Expression of a cDNA for the catalytic subunit of skeletal-muscle phosphorylase kinase in transfected 3T3 cells.
Phosphorylase kinase is a multimeric enzyme of composition (alpha, beta, gamma, delta)4 whose catalytic activity resides in the gamma-subunit. As an approach to understand further its regulation, a cDNA for the gamma-subunit of phosphorylase kinase (gamma PhK) has been cloned into a mammalian expression vector behind the mouse metallothionein-1 promoter. NIH 3T3 cells were co-transfected with this construct (pEV gamma PhK) and pSV2neo, G418-resistant clones were selected, and several were found to have stably incorporated the gamma-subunit cDNA into their genomic DNA. Phosphorylase kinase activity was clearly present in extracts from cultures of pEV gamma PhK-transformed cells and increased several-fold after 24 h of incubation with Zn2+, whereas it was undetectable in the parent 3T3 cells. A significant, but variable, proportion (15-70%) of the activity was Ca2+-dependent. We conclude that the phosphorylase kinase activity expressed by the cells transformed with pEV gamma PhK is due to free gamma-subunit and gamma-subunit associated with cellular calmodulin, which replaces the delta-subunit normally associated with the gamma-subunit in the holoenzyme.
Cawley
K C
KC
Department of Biological Chemistry, School of Medicine, University of California, Davis 95616.
Akita
C G
CG
Walsh
D A
DA
D000818:Animals; D015152:Blotting, Northern; D002384:Catalysis; D002478:Cells, Cultured; D002999:Clone Cells; D004247:DNA / Q000235:genetics*; D004790:Enzyme Induction; D015870:Gene Expression; D009132:Muscles / Q000201:enzymology*; D010764:Phosphorylase Kinase / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids; D006656:Protamine Kinase / Q000378:metabolism; D012313:RNA / Q000032:analysis / Q000302:isolation & purification; D051381:Rats; D014162:Transfection
D012313:RNA; D004247:DNA; D010764:Phosphorylase Kinase; D006656:Protamine Kinase