The regulatory β subunit of phosphorylase kinase interacts with glyceraldehyde-3-phosphate dehydrogenase

Skeletal muscle phosphorylase kinase (PhK) is an (alpha beta gamma delta)(4) hetero-oligomeric enzyme complex that phosphorylates and activates glycogen phosphorylase b (GPb) in a Ca2+-dependent reaction that couples muscle contraction with glycogen breakdown. GPb 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-P-32]ATP with or without Call 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 Ca2+-dependent substrate of PhK, although the rate of phosphorylation is very slow. GAPDH does, however, bind tightly to PhK, inhibiting at low concentrations (IC50 similar to 0.45 mu M) PhK's conversion of GPb. When a short synthetic peptide substrate was substituted for GPb, 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 alpha gamma delta subcomplex, was unable to phosphorylate GAPDH, even though it is catalytically active toward GPb. Moreover, GAPDH had no effect on the conversion of GPb by the alpha gamma delta 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.