Association of Phosphofructokinase-M with Caveolin-3 in Differentiated Skeletal Myotubes

Abstract

Caveolin-3 is a member of the caveolin family of proteins that is primarily expressed in striated muscle cell types (skeletal and cardiac). Here, we show that an approximately 80-kDa protein specifically co-immunoprecipitates with caveolin-3 expressed in differentiated skeletal C2C12 myotubes. Microsequence analysis of this approximately 80-kDa polypeptide revealed its identity as a key regulatory enzyme in the glycolytic pathway, namely phosphofructokinase-M (PFK-M). Pulse-chase experiments demonstrate that PFK-M associates with caveolin-3 with a significant time lag after the biosynthesis of PFK-M. In addition, we show that this interaction is (i) highly regulated by the extracellular concentration of glucose and (ii) can be stabilized by a number of relevant intracellular metabolites, such as fructose 1,6-bisphosphate and fructose 2,6-bisphosphate, which are known allosteric activators of PFK. While the bulk of these experiments were performed in C2C12 cells, identical results were obtained using mouse skeletal muscle extracts. Taken together, our results suggest that glucose-dependent plasma membrane recruitment of activated PFK-M by caveolin-3 could have important implications for understanding the mechanisms that regulate energy metabolism in skeletal muscle fibers.

Metabolic organization in vascular smooth muscle: distribution and localization of caveolin-1 and phosphofructokinase

“…Although these results provide additional clear evidence of some interaction between the proteins, the nature of the interaction between these proteins remains to be elucidated, because PFK could be bound to caveolin directly or through an accessory protein. Moreover, our studies are consistent with studies in differentiated myotubes that have demonstrated, under certain metabolic conditions, that PFK-M forms a stable complex with CAV-3 (50). Therefore, our immunoprecipitation results may represent direct interaction, as has been shown in myotubes, or indirect associations via an intermediary protein.…”

“…Recent studies demonstrated the differential targeting of ␤-adrenergic receptor subtypes to cardiomyocyte caveolae (48,54). Caveolae have also been reported to contain proteins related to glucose metabolism, including PFK (35,50). Therefore, association of caveolin-1 with specific glycolytic enzymes may provide part of the physical basis for compartmentation of glycolysis from related metabolic pathways.…”

Caveolinopathies: from the biology of caveolin-3 to human diseases

“…Indeed, Cav-3 regulates the expression of the insulin receptor on muscle membrane, it modulates the translocation upon insulin induction of the glucose transporter 4, and is required for the cell membrane targeting of phosphofructokinase (PFK), an enzyme that catalyzes a rate-limiting reaction in glycolysis. [23][24][25] Finally, caveolae facilitates free fatty cellular uptake by interacting with several fatty acid transport proteins. 26 In vitro Cav-3 functional characterization determined that this molecule regulates myoblast cell differentiation and survival.…”

Apolipoprotein AII Enrichment of HDL Enhances Their Affinity for Class B Type I Scavenger Receptor but Inhibits Specific Cholesteryl Ester Uptake

“…After washing, cells were incubated for 1 hour at 4°C in HEPES buffer (10 mmol/L HEPES and 150 mmol/L NaCl, pH 8.0) containing 0.5 mmol/L dithio-bis(succinimidyl propionate) (DSP). 30 Cell membranes were prepared as described earlier and analyzed by SDS-PAGE and immunoblotting. After transfer onto nitrocellulose sheets, the SR-BI/ apoAI complexes were revealed either with the monoclonal antibodies against apoAI, or with the polyclonal antibodies against SR-BI.…”