The selective phosphorylation of glycogen phosphorylase (GP) by its only known kinase, phosphorylase kinase (PhK), keeps glycogen catabolism tightly regulated. In addition to the obligatory interaction between the catalytic gamma subunit of PhK and the phosphorylatable region of GP, previous studies have suggested additional sites of interaction between this kinase and its protein substrate. Using short chemical crosslinkers, we have identified direct interactions of GP with the large regulatory alpha and beta subunits of PhK. These newfound interactions were found to be sensitive to ligands that bind PhK. (C) 2016 Elsevier Inc. All rights reserved.