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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The catalytic subunit of phosphorylase kinase is composed of a kinase catalytic core domain (residues 1 to 298), which has a 33% identity with the kinase core of the cyclic AMP-dependent protein kinase, and a C-terminal calmodulin binding domain. The kinase domain of the catalytic subunit has been expressed in Escherichia coli, purified and crystallised in the presence of ATP and magnesium from 5% (w/v) polyethylene glycol 8000, 10% (v/v) glycerol, 50 mM Hepes/NaOH (pH 6.9). A three-fold excess of magnesium to ATP was used for crystal growth. The inclusion of glycerol in the crystallization medium produced a marked reduction in mosaic spread of the diffraction spots from greater than 1 degree to 0.3 degree. The crystals are orthorhombic, space group P2(1)2(1)2(1) with unit cell dimensions a = 47.1 A, b = 69.1 A, c = 112.9 A and one molecule per asymmetric unit. Data to 3 A resolution have been collected and structure determination is in progress.
J Mol Biol 1995 Feb 24
PMID:Expression, purification and crystallisation of phosphorylase kinase catalytic domain. 787 61

Holophosphorylase kinase was digested with Glu-C specific protease; from the peptide mixture calmodulin binding peptides were isolated by affinity chromatography and identified by N-terminal sequence analysis. Two peptides originating from the alpha subunit, having a high tendency to form a positively charged amphiphilic helix and containing tryptophane, were synthesized. Additionally, a homologous region of the beta subunit and a peptide from the alpha subunit present in a region deleted in the alpha' isoform were also selected for synthesis. Binding stoichiometry and affinity were determined by following the enhancement in tryptophane fluorescence occurring upon 1:1 complex formation between these peptides and calmodulin. Finally, Ca2+ binding to calmodulin in presence of peptides was measured. By this way, the peptides alpha 542-566, alpha 547-571, alpha 660-677 and beta 597-614 have been found to bind specifically to calmodulin. Together with previously predicted and synthesized calmodulin binding peptides four calmodulin binding regions have been characterized on each the alpha and beta subunits. It can be concluded that endogenous calmodulin can bind to two calmodulin binding regions in gamma as well as to two regions in alpha and beta. Exogenous calmodulin can bind to two regions in alpha and in beta. A binding stoichiometry of 0.8 mol of calmodulin/alpha beta gamma delta promoter of phosphorylase kinase has been determined by inhibiting the ubiquitination of calmodulin with phosphorylase kinase. Phosphorylase kinase is half maximally activated by 23 nM calmodulin which is in the affinity range of calmodulin binding peptides from beta to calmodulin. Therefore, binding of exogenous calmodulin to beta activates the enzyme. A model for switching endogenous calmodulin between alpha, beta and gamma and modulation of ATP binding to alpha as well as Mg2+/ADP binding to beta by calmodulin is presented.
Mol Cell Biochem 1993 Nov
PMID:Interaction sites on phosphorylase kinase for calmodulin. 793 51

Limited proteolysis of rabbit muscle phosphorylase kinase catalyzed by chymotrypsin generates a 33 kD product whose kinase activity is independent of both calcium and pH over the range of 6.8 to 8.3 (Malencik, D.A. & Fischer, E.H. Calcium and Cell Function III: 161-188, 1982). This active preparation consists of three related species containing residues 1-290, 1-296, and 1-298 of the 44.7 kD gamma-subunit of phosphorylase kinase (Harris, W.R., Malencik, D.A., Johnson, C.M., Carr, S.A., Roberts, G.D., Byles, C.E., Anderson, S.R., Heilmeyer, L.M.G., Fischer, E.H. & Crabb, J.W.J. Biol. Chem. 265:11740-11745, 1991). Good recoveries of catalytic activity--with varying degrees of calcium dependence--result upon the digestion of phosphorylase kinase with assorted proteases. However, especially high yields of the chymotryptic fragment are obtainable, with purification on an Ultrogel-34 column and a DEAE Sepharose CL-6B column giving 23% of the maximum possible protein. Physical characterization shows that the 33 kD chymotryptic fragment is globular, with S20,w = 2.9S, and that it has an isoelectric point of 5.3. Our continuous catalytic assay, based on differences in the binding of the fluorescent dye 1-anilinonaphthalene-8-sulfonate by phosphorylase a and b, shows that, on a molar basis, the activity of the fragment is 2.8 fold greater than that of phosphorylase kinase (Malencik, D.A., Zhao, Z. and Anderson, S.R. Biochem. Biophys. Res. Comm. 174: 344-350, 1991). The active fragment also undergoes autophosphorylation. Incubation with Mg[gamma-P32] ATP results in the reaction of 0.7 mol 32P/mol fragment. When the catalytic subunit of the cAMP-dependent protein kinase is also present, the amount of 32P incorporated increases to 1.1 mol/mol. In the former case, phosphorylation occurs primarily at Ser30 while in the latter an additional reaction takes place at Ser81. The phosphopeptides correspond to sequences occurring in the gamma-subunit of phosphorylase kinase.
Mol Cell Biochem 1993 Nov
PMID:Preparation and functional characterization of a catalytically active fragment of phosphorylase kinase. 793 60

Calmodulin-dependent protein kinases such as myosin light chain kinase (MLCK), calmodulin kinase II, and phosphorylase kinase contain specific sequences responsible for binding calmodulin. These regions are known as calmodulin-binding domains and in many cases are contained within sequences that are short enough to be synthesized by solid-phase techniques. The ability to chemically-synthesize target enzyme calmodulin-binding domains has permitted the use of a variety of biophysical techniques to study the interactions between calmodulin and calmodulin-binding domain peptides. The work reviewed here describes the development and characterization of peptides based on the sequence of the calmodulin-binding domain of skeletal muscle myosin light chain kinase which were labeled with the fluorescent reagent, acrylodan. Data are presented demonstrating the use of fluorescently-labeled peptides to study various aspects of calmodulin-peptide interactions including binding affinity, stoichiometry, specificity, changes in peptide conformation, and thermal stability of the peptide-calmodulin complex. These data indicate the peptides exhibit many of the salient features seen with calmodulin-target enzyme interactions. The fluorescently-labeled peptides should thus serve as useful models for studying calmodulin-target enzyme interactions at the molecular level.
Mol Cell Biochem 1993 Nov
PMID:Development and characterization of fluorescently-labeled myosin light chain kinase calmodulin-binding domain peptides. 793 61

A catalytic fragment, gamma 1-298, derived from limited chymotryptic digestion of phosphorylase b kinase (Harris, W.R. et al., J. Biol. Chem., 265: 11740-11745, 1990), is reported to have about six-fold greater specific activity than does the gamma subunit-calmodulin complex. To test whether there is an inhibitory domain located outside the catalytic core of the gamma subunit, full-length wild-type and seven truncated forms of gamma were expressed in E. coli. Recombinant proteins accumulate in the inclusion bodies and can be isolated, solubilized, renatured, and purified further by ammonium sulfate precipitation and Q-Sepharose column. Four out of seven truncated mutants show similar (gamma 1-353 and gamma 1-341) or less (gamma 1-331 and gamma 1-276) specific activity than does the full-length wild-type gamma, gamma 1-386. Three truncated forms, gamma 1-316, gamma 1-300, and gamma 1-290 have molar specific activities approximately twice as great as those of the full-length wild-type gamma and the nonactivated holoenzyme. All recombinant gamma s exhibit similar Km values for both substrates, i.e., about 18 microM for phosphorylase b and about 75 microM for MgATP. Three truncated gamma s, gamma 1-316, gamma 1-300, and gamma 1-290, have a 1.9- to 2.5-fold greater catalytic efficiency (Vmax/Km) than that of the full-length wild-type gamma and a 3.5- to 4.5-fold greater efficiency than that of the truncated gamma 1-331. This evidence suggests that there is at least one inhibitory domain in the C-terminal region of gamma, which is located at gamma 301-331. gamma 1-290, but not gamma 1-276, which contains the highly conserved kinase domain, is the minimum sequence required for the gamma subunit to exhibit phosphotransferase activity. Both gamma 1-290 and gamma 1-300 have several properties similar to full-length wild-type gamma, including metal ion responses (activation by free Mg2+ and inhibition by free Mn2+), pH dependency, and substrate specificities.
Mol Cell Biochem 1993 Nov
PMID:Expression, purification, characterization, and deletion mutations of phosphorylase kinase gamma subunit: identification of an inhibitory domain in the gamma subunit. 793 63

Transglutaminase, a zero length cross-linker that catalyzes the formation of isopeptide bonds between proximal Gln and Lys side chains, was used as a structural and conformational probe of the hexadecameric phosphorylase kinase molecule (alpha beta gamma delta)4. Brief cross-linking of nonactivated kinase caused formation of alpha-beta dimers, with no cross-linking involving the gamma- and delta-subunits. When the kinase was first activated by autophosphorylation, significant amounts of alpha-alpha dimers were also observed in addition to the alpha-beta, demonstrating the occurrence of a conformational change in the alpha-subunits concomitant with activation. Both dimers resulted from intramolecular cross-linking. Because the COOH-terminal regions of the alpha-subunits are at the lobe tips of this bilobal kinase (Wilkinson D. A. Marion, T. N., Tillman, D. M., Norcum, M. T., Hainfeld, J. F., Seyer, J. M., and Carlson, G. M. (1994) J. Mol. Biol. 235, 974-982), the formation of zero length cross-linked alpha-alpha dimers indicates that the polypeptide backbones of these subunits must stretch from the lobe tips to a more central location where they abut each other. Excess putrescine, as the amine substrate in place of endogenous Lys, was incorporated by transglutaminase predominately into the alpha-subunits of the kinase, with only slight modification of the beta- and gamma-subunits. Exogenous calmodulin (delta'), an activator of the kinase with a binding site on the alpha-subunits (James, P., Cohen, P., and Carafoli, E. (1991) J. Biol. Chem. 266, 7087-7091), was a potent inhibitor of cross-linking. It also inhibited incorporation of putrescine into the alpha-subunits but stimulated incorporation into the beta- and gamma-subunits. Heparin, another activator of the kinase, had the same effects as exogenous calmodulin on cross-linking and putrescine incorporation, suggesting a commonality in the mechanism through which these two effectors activate the holoenzyme, including promoting a conformational change that increases the surface accessibility of target Gln residues on the catalytic gamma-subunit.
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PMID:Zero length conformation-dependent cross-linking of phosphorylase kinase subunits by transglutaminase. 796 56

Microtubule-associated protein tau from Alzheimer brain has been shown to be phosphorylated at several ser/thr-pro and ser/thr-X sites (Hasegawa, M. et al., J. Biol. Chem. 267, 17047-17054, 1992). Several proline-dependent protein kinases (PDPKs) (MAP kinase, cdc2 kinase, glycogen synthase kinase-3, tubulin-activated protein kinase, and 40 kDa neurofilament kinase) are implicated in the phosphorylation of the ser-thr-pro sites. The identity of the kinase(s) that phosphorylate the ser/thr-X sites are unknown. To identify the latter kinase(s) we have compared the phosphorylation of bovine tau by several brain protein kinases. Stoichiometric phosphorylation of tau was achieved by casein kinase-1, calmodulin-dependent protein kinase II, Gr kinase, protein kinase C and cyclic AMP-dependent protein kinase, but not with casein kinase-2 or phosphorylase kinase. Casein kinase-1 and calmodulin-dependent protein kinase II were the best tau kinases, with greater than 4 mol and 3 mol 32P incorporated, respectively, into each mol of tau. With the sequential addition of these two kinases, 32P incorporation approached 6 mol. Peptide mapping revealed that the different kinases largely phosphorylate different sites on tau. After phosphorylation by casein kinase-1, calmodulin-dependent protein kinase II, Gr kinase, cyclic AMP-dependent protein kinase and casein kinase-2, the mobility of tau isoforms as detected by SDS-PAGE was decreased. Protein kinase C phosphorylation did not produce such a mobility shift. Our results suggest that one or more of the kinases studied here may participate in the hyperphosphorylation of tau in Alzheimer disease.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Biochem 1994 Feb 23
PMID:Comparison of the phosphorylation of microtubule-associated protein tau by non-proline dependent protein kinases. 803 84

The binding of phosphorylase kinase to calmodulin-Sepharose 4B was studied by column and batch methods. It was found that the Ca2+ dependence of the interaction strongly depended on the degree of substitution of agarose with calmodulin. Equilibrium adsorption isotherms (i.e., bulk ligand binding functions and lattice site binding functions) of phosphorylase kinase were measured on calmodulin-Sepharose. Sigmoidal bulk ligand binding functions (bulk adsorption coefficients: 1.5-5.8) were found which indicate intermolecular attraction during binding. Hyperbolic lattice site binding functions (lattice adsorption coefficients: 1.0) were obtained thus excluding the existence of a critical surface concentration of immobilized calmodulin and indicating single independent binding sites on the gel surface and on phosphorylase kinase. These findings were combined to optimize the adsorption of phosphorylase kinase on calmodulin-Sepharose, for purification procedures at low Ca2+ concentrations (5-10 microM) minimizing proteolysis by calpains. With this novel method phosphorylase kinase from rabbit and frog skeletal muscle could be purified ca 100- and 200-fold, respectively, in two steps.
J Mol Recognit 1993 Sep
PMID:The binding of phosphorylase kinase to immobilized calmodulin. 806 Jun 69

Eleven Entamoeba histolytica protein-serine/threonine-kinase gene segments were identified using the polymerase chain reaction (PCR) and degenerate oligonucleotide primers to conserved amino acids in subdomains VI and VIII of the catalytic domain of protein-serine/threonine kinases. These ameba gene segments were homologous to myosin light chain kinases, protein kinase C, phosphorylase b kinase, and kinases that regulate glucose repression in yeast and cell growth in mammalian cells. One of these PCR products, which was homologous to the Dictyostelium discoideum protein kinase 2, was used to identify a full-length protein-serine/threonine-kinase gene (Eh rac1) from an E. histolytica genomic library. The open reading frame of Eh rac1 was 409 amino acids long (encoding a 47-kDa protein) and included an amino terminal segment containing 87 mostly charged and polar amino acids and a 322-amino acid carboxyl terminal segment containing the catalytic domain. The catalytic domain of Eh rac1 was homologous to the rac family of protein-serine/threonine-kinases, which are related to cAMP-dependent protein kinases and protein kinase Cs. Southern blots of ameba DNA showed that the Eh rac1 gene was present as a single copy in all strains tested, however pathogenic amebae expressed four times more Eh rac1 mRNAs than did nonpathogenic amebae. These studies suggest that E. histolytica, a primitive unicellular eukaryote, has a complex protein kinase family.
Mol Biochem Parasitol 1993 Aug
PMID:Molecular cloning of a rac family protein kinase and identification of a serine/threonine protein kinase gene family of Entamoeba histolytica. 823 9

X-linked phosphorylase kinase (PHK) deficiency causes X-linked liver glycogenosis (XLG) which is the most frequent liver glycogen storage disorder in man. Recently we assigned XLG to the Xp22 chromosomal region by linkage analysis in two families segregating XLG. In this study a further localization of XLG in Xp22 was performed by extending the number of Xp22 markers, by extension of the number of family members from the two families of our previous study and by linkage analysis in four additional XLG families. Two-point linkage analysis revealed lod scores of 4.60, 5.73, 5.28, 8.62 and 5.14 for linkage between XLG and the DNA markers pXUT23 and pSE3.2-L(DXS16), pD2(DXS43), pTS247-(DXS197) and pPA4B(DXS207), respectively, all at 0% recombination. Linkage heterogeneity was not observed in this set of families. Multipoint linkage analysis increased the lod score for linkage between XLG and Xp22 to 16.79 relative to DXS197/DXS207. The position of the XLG gene was confirmed by analysis of recombinational events locating the XLG gene between DXS85 and DXS41. The XLG gene could not be mapped more precisely in this chromosomal region of approximately 20cM because of the absence of recombinational events between the XLG gene and the Xp22 markers. As we have previously shown that the rabbit liver alpha subunit of PHK (PHKA2) hybridizes to human Xp22, we isolated a human PHKA2 cDNA from a human hepatoma lambda gt11 cDNA library. Fluorescent in situ hybridization mapped human PHKA2 to Xp22. As this physical mapping coincides with the genetic mapping of XLG by linkage analysis, PHKA2 most probably harbours the mutation(s) responsible for XLG.
Hum Mol Genet 1993 May
PMID:X-linked liver glycogenosis: localization and isolation of a candidate gene. 851 97


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