title: Evidence for the location of the allosteric activation switch in the multisubunit phosphorylase kinase complex from mass spectrometric identification of chemically crosslinked peptides.
abstract: Phosphorylase kinase (PhK), an (alphabetagammadelta)(4) complex, regulates glycogenolysis. Its activity, catalyzed by the gamma subunit, is tightly controlled by phosphorylation and activators acting through allosteric sites on its regulatory alpha, beta and delta subunits. Activation by phosphorylation is predominantly mediated by the regulatory beta subunit, which undergoes a conformational change that is structurally linked with the gamma subunit and that is characterized by the ability of a short chemical crosslinker to form beta-beta dimers. To determine potential regions of interaction of the beta and gamma subunits, we have used chemical crosslinking and two-hybrid screening. The beta and gamma subunits were crosslinked to each other in phosphorylated PhK, and crosslinked peptides from digests were identified by Fourier transform mass spectrometry, beginning with a search engine developed "in house" that generates a hypothetical list of crosslinked peptides. A conjugate between beta and gamma that was verified by MS/MS corresponded to crosslinking between K303 in the C-terminal regulatory domain of gamma (gammaCRD) and R18 in the N-terminal regulatory region of beta (beta1-31), which contains the phosphorylatable serines 11 and 26. A synthetic peptide corresponding to residues 1-22 of beta inhibited the crosslinking between beta and gamma, and was itself crosslinked to K303 of gamma. In two-hybrid screening, the beta1-31 region controlled beta subunit self-interactions, in that they were favored by truncation of this region or by mutation of the phosphorylatable serines 11 and 26, thus providing structural evidence for a phosphorylation-dependent subunit communication network in the PhK complex involving at least these two regulatory regions of the beta and gamma subunits. The sum of our results considered together with previous findings implicates the gammaCRD as being an allosteric activation switch in PhK that interacts with all three of the enzyme's regulatory subunits and is proximal to the active site cleft.
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Kansas Medical Center, Kansas City, KA 66209, USA.
authors.forename: Owen W
authors.initials: OW
authors.lastname: Nadeau
authors.forename: David W
authors.initials: DW
authors.lastname: Anderson
authors.forename: Qing
authors.initials: Q
authors.lastname: Yang
authors.forename: Antonio
authors.initials: A
authors.lastname: Artigues
authors.forename: Justin E
authors.initials: JE
authors.lastname: Paschall
authors.forename: Gerald J
authors.initials: GJ
authors.lastname: Wyckoff
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: McClintock
authors.forename: Gerald M
authors.initials: GM
authors.lastname: Carlson
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects*; D000495:Allosteric Site / Q000187:drug effects*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D013058:Mass Spectrometry / Q000379:methods*; D008954:Models, Biological; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D010764:Phosphorylase Kinase / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D017354:Point Mutation / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D025941:Protein Interaction Mapping; D020836:Protein Structure, Quaternary / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D021122:Protein Subunits / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits; D017384:Sequence Deletion / Q000235:genetics; D040681:Structural Homology, Protein; D013388:Succinimides / Q000494:pharmacology
chemical_list: D000596:Amino Acids; D003432:Cross-Linking Reagents; D050505:Mutant Proteins; D010455:Peptides; D021122:Protein Subunits; D013388:Succinimides; D010768:Phosphoserine; C032138:N-(gamma-maleimidobutyryloxy)succinimide; D010764:Phosphorylase Kinase