Dual function of calmodulin (δ) in phosphorylase kinase

Hessová, Zuzana; Varsányi, Magdolna; Heilmeyer, Ludwig

doi:10.1111/j.1432-1033.1985.tb08626.x

Cited by 34 publications

(25 citation statements)

References 42 publications

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“…4A). These results suggest that heparin, which also promotes dissociation of the ␦ subunit (30), activates the enzyme through a different mechanism than ADP, GDP, or phosphorylation.…”

Section: Activators Of Pbk Perturb Its Cross-linking Pattern-be-mentioning

confidence: 92%

Differential Affinity Cross-linking of Phosphorylase Kinase Conformers by the Geometric Isomers of Phenylenedimaleimide

Nadeau

Sacks

Carlson

1997

Journal of Biological Chemistry

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Phosphorylase b kinase (PbK) from skeletal muscle is a highly regulated oligomer consisting of four copies of four distinct subunits (␣␤␥)␦ 4 . The ␥ subunit is catalytic, and the remaining subunits are regulatory. To characterize effector-induced changes in the quaternary structure of the enzyme, we utilized the ortho-, meta, and para-isomers of phenylenedimaleimide (PDM), which in addition to having different geometries, also vary 2.5-fold in their cross-linking spans. Even at concentrations equivalent to the ␣␤␥␦ protomers of PbK, all three isomers caused specific, rapid, and extensive cross-linking of the holoenzyme to form primarily ␣␤ dimers, plus smaller amounts of ␤␥␥ and ␣␥␥ trimers. The formation of these three conjugates was nearly totally inhibited by a 10-fold molar excess over PDM of N-(o-and p-tolyl)succinimide, which are chemically inert structural analogs of PDM. This inhibition suggests that PbK has binding sites for PDM and that PDM acts as an affinity crosslinker in binding to these sites prior to forming crosslinked conjugates. The largest effect on cross-linking in progressing from o-to p-PDM was on the ␣␥␥ trimer, which is preferentially formed by the p-isomer. Activation of the enzyme by either phosphorylation or the allosteric activators ADP and GDP resulted in large increases in the amount of ␣␥␥ formed, small increases in ␤␥␥, and little change in ␣␤. When cross-linked in the presence of the reversibly activating nucleoside diphosphates, PbK remained activated after their removal, indicating that cross-linking had locked it in the active conformation. Our results provide direct evidence for perturbations in the interactions of the catalytic ␥ subunit with the regulatory ␣ and ␤ subunits upon activation of PbK.Through allosteric and covalent modification sites on its three regulatory subunits, phosphorylase b kinase (PbK) 1 integrates neural, hormonal, and metabolic signals to modulate glycogenolytic flux in skeletal muscle (for review, see Refs. 1 and 2). Although the ␣, ␤, and ␦ (calmodulin) regulatory subunits clearly control the activity of the catalytic ␥ subunit, little is known concerning the mechanisms through which they exert this control, including the extent to which their regulatory influence on ␥ is direct versus indirect; this is especially the case for the larger regulatory subunits, ␣ and ␤. Phosphorylation (3) or proteolysis (4) of ␣ causes increased activity of ␥ within the (␣␤␥␦) 4 holoenzyme, but evidence for a direct ␣-␥ interaction that is altered by this activation has not been observed. Likewise, multiple means of activating the holoenzyme cause common conformational changes in the ␤ subunit (5-7); but again, no evidence for alteration of a direct ␤-␥ interaction has been observed. In two previous studies, cross-linking was used successfully to detect changes in the cross-linking patterns of both the ␣ (8) and ␤ (5) subunits upon activation of the holoenzyme; but with the cross-linkers used, the observed changes involved only a second ␣ or ␤ subunit to form homod...

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“…4A). These results suggest that heparin, which also promotes dissociation of the ␦ subunit (30), activates the enzyme through a different mechanism than ADP, GDP, or phosphorylation.…”

Section: Activators Of Pbk Perturb Its Cross-linking Pattern-be-mentioning

confidence: 92%

Differential Affinity Cross-linking of Phosphorylase Kinase Conformers by the Geometric Isomers of Phenylenedimaleimide

Nadeau

Sacks

Carlson

1997

Journal of Biological Chemistry

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“…For example, the stable, native (␣␤␥␦) 4 PhK hexadecamer is capable of further associating through uncharacterized interactions to form even larger complexes (10,11,33,34). Consequently, a positive, nonartifactual, homodimeric interaction between truncated ␣ constructs in the two-hybrid system could potentially represent, disregarding interacting leucine zippers, any of three different possibilities with respect to the native protein: (a) intrasubunit interactions that would normally stabilize that subunit's tertiary structure; (b) intersubunit, intramolecular interactions stabilizing the (␣␤␥␦) 4 parent molecule's quaternary structure; or (c) intersubunit, intermolecular interactions between (␣␤␥␦) 4 molecules.…”

Section: Discussionmentioning

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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“…A search for other common parts of ATP-binding sites in a shows that indeed most of these subdomains can be detected (segments 11 -VII in Table 2). (A); the other part was diluted in 1 mM sodium glycerol 2-phosphate, 0.75 mM dithioerythritol, 100 mM ammonium chloride, 0.01% sodium azide, 5 mg/ml bovine serum albumin (predialyzed against magnesium acctate as described in [16]) for assaying A. activity (B). Conditions for A2 and A0 activity tests were chosen as in [I61 with the exception of varying the ATP concentration in the presence of 4 mM M g z + for both partial activities A2 and A.…”

Section: Discussionmentioning

confidence: 99%

“…Activity assays of phosphorylase kinase were carried out on an autoanalyzer according to Jennissen and Heilmeyer [29]. The conditions for testing the partial activities Az, Al and A. of phosphorylase kinase are described in detail by HessovL et al [16].…”

Section: Methodsmentioning

confidence: 99%

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Reaction of fluorescein isothiocyanate with an ATP‐binding site on the phosphorylase kinase α subunit

Zaman

Varsányi

Heilmeyerm

et al. 1989

European Journal of Biochemistry

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Phosphorylase kinase can be labeled specifically on the c1 subunit with fluorescein 5'-isothiocyanate (FITC) which concomitantly inactivates the enzyme (T. G. Sotiroudis and S. Nikolaropoulus (1984) FEBS Lett. 176, 421 -4251. Labeled peptides have been purified and their primary structure has been determined. The amino acid sequence of the fluorescein-labeled tryptic peptide is Lys-Met-Gln-Asp-Gly-Tyr-Phe-Gly-Gly-Ala-Arg. The environment of this fluorescein-labeled lysine has been determined by sequencing peptides isolated from a Staphylococcus aureus V8 digest and two further cyanogen bromide fragments of the purified ['4C]carboxymethylated u subunit. The partial sequences obtained have then been localized in the primary structure of the CI subunit Proc. Nut1 Acad. Sci. USA 85,2929-29331. Both the incorporation of the fluorescent label and enzymatic inactivation are inhibited by ATP only at pH 7.0; ADP and AMP do not protect. Kinetic analysis reveals a competition between ATP and FITC; a Ki for ATP of 728 100 pM has been determined.Phosphorylase kinase (ATP : phosphorylase phosphotransferase, EC 2.7.1.38) is a key regulatory enzyme in glycogen metabolism which catalyzes the phosphorylation of phosphorylase b to make phosphorylase a (for review see [l] ADP has been demonstrated to be an activator of phosphorylase kinase [18]. This nucleotide binds probably to the fl subunit; however, the structure of its binding domain is still unknown.Corresporzdenct. to L.

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Dual function of calmodulin (δ) in phosphorylase kinase

Cited by 34 publications

References 42 publications

Differential Affinity Cross-linking of Phosphorylase Kinase Conformers by the Geometric Isomers of Phenylenedimaleimide

Differential Affinity Cross-linking of Phosphorylase Kinase Conformers by the Geometric Isomers of Phenylenedimaleimide

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

Reaction of fluorescein isothiocyanate with an ATP‐binding site on the phosphorylase kinase α subunit

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