Mutations of the serine phosphorylated in the protein phosphatase-1-binding motif in the skeletal muscle glycogen-targeting subunit

摘要

pCellular functions of protein phosphatase-1 (PP1) are determined by regulatory subunits that contain the consensus PP1-binding motif, RVXF. This motif was first identified as the site of phosphorylation by cAMP-dependent protein kinase (PKA) in a skeletal muscle glycogen-targeting subunit (GsubM/sub). We reported previously that a recombinant fusion protein of glutathione S-transferase (GST) and the N-terminal domain of GsubM/sub [GST-GsubM/sub-(1-240)] bound PP1 in a pull down assay, and phosphorylation by PKA prevented PP1 binding. Here we report that substitution of either Ala or Val for Ser-67 in the RVSsup67/supF motif in GST-GsubM/sub-(1-240) essentially eliminated PP1 binding. This was unexpected because other glycogen-targeting subunits have a Val residue at the position corresponding to Ser-67. In contrast, a mutation of Ser-67 to Thr (S67T) in GST-GsubM/sub(1-240) gave a protein that bound PP1 the same as wild type and was unaffected by PKA phosphorylation. Full length GsubM/sub tagged with the epitope sequence DYKDDDDK (FLAG) expressed in COS7 cells bound PP1 that was recovered by co-immunoprecipitation, but this association was prevented by treatment of the cells with forskolin. By comparison, PP1 binding with FLAG-GsubM/sub(S67T) was not disrupted by forskolin treatment. Neither FLAG-GsubM/sub(S67A) nor FLAG-GsubM/sub(S67V) formed stable complexes with PP1 in COS7 cells. These results emphasise the unique contribution of Ser-67 in PP1 binding to GsubM/sub. The constitutive PP1-binding activity shown by GsubM/sub(S67T) opens the way for studying the role of GsubM/sub multisite phosphorylation in hormonal control of glycogen metabolism./p