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

Abstract

Skeletal muscle phosphorylase kinase (PhK) is an (αβγδ) 4 hetero-oligomeric enzyme complex that phosphorylates and activates glycogen phosphorylase b (GPb) in a Ca 2+ -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 [γ-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 0 .45 µM) 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 γ subunit. To test this notion, the PhK-GAPDH complex was incubated with a chemical cross-linker, and a dimer between the regulatory β subunit of PhK and GAPDH was formed. This interaction was confirmed by the fact that a subcomplex of PhK missing the β subunit, specifically an αγδ 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 αγδ subcomplex. The interactions described herein between the β subunit of PhK and GAPDH provide a possible mechanism for the direct linkage of glycogenolysis and glycolysis in skeletal muscle.Phosphorylase kinase (ATP:phosphorylase b phosphotransferase, EC 2.7.1.38), hereafter termed PhK, 1 is in fast-twitch skeletal muscle a hexadecameric complex with a subunit composition of (αβγδ) 4 and a mass of 1.3 × 10 6 Da (reviewed in ref 1). The activity of its catalytic γ subunit (44.7 kDa) is controlled by its regulatory α (138.4 kDa), β (125.2 kDa),

A Protein Interaction Network for the Large Conductance Ca2+-activated K+ Channel in the Mouse Cochlea

“…GAPDH and GST were verified by reciprocal coIP in our study, whereas the effect of SOD was demonstrated previously with tempol, a SOD mimetic that interacts with BK␣ in CHO cells, causing an increase in peak current (54). GAPDH is involved in oxidative phosphorylation (55), and its activation is one of the primary effects of Ca 2ϩ influx into the mitochondrion (56 -58). Thus, GAPDH gene expression in the Organ of Corti increases during ischemic conditions (59), a likely result of Ca 2ϩ overload (60).…”

In vivo Phosphoproteome of Human Skeletal Muscle Revealed by Phosphopeptide Enrichment and HPLC−ESI−MS/MS

“…57,58 More recently, GAPDH was shown to be a Ca 2+ -dependent substrate of phosphorylase kinase providing a direct link between glycogenolysis and glycolysis in skeletal muscle. 59 Ganon et al also identified 6 phospholabeled glycolytic enzymes (PKM2, ENO3, ALDOA, LDHA, PGM2, and TPI1) in rat skeletal muscle, and demonstrated an age-dependent change in the phospholabeling of the LDHA and ENO3. 38 Our findings are supported by recent phosphoproteomic studies of human cell lines that have documented serine/threonine phosphorylation of several glycolytic enzymes, and in many cases at the same sites as those identified in the present study.…”

“…Our data indicate that under basal conditions, serine/threonine phosphorylation is more frequent than tyrosine phosphorylation of glycolytic enzymes in human skeletal muscle. Only a few earlier studies of mammalian liver and muscle have provided some evidence for serine/threonine phosphorylation of glycolytic enzymes (PKM2, PFKM), and suggested a role for PKA and CaMK in the regulation of these enzymes. , More recently, GAPDH was shown to be a Ca 2+ -dependent substrate of phosphorylase kinase providing a direct link between glycogenolysis and glycolysis in skeletal muscle . Ganon et al also identified 6 phospholabeled glycolytic enzymes (PKM2, ENO3, ALDOA, LDHA, PGM2, and TPI1) in rat skeletal muscle, and demonstrated an age-dependent change in the phospholabeling of the LDHA and ENO3 .…”

Purification and properties of glyceraldehyde-3-phosphate dehydrogenase from the skeletal muscle of the hibernating ground squirrel,Ictidomys tridecemlineatus

“…Bioinformatic analysis of potential phosphorylation motifs within the GAPDH primary amino acid sequence did, however, indicate a phosphorylation motif that matched the insulin receptor kinase conserved motifs, possibly substantiating some link between GAPDH regulation by tyrosine phosphorylation and fuel metabolism ( Appendix S2 ; Obenauer, Cantley & Yaffe, 2003 ). Additionally, GAPDH serine/threonine phosphorylation has also been well established as an important regulatory mechanism ( Kawamoto & Caswell, 1986 ; Reiss et al, 1996 ; Choudhary, De & Banerjee, 2000 ; Tisdale, 2002 ; Singh et al, 2004 ; Tisdale & Artalejo , 2007 ; Boulatnikov et al, 2008 ; Baba et al, 2010 ; Huang et al, 2011 ). Bioinformatic assessment of the potential serine/threonine phosphorylation sites suggest PKC and CaMK as likely modifiers of GAPDH ( Appendix S2 ), which coincides with the results observed in this study ( Fig.…”