Abstract
Chemical cross-linking as a probe of conformation has consistently shown that activators, including Ca(2+) ions, of the (alphabetagammadelta)(4) phosphorylase kinase holoenzyme (PhK) alter the interactions between its regulatory alpha and catalytic gamma subunits. The gamma subunit is also known to interact with the delta subunit, an endogenous molecule of calmodulin that mediates the activation of PhK by Ca(2+) ions. In this study, we have used two-hybrid screening and chemical cross-linking to dissect the regulatory quaternary interactions involving these subunits. The yeast two-hybrid system indicated that regions near the C termini of the gamma (residues 343-386) and alpha (residues 1060-1237) subunits interact. The association of this region of alpha with gamma was corroborated by the isolation of a cross-linked fragment of alpha containing residues 1015-1237 from an alpha-gamma dimer that had been formed within the PhK holoenzyme by formaldehyde, a nearly zero-length cross-linker. Because the region of gamma that we found to interact with alpha has previously been shown to contain a high affinity binding site for calmodulin (Dasgupta, M., Honeycutt, T., and Blumenthal, D. K. (1989) J. Biol. Chem. 264, 17156-17163), we tested the influence of Ca(2+) on the conformation of the alpha subunit and found that the region of alpha that interacts with gamma was, in fact, perturbed by Ca(2+). The results herein support the existence of a Ca(2+)-sensitive communication network among the delta, gamma, and alpha subunits, with the regulatory domain of gamma being the primary mediator. The similarity of such a Ca(2+)-dependent network to the interactions among troponin C, troponin I, and actin is discussed in light of the known structural and functional similarities between troponin I and the gamma subunit of PhK.
Highlights
Chemical cross-linking as a probe of conformation has consistently shown that activators, including Ca2؉ ions, of the (␣␥␦)4 phosphorylase kinase holoenzyme (PhK) alter the interactions between its regulatory ␣ and catalytic ␥ subunits
The ␥ subunit is known to interact with the ␦ subunit, an endogenous molecule of calmodulin that mediates the activation of PhK by Ca2؉ ions
The C Terminus of PhK ␣ Interacts with the Regulatory Domain of ␥ in the Yeast Two-hybrid System—Our laboratory has previously demonstrated the formation of ␣-␥ complexes through chemical cross-linking of the PhK holoenzyme (4 –7); relatively long cross-linkers were used in those studies, and as a result, it was not unequivocally established that the ␣ and ␥ subunits within the hexadecameric holoenzyme interact, as opposed to being proximal
Summary
PhK, phosphorylase b kinase; CaM, calmodulin; P-b, glycogen phosphorylase-b; BSA, bovine serum albumin; mAb(s), monoclonal antibody(ies); BD, binding domain; AD, actiand most complex enzymes known. Despite the increased availability of structural information regarding PhK, interactions associated with activation and involving specific regions of individual subunits, in particular the ␣ and  subunits, have largely remained uncharacterized. There have been many studies on the activation of PhK by Ca2ϩ, it has not been clear how the binding of Ca2ϩ to the ␦ subunit (CaM) relays structural information to the remainder of the holoenzyme, especially to the ␣ subunit. Our findings reported suggest that the flow of structural information from ␦ to ␣ in the holoenzyme is directly mediated by the C-terminal regulatory domain of ␥ These are the first results to define a specific region of ␥ that interacts with any region of either the ␣ or  subunits of PhK. The finding that the region of ␣-␥ interaction includes a portion of the CaM binding domain of ␥ provides a possible explanation for the previously observed changes in ␣-␥ interactions induced by Ca2ϩ
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