Searching for binding partners for the phosphorylase kinase variant PhK γ181

Abstract

Phosphorylase kinase (Phk), a key regulatory enzyme in glycogenolysis, is a hexadecameric protein that contains 4 different subunits with a stoichiometry (αβλδ)4. Alpha and βare regulatory subunits, γis catalytic and d is an intrinsic molecule of calmodulin. The gene which encodes for the γ subunit, PHKG1, undergoes alternative processing to produce a truncated form of γcontaining 181 residues (γ181) instead of the normal 387 residues. Although much of the kinase helical domain is absent, the truncated form is still catalytic with its activity being influenced by PKC, and its mRNA is expressed in high levels in brain. A natural substrate for γ181 has not yet been identified. In the present study, we are using the LexA based yeast two‐hybrid system to identify binding partners for γ181. Gamma181 was cloned into the pLexA vector and used as bait to screen a human fetal brain cDNA library. Interactions between γ181 and cDNA library proteins were monitored by reporter genes. Characterization of the bait, LexA‐γ181, shows that it is efficiently localized to the nucleus and does not activate the reporter genes. The cDNA library was amplified, titered, and then transformed into EGY48 yeast cells for screening. Following screening for leucine prototrophy and β‐galactosidase activity, we obtained 36 colonies that show positive interactions for leucine prototrophy although only 11 of these colonies are β‐galactosidase positive. Positive clones are currently being sequenced for identification. The discovery of novel binding partners for γ181 is critical in determining the physiological role of this unique kinase in brain.The work was supported in part by NIH grant P20RR16481.