The regulatory beta subunit of phosphorylase kinase interacts with glyceraldehyde-3-phosphate dehydrogenase.
Skeletal muscle phosphorylase kinase (PhK) is an (alphabetagammadelta) 4 hetero-oligomeric enzyme complex that phosphorylates and activates glycogen phosphorylase b (GP b) in a Ca (2+)-dependent reaction that couples muscle contraction with glycogen breakdown. GP b is PhK's only known in vivo substrate; however, given the great size and multiple subunits of the PhK complex, we screened muscle extracts for other potential targets. Extracts of P/J (control) and I/lnJ (PhK deficient) mice were incubated with [gamma- (32)P]ATP with or without Ca (2+) and compared to identify potential substrates. Candidate targets were resolved by two-dimensional polyacrylamide gel electrophoresis, and phosphorylated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified by matrix-assisted laser desorption ionization mass spectroscopy. In vitro studies showed GAPDH to be a Ca (2+)-dependent substrate of PhK, although the rate of phosphorylation is very slow. GAPDH does, however, bind tightly to PhK, inhibiting at low concentrations (IC 50 approximately 0.45 microM) PhK's conversion of GP b. When a short synthetic peptide substrate was substituted for GP b, the inhibition was negligible, suggesting that GAPDH may inhibit predominantly by binding to the PhK complex at a locus distinct from its active site on the gamma subunit. To test this notion, the PhK-GAPDH complex was incubated with a chemical cross-linker, and a dimer between the regulatory beta subunit of PhK and GAPDH was formed. This interaction was confirmed by the fact that a subcomplex of PhK missing the beta subunit, specifically an alphagammadelta subcomplex, was unable to phosphorylate GAPDH, even though it is catalytically active toward GP b. Moreover, GAPDH had no effect on the conversion of GP b by the alphagammadelta subcomplex. The interactions described herein between the beta subunit of PhK and GAPDH provide a possible mechanism for the direct linkage of glycogenolysis and glycolysis in skeletal muscle.
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160, USA.
Igor G
IG
Boulatnikov
Owen W
OW
Nadeau
Patrick J
PJ
Daniels
Jessica M
JM
Sage
Marina D
MD
Jeyasingham
Maria T
MT
Villar
Antonio
A
Artigues
Gerald M
GM
Carlson
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004800:Enzymes, Immobilized / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D051379:Mice; D018482:Muscle, Skeletal / Q000187:drug effects / Q000201:enzymology; D010764:Phosphorylase Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D021122:Protein Subunits / Q000378:metabolism*; D011817:Rabbits; D013388:Succinimides / Q000494:pharmacology; D014020:Tissue Extracts
D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D021122:Protein Subunits; D013388:Succinimides; D014020:Tissue Extracts; C032138:N-(gamma-maleimidobutyryloxy)succinimide; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010764:Phosphorylase Kinase; D002118:Calcium