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

Jun Liu, Jun Wu, Carey Oliver, Shirish Shenolikar, David L. Brautigan

Research output: Contribution to journalArticlepeer-review

27 Scopus citations


Cellular 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 (G(M)). We reported previously that a recombinant fusion protein of glutathione S-transferase (GST) and the N-terminal domain of G(M) [GST-G(M)(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 RVS67F motif in GST-G(M)(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-G(M)(1-240) gave a protein that bound PP1 the same as wild type and was unaffected by PKA phosphorylation. Full length G(M) 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-G(M)(S67T) was not disrupted by forskolin treatment. Neither FLAG-G(M)(S67A) nor FLAG-G(M)(S67V) formed stable complexes with PP1 in COS7 cells. These results emphasise the unique contribution of Ser-67 in PP1 binding to G(M). The constitutive PP1-binding activity shown by G(M)(S67T) opens the way for studying the role of G(M) multisite phosphorylation in hormonal control of glycogen metabolism.

Original languageEnglish (US)
Pages (from-to)77-82
Number of pages6
JournalBiochemical Journal
Issue number1
StatePublished - Feb 15 2000


  • Forskolin
  • PP1 binding protein
  • RVXF binding site
  • cAMP-dependent protein kinase

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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