Effector-sensitive cross-linking of phosphorylase b kinase by the novel cross-linker 4-phenyl-1,2,4-triazoline-3,5-dione

Ayers, Nancy A.; Nadeau, Owen W.; Read, Mark W.; Rây, Prafulla Chandra; Carlson, Gerald M.

doi:10.1042/bj3310137

Cited by 21 publications

(19 citation statements)

References 37 publications

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“…From this it was concluded that, even though ''the activated conformation induced by Ca 2+ shares structural features in common with conformations of PhK induced by other activators, it also has features that are clearly distinct'' (Nadeau et al 1997, 1999). That conclusion is consistent with the results observed using other chemical cross-linkers as conformational probes (Nadeau et al 1997;Ayers et al 1998), with the results herein, and with the fact that, no matter how it is otherwise activated, PhK still requires Ca 2+ for catalytic activity. The deprotonation event(s) leading to a less negative zeta potential (Fig.…”

Section: +supporting

confidence: 82%

Physicochemical changes in phosphorylase kinase associated with its activation

2008

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Phosphorylase kinase (PhK) regulates glycogenolysis through its Ca 2+ -dependent phosphorylation and activation of glycogen phosphorylase. The activity of PhK increases dramatically as the pH is raised from 6.8 to 8.2 (denoted as [pH), but Ca 2+ dependence is retained. Little is known about the structural changes associated with PhK's activation by [pH and Ca 2+ , but activation by both mechanisms is mediated through regulatory subunits of the (abgd) 4 PhK complex. In this study, changes in the structure of PhK induced by [pH and Ca 2+ were investigated using second derivative UV absorption, synchronous fluorescence, circular dichroism spectroscopy, and zeta potential analyses. The joint effects of Ca 2+ and [pH on the physicochemical properties of PhK were found to be interdependent, with their effects showing a strong inflection point at pH ;7.6. Comparing the properties of the conformers of PhK present under the condition where it would be least active (pH 6.8 À Ca 2+ ) versus that where it would be most active (pH 8.2 + Ca 2+ ), the joint activation by [pH and Ca 2+ is characterized by a relatively large increase in the content of sheet structure, a decrease in interactions between helix and sheet structures, and a dramatically less negative electrostatic surface charge. A model is presented that accounts for the interdependent activating effects of [pH and Ca 2+ in terms of the overall physicochemical properties of the four PhK conformers described herein, and published data corroborating the transitions between these conformers are tabulated.Keywords: phosphorylase kinase; Ca 2+ ; Tyr environment; secondary structure; tertiary structure; surface electrostatics; zeta potential Phosphorylase kinase (PhK), a complex regulatory enzyme in the cascade activation of glycogenolysis, is the only known kinase to phosphorylate and activate glycogen phosphorylase. By far, the most is known about the PhK from skeletal muscle (for reviews, see Heilmeyer Jr. 1991; Brushia and Walsh 1999), and that is the subject of this study. This PhK is a 1.3-MDa hexadecameric complex composed of four copies each of a single catalytic protein kinase subunit (g, 44.7 kDa) and three different regulatory subunits (a, 138.4 kDa; b, 125.2 kDa; and d, 17.7 kDa). Thus, its regulatory subunits account for 86% of the mass of the (abgd) 4 PhK complex. The a and b subunits are homologous and inhibitory, whereas the d subunits, which are endogenous molecules of calmodulin, are stimulatory. It is through allosteric sites on these regulatory subunits that PhK integrates metabolic (ADP), hormonal (cAMP and Ca 2+ ), and neural (Ca 2+ ) signals to control the rate of glycogen breakdown through large increases in the kinase activity of its catalytic g subunits. Ca 2+ ions presumably exert their stimulatory effect through binding to the d subunits, which in muscle couples contraction with energy production to supply

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Section: +supporting

confidence: 82%

Physicochemical changes in phosphorylase kinase associated with its activation

2008

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“…It should be noted that assessment of the influence of these serine residues, or their mutants, on β-β interactions as measured in two-hybrid assays may not fully correspond to their role when they are part of the entire PhK complex, in that the remaining α, γ and δ subunits have all been shown to influence the structure of the β subunits. 19,34,38,39 Regardless, the overall results from two-hybrid screens of truncation or mutation constructs of the N terminus of β indicate that this region of the subunit inhibits its self-association and suggest that perturbations in the structure of this region attenuate that inhibition. That Nβ1, but not Nβ2, inhibits PhK A and perturbs crosslinking of β-γ and β-β dimers by GMBS and DFDNB, respectively, suggests that the inhibitory …”

Section: Resultsmentioning

confidence: 89%

Evidence for the Location of the Allosteric Activation Switch in the Multisubunit Phosphorylase Kinase Complex from Mass Spectrometric Identification of Chemically Crosslinked Peptides

Nadeau

Anderson

Yang

et al. 2007

Journal of Molecular Biology

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Phosphorylase kinase (PhK), an (alphabetagammadelta)(4) complex, regulates glycogenolysis. Its activity, catalyzed by the gamma subunit, is tightly controlled by phosphorylation and activators acting through allosteric sites on its regulatory alpha, beta and delta subunits. Activation by phosphorylation is predominantly mediated by the regulatory beta subunit, which undergoes a conformational change that is structurally linked with the gamma subunit and that is characterized by the ability of a short chemical crosslinker to form beta-beta dimers. To determine potential regions of interaction of the beta and gamma subunits, we have used chemical crosslinking and two-hybrid screening. The beta and gamma subunits were crosslinked to each other in phosphorylated PhK, and crosslinked peptides from digests were identified by Fourier transform mass spectrometry, beginning with a search engine developed "in house" that generates a hypothetical list of crosslinked peptides. A conjugate between beta and gamma that was verified by MS/MS corresponded to crosslinking between K303 in the C-terminal regulatory domain of gamma (gammaCRD) and R18 in the N-terminal regulatory region of beta (beta1-31), which contains the phosphorylatable serines 11 and 26. A synthetic peptide corresponding to residues 1-22 of beta inhibited the crosslinking between beta and gamma, and was itself crosslinked to K303 of gamma. In two-hybrid screening, the beta1-31 region controlled beta subunit self-interactions, in that they were favored by truncation of this region or by mutation of the phosphorylatable serines 11 and 26, thus providing structural evidence for a phosphorylation-dependent subunit communication network in the PhK complex involving at least these two regulatory regions of the beta and gamma subunits. The sum of our results considered together with previous findings implicates the gammaCRD as being an allosteric activation switch in PhK that interacts with all three of the enzyme's regulatory subunits and is proximal to the active site cleft.

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“…Using various cross-linkers as structural probes of the hexadecamer, the ␣ subunit forms cross-linked complexes with the catalytic ␥ subunit, and these conjugates are substantially increased upon activation of the enzyme (30,41). One region of ␣ cross-linked to the ␥ subunit has been delineated to lie C-terminal to residue 1015 (41), which overlaps the region containing the second association domain identified herein.…”

Section: Discussionmentioning

confidence: 99%

“…Potential ␣-␣ interactions have not been previously observed with the nonactivated conformer of the PhK holoenzyme. We selected chemical crosslinking at the nonactivating pH of 6.8 as the method to probe for these interactions, because the unambiguous identification of cross-linked conjugates is now possible using PhK subunitspecific mAbs (30). From screening a variety of bifunctional reagents, the heterobifunctional cross-linker sulfo-GMBS was observed to form an ␣-␣ dimer (268.5 kDa, 3.0% error) (Fig.…”

Section: -1237mentioning

confidence: 99%

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

Ayers¹,

Wilkinson²,

Fitzgerald³

et al. 1999

Journal of Biological Chemistry

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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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Effector-sensitive cross-linking of phosphorylase b kinase by the novel cross-linker 4-phenyl-1,2,4-triazoline-3,5-dione

Cited by 21 publications

References 37 publications

Physicochemical changes in phosphorylase kinase associated with its activation

Physicochemical changes in phosphorylase kinase associated with its activation

Evidence for the Location of the Allosteric Activation Switch in the Multisubunit Phosphorylase Kinase Complex from Mass Spectrometric Identification of Chemically Crosslinked Peptides

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

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