The cytoskeletal organizing protein Cdc42-interacting protein 4 associates with phosphorylase kinase in skeletal muscle.
Phosphorylase kinase is a key enzyme in regulating glycogenolytic flux in skeletal muscle in response to changing energy demands. In the present study, we sought to identify interacting proteins of phosphorylase kinase by yeast two-hybrid screening. Screening a rabbit skeletal muscle cDNA library with the exposed C-terminus of the alpha subunit (residues 1060-1237), we identified eight independent, yet overlapping, constructs of cdc42-interacting protein 4 (CIP4). Immunocytochemistry indicated that CIP4 colocalized with phosphorylase kinase in vivo, and the cognate binding domain on CIP4 was determined to lie between residues 398 and 545. While this region of CIP4 does contain a known src homology 3 domain, transient transfections and coimmunoprecipitation experiments showed that this domain is not responsible for the dimeric interaction. Based upon sequence analysis the association is inferred to be mediated by two proline-rich sequences in CIP4, residues 436-439 and 441-444, that bind to a cognate WW domain found between residues 1107 and 1129 of PhKalpha.
Archila
Soleil
S
Department of Biology, Western Kentucky University, Bowling Green, 42101-1080, USA.
King
Mark A
MA
Carlson
Gerald M
GM
Rice
Nancy A
NA
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D006801:Humans; D047468:Immunoprecipitation; D007763:Lac Operon / Q000235:genetics; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D015778:Minor Histocompatibility Antigens; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism*; D010764:Phosphorylase Kinase / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D020798:Two-Hybrid System Techniques; D015003:Yeasts / Q000235:genetics; D001616:beta-Galactosidase / Q000378:metabolism; D018909:src Homology Domains / Q000235:genetics
D008869:Microtubule-Associated Proteins; D015778:Minor Histocompatibility Antigens; D021122:Protein Subunits; C485787:Trip10 protein, mouse; D010764:Phosphorylase Kinase; D001616:beta-Galactosidase