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PubMed1989

Purification and partial characterization of protein phosphatases from rat thymus.

Protein phosphatases assayed with phosphorylase alpha are present in the soluble and particulate fractions of rat thymocytes. Phosphorylase phosphatase activity in the cytosol fraction was resolved by heparin-Sepharose chromatography into type-1 and type-2A enzymes. Similarities between thymocyte and muscle or liver protein phosphatase-1 included preferential dephosphorylation of the beta subunit of phosphorylase kinase, inhibition by inhibitor-2 and retention by heparin-Sepharose. Similarities between thymocyte and muscle or liver protein phosphatase-2A included specificity for the alpha subunit of phosphorylase kinase, insensitivity to the action of inhibitor-2, lack of retention by heparin-Sepharose and stimulation by polycationic macromolecules such as polybrene, protamine and histone H1. Protein phosphatase-1 from the cytosol fraction of thymocytes had an apparent molecular mass of 120 kDa as determined by gel filtration. The phosphatase-2A separated from the cytosol of thymocytes may correspond to phosphatase-2A0, since it was completely inactive (latent) in the absence of polycation and had activity only in the presence of polycations. The apparent molecular mass of phosphatase-2A0 from thymocytes was 240 kDa as determined by gel filtration. The catalytic subunit of thymocyte type-1 protein phosphatase was purified with heparin-Sepharose chromatography followed by gel filtration and fast protein liquid chromatography on Mono Q column. The purified type-1 catalytic subunit exhibited a specific activity of 8.2 U/mg and consisted of a single protein of 35 kDa as judged by SDS-gel electrophoresis. The catalytic subunit of type-2A phosphatase from thymocytes appearing in the heparin-Sepharose flow-through fraction was further purified on protamine-Sepharose, followed by gel filtration. The specific activity of the type-2A catalytic subunit was 2.1 U/mg and consisted of a major protein of 34.5 kDa, as revealed by SDS-gel electrophoresis.

Animals↗

PubMed1993

Identification of a serine/threonine-specific protein phosphatase from the archaebacterium Sulfolobus solfataricus.

We have observed that soluble extracts from the extreme acidothermophilic archaebacterium Sulfolobus solfataricus contained protein phosphatase activity that was greatly stimulated by the divalent metal ions Mn2+, Mg2+, Ni2+, or Co2+. This activity apparently arose from a single enzyme since (a) stimulation by these divalent metal ions was not additive and (b) protein phosphatase activity eluted as a single peak from both a DE52 ion-exchange column and a Sephadex G-100 gel filtration column. Its apparent molecular mass was approximately 28,000 daltons. The enzyme dephosphorylated a variety of phosphoserine-containing substrates including casein, histone H2a, phosphorylase kinase, or glycogen phosphorylase. The enzyme would not dephosphorylate either histone H1 or a number of phosphotyrosine-containing compounds. It removed only half the phosphate bound to histone H2b, which is phosphorylated at two sites by the cAMP-dependent protein kinase. Protein phosphatase activity was inhibited by EDTA, Cu2+, Zn2+, NaF, inorganic phosphate, or pyrophosphate; but was unaffected by other potential activators and inhibitors such as microcystin, okadaic acid, vanadate, polyamines, or sulfhydryl modifying reagents. This enzyme represents the first protein phosphatase to be identified in any member of the third and oldest phylogenetic kingdom in nature, the archaebacteria.

Enzyme Activation↗

PubMed1976

Protein phosphorylation and hormone action.

Although the scheme hormone leads to raised cyclic AMP levels leads to activated protein kinase leads to phosphorylated protein leads to physiological response may represent an outline for the action of several hormones, in the best understood example, namely regulation of glucogen metabolism in mammalian muscle, the picture is more complex. Modification of phosphorylase kinase by cyclic AMP-dependent protein kinase, after stimulation by adrenaline, leads to phosphorylation of the enzyme at two sites. Activation is associated exclusively with the phosphorylation of the primary site, but the secondary phosphorylation indirectly antagonizes the primary phosphorylation in that it is necessary to render the primary site susceptible to dephosphorylation. The recent separation of two distinct phosphorylase kinase phosphatases specific for the two sites shows that reversal of the hormonal stimulation is controlled by the relative activities of two enzymes with opposing functions. Glycogen synthetase, which is phosphorylated and inactivated by cyclic AMP-dependent protein kinase, is also under the control of insulin. Although insulin appears to stimulate glycogen synthetase by reversal of the inactivation catalysed by the cyclic AMP-dependent protein kinase, tissue cyclic AMP concentrations do not alter. The recent identification of a second glycogen synthetase kinase, unaffected by cyclic AMP, therefore raises the possibility that insulin action may also be mediated through phosphorylation-dephosphorylation mechanisms, which antagonize those mediated through cyclic AMP-dependent protein kinase.

Adenosine Triphosphate↗

PubMed2003

Effects of chronic ethanol treatment on the angiotensin II-mediated p42/p44 mitogen-activated protein kinase and phosphorylase a activation in rat hepatocytes.

We have demonstrated previously that 24 h of ethanol treatment potentiates angiotensin II (ANG II)-stimulated p42/p44 mitogen-activated protein kinase (MAPK) activity in hepatocytes. This potentiation of p42/p44 MAPK by ethanol exhibited agonist selectivity. To compare the effects of acute (24 h) versus chronic (6 weeks) ethanol treatment, ANG II-induced intracellular signaling was examined in (1) rat hepatocytes treated with ethanol for 24 h and (2) hepatocytes obtained from rats fed ethanol for 6 weeks. In hepatocytes obtained from rats fed ethanol for 6 weeks, ANG II-stimulated phosphorylase a was reduced, and this activity was calcium dependent and p42/p44 MAPK independent. Surprisingly, ANG II-stimulated p42/p44 MAPK activation was not affected in hepatocytes obtained from rats fed ethanol chronically (6 weeks). However, chronic (6 weeks) ethanol treatment decreased ethanol potentiation of p42/p44 MAPK by about 56.3% +/- 3.6% for p42 MAPK and 61.3% +/- 11.7% for p44 MAPK. Furthermore, ethanol had no effect on the expression of angiotensinogen and c-myc mRNA in hepatocytes. A decrease in ANG II-activated phosphorylase a, but not in p42/p44 MAPK activation, after chronic (6 weeks) ethanol treatment leads to the conclusion that they may not be dependent on each other.

Angiotensin II↗

PubMed1986

Detection of a metalloproteinase in patients with acute and chronic renal failure.

The effect of different dialyzer membrane materials (cuprophan, cellulose hydrate, polyacrylonitrile, polymethylmethacrylate, ethylene-vinyl alcohol copolymer) on the ultrafiltrate proteinase activity was investigated in 26 patients with acute renal failure (ARF) and 40 patients undergoing regular hemodialysis treatment (RDT). Furthermore, the proteinase activity was characterized in vitro using azocasein and phosphorylase kinase as substrates in the absence and presence of different proteinase inhibitors. Proteinase activity of ultrafiltrates obtained from ARF patients was significantly enhanced with the dialyzer KF 101 (ethylene-vinyl alcohol copolymer). The digestion pattern of phosphorylase kinase revealed an identical type of proteinases in ultrafiltrates of ARF and RDT patients. The pH optimum of this proteinase was at alkaline pH. The proteinase activity could be inhibited in the presence of EDTA, whereas serine proteinase inhibitors were ineffective. Furthermore, the inactivated proteinase after Sephadex G-10 chromatography (in order to separate ultrafiltrate electrolytes and trace elements from protein) could be reactivated after the addition of Mg++ and/or Ca++. We conclude that a metalloproteinase can be found in ARF and RDT patients, and that KF 101 is more effectively eliminating the proteinase activity in ARF patients than other dialyzer membranes.

Acute Kidney Injury↗

PubMed1994

Comparison of the phosphorylation of microtubule-associated protein tau by non-proline dependent protein kinases.

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)

Animals↗

PubMed1993

Development and characterization of fluorescently-labeled myosin light chain kinase calmodulin-binding domain peptides.

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.

Amino Acid Sequence↗

PubMed1987

Calcium . calmodulin-dependent protein kinase II and calcium . phospholipid-dependent protein kinase activities in rat tissues assayed with a synthetic peptide.

Rat tissue levels of Ca2+ . calmodulin-dependent protein kinase II (protein kinase II) and Ca2+ . phospholipid-dependent protein kinase (protein kinase C) were selectively assayed using the synthetic peptide syntide-2 as substrate. The sequence of syntide-2 (pro-leu-ala-arg-thr-leu-ser-val-ala-gly-leu-pro-gly-lys-lys) is homologous to phosphorylation site 2 in glycogen synthase. The relative Vmax/Km ratios of the known Ca2+-dependent protein kinases for syntide-2 were determined to be as follows: protein kinase II, 100; protein kinase C, 22; phosphorylase kinase, 2; myosin light chain kinase, 0.005. Levels of protein kinase II were highest in cerebrum (3.36 units/g tissue) and spleen (0.85 units/g) and lowest in testis (0.05 units/g) and kidney (0.04 units/g). Protein kinase II activity was localized predominantly in the 100,000g particulate fraction of cerebrum and testis, in the supernatant fraction of heart, liver, adrenal, and kidney, and about equally distributed between particulate and supernatant in spleen and lung. Likewise, protein kinase C activity was highest in cerebrum (0.56 units/g) and spleen (0.47 units/g), and the majority of activity was present in the cytosolic fraction for all tissues measured except for cerebrum and testis in which the kinase activity was equal in both fractions. Finally, the ratios of protein kinase II to protein kinase C were different in various rat tissues and between particulate and supernatant fractions. These results suggest somewhat different functions for these two Ca2+-regulated, multifunctional protein kinases.

Animals↗

PubMed1986

A multisubstrate Ca2+ and cyclic nucleotide independent kinase from vascular smooth muscle: modulation of activity by polycations.

A multisubstrate Ca2+ and cyclic nucleotide independent kinase (Mr = 47,000) was purified from bovine aortic smooth muscle. Phosphorylation of glycogen synthase by this enzyme was polycation modulable. Low concentrations of polylysine (0.04-0.16 microM) stimulated phosphorylation 2-7 fold, whereas higher concentrations suppressed phosphorylation. Glycogen synthase converted to its glucose 6-PO4 dependent form following phosphorylation in either the presence (7 mol 32P/mol synthase) or absence (4 mol 32P/mol synthase) of polylysine: extent of conversion correlated to extent of phosphorylation. Seven of 14 potential substrates tested were phosphorylated: kinase activity was greatest for phosvitin followed by casein, the receptor protein from type 2 cAMP-kinase, histone H2b, phosphorylase kinase, glycogen synthase, and myocardial myosin light chains. Phosphorylation of phosvitin or synthase was inhibited by heparin (1/2 maximally by 0.5 microgram/ml without salt and 37 micrograms/ml with 150 mM NaCl). The results suggest that the enzyme may participate in regulating arterial glycogen metabolism and that such regulation may be modulated by polycationic and polyanionic effectors.

Animals↗

PubMed1990

A synthetic peptide substrate for selective assay of protein kinase C.

Among various phosphate acceptor proteins and peptides so far tested, a synthetic peptide having the sequence surrounding Ser(8) of myelin basic protein, Gln-Lys-Arg-Pro-Ser(8)-Gln-Arg-Ser-Lys-Tyr-Leu, (MBP4-14), is the most specific and convenient substrate which can be used for selective assay of protein kinase C. This peptide is not phosphorylated by cyclic AMP-dependent protein kinase, casein kinases I and II, Ca2+/calmodulin-dependent protein kinase II, or phosphorylase kinase, and can be routinely used for the assay of protein kinase C with low background in the crude tissue extracts. The Km value is considerably low (7 microM) with a Vmax value of twice as much as that for H1 histone.

Amino Acid Sequence↗

PubMed1990

Development and characterization of polyclonal antibodies against a conserved sequence in the catalytic domain of protein kinases.

Using a synthetic oligopeptide (CGGGTPEYLAPEGGK) crosslinked to keyhole limpet hemocyanin we have raised polyclonal rabbit antibodies against a 9 residue homologous region found in the catalytic domain of most protein kinases. These antibodies reacted during Western immunoblotting with cAMP dependent protein kinase catalytic subunit, phosphorylase kinase gamma subunit and calcium calmodulin dependent protein kinase II which have homologous sequences of GTPEYLAPE, GTPSYLAPE and GTPGYLSPE, respectively. Five other protein kinases did not react with anti-GTPEYLAPE antibodies during Western immunoblotting. Affinity-purified antibodies were able to detect as little as 50 ng of cAMP dependent protein kinase and 200 ng of Ca2+/calmodulin dependent protein kinase II. Immunoblotting of A431 cell plasma membrane vesicles indicated the presence of an approximately 55 kDa protein that contains the conserved sequence and is likely to be a protein kinase. Antibodies directed against conserved sequences present in protein kinases, or possibly other enzymes, may be useful in identifying previously uncharacterized enzymes at the protein level.

Amino Acid Sequence↗

PubMed1985

NAD+ kinase--a review.

NAD+ kinase catalyzes the only (known) biochemical reaction leading to the production of NADP+ from NAD+. Most evidence indicates it is found in the cytoplasm, but reports of its presence in (other) cell bodies can not be discounted. Viewed as a protein, our knowledge of NADK composition and architecture is rudimentary. Though recognized as a large multimeric protein, no agreement is evident for the molecular weight (Mr = approximately 4-65 X 10(4] of the native protein. Is calmodulin an integral subunit of (some, all) NAD+ kinases (analogous to phosphorylase kinase in skeletal muscle)? Or is it an external modulator? Consensus is evident that a subunit of molecular weight 30-35 X 10(3) is a component of the mammalian and yeast kinase. In one case (rabbit liver) two types of subunits are reported to give rise to oligomers differing in molecular weight and catalytic activities. Viewed as an enzyme it is not known why such a complex aggregate is needed for what might otherwise appear to a routine phosphorylation reaction. Rapid equilibrium random (for pigeon liver and C. utilis preparations) and ping-pong (for A. vinelandii kinase) mechanisms have been proposed for the reaction, with multiple reactant binding sites indicated for the random cases. From the perspective of enzyme modulation, the demonstration that green plant and sea urchin egg kinases are targets for calmodulin regulation by intracellular Ca2+ links NADP+ production in these sources to the multi-level discriminatory control functions inherent to this Ca2+-protein complex. Significant questions arise from the results of various investigators considered in this review. These queries offer fertile ground for the selective design of key experiments directed to a better understanding of NAD+ kinase function and pyridine nucleotide biochemistry.

Calmodulin↗

PubMed1991

A phosphatase resistant substrate for the assay of protein kinase C in crude tissue extracts.

Protein kinase C (PKC) is routinely assayed, after it is partially purified over DEAE-cellulose chromatography to eliminate any interfering protein kinases and phosphatases, by measuring the transfer of gamma-phosphate of [gamma-32P]ATP to H1 histone. Recently, it has been shown that a synthetic peptide, comprising residues 4-14 of myelin basic protein (MBP4-14), is a very selective PKC substrate which is not phosphorylated effectively by cyclic AMP-dependent protein kinase, casein kinase I and II, Ca2+/calmodulin dependent protein kinase II or phosphorylase kinase [Yasuda, I., Kishimoto, A., Tanaka, S-I., Tominaga, M., Sakurai, A. and Nishizuka, Y. (1990) BBRC 166, 1220-1227]. We report here that once MBP4-14 is phosphorylated, it is not dephosphorylated by okadaic acid-sensitive phosphatases (protein phosphatases 1, 2A and 3) or other protein phosphatases such as calcineurin and/or PP 2C present in hippocampal homogenates. Therefore, MBP4-14 can be used for PKC assay in crude extracts of neural tissue.

Adenosine Triphosphate↗

PubMed2003

Alternative binding modes of an inhibitor to two different kinases.

Protein kinases are targets for therapeutic agents designed to intervene in signaling processes in the diseased state. Most kinase inhibitors are directed towards the conserved ATP binding site. Because the essential features of this site are conserved in all eukaryotic protein kinases, it is generally assumed that the same compound will bind in a similar manner to different protein kinases. The inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) is a selective inhibitor for the protein kinase CK2 (IC50 1.6 micro m) (Sarno et al. (2001) FEBS Letts.496, 44-48). Three other kinases [cyclin-dependent protein kinase 2 (CDK2), phosphorylase kinase and glycogen synthase kinase 3beta] exhibit approximately 10-fold weaker affinity for TBB than CK2. We report the crystal structure of TBB in complex with phospho-CDK2-cyclin A at 2.2 A resolution and compare the interactions with those observed for TBB bound to CK2. TBB binds at the ATP binding site of both kinases. In CDK2, each of the four bromine atoms makes polar contacts either to main chain oxygens in the hinge region of the kinase or to water molecules, in addition to several van der Waals contacts. The mode of binding of TBB to CDK2 is different from that to CK2. TBB in CDK2 is displaced more towards the hinge region between the N- and C-terminal lobes and rotated relative to TBB in CK2. The ATP binding pocket is wider in CDK2 than in CK2 resulting in fewer van der Waals contacts but TBB in CK2 does not contact the hinge. The structures show that, despite the conservation of the ATP binding pocket, the inhibitor is able to exploit different recognition features so that the same compound can bind in different ways to the two different kinases.

Adenosine Triphosphate↗

PubMed1985

The protein phosphatases involved in cellular regulation. Influence of polyamines on the activities of protein phosphatase-1 and protein phosphatase-2A.

The effects of polyamines on the oligomeric forms of protein phosphatase-1 (1G), protein phosphatase-2A (2A0, 2A1 and 2A2) and their free catalytic subunits (1C and 2AC) has been studied using homogeneous enzymes isolated from rabbit skeletal muscle. Spermine increased the activity of protein phosphatase-2A towards eight of nine substrates tested. Half-maximal activation was observed at 0.2 mM with optimal effects at 1-2 mM. Above 2 mM, spermine became inhibitory. The most impressive activation of protein phosphatase-2A was obtained with glycogen synthase, especially when phosphorylated at sites-3 (8-15-fold with protein phosphatase-2A1) and phenylalanine hydroxylase (6-7-fold with protein phosphatase-2A1) as substrates. Activation of protein phosphatases 2A0, 2A1 and 2A2 was greater than that observed with 2AC. Spermine was a more potent activator than spermidine, while putrescine had only a small effect. Qualitatively similar results were obtained with five other substrates, although maximal activation was much less (1.3-3-fold with protein phosphatase-2A1). The rate of dephosphorylation of glycogen phosphorylase was decreased by spermine, inhibition being more pronounced with protein phosphatase-2AC than with 2A0, 2A1 and 2A2. Spermine (I50 = 0.1 mM with protein phosphatase-2AC) was a more potent inhibitor than spermidine (I50 = 0.9 mM) or putrescine (I50 = 8 mM). Partially purified preparations of protein phosphatases-2A0, 2A1 and 2A2 from from rat liver were affected by spermine in a similar manner to the homogeneous enzymes from rabbit skeletal muscle. Spermine did not activate protein phosphatase-1 to the same extent as protein phosphatase-2A. Greatest stimulation (2.5-fold) was again observed with glycogen synthase labelled in sites-3, with half-maximal activation at 0.2 mM and optimal effects at 1-2 mM spermine. Spermine was a much more effective stimulator than spermidine, while putrescine was ineffective. Very similar results were obtained with protein phosphatases 1G and 1C. With four other substrates maximal activation by spermine was less than 1.5-fold, while the dephosphorylation of glycogen synthase (labelled in site-2), phosphorylase kinase, pyruvate kinase and glycogen phosphorylase were inhibited. Spermine (I50 = 0.04 mM) was a more potent inhibitor of the dephosphorylation of glycogen phosphorylase than spermidine (I50 = 0.9 mM) or putrescine (I50 = 9 mM).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1979

Stimulation of glycogen synthase phosphorylation by calcium-dependent regulator protein.

Phosphorylation of skeletal muscle glycogen synthase catalyzed by a protein kinase is stimulated up to 10-fold by the calcium-dependent regulator (CDR) protein. Half-maximal stimulation requires about 1 microgram of CDR/ml. Phosphorylation by the CDR-dependent synthase kinase is more rapid at pH 8.6 than at pH 6.8 and is blocked by ethylene glycol bis(beta-aminoethyl-ether)N,N'-tetraacetic acid and trifuloperazine. Approximately 60 to 70% of the phosphate is incorporated into the trypsin-insensitive region of glycogen synthase resulting in conversion of the a form to the b form of the enzyme. The CDR-dependent synthase kinase is not myosin light chain kinase, as this enzyme does not phosphorylate glycogen synthase. Furthermore, synthase phosphorylation by the cAMP-dependent protein kinase catalytic subunit is not affected by CDR. The possibility that CDR-dependent synthase kinase may be phosphorylase kinase is being investigated.

Animals↗

PubMed1991

Role of acidic residues as substrate determinants for casein kinase I.

Sites phosphorylated by casein kinase I have been characterized by the presence of acidic amino acids NH2-terminal to the modified residue. Recently, phosphoserine was shown to be a particularly effective determinant for casein kinase I action when present in the motif -S(P)-X-X-S- (Flotow, H., Graves, P. R., Wang, A., Fiol, C. J., Roeske, R. W., and Roach, P. J. (1990) J. Biol. Chem. 265, 14264-14269). Nonetheless, nonphosphorylated substrates for casein kinase I are well documented. In this study, we examined the efficacy of Asp and Glu residues as determinants of casein kinase I action using synthetic peptide substrates. Peptides with runs of Asp residues in the motif Dn-X-X-S- were substrates for casein kinase I. Peptides with n = 3 or 4 were the most effective substrates, much better than n = 2. The peptide with n = 1, a single Asp residue, was a very poor substrate. A block of 4 Glu residues was a little less effective as a substrate determinant than 4 Asp residues in an otherwise identical peptide. The most effective substrate, with the motif -D-D-D-D-X-X-S-, was specific for casein kinase I and was not detectably phosphorylated by cyclic AMP-dependent protein kinase, casein kinase II, glycogen synthase kinase 3, or phosphorylase kinase and thus will be useful for the specific assay of casein kinase I. This peptide was nonetheless significantly worse as a substrate than peptides in which casein kinase I action was determined by phosphoserine in the -3 position. Still, the fact that Asp or Glu residues can specify a casein kinase I substrate suggests that acidic character has a role in substrate selection by this protein kinase.

Amino Acid Sequence↗

PubMed1983

Time course of alpha1-adrenergic and vasopressin actions on phosphorylase activation, calcium efflux, pyridine nucleotide reduction, and respiration in hepatocytes.

Calcium efflux from rat liver perfused with nonrecirculating medium was observed at 1.4 s following 10(-6) M (-)epinephrine infusion, when the perfusate Ca2+ was 60 microM. Net calcium efflux was also seen in livers perfused with 1.3 microM Ca2+ at approximately 8 s. In isolated rat hepatocytes, phosphorylase, a cytosolic enzyme, was activated significantly at 3 s and maximally at approximately 15 s by phenylephrine (10(-5) M), epinephrine (10(-6) M), and vasopressin (10(-8) M). Hexose phosphates were elevated at between 3 and 6 s with vasopressin. Phenylephrine and vasopressin stimulated hepatocyte respiration relatively slowly. The effects took 10 s to become evident, were dependent on the presence of Ca2+, and were probably the result of increased total cellular reduced pyridine nucleotide observed at 5 s. The slowness of the increase in respiration indicates that it cannot be the cause of the Ca2+ mobilization, but is more likely to be a consequence of it. From these studies, it is proposed that, following binding of catecholamines to alpha 1-adrenergic receptors, Ca2+ is first mobilized from the plasma membrane resulting in an elevation of the free Ca2+ ion concentration in the cytosol (Charest, R., Blackmore, P. F., Berthon, B., and Exton, J. H. (1983) J. Biol. Chem. 258, 8769-8773) which stimulates phosphorylase kinase and, hence, phosphorylase. These events begin to occur within the first 2 to 3 s. Following this, the concentration of reduced pyridine nucleotide(s) increases at 5 s resulting in the stimulation of respiration seen at 10 s. These events occur more slowly than the mobilization of cell Ca2+ and activation of phosphorylase, and may be secondary to the rise in cytosolic Ca2+. The time at which mitochondrial Ca2+ decreases is not known, but it accounts for most of the Ca2+ mobilized.

Animals↗