The regulatory alpha subunit of phosphorylase kinase may directly participate in the binding of glycogen phosphorylase.
The yeast two-hybrid screen has been used to identify potential regions of interaction of the largest regulatory subunit, alpha, of phosphorylase kinase (PhK) with two fragments of its protein substrate, glycogen phosphorylase b (Phb). One fragment, corresponding to residues 17-484 (PhbN'), contained the regulatory domain of the protein, but in missing the first 16 residues was devoid of the sole phosphorylation site of Phb, Ser14; the second fragment corresponded to residues 485-843 (PhbC) and contained the catalytic domain of Phb. Truncation fragments of the alpha subunit were screened for interactions against these two substrate fragments. PhbC was not found to interact with any alpha constructs; however, PhbN' interacted with a region of alpha (residues 864-1014) that is near the phosphorylatable region of that subunit. PhbN' was also screened for interactions against a variety of fragments of the catalytic gamma subunit of PhK; however, no interactions were detected, even with full-length gamma. Our results support the idea that amino acid residues proximal to the convertible serine of Phb are important for its specific interaction with the catalytic subunit of PhK, but that regions distinct from the convertible serine residue of Phb and from the catalytic domain of PhK may also be involved in the interaction of these two proteins.
Andreeva
I E
IE
Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, 117071 Russia. i_andreeva@yahoo.com
Rice
N A
NA
Carlson
G M
GM
D020134:Catalytic Domain; D024981:Glycogen Phosphorylase / Q000378:metabolism*; D010764:Phosphorylase Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010763:Phosphorylase b / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism*; D013379:Substrate Specificity; D020798:Two-Hybrid System Techniques
D021122:Protein Subunits; D024981:Glycogen Phosphorylase; D010763:Phosphorylase b; D010764:Phosphorylase Kinase