Physicochemical properties of rabbit skeletal muscle phosphorylase kinase

Hayakawa, Taro; Perkins, John P.; Walsh, Donal A.; Krebs, Edwin G.

doi:10.1021/bi00728a001

Cited by 179 publications

(44 citation statements)

References 24 publications

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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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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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“…Skeletal muscle phosphorylase kinase is formed from three types of polypeptide chain, termed a, fl and y [3,4], the smallest active species of the enzyme (molecular weight 1 280000) having the structure (a/3y)4 [4,5]. When the enzyme is incubated with cyclic-AMP-dependent protein kinase, cyclic AMP, magnesium ions and [ Y -~~P I A T P , 32P-radioactivity becomes attached covalently to both the a and Abbreviation.…”

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confidence: 99%

Separation of Two Phosphorylase Kinase Phosphatases from Rabbit Skeletal Muscle

1976

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The dephosphorylation of phosphorylase kinase has been shown to be catalysed by two distinct enzymes, termed a-phosphorylase kinase phosphatase and P-phosphorylase kinase phosphatase.These two enzymes show essentially absolute specificity towards the a and p subunits respectively.The two phosphatases copurified through ethanol fractionation, DEAE-cellulose chromatography and ammonium sulphate precipitation, but were separated from each other by gel filtration on Sephadex G-200. a-Phosphorylase kinase phosphatase was purified 500-fold from the ethanol precipitation step, and p-phosphorylase kinase phosphatase 320-fold. The molecular weights estimated by gel filtration were 170-180000 for a-phosphorylase kinase phosphatase and 75-80000 for Bphosphorylase kinase phosphatase.Since the activity of phosphorylase kinase correlates with the state of phosphorylation of the p subunit (Cohen, P. (1974)), p-phosphorylase kinase phosphatase is the enzyme which reverses the activation of phosphorylase kinase. a-Phosphorylase kinase phosphatase is an enzyme activity that has not been recognised previously. Since the role of the a-subunit phosphorylation is to stimulate the rate of dephosphorylation of the /3 subunit (Cohen, P. (1974)), a-phosphorylase kinase phosphatase can be regarded as the enzyme which inhibits the reversal of the activation of phosphorylase kinase. The implications of these findings for the hormonal control of phosphorylase kinase activity by multisite phosphorylation are discussed.

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“…The reaction was carried out at 300 for 20 min. cAMP formed in the assay system was chromatographically isolated and was estimated by the protein binding assay of Gilman (6) mediately into a beaker containing 200 ml of cold 10% trichloroacetic acid (7). Following two rinses with the same acid and two rinses with methanol, the radioactivity of the filter paper was estimated in a Packard Tri-Carb liquid scintillation spectrometer (model 3390) in 10 ml of scintillation medium with toluene-Triton X-100 as solvent (8).…”

Section: Methodsmentioning

confidence: 99%