Phosphorylase Kinase ß Represents a Novel Prognostic Biomarker and Inhibits Malignant Phenotypes of Liver Cancer Cell.

Glycogen phosphorylase kinase ß-subunit (PHKB) is a regulatory subunit of phosphorylase kinase (PHK), involving in the activation of glycogen phosphorylase (GP) and the regulation of glycogen breakdown. Emerging evidence suggests that PHKB plays a role in tumor progression. However, the function of PHKB in HCC progression remains elusive. Here, our study revealed that the expression of PHKB significantly decreased in HCC tissues, and the low expression of PHKB could serve as an independent indicator for predicting poor prognosis in HCC. Functional experiments showed that PHKB knockdown significantly promoted cell proliferation both in vitro and in vivo, whereas PHKB overexpression resulted in opposing effects. Additionally, in vitro assays revealed that the over (or high) expression of PHKB greatly hindered HCC cell invasion and increased apoptosis rates. Also, we found that the over (or high) expression of PHKB effectively suppressed the epithelial-mesenchymal transition, which was further confirmed by our clinical data. Intriguingly, the biological function of PHKB in HCC was independent of glycogen metabolism. Mechanically, PHKB could inhibit AKT and STAT3 signaling pathway activation in HCC. Collectively, our data demonstrate that PHKB acts as a novel prognostic indicator for HCC, which exerts its suppression function via inactivating AKT and STAT3. Our data might provide novel insights into progression and facilitate the development of a new therapeutic strategy for HCC.

Phosphorylase kinase ß affects colorectal cancer cell growth and represents a novel prognostic biomarker.

PURPOSE: To study the expression and intracellular localization of phosphorylase kinase ß (PHKß) protein in colorectal cancers (CRCs), analyze its correlation with clinicopathological features and prognosis, and study the biological roles and mechanism-of-action of PHKß in CRC cell lines. METHODS: Quantitative polymerase chain reaction (qPCR) and western blot assays were performed to compare the expressions of PHKß mRNA and protein in CRC tissues and matched normal mucosa. Tissue microarrays and immunohistochemical staining were performed to detect the expression and intracellular location of PHKß protein and analyze its correlation with the clinicopathological characteristics and prognosis in CRC patients. Proliferation, cell cycle, wound healing, and xenograft models were used to elucidate the potential role of PHKß in vitro and in vivo. RESULTS: PHKß mRNA and protein were found to be overexpressed in CRC tissue compared to the levels in normal mucosa. Positive expression of PHKß was significantly correlated with TNM stage and distal metastasis, and elevated expression of PHKß was an independent prognostic factor in patients with CRC. PHKß knockdown impaired proliferation of CRC in vitro and in vivo and induced cell cycle arrest. CONCLUSIONS: PHKß affects CRC cell growth and represents a novel prognostic biomarker.

The role of molecular regulation and targeting in regulating calcium/calmodulin stimulated protein kinases.

Calcium/calmodulin-stimulated protein kinases can be classified as one of two types - restricted or multifunctional. This family of kinases contains several structural similarities: all possess a calmodulin binding motif and an autoinhibitory region. In addition, all of the calcium/calmodulin-stimulated protein kinases examined in this chapter are regulated by phosphorylation, which either activates or inhibits their kinase activity. However, as the multifunctional calcium/calmodulin-stimulated protein kinases are ubiquitously expressed, yet regulate a broad range of cellular functions, additional levels of regulation that control these cell-specific functions must exist. These additional layers of control include gene expression, signaling pathways, and expression of binding proteins and molecular targeting. All of the multifunctional calcium/calmodulin-stimulated protein kinases examined in this chapter appear to be regulated by these additional layers of control, however, this does not appear to be the case for the restricted kinases.

Calcineurin B-like domains in the large regulatory alpha/beta subunits of phosphorylase kinase.

Phosphorylase kinase (PhK) is a large hexadecameric complex that catalyzes the phosphorylation and activation of glycogen phosphorylase (GP). It consists in four copies each of a catalytic subunit (gamma) and three regulatory subunits (alpha beta delta). Delta corresponds to endogenous calmodulin, whereas little is known on the molecular architecture of the large alpha and beta subunits, which probably arose from gene duplication. Here, using sensitive methods of sequence analysis, we show that the C-terminal domain (named domain D) of these alpha and beta subunits can be significantly related to calcineurin B-like (CBL) proteins. CBL are members of the EF-hand family that are involved in the regulation of plant-specific kinases of the CIPK/PKS family, and relieve autoinhibition of their target kinases by binding to their regulatory region. The relationship highlighted here suggests that PhK alpha and/or beta domain D may be involved in a similar regulation mechanism, a hypothesis which is supported by the experimental observation of a direct interaction between domain D of PhKalpha and the regulatory region of the Gamma subunit. This finding, together the identification of significant similarities of domain D with the preceding domain C, may help to understand the molecular mechanism by which PhK alpha and/or beta domain D might regulate PhK activity.

Structural evidence for co-evolution of the regulation of contraction and energy production in skeletal muscle.

Skeletal muscle phosphorylase kinase (PhK) is a Ca(2+)-dependent enzyme complex, (alpha beta gamma delta)(4), with the delta subunit being tightly bound endogenous calmodulin (CaM). The Ca(2+)-dependent activation of glycogen phosphorylase by PhK couples muscle contraction with glycogen breakdown in the "excitation-contraction-energy production triad." Although the Ca(2+)-dependent protein-protein interactions among the relevant contractile components of muscle are well characterized, such interactions have not been previously examined in the intact PhK complex. Here we show that zero-length cross-linking of the PhK complex produces a covalent dimer of its catalytic gamma and CaM subunits. Utilizing mass spectrometry, we determined the residues cross-linked to be in an EF hand of CaM and in a region of the gamma subunit sharing high sequence similarity with the Ca(2+)-sensitive molecular switch of troponin I that is known to bind actin and troponin C, a homolog of CaM. Our findings represent an unusual binding of CaM to a target protein and supply an explanation for the low Ca(2+) stoichiometry of PhK that has been reported. They also provide direct structural evidence supporting co-evolution of the coordinate regulation by Ca(2+) of contraction and energy production in muscle through the sharing of a common structural motif in troponin I and the catalytic subunit of PhK for their respective interactions with the homologous Ca(2+)-binding proteins troponin C and CaM.

Phosphorylase kinase: the complexity of its regulation is reflected in the complexity of its structure.

Intracellular glycogen stores are used to maintain blood-glucose homeostasis during fasting, are a source of energy for muscle contraction, and are used to support a broad range of cellular activities in most tissues. A diversity of signals accelerate glycogen degradation that are mediated by phosphorylase b kinase (Phk), which phosphorylates and thereby activates glycogen phosphorylase. Phk is among the most complex of the protein kinases so far elucidated. It has one catalytic (gamma) subunit and three different regulatory (alpha, beta, and delta) subunits, a molecular mass of 1.3 X 106 daltons, and each holoenzyme molecule is presumed to contain four molecules of each subunit. The three regulatory subunits inhibit the phosphotransferase activity of the gamma subunit. Ca2+ relieves inhibition via the delta subunit, which is identical to calmodulin but remains an integral component of the holoenzyme even when the [Ca2+] is lowered to nanomolar levels. Phosphorylation of the alpha and beta subunits by the 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) also relieves inhibition of the gamma subunit and thereby activates the enzyme. The stimulatory effects of Ca2+ and phosphorylation appear to be structurally coupled and are cooperative. In addition, Phk is activated in vitro by autophosphorylation, limited proteolysis of the regulatory subunits, and various allosteric effectors and these may also be mechanisms of physiological importance. The molecular mechanisms of regulation are currently poorly understood, but new insights are beginning to emerge. This review discusses current knowledge and concepts of the structure, function and regulation of Phk.

The testis isoform of the phosphorylase kinase catalytic subunit (PhK-gammaT) plays a critical role in regulation of glycogen mobilization in developing lung.

In order to identify the form of phosphorylase kinase catalytic subunit expressed in developing lung, degenerate polymerase chain reaction primers were designed based on conserved domains of the two known catalytic subunits, expressed primarily in muscle and testis. Amplification of cDNA from day 19 fetal rat lung followed by cloning and sequence analyses indicated that only the testis isoform of phosphorylase kinase (PhK-gammaT) was detectable in fetal lung. In situ hybridization analyses indicated that expression of PhK-gammaT RNA in developing lung tissue was widespread and not restricted to Type II epithelial cells; PhK-gammaT protein expression was temporally and spatially correlated with expression of PhK-gammaT RNA. PhK-gammaT RNA and protein expression was also characterized in the PhK-deficient glycogen storage disease (gsd) rat. PhK-gammaT RNA levels were similar in Type II cells isolated from wild type and gsd/gsd fetuses; in contrast, PhK-gammaT protein was virtually undetectable in gsd/ gsd Type II cells and enzyme activity was very low. These results suggest that PhK-gammaT plays a critical role in mobilization of glycogen during fetal lung development and that failure to catabolize glycogen in the gsd/gsd rat is related to an untranslatable PhK-gammaT RNA or unstable protein.

DPhK-gamma, a putative Drosophila kinase with homology to vertebrate phosphorylase kinase gamma subunits: molecular characterisation of the gene and phenotypic analysis of loss of function mutants.

Partial and total loss of function mutant alleles of a putative Drosophila homologue (DPhK-gamma) of the vertebrate phosphorylase kinase gamma-subunit gene have been isolated. DPhK-gamma is required in early embryonic processes, such as gastrulation and mesoderm formation; however, defects in these processes are seen only when both the maternal and zygotic components of DPhK-gamma expression are eliminated. Loss of zygotic expression alone does not appear to affect normal embryonic and larval development; some pupal lethality is observed but the majority of mutant animals eclose as adults. Many of these adults show defects in their leg musculature (e.g. missing and degenerating muscles), in addition to exhibiting melanised "tumours" on their leg joints. Loss of only the maternal component has no obvious phenotypic consequences. The DPhK-gamma gene has been cloned and sequenced. It has an open reading frame (ORF) of 1680 bp encoding a 560 amino acid protein. The predicted amino acid sequence of DPhK-gamma has two conserved domains, the catalytic kinase and calmodulin-binding domains, separated by a linker sequence. The amino acid sequence of DPhK-gamma is homologous to that of mammalian PhK-gamma proteins but differs in the length and amino acid composition of its linker sequence. The expression of DPhK-gamma mRNA is developmentally regulated. We discuss the implications of these observations.

Regulation of the (Ca(2+)+Mg2+)-ATPase of basolateral membranes from kidney proximal tubules by kinase-mediated phosphorylation and by insulin.

Membrane vesicles derived from the basolateral aspect of kidney proximal tubule cells are phosphorylated by ATP in the absence of Ca2+. This Mg(2+)-dependent, hydroxylamine-resistant phosphorylation was associated with a 50% inhibition of the (Ca(2+)+Mg2+)-ATPase activity measured upon addition of micromolar Ca2+ concentrations, enough to saturate the high-affinity sites of the Ca2+ pump. The presence of either the protein kinase inhibitor H7 or insulin during phosphorylation virtually eliminated the inhibitory effect associated with phosphorylation. However, insulin itself inhibited ATP hydrolysis by the (Ca(2+)+Mg2+)-ATPase when it was present in the assay medium containing buffer, ATP, Mg2+ and Ca2+, the hydrolytic activity being initiated by addition of the membranes without prior phosphorylation. These results suggest that insulin may play a role in regulating transepithelial Ca2+ transport in renal proximal tubules, and that its effects may be linked with a kinase-mediated process that depends on the functional state of the (Ca(2+)+Mg2+)-ATPase.

Ca2+-dependent and cAMP-dependent control of nicotinic acetylcholine receptor phosphorylation in muscle cells.

Mouse BC3H1 myocytes were incubated with 32Pi before acetylcholine receptors were solubilized, immunoprecipitated, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. More than 90% of the 32P found in the receptor was bound to the delta subunit. Two phosphorylation sites in this subunit were resolved by reverse phase high performance liquid chromatography after exhaustive proteolysis of the protein with trypsin. Sites 1 and 2 were phosphorylated to approximately the same level in control cells. The divalent cation ionophore, A23187, increased 32P in site 1 by 40%, but did not affect the 32P content of site 2. In contrast, isoproterenol increased 32P in site 2 by more than 60%, while increasing 32P in site 1 by only 20%. When dephosphorylated receptor was incubated with [gamma-32P]ATP and the catalytic subunit of cAMP-dependent protein kinase, the delta subunit was phosphorylated to a maximal level of 1.6 phosphates/subunit. Approximately half of the phosphate went into site 2, with the remainder going into a site not phosphorylated in cells. The alpha subunit was phosphorylated more slowly, but phosphorylation of both alpha and delta subunits was blocked by the heat-stable protein inhibitor of cAMP-dependent protein kinase. Phosphorylation of the receptor was also observed with preparations of phosphorylase kinase. In this case phosphorylation occurred in the beta subunit and site 1 of the delta subunit, neither of which were phosphorylated by cAMP-dependent protein kinase. The rate of receptor phosphorylation by phosphorylase kinase was slow relative to that catalyzed by cAMP-dependent protein kinase. Therefore, it can not yet be concluded that phosphorylase kinase phosphorylates the beta subunit and the delta subunit site 1 in cells. However, the results strongly support the hypothesis that phosphorylation by cAMP-dependent protein kinase accounts for phosphorylation of the alpha subunit and the delta subunit site 2 in response to elevations in cAMP.

[Molecular mechanisms of the regulation of phosphorylase kinase from skeletal muscles of mammals and birds]. / Molekuliarnye mekhanizmy reguliatsii aktivnosti kinazy fosforilazy iz myshts mlekopitaiushchikh i ptits.

Red and white avian skeletal muscles (chicken and pigeon) contain the same alpha'-isoenzyme of phosphorylase kinase. According to data from gradient polyacrylamide slab electrophoresis in the presence of SDS, the molecular masses of beta- and gamma-subunits of phosphorylase kinase from rabbit, chicken and pigeon muscles are not identical. Electron microscopy data suggest that the quaternary structure of chicken and pigeon phosphorylase kinase is of the same type. The alpha'-isozyme of chicken and pigeon phosphorylase kinase is strongly activated by calmodulin and troponin C. Avian phosphorylase kinase is activated 2--3-fold by phosphorylation with cAMP-dependent protein kinase and by autophosphorylation. This activation is associated with the phosphorylation of both alpha'- and beta-subunits. The affinity of pigeon phosphorylase kinase a for Ca2+ is 20 times as high as that of phosphorylase kinase b.

Calcium-dependent phosphorylation of bovine cardiac C-protein by phosphorylase kinase.

Phosphorylase kinase catalyzed the calcium-dependent phosphorylation of bovine cardiac C-protein. Phosphorylation of C-protein by phosphorylase kinase reached nearly 2 mol [32P]/mol C-protein. Tryptic phosphopeptide mapping and phosphoamino acid analysis indicated that phosphorylase kinase maybe phosphorylating some of the same seryl residues that undergo phosphorylation by cAMP-dependent protein kinase and that C-protein from bovine and chicken heart are structurally different. Bovine cardiac C-protein was not a substrate for a number of calcium and cyclic nucleotide-independent protein kinases, suggesting that phosphorylation of cardiac C-protein is restricted to protein kinases which are modulated by calcium and cAMP.

A new variant of glycogen storage disease. Type IXc.

Three siblings, a body and two girls, had clinical, laboratory, and morphologic findings that were suggestive of glycogen storage disease (GSD) type IXa. Patients of both sexes with phosphorylase kinase (PK) deficiency usually have an excessive glycogen content only in the liver and normal glycogen content and PK activity in muscle. The siblings in this study had an increased glycogen content in the liver but also in muscle and reduced PK activity in liver, muscle, erythrocytes, and leukocytes. This condition should be labeled as GSD type IXc.