Specific association of calmodulin-dependent protein kinase and related substrates with the junctional sarcoplasmic reticulum of skeletal muscle

Abstract

A systematic study of protein kinase activity and phosphorylation of membrane proteins by ATP was carried out with vesicular fragments of longitudinal tubules (light SR) and junctional terminal cisternae (JTC) derived from skeletal muscle sarcoplasmic reticulum (SR). Following incubation of JTC with ATP, a 170,000-Da glycoprotein, a 97,500-Da protein (glycogen phosphorylase), and a 55,000-60,000-Da doublet (containing calmodulin-dependent protein kinase subunit) underwent phosphorylation. Addition of calmodulin in the presence of Ca2+ (with no added protein kinase) produced a 10-fold increase of phosphorylation involving numerous JTC proteins, including the large (approximately 450,000 Da) ryanodine receptor protein. Calmodulin-dependent phosphorylation of the ryanodine receptor protein was unambiguously demonstrated by Western blot analysis. The specificity of these findings was demonstrated by much lower levels of calmodulin-dependent phosphorylation in light SR as compared to JTC, and by much lower cyclic AMP dependent kinase activity in both JTC and light SR. These observations indicate that the purified JTC contain membrane-bound calmodulin-dependent protein kinase that undergoes autophosphorylation and catalyzes phosphorylation of various membrane proteins. Protein dephosphorylation was very slow in the absence of added phosphatases, but was accelerated by the addition of phosphatase 1 and 2A (catalytic subunit) in the absence of Ca2+, and calcineurin in the presence of Ca2+. Therefore, in the muscle fiber, dephosphorylation of SR proteins relies on cytoplasmic phosphatases. No significant effect of protein phosphorylation was detected on the Ca2(+)-induced Ca2+ release exhibited by isolated JTC vesicles. However, the selective and prominent association of calmodulin-dependent protein kinase and related substrates with junctional membranes, its Ca2+ sensitivity, and its close proximity to the ryanodine and dihydropyridine receptor Ca2+ channels suggest that this phosphorylation system is involved in regulation of functions linked to these structures.

Dual regulation of Ca2+/calmodulin-dependent kinase II activity by membrane voltage and by calcium influx.

“…The unique spatial pattern of CaMKII activation fits nicely with the identified functional role ofCaMKII in relating the sarcolemmal Ca2+ channels that we demonstrated. Biochemical studies in other tissues also suggest that the intracellular CaMKII distribution may be related to its target proteins in a tissue-specific manner (21,22). The profile of CaMKII activation under different experimental conditions is, as discussed above, qualitatively in agreement with the electrophysiological data on the CaMKII-mediated change inIca and is generally consistent with the biochemical results obtained from neural tissues (23,24).…”

Colocalization of the Dihydropyridine Receptor, the Plasma‐Membrane Calcium ATPase Isoform 1 and the Sodium/Calcium Exchanger to the Junctional‐Membrane Domain of Transverse Tubules of Rabbit Skeletal Muscle

“…The isolated terminal cisternae (the R 4 fraction from Saito's gradient) should be expected to contain about 10 % of junctional-transverse-tubule contaminants, whereas, on a protein basis (Chu et al, 1990), this fraction was found to contain only trace amounts of the DHP a-I subunit (Fig. 1).…”

Roles of the calcineurin and CaMK signaling pathways in fast-to-slow fiber type transformation of cultured adult mouse skeletal muscle fibers

“…Calcineurin has been shown to respond to the slow fiber-type pattern of electrical stimulation and thus to require elevated [Ca 2ϩ ] i (6,13). In contrast, other studies show that [Ca 2ϩ ] i as low as 100 nM can activate CaMKII and induce phosphorylation of RyR complexes (9,14,17). Furthermore, autophosphorylation of CaMKII in the presence of Ca 2ϩ -CaM can cause it to remain active independent of Ca 2ϩ -CaM for a long period (5,22).…”