A conserved domain for glycogen binding in protein phosphatase-1 targeting subunits.
The skeletal muscle glycogen-binding subunit (GM) of protein phosphatase-1 (PP1) is the founding member of a family of proteins that tether the PP1 catalytic subunit (PP1C) to glycogen and promote the dephosphorylation of glycogen synthase. A hydrophobic sequence (called here the VFV motif) is conserved among GM, the liver subunit GL, and the widely expressed subunits, PTG, R5 and U5. This study analyzed the role of this VFV motif in binding to glycogen and PP1C. Glutathione S-transferase (GST) fusions with the N-terminal domain of GM (GST-GM(1-240)) and with the full length R5 protein (GST-R5) both bound to glycogen in a co-sedimentation assay. In contrast, GST itself did not bind to glycogen. A single residue substitution in GST-GM(1-240), F155A, reduced glycogen binding by 40%. Double residue substitutions V150A/F155A and F155A/V159A resulted in greater reductions (60-70%) in glycogen binding, showing these hydrophobic residues influenced the protein-glycogen interaction. The wild type and V150A/ F155A fusion proteins were digested by trypsin into the same sized fragments at the same rate. Furthermore, the wild type and mutated GST-GM proteins as well as GST-R5 bound equivalent amounts of PP1C, in either pull-down or far-Western assays. These results demonstrated retention of overall tertiary structure by the mutated fusion proteins, and indicated that glycogen and PP1C binding are independent of one another. A 68 residue segment of R5 encompassing the VFV motif was sufficient to produce glycogen binding when fused to GST. This motif, that is in bacterial and fungal starch metabolizing enzymes, probably has been conserved during evolution as a functional domain for binding glycogen and starch.
Wu
J
J
Center for Cell Signaling, University of Virginia, Health Sciences Center, Charlottesville 22908, USA.
Liu
J
J
Thompson
I
I
Oliver
C J
CJ
Shenolikar
S
S
Brautigan
D L
DL
D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D017124:Conserved Sequence; D005982:Glutathione Transferase / Q000378:metabolism; D006003:Glycogen / Q000378:metabolism*; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology* / Q000378:metabolism; D009154:Mutation; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D054645:Protein Phosphatase 1; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D011993:Recombinant Fusion Proteins; D006003:Glycogen; D005982:Glutathione Transferase; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1