Self-association of the α Subunit of Phosphorylase Kinase as Determined by Two-hybrid Screening

Abstract

The structural organization of the (␣␤␥␦) 4 phosphorylase kinase complex has been studied using the yeast two-hybrid screen for the purpose of elucidating regions of ␣ subunit interactions. By screening a rabbit skeletal muscle cDNA library with residues 1-1059 of the ␣ subunit of phosphorylase kinase, we have isolated 16 interacting, independent, yet overlapping transcripts of the ␣ subunit containing its C-terminal region. Domain mapping of binary interactions between ␣ constructs revealed two regions involved in the self-association of the ␣ subunit: residues 833-854, a previously unrecognized leucine zipper, and an unspecified region 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 domains contained within the latter two, overlapping regions may be further narrowed to the stretches from 1057 to 1237 and from 864 to 971. Cross-linking of the nonactivated holoenzyme with N-(␥-maleimidobutyroxy)sulfosuccinimide ester produced intramolecularly cross-linked ␣-␣ dimers, consistent with portions of two ␣ subunits in the holoenyzme being in sufficient proximity to associate. This is the first report to identify potential areas of contact between the ␣ subunits of phosphorylase kinase. Additionally, issues regarding the general utility of twohybrid screening as a method for studying homodimeric interactions are discussed.Phosphorylase b kinase (PhK) 1 is among the largest and most complex of the protein kinases, with a mass of 1.3 ϫ 10 6 Da and a subunit stoichiometry of (␣␤␥␦) 4 (reviewed in Refs. 1 and 2). The catalytic ␥ subunit of this complex oligomer is allosterically controlled through alterations in quaternary structure initiated by the regulatory ␣, ␤, and ␦ subunits, the latter being an intrinsic molecule of calmodulin (3). Studies have shown that activation of PhK by multiple effectors occurs concomitantly with common conformational changes in the ␣ and ␤ subunits (4 -6); moreover, the activity of free ␥ subunit is inhibited by the ␣ and ␤ subunits (7,8). It has been proposed that the ␣ and ␤ subunits of PhK impose steric constraint upon ␥ that, when removed, leads to activation of the kinase (7-10).We have chosen to study the largest of the regulatory subunits, ␣, because relatively few studies have been devoted to understanding its role in the structure and function of PhK. Previous studies using electron microscopy and immunoelectron microscopy have suggested that the ␣ subunit is peripherally located in the holoenzyme (11, 12), which is consistent with its high susceptibility to proteolysis by a wide variety of proteases (5). Based upon the probable exposed location of ␣ within the PhK hexadecamer, we hypothesized that this subunit may interact with other muscle proteins and investigated potential PhK ␣ interactions by screening a skeletal muscle cDNA library for potential binding partners of ␣ using the yeast two-hybrid system.The two-hybr...

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“…The most straightforward explanation for those two-hybrid results would be that the full-length wild type ␤ subunit is unable to self-associate because its N terminus interacts with its C terminus, thus blocking self-association of the latter. Such a conclusion could not be reached from two-hybrid data, however, because observed homodimeric interactions could represent either intrasubunit or intersubunit interactions, which would be indistinguishable unless a single small region selfassociated (47). The possibility of intersubunit interactions is of special concern with PhK because ␤␤ dimers can be readily formed in activated PhK by a short cross-linker (42).…”

Amino Acid Substitutions of Coiled-Coil Protein Tpr Abrogate Anchorage to the Nuclear Pore Complex but Not Parallel, In-Register Homodimerization

“…However, whether these regions may reflect preferential assembly sites for Tpr monomers remains uncertain because yeast reporter gene activity is not necessarily equally proportional to the actual interactions between different pairs of fusion proteins. It can be modulated by various factors, including fusion protein stability and nuclear import and sterical constraints (Ye and Worman, 1995;Ayers et al, 1999).…”

The Calmodulin-binding Domain of the Catalytic γ Subunit of Phosphorylase Kinase Interacts with Its Inhibitory α Subunit

“…Protein expression of all ␣ and ␥ constructs was verified by Western analysis, with minor modification, as previously described (24) using either a DNA BD crossreactive LexA polyclonal Ab (kindly provided by Dr. Erica Golemis, Fox Chase Cancer Center) or an AD cross-reactive hemagglutinin mAb (Roche Molecular Biochemicals). Positive associations between ␣ and ␥ constructs were monitored by transcriptional activation of the LEU2 gene by growth on defined media lacking leucine (Leu Ϫ ) and of the lacZ reporter gene by liquid ␤-galactosidase assays using o-nitrophenyl galactopyranoside as substrate (22,25).…”

“…Two-hybrid Plasmid Construction-All PhK ␣ constructs were engineered as previously described (22). Rabbit skeletal muscle PhK ␥ cDNA was kindly provided by Dr. Donald J. Graves (Iowa State University) and used as the template for the preparation of all ␥ constructs.…”

“…Two-hybrid Library Screening-A rabbit skeletal muscle cDNA library (22) was used to screen for interactors of PhK␥ by using ␥300C and ␥FL constructs (described above) as bait. Yeast library transformants containing either of the DNA-BD bait plasmids and the AD cDNA library were generated as previously described (22).…”