Self-association of the alpha subunit of phosphorylase kinase as determined by two-hybrid screening

The structural organization of the (alpha beta gamma delta)(4) phosphorylas e kinase complex has been studied using the yeast two-hybrid screen for the purpose of elucidating regions of alpha subunit interactions. By screening a rabbit skeletal muscle cDNA library with residues 1-1059 of the alpha su bunit of phosphorylase kinase, we have isolated 16 interacting, independent , yet overlapping transcripts of the a subunit containing its C-terminal re gion. Domain mapping of binary interactions between alpha constructs reveal ed two regions involved in the self-association of the alpha subunit: resid ues 833-854, a previously unrecognized leucine zipper, and an unspecified r egion within residues 1015-1237, The cognate binding partner for the latter domain has been inferred to lie within the stretch from residues 864-1059. Indirect evidence from the literature suggests that the interacting domain s contained within the latter two, overlapping regions may be further narro wed to the stretches from 1057 to 1237 and from 864 to 971. Cross-linking o f the nonactivated holoenzyme with N-(gamma-maleimidobutyroxy) sulfosuccini mide ester produced intramolecularly cross-linked alpha-alpha dimers, consi stent with portions of two alpha subunits in the holoenyzme being in suffic ient proximity to associate. This is the first report to identify potential areas of contact between the alpha subunits of phosphorylase kinase. Addit ionally, issues regarding the general utility of two-hybrid screening as a method for studying homodimeric interactions are discussed.