A conserved domain for glycogen binding in protein phosphatase‐1 targeting subunits

FEBS Letters

Brautigan, David L¹  Wu, Jun¹  Thompson, Irene¹  Shenolikar, Shirish²  Oliver, Carey J²  Liu, Jun¹ 

[1] Center for Cell Signaling, University of Virginia, Health Sciences Center, Box 577, Charlottesville, VA 22908, USA;Department of Pharmacology and Cancer Biology, Duke University Medical Center, Box 3813, Durham, NC 27710, USA

关键词: Protein sequence;    Evolution;    Hydrophobic residue;    Starch binding;    GM;    the skeletal muscle glycogen targeting subunit for PP1C (also called RGL);    GL;    the liver glycogen targeting subunit for PP1C;    PTG;    protein targeting to glycogen subunit of PP1C;    R5 and U5;    targeting subunits of PP1C;    PP1Cα;    the α isoform of the type-1 protein phosphatase catalytic subunit;    GST;    glutathione S-transferase;    GST-GM(1–240);    a fusion of GST to the first 240 residues of the GM subunit;    GST-R5;    a fusion of GST to full length R5;    GST-VFV(156–223);    a fusion of GST to residues 156–223 of R5;   

DOI  :  10.1016/S0014-5793(98)01371-4

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The skeletal muscle glycogen-binding subunit (G_M) 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 G_M, the liver subunit G_L, 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 G_M (GST-G_M(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-G_M(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-G_M 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.

【 授权许可】

Unknown