The Muscle-specific Calmodulin-dependent Protein Kinase Assembles with the Glycolytic Enzyme Complex at the Sarcoplasmic Reticulum and Modulates the Activity of Glyceraldehyde-3-phosphate Dehydrogenase in a Ca2+/Calmodulin-dependent Manner

Abstract

The skeletal muscle specific Ca 2؉ /calmodulin-dependent protein kinase (CaMKII␤ M ) is localized to the sarcoplasmic reticulum (SR) by an anchoring protein, ␣KAP, but its function remains to be defined. Protein interactions of CaMKII␤ 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. CaMKII␤ 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 CaMKII␤ M , and their phosphorylation was enhanced in a Ca 2؉ -and calmodulin (CaM)-dependent manner. The CaMKII␤ M could directly phosphorylate GAPDH and markedly increase (ϳ3.4-fold) its activity in a Ca 2؉ /CaMdependent manner. These data suggest that the muscle CaMKII␤ M isoform may serve to assemble the glycogenmobilizing and glycolytic enzymes at the SR membrane and specifically modulate the activity of GAPDH in response to calcium signaling. Thus, the activation of CaMKII␤ 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.Free calcium (Ca 2ϩ ) regulates diverse cellular functions by acting as an intracellular second messenger. A large part of these cellular functions are mediated by CaM, 1 which is the ubiquitous intracellular Ca 2ϩ receptor. The Ca 2ϩ ⅐CaM complex allosterically activates numerous proteins, including Ca 2ϩ /CaMdependent protein kinase II (CaMKII) (1). CaMKII is a multifunctional enzyme that is highly expressed in brain and muscle. The kinase is believed to serve important roles in synaptic transmission (2, 3), gene transcription (4, 5), cell growth (6), and control of excitation-contraction coupling (7-9).The subcellular distribution of CaMKII indicates cytosolic and membrane localizations in different tissues (10). In skeletal muscle, an isoform of CaMKII is targeted to the SR membrane by a non-kinase protein, ␣KAP (11, 12). Different studies have been conducted to determine whether membrane-bound CaM kinase could phosphorylate different substrate proteins by virtue of its proximity effects and thereby regulate SR function. Although the calcium release channel/ryanodine receptor (RyR) and the calcium pump/Ca 2ϩ -ATPase in skeletal muscle SR were shown to be substrates of CaMKII␤ (13, 14), there does not appear to be any clear effects on the regulation of functional activity of these proteins induced by such phosphorylation (7,8,(15)(16)(17). Moreover, there is clear evidence that the RyR and calcium pump are regulated by local ATP, Ca 2ϩ , and CaM through direct ligand binding (15)(16)(17). In this regard, both Ca 2ϩ and CaM are present at the SR, and the level of ATP is believed to be tightly controlled through a membrane-bound glycolytic mac...

Energy Management by Enhanced Glycolysis in G1-phase in Human Colon Cancer Cells In Vitro and In Vivo

“…After sonication for 4 separate periods of 30 seconds at 200 W on ice, the suspensions were centrifuged at 20,000 Â g for 10 minutes, and the supernatants were saved. Activities of phosphofructokinase (PFK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and pyruvate kinase (PK) were determined by monitoring changes in absorbance at 340 nm as described previously (5,30,31).…”

α-Kinase Anchoring Protein αKAP Interacts with SERCA2A to Spatially Position Ca2+/Calmodulin-dependent Protein Kinase II and Modulate Phospholamban Phosphorylation

“…Data also suggest that ␣KAP along with the novel CaMKII␤ 4 are enriched in cardiac SR membranes, implying a common regulatory role for these molecules in these two muscle types (13)(14)(15). Further, studies suggest that CaMKII␤ 4 can recruit the glycolytic machinery to the SR membrane in cardiac and skeletal muscle and potentially serve to spatially modulate the supply of ATP for the calcium transport process (13,14). In view of the emerging concept of spatial and temporal control of signal transduction through kinase-anchoring proteins, we investigated further the role of ␣KAP at the SR membrane and found that it directly interacts with the calcium ATPase and serves to recruit CaMKII isoforms and modulate the phosphorylation of PLN at Thr-17, which is known to critically regulate calcium uptake and muscle relaxation (18).…”

“…Data also suggest that ␣KAP along with the novel CaMKII␤ 4 are enriched in cardiac SR membranes implying a common regulatory role for these molecules in these two muscle types (13)(14)(15). Further, studies suggest a significant level of a muscle-specific CaMKII ␤ isoform (CaMKII␤ 4 ) in cardiac and skeletal muscle (14 -16).…”

“…Data also suggest that ␣KAP along with the novel CaMKII␤ 4 are enriched in cardiac SR membranes, implying a common regulatory role for these molecules in these two muscle types (13)(14)(15). Further, studies suggest that CaMKII␤ 4 can recruit the glycolytic machinery to the SR membrane in cardiac and skeletal muscle and potentially serve to spatially modulate the supply of ATP for the calcium transport process (13,14).…”

“…Although four different isoforms of CaMKII (␣, ␤, ␦, and ␥) are expressed in a tissuespecific manner, cardiac tissue is shown to have predominance of CaMKII␦ C (cytosolic) and CaMKII␦ B (nuclear) isoforms, which serve roles in excitation-contraction coupling and cell growth, respectively (7,12). Studies have also revealed a significant level of a muscle-specific CaMKII ␤ isoform (CaMKII␤ 4 ) in skeletal and cardiac muscle (11,(13)(14)(15). In addition, the CAMK2A gene that encodes CaMKII␣ kinase in brain expresses an alternatively spliced non-kinase polypeptide designated ␣KAP in cardiac and skeletal muscle (14 -16).…”