Caveolin-1 functions as a scaffolding protein for phosphofructokinase in the metabolic organization of vascular smooth muscle.
Using confocal microscopy, we have demonstrated a similar distribution of phosphofructokinase (PFK) with caveolin-1 (CAV-1) mainly at the periphery (membrane) in freshly isolated vascular smooth muscle (VSM) cells and in cultured A7r5 VSM cells. Co-immunoprecipitation analysis validated the interaction between the proteins. To further test the hypothesis that PFK and CAV-1 are colocalized, we used small interfering RNA (siRNA) to downregulate CAV-1 expression and disrupt the protein-protein interactions between PFK and CAV-1. Transfection of cultured A7r5 cells with CAV-1 siRNA resulted in a decreased level of immunoreactive CAV-1 and a consequent shift in the distribution of PFK with less localization of PFK to the periphery of the cells and increased immunoreactivity at the perinuclear region as compared to control. Analysis of the average PFK intensity across cultured A7r5 cells demonstrated a higher central:peripheral intensity ratio (CPI ratio) in siRNA-treated cells than in the control. These results validate the possible role of CAV-1 as a scaffolding protein for PFK as evidenced by the significant redistribution of PFK after CAV-1 downregulation. We therefore conclude that CAV-1 may function as a scaffolding protein for PFK and that this contributes to the compartmentation of glycolysis from other metabolic pathways in VSM.
Vallejo
Johana
J
Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, Missouri 65212, USA.
Hardin
Christopher D
CD
D000818:Animals; D051242:Caveolin 1; D022461:Caveolins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D025221:Phosphofructokinases / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D014162:Transfection
C495537:Cav1 protein, rat; D051242:Caveolin 1; D022461:Caveolins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D025221:Phosphofructokinases