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The Regulatory alpha Subunit of Phosphorylase Kinase May Directly Participate in the Binding of Glycogen Phosphorylase

I. E. Andreeva1,2*, N. A. Rice3,4, and G. M. Carlson3

1Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky pr. 33, Moscow, 117071 Russia; fax: (095) 954-2732; E-mail: i_andreeva@yahoo.com

2Current address: Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030 USA; fax: (713) 798-3475; E-mail: andreeva@bcm.tmc.edu

3Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, Missouri 64110-2499 USA; fax: (816) 235-5595; E-mail: carlsonGM@umkc.edu

4Current address: Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, 80309 USA; E-mail: ayersn@stripe.colorado.edu

* To whom correspondence should be addressed.

Received April 17, 2002; Revision received June 5, 2002
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.
KEY WORDS: glycogen phosphorylase b, phosphorylase kinase, yeast two-hybrid system, protein-protein interaction