PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “PHOSPHORYLASE KINASE”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 271 records · Page 15Linked to original sources
PubMed1985

Stimulation of autophosphorylation of rabbit skeletal muscle phosphorylase kinase by glycogen synthase on glycogen particles.

Glycogen synthase stimulated the autophosphorylation and autoactivation of phosphorylase kinase from rabbit skeletal muscle. This stimulation was additive to that by glycogen and the reaction was dependent on Ca2+. The effect by glycogen synthase was maximum within the activity ratio (the activity of enzyme without glucose-6-P divided by the activity with 10 mM glucose-6-P) of 0.3 and over 0.3 it was rather inhibitory. The results suggest that autophosphorylation of phosphorylase kinase in the presence of glycogen synthase on glycogen particles may be an important regulatory mechanism of glycogen metabolism in skeletal muscle.

Animals↗
PubMed2001

An inhibitory segment of the catalytic subunit of phosphorylase kinase does not act as a pseudosubstrate.

The C terminus of the catalytic gamma subunit of phosphorylase kinase contains two autoinhibitory calmodulin binding domains designated PhK13 and PhK5. These peptides inhibit truncated gamma(1-300). Previous data show that PhK13 (residues 302-326) is a competitive inhibitor with respect to phosphorylase b, with a K(i) of 1.8 microm. This result suggests that PhK13 may bind to the active site of truncated gamma(1-300). Variants of PhK13 were prepared to localize the determinants for interaction with the catalytic fragment gamma(1-300). PhK13-1, containing residues 302-312, was found to be a competitive inhibitor with respect to phosphorylase b with a K(i) of 6.0 microm. PhK13 has been proposed to function as a pseudosubstrate inhibitor with Cys-308 occupying the site that normally accommodates the phosphorylatable serine in phosphorylase b. A PhK13-1 variant, C308S, was synthesized. Kinetic characterization of this peptide reveals that it does not serve as a substrate but is a competitive inhibitor. Additional variants were designed based on previous knowledge of phosphorylase kinase substrate determinants. Variants were analyzed as substrates and as inhibitors for truncated gamma(1-300). Although PhK13-1 does not appear to function as a pseudosubstrate, several specificity determinants employed in the recognition of phosphorylase b as substrate are utilized in the recognition of PhK13-1 as an inhibitor.

Adenosine Triphosphatases↗
PubMed1977

The phosphorylation sites of troponin T from white skeletal muscle and the effects of interaction with troponin C on their phosphorylation by phosphorylase kinase.

1. The phosphorylation of troponin T from rabbit white sketetal muscle is catalysed by phosphorylase kinase, but not at a significant rate by bovine 3':5'-cyclic AMP-dependent protein kinase. 2. The amino acid sequences adjacent to the three major phosphorylation sites of troponin T were determined. 3. The serine in the N-terminal peptide (Asx,SerP, Glx)Glu-Val-Glu, is that phosphorylated (SerP, phosphoserine) when the troponin complex is isolated. 4. The other two sites of phosphorylation are located in the sequence Ala-Leu-(Ser, SerP)-Met-Gly-Ala-Asn-Tyr(Ser,SerP)Tyr. 5. When troponin T is phosphorylated in the presence of troponin C, the extent of phosphorylation at each site is considerably decreased. 6. CNBr fragments of troponin T are also phosphorylated by phosphorylase kinase, but the rate of phosphorylation at each site in the CNBr fragments is considerably slower than in the native protein. 7. From these studies it is suggested that troponin C interacts with troponin T in the region containing the two closely situated phosphorylation sites.

Amino Acid Sequence↗
PubMed1990

Pest sequences present in the subunits of phosphorylase kinase.

We report that the amino acid sequences of all four subunits of rabbit skeletal muscle phosphorylase kinase possess one or more regions rich in proline (P), glutamic acid (E), serine (S) and threonine (T). alpha and beta subunits contain strong PEST sequences, showing PEST scores greater than 0 (Rogers et al. (1986) Science 234, 364-368), while gamma and delta subunits contain weak PEST regions (negative PEST scores greater than -5.3). In addition to PEST sequences, alpha, beta and gamma subunits contain clusters of arginine pairs. The above sequence characteristics may serve to signal rapid turnover of phosphorylase kinase.

Amino Acid Sequence↗
PubMed1980

Calcium control of muscle phosphorylase kinase through the combined action of calmodulin and troponin.

Although it has been believed for several years that Ca2+ are the means by which glycogenolysis and muscle contraction are synchronized, it is only over the past two years that this concept has started to be placed on a firm molecular basis. The current evidence suggests that the regulation of phosphorylase kinase by Ca2+ in vivo is achieved through the interaction of this divalent cation with calmodulin (the delta subunit) and troponin C, and that the relative importance of these two calcium binding proteins depends on the state of phosphorylation of the enzyme [FIGURE 1]. In the low-activity dephosphorylated b form, increasing Ca2+ from 0.1 microM to concentrations in the microM range produces a 5-10-fold activation through the binding of Ca2+ to the delta subunit, and a further 15-25-fold activation through the binding of Ca2+ to troponin C [TABLE 1]. Troponin C rather than the delta subunit is therefore the dominant calcium dependent regulator of the b form, providing an attractive mechanism for coupling glycogenolysis and muscle contraction. On the other hand, the high-activity phosphorylated a form is only activated very slightly by troponin [Section 7]. The delta subunit is therefore the dominant calcium dependent regulator of the hormonally activated state of the enzyme. It has recently become clear that phosphorylase kinase not only phosphorylates and activates phosphorylase, but also phosphorylates glycogen synthase, decreasing its activity. The regulation of phosphorylase kinase by Ca2+ may therefore also provide a mechanism for achieving synchronous control of the pathways of glycogenolysis and glycogen synthesis.

Animals↗
PubMed1986

Stimulation of glycogen phosphorylase kinase by phospholipids.

The acidic phospholipids phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-biphosphate (PIP2) and the neutral phospholipid lysophosphatidylcholine (LPC) were found to stimulate (3 to 8-fold) the activity of nonactivated rabbit skeletal muscle phosphorylase kinase at pH 6.8, without significantly affecting the activity at pH 8.2. In this respect, phosphatidylcholine and phosphatidylethanolamine were ineffective, while the anionic detergent sodium dodecyl sulfate (SDS) and the anionic steroid dehydroisoandrosterone sulfate (DIAS) were able to mimic the action of phospholipids. SDS was also found to be a very efficient activator of the autophosphorylation of phosphorylase kinase (20-fold activation at 200 microM). The activating effect of phospholipids largely depends on the size of lipid vesicles, which is connected with the procedure of their preparation. These results suggest that phosphorylase kinase belongs to the class of Ca2+-dependent enzymes, which are sensitive to stimulation by calmodulin, limited proteolysis and anionic amphiphiles.

Animals↗
PubMed1995

Human cDNA encoding the muscle isoform of the phosphorylase kinase gamma subunit (PHKG1).

Muscle glycogenosis caused by phosphorylase kinase (Phk) deficiency may lead to exercise intolerance, weakness and musculatur atrophy. The gene encoding the muscle isoform of the Phk gamma subunit (gamma M) is one of the candidate genes in which mutations responsible for this condition should be sought. Here, we report the cDNA sequence and the predicted primary structure of the human gamma M subunit.

Amino Acid Sequence↗
PubMed1989

Ca2+-independent interaction of the gamma subunit of phosphorylase kinase with dansyl-calmodulin.

A strong Ca2+-independent interaction between the isolated, active gamma subunit of phosphorylase kinase and dansyl-calmodulin (dansyl-CaM) was observed by monitoring changes in fluorescence intensity in the absence of calcium ion. The pure, active gamma subunit of phosphorylase kinase was simply prepared by dialyzing the HPLC-purified, inactive gamma subunit against 8 M urea, containing 0.1 mM DTT, 0.1 M Hepes at pH 6.8 or 0.1 M Tris at pH 8.2, followed by dilution of urea with pH 6.8 or 8.2 buffer. The dissociation constants determined by fluorescence spectroscopy for the gamma subunit to dansyl-CaM are 25.7 +/- 0.6 and 104 +/- 12 nM at pH 6.8 in the presence and absence of CaCl2. At pH 8.2, these values are 4.9 +/- 0.3 and 29 +/- 8 nM in the presence and absence of CaCl2. As the free Ca2+ decreases to as low as 10(-9) M, the fluorescence intensity and the fluorescence polarization of the gamma subunit and dansyl-CaM complex do not decrease in parallel, indicating that the complex does not come apart at low Ca2+ concentration. The presence of Mg2+ affects the interaction between dansyl-CaM and the gamma subunit, as indicated by the increase in the polarization of fluorescence of dansyl-CaM. Mn2+ interferes with the interaction of the gamma subunit and dansyl-CaM. Free ATP has little effect.

Adenosine Triphosphate↗
PubMed1994

Baculovirus-directed expression of the gamma-subunit of phosphorylase kinase: purification and calmodulin dependence.

A recombinant baculovirus containing a cDNA encoding the gamma-subunit of phosphorylase kinase from mouse skeletal muscle was constructed. Cultures of Sf-9 insect cells infected with the gamma-baculovirus produce an intact and soluble gamma-protein. A purification procedure is presented that yields a sample of gamma-protein which is devoid of interfering enzyme activity and which is not associated with calmodulin from the insect cells. The isolated gamma sample has a Km for phosphorylase b of 36 (+/- 6, S.E.M) microM at pH 8.2 and 140 (+/- 25) microM at pH 6.8. These values are similar to those reported for the activated phosphorylase kinase holoenzyme isolated from skeletal muscle tissue. However, the Vmax. of the baculovirus-expressed gamma is 65 and 80% of that of the activated holoenzyme at pH 6.8 and 8.2 respectively. These results indicate that one or more of the regulatory subunits alpha, beta, or calmodulin stimulate the activity of the catalytic subunit gamma in the activated holoenzyme. Addition of calmodulin to the baculovirus-expressed gamma stimulates its activity 1.5-2.0 fold at pH 6.8 in both the presence and absence of calcium. At pH 8.2, calmodulin has only minor stimulatory affects. The stimulation by calmodulin at pH 6.8 results from an increase in the Vmax of gamma with little effect on its Km. This result is unlike that for most calmodulin-stimulated kinases which bind calmodulin only in the presence of calcium and exhibit a decrease in their Km upon binding calmodulin. The change in Vmax. of gamma in the presence of calmodulin and in the absence of calcium presents a novel mechanism for the regulation of a calmodulin-stimulated kinase.

Animals↗
PubMed1995

Phosphorylation/activation of phosphorylase b kinase by cAMP/Ca2(+)-independent, autophosphorylation-dependent protein kinase.

Phosphorylase b kinase from rabbit skeletal muscle can be phosphorylated and activated by a cyclic nucleotide- and Ca2(+)-independent protein kinase previously identified as an autophosphorylation-dependent multifunctional protein kinase (auto-kinase) from brain and liver (Yang et al., J. Biol. Chem. 262, 7034-7040 (1987) and Yang et al. J. Biol. Chem. 262, 9421-9427 (1987)). This independent kinase phosphorylates both alpha and beta subunits of phosphorylase b kinase and results in a approximately 5-fold activation of the kinase when 0.55 and 0.5 mol of phosphate are incorporated into the alpha and beta subunits, respectively. Activation of phosphorylase b kinase catalyzed by auto-kinase is about 70% of that observed with cAMP-dependent protein kinase. Analysis of phosphopeptide maps of alpha and beta subunits further reveals that both kinases phosphorylate almost the same sites on both alpha and beta subunits, suggesting that activation of phosphorylase b kinase by the two kinases may be through a common molecular action mechanism. Taken together with the previous result that auto-kinase can inactivate glycogen synthase, the present study provides initial evidence that a coordinate control mechanism for simultaneous regulation of glycogenolysis and glycogenesis can be modulated by autophosphorylation-dependent protein kinase in a cAMP- and Ca2(+)-independent pathway, representing a new mode of control mechanism for the regulation of glycogen metabolism in cells.

Animals↗
PubMed1985

Adenosine 5'-diphosphate as an allosteric effector of phosphorylase kinase from rabbit skeletal muscle.

Equilibrium binding and activity studies indicate that adenosine 5'-diphosphate binds to phosphorylase kinase with high affinity at a site, or sites, distinct from the catalytic site. Equilibrium dialysis at pH 6.8 and 8.2, with and without Mg2+, and with phosphorylated and nonphosphorylated enzyme preparations revealed approximately 8 ADP binding sites per alpha 4 beta 4 gamma 4 delta 4 hexadecamer, with Kd values ranging from 0.26 to 17 microM. Decreasing the pH from 8.2 to 6.8 or removing the Mg2+ enhanced the affinity for ADP. At pH 6.8, ADP stimulated the phosphorylase conversion and autophosphorylation activities of the nonactivated enzyme. Analogs of ADP with modifications at the 2'-, 3'-, and 5'-positions allowed determination of structural requirements for the stimulation of activity. ADP seems to alter the conformation of the beta subunit because addition of the nucleotide inhibits its dephosphorylation by phosphoprotein phosphatase and its chemical cross-linking by 1,5-difluoro-2,4-dinitrobenzene. The binding affinities and effects of ADP suggest that it may function physiologically as an allosteric effector of phosphorylase kinase.

Adenosine Diphosphate↗
PubMed1977

Phosphorylation of the inhibitory subunit of troponin in perfused hearts of mice deficient in phosphorylase kinase. Evidence for the phosphorylation of troponin by adenosine 3':5'-phosphate-dependent protein kinase in vivo.

When hearts from control and phosphorylase kinase-deficient (I strain) mice were perfused with 0.1 micrometer-DL-isoprenaline, there was a parallel increase in contraction, cyclic AMP concentration and troponin I phosphorylation. However, there was no increase in phosphorylase a in the I-strain hearts, whereas the control hearts showed a large increase. Assays of I-strain heart extracts showed a normal cyclic AMP-dependent protein kinase activity but no phosphorylase kinase activity. It is concluded that troponin I is phosphorylated in intact hearts by protein kinase and not phosphorylase kinase.

Animals↗
PubMed1986

[The role of calmodulin (delta-subunit) in the activation of phosphorylase kinase from rabbit skeletal muscles].

A comparative study on the structure of nonactivated and activated forms of phosphorylase kinase was carried out. The enzyme was activated by incubation in alkaline medium (pH 8.5), by phosphorylation with cAMP-dependent protein kinase and by limited proteolysis. The comparative analysis was based on the use of hydrophobic chromatography on phenyl-sepharose and electrophoresis in polyacrylamide gel density gradient. Activation of the enzyme was accompanied by separation of a low molecular weight component (Mr about 17 000). Using chromatography on phenyl-sepharose, this low molecular weight protein was obtained in a homogeneous state. It was found that the properties of the protein are close to those of calmodulin. The presence of calmodulin in phosphorylase kinase preparations was judged upon by the activation of the calmodulin-dependent form of phosphodiesterase. The boiled and subtilisin-treated kinase activates phosphodiesterase in the same way as does bovine brain calmodulin. The experimental results suggest that the delta-subunit is a protein inhibitor of the enzyme.

Animals↗
PubMed1995

Does phosphorylase kinase control glycogen biosynthesis in skeletal muscle?

Immunoblotting as well as enzyme assays demonstrate the presence of the self-glucosylating protein, glycogenin, in the protein-glycogen complex, in the sarcoplasmic reticulum and in phosphorylase kinase. In all three compartments glycogenin occurs in different, albeit, defined glucosylated forms, which upon deglucosylation are converted into a 42 kDa form. We suggest that phosphorylase kinase might have a dual function in glycogen biogenesis: firstly, control of glycogen degradation in the protein-glycogen complex via phosphorylation of glycogen phosphorylase b; secondly, regulation of glycogen biosynthesis on the sarcoplasmic reticular membranes via phosphorylation and thereby inhibition of glycogen synthase.

Animals↗
PubMed1995

Identification of the substrate and pseudosubstrate binding sites of phosphorylase kinase gamma-subunit.

Using site-directed mutagenesis, we proposed that an autoinhibitory domain(s) is located at the C-terminal region (301-386) of the phosphorylase kinase gamma-subunit (Huang, C.-Y.F., Yuan C.-J., Livanova, N.B., and Graves, D.J. (1993) Mol. Cell. Biochem. 127/128, 7-18). Removal of the putative inhibitory domain(s) by truncation results in the generation of a constitutively active and calmodulin-independent form, gamma 1-300. To probe the structural basis of autoinhibition of gamma-subunit activity, two synthetic peptides, PhK13 (gamma 303-327) and PhK5 (gamma 343-367), corresponding to the two calmodulin-binding regions, were assayed for their ability to inhibit gamma 1-300. Competitive inhibition of gamma 1-300 by PhK13 was found versus phosphorylase b (Ki = 1.8 microM) and noncompetitive inhibition versus ATP. PhK5 showed noncompetitive inhibition with respect to both phosphorylase b and ATP. Calmodulin released the inhibition caused by both peptides. These results indicate that there are two distinct auto-inhibitory domains within the C terminus of the gamma-subunit and that these two domains overlap with the calmodulin-binding regions. Two mutant forms of gamma 1-300, E111K and E154R, were used to probe the enzyme-substrate-binding region using peptide substrate analogs corresponding to residues 9-18 of phosphorylase b (KRK11Q12ISVRGL). The data suggest that Glu111 interacts with the P-3 position of the substrate (Lys11) and Glu154 interacts with the P-2 site (Gln12). Both E111K and E154R were competitively inhibited with respect to phosphorylase b by PhK13, with 14- and 8-fold higher Ki values, respectively, than that observed with the wild-type enzyme. These data are consistent with a model for the regulation of the gamma-subunit of phosphorylase kinase in which PhK13 acts as a competitive pseudosubstrate that directly binds the substrate binding site of the gamma-subunit (Glu111 and Glu154).

Amino Acid Sequence↗
PubMed1995

Two structures of the catalytic domain of phosphorylase kinase: an active protein kinase complexed with substrate analogue and product.

BACKGROUND: Control of intracellular events by protein phosphorylation is promoted by specific protein kinases. All the known protein kinase possess a common structure that defines a catalytically competent entity termed the 'kinase catalytic core'. Within this common structural framework each kinase displays its own unique substrate specificity, and a regulatory mechanism that may be modulated by association with other proteins. Structural studies of phosphorylase kinase (Phk), the major substrate of which is glycogen phosphorylase, may be expected to shed light on its regulation. RESULTS: We report two crystal structures of the catalytic core (residues 1-298; Phk gamma trnc) of the gamma-subunit of rabbit muscle phosphorylase kinase: the binary complex with Mn2+/beta-gamma-imidoadenosine 5'-triphosphate (AMPPNP) to a resolution of 2.6 A and the binary complex with Mg2+/ADP to a resolution of 3.0 A. The structures were solved by molecular replacement using the cAMP-dependent protein kinase (cAPK) as a model. CONCLUSIONS: The overall structure of Phk gamma trnc is similar to that of the catalytic core of other protein kinases. It consists of two domians joined on one edge by a 'hinge', with the catalytic site located in the cleft between the domains. Phk gamma trnc is constitutively active, and lacks the need for an activatory phosphorylation event that is essential for many kinases. The structure exhibits an essentially 'closed' conformation of the domains which is similar to that of cAPK complexed with substrates. The phosphorylated residue that is located at the domain interface in many protein kinases and that is believed to stabilize an active conformation is substituted by a glutamate in Phk gamma trnc. The glutamate, in a similar manner to the phosphorylated residue in other protein kinases, interacts with an arginine adjacent to the catalytic aspartate but does not participate in interdomain contacts. The interactions between the enzyme and the nucleotide product of its activity, Mg2+/ADP, explain the inhibitory properties of the nucleotides that are observed in kinetic studies.

Adenosine Diphosphate↗
PubMed1986

Activation of phosphorylase kinase through autophosphorylation by membrane component phospholipids.

Phosphatidic acid (PtdOH) has been shown not only to stimulate autophosphorylation and autoactivation of phosphorylase kinase of rabbit skeletal muscle but also to decrease the apparent Ka for Ca2+ on autophosphorylation sharply [Negami et al. (1985) Biochem. Biophys. Res. Commun. 131, 712-719]. In this study we investigated the interaction between PtdOH and other phospholipids on autophosphorylation and autoactivation of this enzyme. Acidic phospholipids, such as phosphatidylserine (PtdSer), phosphatidylinositol (PtdIns) and PtdOH, stimulated this reaction about 2-4-fold, and the approximate Ka values of this reaction were 10 micrograms/ml, 6.3 micrograms/ml and 30 micrograms/ml respectively. The molar ratio of PtdIns and PtdSer with maximal effect on autophosphorylation was about 1:1. Under these conditions PtdOH stimulated the initial velocity of autophosphorylation about 5.2-fold. When fully autophosphorylated, about 12-13 mol phosphate per tetramer (alpha beta gamma delta) were incorporated in the presence of mixed acidic phospholipids (PtdOH:PtdIns:PtdSer = 2:1:1), which was about twice as much as values observed without effectors. In the presence of mixed acidic phospholipids there was a concomitant enhancement of kinase activity, about 30-40-fold at pH 6.8 and 2.5-3-fold at pH 8.2. Mixed acidic phospholipids sharply decreased an apparent Ka for Ca2+ from 4 X 10(-5) M to 8 X 10(-7) M. With mixed acidic phospholipids as effectors this autophosphorylation occurred through an intramolecular mechanism. Based on these results, autophosphorylation and autoactivation of phosphorylase kinase in the presence of acidic phospholipids may account for an important regulatory mechanism of glycogenolysis in muscle contraction.

Adenosine Triphosphate↗