Complementing these results, anti-CaM kinase II antibodies specifically stained purified DGC immobilized on nitrocellulose membranes. Renaturation of electrophoretically resolved DGC proteins revealed a single protein kinase band (M(r) approximately 60,000) that, like CaM kinase II, underwent Ca(2+)-calmodulin dependent autophosphorylation. Based on these observations, we conclude DGC-PK represents a dystrophin-/syntrophin-phosphorylating skeletal muscle isoform of CaM kinase II. We also show that phosphorylation of the dystrophin C terminal domain sequences inhibits their syntrophin binding in vitro, suggesting a regulatory role for phosphorylation.
Madhavan
R
R
Department of Biochemistry, University of Tennessee-Memphis, 858 Madison Ave., Memphis, TN 38163, USA.
Jarrett
H W
HW
D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D002135:Calcium-Binding Proteins; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D003598:Cytoskeletal Proteins / Q000737:chemistry; D016189:Dystrophin / Q000737:chemistry*; D007527:Isoenzymes / Q000737:chemistry / Q000276:immunology; D008562:Membrane Glycoproteins / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D009124:Muscle Proteins / Q000737:chemistry*; D018482:Muscle, Skeletal / Q000201:enzymology; D020388:Muscular Dystrophy, Duchenne / Q000201:enzymology; D010766:Phosphorylation; D011817:Rabbits; D049031:Sarcoglycans; D015398:Signal Transduction; D014357:Trypsin
D000906:Antibodies; D002135:Calcium-Binding Proteins; D003598:Cytoskeletal Proteins; D016189:Dystrophin; D007527:Isoenzymes; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009124:Muscle Proteins; D049031:Sarcoglycans; C091825:syntrophin alpha1; D000255:Adenosine Triphosphate; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D014357:Trypsin