A CONSERVED DOMAIN FOR GLYCOGEN BINDING IN PROTEIN PHOSPHATASE-1 TARGETING SUBUNITS

The skeletal muscle glycogen-binding subunit (G(M)) of protein phospha tase-1 (PP1) is the founding member of a family of proteins that tethe r the PP1 catalytic subunit (PP1C) to glycogen and promote the dephosp horylation of glycogen synthase. A hydrophobic sequence (called here t he VFV motif) is conserved among G(M), the liver subunit G(L), and the widely expressed subunits, PTG, R5 and U5. This study analyzed the ro le of this VFV motif in binding to glycogen and PP1C. Glutathione S-tr ansferase (GST) fusions with the N-terminal domain of G(M) (GST-G(M(1- 240))) and with the full length R5 protein (GST-RS) both bound to glyc ogen in a co-sedimentation assay. In contrast, GST itself did not bind to glycogen. A single residue substitution in GST-G(M(1-240)), F155A, reduced glycogen binding by 40%. Double residue substitutions V150A/F 155A and F155A/V159A resulted in greater reductions (60-70%) in glycog en binding, showing these hydrophobic residues influenced the protein- glycogen interaction. The wild type and V150A/F155A fusion proteins we re digested by trypsin into the same sized fragments at the same rate. Furthermore, the wild type and mutated GST-G(M) proteins as well as G ST-R5 bound equivalent amounts of PP1C, in either pull-down or far-Wes tern 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 bi nding when fused to GST. This motif, that is in bacterial and fungal s tarch metabolizing enzymes, probably has been conserved during evoluti on as a functional domain for binding glycogen and starch. (C) 1998 Fe deration of European Biochemical Societies.