Characterization of the recombinant C-terminal domain of dystrophin: phosphorylation by calmodulin-dependent protein kinase II and dephosphorylation by type 2B protein phosphatase.
We report that the C-terminal domain of skeletal muscle dystrophin expressed as a fusion protein with glutathione S-transferase (designated GST-CT-1) is a substrate for Ca2+/calmodulin-dependent phosphorylation and dephosphorylation. GST-CT-1 and GST-CT-1F (GST-CT-1 truncated by 20-25 residues) were phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). The stoichiometries of phosphorylation by CaM kinase II were 1.65 mol of Pi/mol of GST-CT-1 and 0.39 mol of Pi/mol of GST-CT-1F, respectively, suggesting that the principal site(s) of phosphorylation is (are) located in the C-terminal 20-25 residues that are missing from GST-CT-1F. The GST-CT-1 fusion protein was phosphorylated on both serine and threonine residues, whereas GST-CT-1F was phosphorylated only on serine. CaM kinase II-phosphorylated GST-CT-1 and GST-CT-1F were efficiently dephosphorylated by calcineurin, a Ca2+/calmodulin-dependent protein phosphatase (type 2B protein phosphatase). Importantly, calcineurin was found to be associated with a purified sarcolemmal membrane preparation enriched in dystrophin. Type 2A protein phosphatase isolated from smooth muscle (SMP-I) and its catalytic subunit (SMP-ic) also dephosphorylated GST-CT-1, but were less active toward these substrates than was calcineurin. Type 2C phosphatase (SMP-II) and type 1 protein phosphatases [SMP-III, SMP-IV, and myosin-associated phosphatase (PP1M) of smooth muscle and skeletal muscle protein phosphatase 1c] were ineffective in dephosphorylating the C-terminal region of dystrophin.(ABSTRACT TRUNCATED AT 250 WORDS)
Walsh
M P
MP
Department of Medical Biochemistry, Faculty of Medicine, University of Calgary, Alberta, Canada.
Busaan
J L
JL
Fraser
E D
ED
Fu
S Y
SY
Pato
M D
MD
Michalak
M
M
D000818:Animals; D001921:Brain / Q000378:metabolism; D019703:Calcineurin; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002147:Calmodulin / Q000302:isolation & purification / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D002417:Cattle; D002645:Chickens; D016189:Dystrophin / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D005895:Gizzard, Avian / Q000378:metabolism; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D016189:Dystrophin; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases