Abstract
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.
Highlights
Phosphorylase b kinase (PhK)1 is among the largest and most complex of the protein kinases, with a mass of 1.3 ϫ 106 Da and a subunit stoichiometry of (␣␥␦
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
Given that the ␣ subunit is farnesylated at Cys-1234 [26], potentially directing it to membranes, we chose to use only amino acids 1–1059 of ␣ fused to the LexA DNA binding domain (BD) protein (␣1059C-BD) as a molecular bait for screening a rabbit skeletal muscle cDNA library, in order to improve the chances for nuclear import of the bait
Summary
PhK, phosphorylase b kinase; BD, binding domain; AD, activation domain; pLexA, a 2 HIS3 plasmid containing the DNA binding domain of LexA; pB42AD, a 2 TRP1 plasmid containing a B42 activation domain; SD ϪHisϪTrpϪUra, synthetic medium containing 2% glucose and lacking histidine, tryptophan, and uracil; SD Gal/Raf ϪHisϪTrpϪUra, synthetic medium containing 2% galactose and 1% raffinose and lacking histidine, tryptophan, and uracil; mAb, monoclonal antibody; sulfo-GMBS, N-(␥-maleimidobutyroxy)sulfosuccinimide ester; DHFR, dihydrofolate reductase; ELISA, enzyme-linked immunosorbent assay; ␣FL, full-length ␣. 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-hybrid system is based on the modularity of eukaryotic transcription factors possessing functionally separate DNA binding and activation domains that need not be covalently linked to activate transcription (reviewed in Ref. 13). This attribute has been exploited to directly evaluate potential interactions between proteins fused to either DNA binding domain (BD) or activation domain (AD) modules. Cross-linked ␣-␣ dimers have previously been reported to be formed during cross-linking of PhK under conditions in which it is activated [14, 15], the data presented are the first to identify regions of potential ␣-␣ contact
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