Self-association of the alpha subunit of phosphorylase kinase as determined by two-hybrid screening.
The structural organization of the (alphabetagammadelta)(4) phosphorylase 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 subunit of phosphorylase kinase, we have isolated 16 interacting, independent, yet overlapping transcripts of the alpha subunit containing its C-terminal region. Domain mapping of binary interactions between alpha constructs revealed two regions involved in the self-association of the alpha 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-(gamma-maleimidobutyroxy)sulfosuccin-imide ester produced intramolecularly cross-linked alpha-alpha dimers, consistent with portions of two alpha subunits in the holoenyzme being in sufficient proximity to associate. This is the first report to identify potential areas of contact between the alpha subunits of phosphorylase kinase. Additionally, issues regarding the general utility of two-hybrid screening as a method for studying homodimeric interactions are discussed.
Ayers
N A
NA
Division of Molecular Biology, School of Biological Sciences, University of Missouri, Kansas City, Missouri 64110-2499, USA.
Wilkinson
D A
DA
Fitzgerald
T J
TJ
Carlson
G M
GM
D000595:Amino Acid Sequence; D000818:Animals; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018076:DNA, Complementary; D015723:Gene Library; D016350:Leucine Zippers; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology*; D016296:Mutagenesis; D010446:Peptide Fragments / Q000737:chemistry; D010764:Phosphorylase Kinase / Q000737:chemistry* / Q000378:metabolism*; D055503:Protein Multimerization; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques
D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D010764:Phosphorylase Kinase