Dystrophin is phosphorylated by endogenous protein kinases

Abstract

Dystrophin, the protein coded by the gene missing in Duchenne muscular dystrophy, is assumed to be a component of the membrane cytoskeleton of skeletal muscle. Like other cytoskeletal proteins in different cell types, dystrophin bound to sarcolemma membranes was found to be phosphorylated by endogenous protein kinases. The phosphorylation of dystrophin was activated by cyclic AMP, cyclic GMP, calcium and calmodulin, and was inhibited by cyclic AMP-dependent protein kinase peptide inhibitor, mastoparan and heparin. These results suggest that membrane-bound dystrophin is a substrate of endogenous cyclic AMP- and cyclic GMP-dependent protein kinases, calcium/calmodulin-dependent kinase and casein kinase II. The possibility that dystrophin could be phosphorylated by protein kinase C is suggested by the inhibition of phosphorylation by staurosporin. On the other hand dystrophin seems not to be a substrate for protein tyrosine kinases, as shown by the lack of reaction of phosphorylated dystrophin with a monoclonal antiphosphotyrosine antibody. Sequence analysis indicates that dystrophin contains seven potential phosphorylation sites for cyclic AMP- and cyclic GMP-dependent protein kinases (all localized in the central rod domain of the molecule) as well as several sites for protein kinase C and casein kinase II. Interestingly, potential sites of phosphorylation by protein kinase C and casein kinase II are located in the proximity of the actin-binding site. These results suggest, by analogy with what has been demonstrated in the case of other cytoskeletal proteins, that the phosphorylation of dystrophin by endogenous protein kinases may modulate both self assembly and interaction of dystrophin with other cytoskeletal proteins in vivo.

Ecto-ATPase Activity of α-Sarcoglycan (Adhalin)

“…Furthermore, like other ecto-ATPases (33,45,46), the ATPase activity of dystrophinDAPs preparations was not inhibited by inhibitors of ion-translocating ATPases such as thapsigargin, cyclopiazonic acid, and vanadate (data not shown). It appears likely that the relatively low specific activity is because of the presence of digitonin, used to purified the DAP complex (22,23). Indeed, it has been demonstrated that detergents inhibit the activity of other ectoATPases (31,33).…”

“…isolated as described previously (22). The purification of the dystrophinDAPs complex was performed according to the digitonin, 0.5 M NaCl, wheat germ agglutinin protocol of Ervasti et al (23), as described previously (24), with the only difference being that the dystrophinDAPs purification was terminated after the DEAE-cellulose column chromatography.…”

Expression of Sarco(endo)Plasmic Reticulum Ca2+‐Atpase Slow (SERCA2) Isoform in Regenerating Rat Soleus Skeletal Muscle Depends on Nerve Impulses

“…Triads were prepared using the method of Mitchell et al (1983), with slight modifications (Luise et al 1993). Production of these monoclonal antibodies was carried out by immunizing Balb/C mice (Charles River, Italy) with the triad membrane fraction, according to established procedures.…”

Functional roles of dystrophin and of associated proteins. New insights for the sarcoglycans

“…Two Ca 2+ -calmodulin binding sites at the N-terminal domain of dystrophin appear to control 376 both polymerization [73] and interaction with F-actin [74]. Dystrophin is phosphorylated by endogenous and exogenous protein kinase [75,76] and phosphorylation modulates its actin-binding activity [77].…”