The Regulatory β Subunit of Phosphorylase Kinase Interacts with Glyceraldehyde‐3‐phosphate Dehydrogenase

Abstract

Skeletal muscle phosphorylase kinase (PhK), a Ca2+-dependent (αβγδ)4 complex, phosphorylates glycogen phosphorylase (GP), its only known in vivo substrate. Given PhK's size and complexity, we screened muscle extracts for other potential targets. Extracts of P/J (control) and I/lnJ (PhK deficient) mice were incubated with [γ-32P]ATP ± Ca2+ and compared to identify potential substrates. Candidate targets were resolved by 2D-PAGE and phosphorylated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified by MALDI-MS. In vitro studies showed GAPDH to be a Ca2+-dependent substrate of PhK, although the rate and stoichiometry of phosphorylation were low. GAPDH did, however, inhibit at low concentrations (IC50 = 0.45μM) PhK's conversion of GP. When a short synthetic peptide substrate was substituted for GP, the inhibition was negligible, suggesting that GAPDH may inhibit predominantly by binding to PhK at a locus distinct from the active site on the γ subunit. To test this notion, the PhK/GAPDH complex was incubated with chemical crosslinkers and crosslinking of the regulatory β subunit of PhK to GAPDH was shown. This interaction was confirmed by the finding that GAPDH had no effect on GP conversion by a subcomplex of PhK missing the β subunit, an αγδ trimer. Interactions between PhK and GAPDH provide a possible mechanism for linking glycogenolysis and glycolysis in muscle. (Supported by NIH grant DK32953).