Abstract
The skeletal muscle specific Ca(2)+/calmodulin-dependent protein kinase (CaMKIIbeta(M)) is localized to the sarcoplasmic reticulum (SR) by an anchoring protein, alphaKAP, but its function remains to be defined. Protein interactions of CaMKIIbeta(M) indicated that it exists in complex with enzymes involved in glycolysis at the SR membrane. The kinase was found to complex with glycogen phosphorylase, glycogen debranching enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and creatine kinase in the SR membrane. CaMKIIbeta(M) was also found to assemble with aldolase A, GAPDH, enolase, lactate dehydrogenase, creatine kinase, pyruvate kinase, and phosphorylase b kinase from the cytosolic fraction. The interacting proteins were substrates of CaMKIIbeta(M), and their phosphorylation was enhanced in a Ca(2+)- and calmodulin (CaM)-dependent manner. The CaMKIIbeta(M) could directly phosphorylate GAPDH and markedly increase ( approximately 3.4-fold) its activity in a Ca(2+)/CaM-dependent manner. These data suggest that the muscle CaMKIIbeta(M) isoform may serve to assemble the glycogen-mobilizing and glycolytic enzymes at the SR membrane and specifically modulate the activity of GAPDH in response to calcium signaling. Thus, the activation of CaMKIIbeta(M) in response to calcium signaling would serve to modulate GAPDH and thereby ATP and NADH levels at the SR membrane, which in turn will regulate calcium transport processes.
Highlights
Allosterically activates numerous proteins, including Ca2ϩ/CaMdependent protein kinase II (CaMKII) [1]
A number of the interacting proteins were phosphorylated in a Ca2ϩ/CaM-dependent manner in cytosolic (Fig. 3, panel b) and sarcoplasmic reticulum (SR) membrane (Fig. 3, panel c) fractions of skeletal muscle tissue. These substrate proteins correspond to the polypeptides of ϳ55, 47, 44, 39, and 36 kDa from the cytosol (Fig. 3, panel b), which were identified as pyruvate kinase, enolase, creatine kinase, aldolase, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively, and the ϳ97, 61, and 36-kDa polypeptides from the SR membrane extract (Fig. 3, panel c), which correspond to glycogen phosphorylase b, CaMKII, and GAPDH, respectively
A CaMKII isoform referred to as a muscle-specific CaMKIIM was shown to be targeted to the SR membrane in skeletal muscle by a non-kinase protein, ␣-KAP [11, 12], but the role of this kinase in SR function remains unknown
Summary
CaM, calmodulin; CaM kinase, calcium calmodulin-dependent protein kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SR, sarcoplasmic reticulum; MALDI-TOF, matrix-assisted laser desorption ionization/time-of-flight; RyR, ryanodine receptor; GST, glutathione S-transferase; PBS, phosphate-buffered saline; PBST, PBS with Tween 20; MOPS, 4-morpholinepropanesulfonic acid; GAP, glyceraldehyde 3-phosphate; LDH, lactate dehydrogenase; TBS, Tris-buffered saline; PGK, 3-phosphoglycerate kinase; PVDF, polyvinylidene difluoride. There is clear evidence that the RyR and calcium pump are regulated by local ATP, Ca2ϩ, and CaM through direct ligand binding [15,16,17] In this regard, both Ca2ϩ and CaM are present at the SR, and the level of ATP is believed to be tightly controlled through a membrane-bound glycolytic machinery involving phosphorylase b, creatine kinase, and GAPDH (18 –20). In view of the previous studies that implicate a role for local ATP and NADH in the regulation of SR function and excitation-contraction coupling, our results suggest that. The membrane-bound CaMKIIM may be important for targeting the glycolytic machinery to the SR and modulate the local levels of NADH and ATP in response to calcium signaling in skeletal muscle
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