Alpha Subunit Of Phosphorylase Kinase Research Articles

Abstract 1040: Differential levels of glycogen in breast cancer cell lines: A potential new target

Abstract Cancer cells have been known to alter their metabolic processes in order to survive and proliferate. Normally in muscle and liver, excess glucose is stored within the cells as glycogen. Elevated levels of glycogen have also been found in various cancers, including breast cancers. Recent studies have implicated glycogen metabolism as important in promoting survival of cancer cells, suggesting targeting of glycogen metabolism as a possible treatment to inhibit cancer cell growth. In general, modulation of cancer metabolism is believed to be an attractive adjunct strategy to conventional or targeted therapies. Here we set out to investigate glycogen levels as well as levels of proteins involved in glycogen synthesis and degradation vary across different breast cancer cell lines. A glucose metabolism qPCR array found differential levels of the alpha subunit of phosphorylase kinase 1, a key enzyme involved in glycogen degradation among three different breast cancer cell lines. Expression levels of glycogen synthesis and degradation enzymes were assessed using qPCR and immunoblot in various breast cancer cell lines. Glycogen levels in these breast cancer cell lines were quantified using an amyloglucosidase reaction coupled with other enzymatic reactions to produce a fluorescent product. It was found that MDA-MB-231, SUM149, and MCF7 cell lines had increased levels of glycogen, between 6.5 and 23.5 μg glycogen per mg protein, whereas SUM190 and normal-like breast epithelial cell line MCF10A had undetectable levels of glycogen. These findings demonstrate that glycogen metabolism can vary widely amongst cancer types, indicating that therapies targeted to disrupt glycogen degradation may produce differential results and that further study of the role of glycogen metabolism in cancer is warranted. Citation Format: Joel A. Yates, Megan Altemus, Zhifen Wu, Michelle L. Wynn, Sofia D. Merajver. Differential levels of glycogen in breast cancer cell lines: A potential new target. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1040.

Clinical application of massively parallel sequencing in the molecular diagnosis of glycogen storage diseases of genetically heterogeneous origin

Glycogen storage diseases are a group of inborn errors of glycogen synthesis or catabolism. The outcome for untreated patients can be devastating. Given the genetic heterogeneity and the limited availability of enzyme study data, the definitive diagnosis of glycogen storage diseases is made on the basis of sequence analysis of selected potentially causative genes. A massively parallel sequencing test was developed for simultaneous sequencing of 16 genes known to cause muscle and liver forms of glycogen storage diseases: GYS2, GYS1, G6PC, SLC37A4, GAA, AGL, GBE1, PYGM, PYGL, PFKM, PHKA2, PHKB, PHKG2, PHKA1, PGAM2, and PGM1. All the nucleotides in the coding regions of these 16 genes have been enriched with sufficient coverage in an unbiased manner. Massively parallel sequencing demonstrated 100% sensitivity and specificity as compared with Sanger sequencing. Massively parallel sequencing correctly identified all types of mutations, including single-nucleotide substitutions, small deletions and duplications, and large deletions involving one or more exons. In addition, we have confirmed the molecular diagnosis in 11 of 17 patients in whom glycogen storage diseases were suspected. This report demonstrates the clinical utility of massively parallel sequencing technology in the diagnostic testing of a group of clinically and genetically heterogeneous disorders such as glycogen storage diseases, in a cost- and time-efficient manner.

X-linked Liver Glycogenosis in a Taiwanese Family: Transmission From Undiagnosed Males

X-linked liver glycogenosis (XLG), also known as glycogen storage disease type-lXa, is characterized by hepatomegaly, abnormal liver functions and growth retardation. It is caused by mutations in the PHKA2 gene that encodes the alpha-subunit of phosphorylase kinase (PHK). XLG can be divided into two subtypes: XLG-I, with a deficiency in PHK activity in peripheral blood cells and the liver; and XLG-II, with normal PHK activity in vitro. This report describes two boys who presented with hepatomegaly and abnormal liver function. Pedigree analysis revealed them to be fifth-degree relatives, with the disease transmitted through undiagnosed grandfathers. Liver histology confirmed GSD diagnosis, and both cases had a deficiency in PHK activity in red blood cells and liver tissues. This is the first report of XLG-I in the ethnic-Chinese population in Taiwan. This report indicates that XLG may be undiagnosed or underestimated. A correct diagnosis is necessary for proper management and genetic counseling.

3D mapping of glycogenosis-causing mutations in the large regulatory alpha subunit of phosphorylase kinase

Mutations in the liver isoform of the Phosphorylase Kinase (PhK) α subunit (PHKA2 gene) cause X-linked liver glycogenosis (XLG), the most frequent type of PhK deficiency (glycogen-storage disease type IX). XLG patients can be divided in two subgroups, with similar clinical features but different activity of PhK (decreased in liver and blood cells for XLG-I and low in liver but normal or enhanced in blood cells for XLG-II). Here, we show that the PHKA2 missense mutations and small in-frame deletions/insertions are concentrated into two domains of the protein, which were recently described. In the N-terminal glucoamylase domain, mutations (principally leading to XLG-II) are clustered within the predicted glycoside-binding site, suggesting that they may have a direct impact on a possible hydrolytic activity of the PhK α subunit, which remains to be demonstrated. In the C-terminal calcineurin B-like domain (domain D), mutations (principally leading to XLG-I) are clustered in a region predicted to interact with the regulatory region of the PhK catalytic subunit and in a region covering this interaction site. Altogether, these results show that PHKA2 missense mutations or small in-frame deletions/insertions may have a direct impact on the PhK α functions and provide a framework for further experimental investigation.

Myopathy and phosphorylase kinase deficiency caused by a mutation in thePHKA1 gene

Phosphorylase kinase (PhK) deficiency is the underlying cause of variable clinical symptoms depending on the tissues involved. Until today, only a few cases of myopathy associated with muscle PhK deficiency caused by a mutation in the gene encoding the alpha subunit of phosphorylase kinase (PHKA1) have been reported. We describe a male patient with myopathy and absent muscle PhK activity caused by a frameshift mutation in the gene encoding the alpha subunit of PhK on chromosome Xq12-q13.

The regulatory alpha subunit of phosphorylase kinase may directly participate in the binding of glycogen phosphorylase.

The yeast two-hybrid screen has been used to identify potential regions of interaction of the largest regulatory subunit, alpha, of phosphorylase kinase (PhK) with two fragments of its protein substrate, glycogen phosphorylase b (Phb). One fragment, corresponding to residues 17-484 (PhbN'), contained the regulatory domain of the protein, but in missing the first 16 residues was devoid of the sole phosphorylation site of Phb, Ser14; the second fragment corresponded to residues 485-843 (PhbC) and contained the catalytic domain of Phb. Truncation fragments of the alpha subunit were screened for interactions against these two substrate fragments. PhbC was not found to interact with any alpha constructs; however, PhbN' interacted with a region of alpha (residues 864-1014) that is near the phosphorylatable region of that subunit. PhbN' was also screened for interactions against a variety of fragments of the catalytic gamma subunit of PhK; however, no interactions were detected, even with full-length gamma. Our results support the idea that amino acid residues proximal to the convertible serine of Phb are important for its specific interaction with the catalytic subunit of PhK, but that regions distinct from the convertible serine residue of Phb and from the catalytic domain of PhK may also be involved in the interaction of these two proteins.

Muscle phosphorylase kinase is not a substrate of AMP-activated protein kinase.

AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (cAMPK) have been reported to phosphorylate sites on phosphorylase kinase (PhK). Their target residues Ser 1018 and Ser 1020, respectively, are located in the so-called multi-phosphorylation domain in the PhK alpha subunit. In PhK preparations, only one of these serines is phosphorylated, but never both of them. The aim of this study was to determine whether phosphorylation by cAMPK or AMPK would influence subsequent phosphorylation by the other kinase. Surprisingly, employing four different PhK substrates, it could be demonstrated that, in contradiction to previous reports, PhK is not phosphorylated by AMPK.

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

The structural organization of the (alphabetagammadelta)(4) phosphorylase kinase complex has been studied using the yeast two-hybrid screen for the purpose of elucidating regions of alpha subunit interactions. By screening a rabbit skeletal muscle cDNA library with residues 1-1059 of the alpha subunit of phosphorylase kinase, we have isolated 16 interacting, independent, yet overlapping transcripts of the alpha subunit containing its C-terminal region. Domain mapping of binary interactions between alpha constructs revealed two regions involved in the self-association of the alpha 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-(gamma-maleimidobutyroxy)sulfosuccin-imide ester produced intramolecularly cross-linked alpha-alpha dimers, consistent with portions of two alpha subunits in the holoenyzme being in sufficient proximity to associate. This is the first report to identify potential areas of contact between the alpha subunits of phosphorylase kinase. Additionally, issues regarding the general utility of two-hybrid screening as a method for studying homodimeric interactions are discussed.

Genetic deficiencies of the glycogen phosphorylase system.

Several types of glycogen storage disease attributable to a deficiency of phosphorylase or phosphorylase kinase have been described. These diseases have been divided according to clinical symptoms, mode of inheritance, and affected tissue. However, this classification is questionable, as the clinical symptoms of these different diseases are similar, the mode of inheritance is often difficult to establish, and the biochemical assays are subject to several technical problems. A better classification would be based upon the identification of mutations in the respective disease genes. The molecular heterogeneity, however, is large, and at least 10 genes are involved. Mutations have been found in the muscle phosphorylase gene in patients with muscle phosphorylase deficiency, in the gene encoding the liver alpha subunit of phosphorylase kinase in patients with X-linked liver glycogenosis, and in the gene for the muscle alpha subunit of phosphorylase kinase in a patient with muscle phosphorylase kinase deficiency. We review here the different deficiencies of the phosphorylase system.

Mutation hotspots in the PHKA2 gene in X-linked liver glycogenosis due to phosphorylase kinase deficiency with atypical activity in blood cells (XLG2).

In five cases of X-linked liver glycogenosis subtype 2 (XLG2), we have identified mutations in the gene encoding the liver isoform of the phosphorylase kinase alpha subunit (PHKA2). XLG2 is a rare variant of X-linked phosphorylase kinase (Phk) deficiency of the liver. Whereas in the more common form of X-linked hepatic Phk deficiency, XLG1, the enzyme's activity is decreased both in liver and in blood cells, Phk activity in XLG2 is low in liver but normal or even enhanced in blood cells. Although missense, nonsense and splicesite mutations in the PHKA2 gene were recently identified in several cases of XLG1, no mutations have yet been described for XLG2 and a molecular explanation for the peculiar biochemical phenotype of XLG2 has been lacking. All mutations found in the present study result in non-conservative amino acid replacements of residues that are absolutely conserved between the alpha L, alpha M and beta subunits of Phk [H132P, H132Y, R186H (twice) and D299G]. Strikingly, in two pairs of cases the mutations affect the same codon. These results demonstrate that: (i) XLG2 is caused by mutations in PHKA2 and is therefore allelic with XLG1; and (ii) XLG2 mutations appear to cluster in limited sequence regions or even individual codons.

Dinucleotide repeat polymorphism within the PHKA1 gene at Xq12-q13

A polymorphic complex repeat including two (TG)n stretches was identified in the intron following codon 26 of the human gene encoding the muscle isoform of the phosphorylase kinase alpha subunit (PHKA1). It should be a useful marker for linkage analysis of families with heritable phosphorylase kinase deficiency and for gene mapping in the vicinity of the X inactivation center.

Expression and biochemical properties of a protein serine/threonine phosphatase encoded by bacteriophage lambda.

The predicted amino acid sequence encoded by the open reading frame 221 (orf221) of bacteriophage lambda exhibited a high degree of similarity to the catalytic subunits of a variety of protein serine/threonine phosphatases belonging to PP1, PP2A, and PP2B groups. Cloning and expression of the orf221 gene in Escherichia coli provided direct evidence that the gene codes for a protein serine/threonine phosphatase. The single-subunit recombinant enzyme was purified in soluble form and shown to possess a unique repertoire of biochemical properties--e.g., an absolute requirement for Mn2+, resistance to okadaic acid, inhibitors 1 and 2, and ability to dephosphorylate casein, adenovirus E1A proteins, and the alpha subunit of phosphorylase kinase. No phosphotyrosine phosphatase activity was observed. Mutational and biochemical analyses identified the conserved residues 73-77 and Cys138 to be important for activity. The name PP-lambda is proposed for this unusual prokaryotic enzyme.

The multiphosphorylation domain of the phosphorylase kinase alpha M and alpha L subunits is a hotspot of differential mRNA processing and of molecular evolution.

We have cloned and sequenced human cDNAs encoding the complete phosphorylase kinase alpha subunit muscle isoform (alpha M). The predicted polypeptide is highly similar to the sequence known from rabbit muscle but lacks a major part of its multiphosphorylation domain, including the main phosphorylation site for cAMP-dependent protein kinase (PKA). Analysis of this region by reverse-transcribed polymerase chain reaction (RT-PCR) in several human and rabbit tissues demonstrates that it is subject to elaborate differential mRNA splicing. Amino acids 1012-1024 of the full-length rabbit sequence, including the major PKA phosphorylation site, and amino acids 1025-1041, which harbor at least one endogenous phosphorylation site, can be deleted from the predicted polypeptide individually or in combination. Molecules lacking one or both of these segments constitute a major part of the alpha M subunit population in many rabbit tissues and constitute the vast majority in all human tissues analyzed. Similar, tissue-dependent differential splicing events could be detected by RT-PCR in the human alpha subunit isoform from liver (alpha L). The expression of the differentially spliced alpha M subtypes differs markedly between corresponding human and rabbit tissues. Sequence divergence in this region is particularly high, not only between the muscle and liver isoforms, but also between alpha M sequences from four different animal species. Moreover, a duplication of the exon encoding the main PKA phosphorylation site was discovered in the mouse. Thus, the multiphosphorylation domain of the phosphorylase kinase alpha subunit isoforms is subject to pronounced structural variation not only between different tissues of one organism via differential splicing, but also in the course of evolution.

Dephosphorylation of phosphorylated atrial natriuretic peptide by protein phosphatase 2A.

A phosphatase which exhibits strong activity toward phosphorylated atrial natriuretic peptide (ANP) was identified in the soluble fraction of rat brain homogenate. This ANP phosphatase has a neutral pH optimum, does not require divalent cations for activity, is inhibited by low concentrations of okadaic acid (50% inhibition at 1 nM) and preferentially dephosphorylates the alpha subunit of phosphorylase kinase. These properties are characteristic of serine/threonine protein phosphatase type 2A (PP2A). The apparent molecular mass of the ANP phosphatase (160 kDa), as estimated by gel filtration, is similar to that of the native heterotrimeric form of PP2A. In addition, phosphorylated ANP is an excellent substrate for the purified catalytic subunit of PP2A (Km = 42 microM, Vmax = 10.3 mumol x min-1 x mg-1). In contrast, protein phosphatase 2B (PP2B) has only very low ANP phosphatase activity (Km = 2.5 microM, Vmax = 0.008 mumol x min-1 x mg-1), and the catalytic subunit of protein phosphatase type 1 (PP1) as well as purified protein phosphatase type 2C (PP2C) are essentially inactive on ANP. These findings are consistent with the observation that PP2A-like activity accounts for virtually all ANP dephosphorylation in brain homogenate. While the phosphorylation of ANP in vitro by cAMP-dependent protein kinase is well documented, this is a first report on a phosphatase that efficiently can reverse this modification.

Characterization of neuronal protein phosphatases in Aplysia californica.

Biochemical properties of neuronal protein phosphatases from Aplysia californica were characterized. Dephosphorylation of phosphorylase alpha by extracts of abdominal ganglia and clusters of sensory neurons from pleural ganglia was demonstrated. Type-1 protein phosphatase (PrP-1) was identified in these extracts by the dephosphorylation of the beta-subunit of phosphorylase kinase and its inhibition by the protein, inhibitor-2. Type-2A protein phosphatase (PrP-2A) was demonstrated by the dephosphorylation of the alpha-subunit of phosphorylase kinase, which was insensitive to inhibitor-2. As in vertebrate tissues, only four enzymes, PrP-1 (47%), PrP-2A (42%), PrP-2B (11%), and PrP-2C (less than 1%), accounted for all the cellular protein phosphatase activity dephosphorylating phosphorylase kinase. Aplysia PrP-1 and PrP-2A were potently inhibited by okadaic acid, with PrP-1 being approximately 20-fold more sensitive than PrP-2A. By comparison, purified PrP-2A from rabbit skeletal muscle was 15- to 20-fold more sensitive to okadaic acid than PrP-1 from the same source. Only PrP-1 was associated with the particulate fractions from Aplysia neurons, whereas PrP-1 and PrP-2A, -2B, and -2C were all present in the cytosol. Extraction of the particulate PrP-1 decreased its sensitivity to okadaic acid by sixfold, suggesting that cellular factor(s) affect its sensitivity to this inhibitor. In most respects, protein phosphatases from Aplysia neurons resemble their mammalian counterparts, and their biochemical characterization sets the stage for examining the role of these enzymes in neuronal plasticity, and in learning and memory.

Mapping of the phosphorylase kinase alpha subunit gene on the mouse X chromosome

Phosphorylase kinase is a glycogenolytic enzyme in several animal tissues. Within the last few years all four subunits of the enzyme have been cloned. The beta, gamma, and delta subunits are known to be autosomal. We have mapped the alpha subunit of phosphorylase kinase, recently cloned by Zander et al. (1988), in an interspecific mouse pedigree and localized it on the X chromosome, where it maps between the X-linked zinc finger protein and phosphoglycerate kinase genes, close to the latter. In man and mouse several X-linked disorders of this enzyme have been described. Although the X-linked phosphorylase kinase deficiency in mice may be caused by a mutation in the structural gene for the alpha subunit, mapped here, the existence of a separate regulatory locus, important in the normal expression or function of the enzyme in muscle, still remains a possibility.

Regulation of protein phosphatase‐1G from rabbit/skeletal muscle

The glycogen-associated form of protein phosphatase-1 (PP-1G) is a heterodimer comprising a 37-kDa catalytic (C) subunit and a 161-kDa glycogen-binding (G) subunit, the latter being phosphorylated by cAMP-dependent protein kinase at two serine residues (site 1 and site 2). Here the amino acid sequence surrounding site 2 has been determined and this phosphoserine shown to lie 19 residues C-terminal to site 1 in the primary structure. The sequence in this region is: (sequence; see text) At physiological ionic strength, phosphorylation of glycogen-bound PP-1G was found to release all the phosphatase activity from glycogen. The released activity was free C subunit, and not PP-1G, while the phospho-G subunit remained bound to glycogen. Dissociation reflected a greater than or equal to 4000-fold decrease in affinity of C subunit for G subunit and was readily reversed by dephosphorylation. Phosphorylation and dephosphorylation of site 2 was rate-limiting for dissociation and reassociation of C subunit. Release of C subunit was also induced by the binding of anti-site-1 Fab fragments to glycogen-bound PP-1G. At near physiological ionic strength, PP-1G and glycogen concentration, site 2 was autodephosphorylated by PP-1G with a t0.5 of 2.6 min at 30 degrees C, approximately 100-fold slower than the t0.5 for dephosphorylation of glycogen phosphorylase under the same conditions. Site 2 was a good substrate for all three type-2 phosphatases (2A, 2B and 2C) with t0.5 values less than those toward the alpha subunit of phosphorylase kinase. At the levels present in skeletal muscle, the type-2A and type-2B phosphatases are potentially capable of dephosphorylating site 2 in vivo within seconds. Site 1 was at least 10-fold less effective than site 2 as a substrate for all four phosphatases. In conjunction with information presented in the following paper in this issue of this journal, the results substantiate the hypothesis that PP-1 activity towards the glycogen-metabolising enzymes is regulated in vivo by reversible phosphorylation of a targetting subunit (G) that directs the C subunit to glycogen--protein particles. The efficient dephosphorylation of site 2 by the Ca2+/calmodulin-stimulated protein phosphatase (2B) provides a potential mechanism for regulating PP-1 activity in response to Ca2+, and represents an example of a protein phosphatase cascade.

Identification of high levels of type 1 and type 2A protein phosphatases in higher plants

Extracts of Brassica napus (oilseed rape) seeds contain type 1 and type 2A protein phosphatases whose properties are indistinguishable from the corresponding enzymes in mammalian tissues. The type 1 activity dephosphorylated the beta-subunit of phosphorylase kinase selectively and was inhibited by the same concentrations of okadaic acid [IC50 (concentration causing 50% inhibition) approximately 10 nM], mammalian inhibitor 1 (IC50 = 0.6 nM) and mammalian inhibitor 2 (IC50 = 2.0 nM) as the rabbit muscle type 1 phosphatase. The plant type 2A activity dephosphorylated the alpha-subunit of phosphorylase kinase preferentially, was exquisitely sensitive to okadaic acid (IC50 approximately 0.1 nM), and was unaffected by inhibitors 1 and 2. As in mammalian tissues, a substantial proportion of plant type 1 phosphatase activity (40%) was particulate, whereas plant type 2A phosphatase was cytosolic. The specific activities of the plant type 1 and type 2A phosphatases were as high as in mammalian tissue extracts, but no type 2B or type 2C phosphatase activity was detected. The results demonstrate that the improved procedure for identifying and quantifying protein phosphatases in animal cells is applicable to higher plants, and suggests that okadaic acid may provide a new method for identifying plant enzymes that are regulated by reversible phosphorylation.

Reaction of fluorescein isothiocyanate with an ATP-binding site on the phosphorylase kinase alpha subunit

Phosphorylase kinase can be labeled specifically on the alpha subunit with fluorescein 5'-isothiocyanate (FITC) which concomitantly inactivates the enzyme (T. G. Sotiroudis and S. Nikolaropoulus (1984) FEBS Lett. 176, 421-425). 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 [14C]carboxymethylated alpha subunit. The partial sequences obtained have then been localized in the primary structure of the alpha subunit [Zander et al. (1988) Proc. Natl Acad. Sci. USA 85, 2929-2933]. 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 microM has been determined.

The alpha and beta subunits of phosphorylase kinase are homologous: cDNA cloning and primary structure of the beta subunit.

We have cloned cDNA molecules encoding the beta subunit of phosphorylase kinase (ATP:phosphorylase-b phosphotransferase; EC 2.7.1.38) from rabbit fast-twitch skeletal muscle and have determined the complete primary structure of the polypeptide by a combination of peptide and DNA sequencing. In the mature beta subunit, the initial methionine is replaced by an acetyl group. The subunit is composed of 1092 amino acids and has a calculated molecular mass of 125,205 Da. Alignment of its sequence with the alpha subunit of phosphorylase kinase reveals extensive regions of homology, but each molecule also possesses unique sequences. Two of the three phosphorylation sites known for the beta subunit and all seven phosphorylation sites known for the alpha subunit are located in these unique domains.