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-1(F) (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 P-i/mol of GST-CT-1 and 0.39 mol of P-i/mol of GST-CT-1(F), 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-1(F). The GST-CT-1 fusion protein was phosphorylated on both serine, and threonine residues, whereas GST-CT-1(F) was phosphorylated only on serine, CaM kinase II-phosphorylated GST-CT-1 and GST-CT-1(F) 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-i(c)) 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. We conclude that calmodulin-dependent phosphorylation-dephosphorylation of the C-terminal domain of dystrophin may play a role in regulating dystrophin-membrane interactions and/or transducing signals from the extracellular matrix via the dystrophin molecule to the cytoskeleton.