Protein phosphatase type-1 and glycogen bind to a domain in the skeletal muscle regulatory subunit containing conserved hydrophobic sequence motif.
This study identifies a 100-residue domain within the rabbit skeletal muscle regulatory subunit (PP1G) that binds both type-1 protein phosphatase (PP1C) and glycogen. An N-terminal portion of PP1G was cloned by RT-PCR, and different sized fragments were expressed in bacteria as glutathione S-transferase (GST) fusion proteins. A GST-PP1G fusion containing residues 51-240 bound both PPIC and glycogen, whereas GST alone or fusions containing residues 51-140 or 241-360 bound neither PP1C nor glycogen. The PPIC in whole cell lysates or partially purified PP1C from skeletal muscle, or a complex of PP1C-MCLR-biotin, all bound more effectively than Mn(2+)-activated, recombinant PP1C purified from bacteria. Binding was enhanced by increasing the ionic strength and was disrupted by ethylene glycol, consistent with hydrophobic interactions being critical for stable association. Phosphorylation of the GST-PP1G fusion by cAMP-dependent protein kinase prevented completely association of PP1C. This domain of PP1G, from residues 141-240, contains two sequence motifs of hydrophobic residues: Gx8FEKx10W and DxFxFxIxL, that are conserved among the known glycogen-binding PP1 regulatory subunits. These segments are predicted to form an alpha helix and a beta sheet, and we propose that they are the sites for association with PP1C and glycogen, respectively.
Wu
J
J
Center for Cell Signaling, University of Virginia, Charlottesville 22908, USA.
Kleiner
U
U
Brautigan
D L
DL
D016475:3T3 Cells / Q000737:chemistry; D000818:Animals; D015153:Blotting, Western; D002457:Cell Extracts / Q000494:pharmacology; D003001:Cloning, Molecular; D017124:Conserved Sequence / Q000235:genetics*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006003:Glycogen / Q000378:metabolism*; D051379:Mice; D018482:Muscle, Skeletal / Q000737:chemistry; D009994:Osmolar Concentration; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D054645:Protein Phosphatase 1; D017433:Protein Structure, Secondary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014461:Ultracentrifugation
D002457:Cell Extracts; D011993:Recombinant Fusion Proteins; D006003:Glycogen; D017868:Cyclic AMP-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1