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PubMed1978

Site-specific phosphorylation of beta-casein by proetin kinases from rabbit reticulocytes.

The B variant of beta-casein was phosphorylated with [gamma-32P]ATP using four different protein kinases isolated from rabbit reticulocytes. Casein was maximally phosphorylated by the individual protein kinase activities and subjected to chymotrptic digestion. The peptides were separated by a two-dimensional peptide fingerprinting technique, and the phosphorylated peptides were identified by autoradiography, The two phosphorylated peptides obtained from the action of casein kinase I were shown to have different migration patterns from those obtained with casein kinase II. The cAMP-regulated protein kinases had the same substrate specificity with beta-casein B, and the two phosphorylated peptides obtained using these enzymes were distinct from those phosphorylated by the cAMP-independent enzymes. Thus, the different protein kinases can be identified by substrate specificity using beta-casein.

Animals↗

PubMed1975

Multiple forms of casein kinase from rabbit erythrocytes.

Two rabbit erythrocyte casein kinases, GTP:casein kinase I and GTP:casein kinase II, have been purified 29 000- and 47 000-fold, respectively. Studies employing sucrose density gradient centrifugation indicate that kinase I has a molecular weight of about 9.5 - 10(5) (25 S) and kinase II about 1.4 - 10(6) (32 S). These enzymes can utilize either ATP or GTP as the phosphoryl donor. Among various protein substrates examined, these kinases catalyze the phosphorylation of casein greater than 50% dephosphorylated phosvitin congruent to 50% dephosphorylated casein greater than phosvitin. Histones, protamine and bovine serum albumin are poor phosphoryl acceptors. Kinetic data indicate that both enzymes are inhibited by high casein substrate concentrations which may be partially relieved by NaCl. Both phosphotransferases require Mg(2+) for activity and are optimally active at pH 9.0. The enzymes have apparent Km values of 2.5 - 10(-5) M for GTP, 2 - 10(-5) M for ATP, and 0.4--0.6 mg/ml for casein. The incorporation of the terminal phosphate of GTP into casein as catalyzed by these enzymes is inhibited to varying degrees by ATP, ITP, ADP, and GDP but not by UTP, CTP, GMP, adenosine 3':5'-cyclic monophosphate, and guanosine 3':5'-cyclic monophosphate. In addition, NaF and 2,3-diphosphoglyceric acid are also found to inhibit the activity of both kinases. The effect of 2,3-diphosphoglycerate is interesting and suggests that this metabolite may regulate the activity of the casein kinases in the red blood cells.

Animals↗

PubMed2024

The ARK2N-CK2 complex initiates transcription-coupled repair through enhancing the interaction of CSB with lesion-stalled RNAPII.

Transcription is extremely important for cellular processes but can be hindered by RNA polymerase II (RNAPII) pausing and stalling. Cockayne syndrome protein B (CSB) promotes the progression of paused RNAPII or initiates transcription-coupled nucleotide excision repair (TC-NER) to remove stalled RNAPII. However, the specific mechanism by which CSB initiates TC-NER upon damage remains unclear. In this study, we identified the indispensable role of the ARK2N-CK2 complex in the CSB-mediated initiation of TC-NER. The ARK2N-CK2 complex is recruited to damage sites through CSB and then phosphorylates CSB. Phosphorylation of CSB enhances its binding to stalled RNAPII, prolonging the association of CSB with chromatin and promoting CSA-mediated ubiquitination of stalled RNAPII. Consistent with this finding, Ark2n-/- mice exhibit a phenotype resembling Cockayne syndrome. These findings shed light on the pivotal role of the ARK2N-CK2 complex in governing the fate of RNAPII through CSB, bridging a critical gap necessary for initiating TC-NER.

DNA Repair Enzymes↗

PubMed2026

CK2α restriction of STING accumulation underlies systemic aging.

Chronic activation of the cGAS-STING pathway drives inflammaging and cellular senescence. Although nuclear envelope (NE) barrier failure leading to cytoplasmic chromatin leakage is a key trigger, the molecular mechanisms governing STING activity at the NE during aging remain poorly understood. Here, we identify lamin A/C (LMNA) as a critical NE scaffold that orchestrates STING regulation by recruiting both STING and Casein Kinase 2 (CK2α). We demonstrate that LMNA facilitates the phosphorylation of STING at Ser366 by CK2α, which promotes STING turnover and restricts its accumulation, thereby attenuating pathway activation and mitigating senescence in myeloid cells as well as systemic aging. Strikingly, pharmacologic STING inhibition in vivo robustly rescues progeroid phenotypes-including loss of bone density and multi-tissue senescence-and extends lifespan in progeroid mouse models. Moreover, H-151 treatment also ameliorates the premature aging phenotypes induced by myeloid-specific CK2α ablation. In contrast, constitutive STING ablation yields limited survival benefits, revealing that controlled attenuation of STING signaling, rather than complete elimination, drives therapeutic efficacy. Our findings establish the LMNA-CK2-STING axis as a key biochemical mechanism that suppresses innate immune activation at the NE, offering a promising strategy for ameliorating aging and progeroid pathologies.

Animals↗

PubMed2025

Genetic analysis of four cases of Poirier Bienvenu neurodevelopmental syndrome associated with CSNK2B variant.

BACKGROUND: CSNK2B deficiency underlies the pathogenesis of Poirier-Bienvenu neurodevelopmental syndrome (POBINDS). In this study, we present four cases of pediatric seizures caused by de novo variants in CSNK2B, with the aim to reinforce the clinical and variant data pertaining to early genetic factors associated with epilepsy. METHODS: Trio whole exome sequencing were used to detect variants in the proband and her family members, and bioinformatics annotation was performed for the variant. Sanger sequencing and CSNK2B cDNA sequencing were employed to ascertain the carrier status of additional family members and evaluate the potential impact of variants on splicing. RESULTS: All four cases presented with epilepsy as the initial manifestation, accompanied by global developmental delay, particularly in language and motor developmental delay. Cases 1, 3 and 4 exhibited full-scale tonic-clonic seizures, while case 2 displayed myoclonic and typical absence seizures. Furthermore, case 2 demonstrated delayed growth and development compared to age-matched peers. No abnormality was detected in the head magnetic resonance imaging (MRI). Genetic analysis revealed novel heterozygous variants in the CSNK2B gene in all four cases, including c.175 + 1G > A, c.73-2A > G, c.291 + 1G > A and c.481delA. In case 2, reverse transcription analysis of CSNK2B mRNA revealed the retention of the 3' end sequence of Intron 2 and deletion of the 5' end sequence of Exon 3. In treatment, four case received a combination of one to three types of antiseizure medication and rehabilitation training individually. Case 1 continued to experience seizures to varying degrees, while cases 2-4 demonstrated effective seizure control. Overall motor and intellectual development improved in all four cases, however, there was slow recovery in language function. CONCLUSION: This study elucidates the molecular etiology of epilepsy in four cases with POBINDS and expands the mutational spectrum of pathogenic variants in the CSNK2B, highlighting their impact on splicing. The highly genetic heterogeneous phenotype of POBINDS relies on the detection of pathogenic variants in CSNK2B. Conventional antiseizure medication effectively control seizures, while rehabilitation treatment can significantly improve intelligence and motor function to varying degrees; however, language recovery tends to be relatively slow.

Humans↗

PubMed1979

[Human erythrocyte protein kinases (author's transl)].

Cyclic AMP dependent and independent protein kinase activities are present in membrane as well as cytosol of human erythrocyte. Red cell membrane contains at least: 1) a cAMP dependent histone kinase type I using ATP as phosphoryl donor; 2) a cAMP independent casein kinase using ATP or GTP as phosphoryl donor; this kinase probably phosphorylates erythrocyte spectrin. In lysate of human red cell there are two or three cAMP dependent protein kinase type I and II and one cAMP independent casein kinase. Characteristics of these protein kinase activities are reviewed. Membrane substrates are numbered taking in account that the activity assays are often of dubious value. Several phosphorylation abnormalities previously reported in hematologic and non-hematologic disorders are still uncertain and at present time not any red cell protein kinase deficiency has been demonstrated.

Animals↗

PubMed1977

Purification and characterization of Novikoff ascites tumor protein kinase.

A protein kinase, designed KII, has been purified 5000-fold from Novikoff ascites tumor cells. The purification procedure also allows for the purification of a second major protein kinase, designated KI, as well as RNA polymerase I and II. Purified KII has a sedimentation constant of 7.6 S and a Stokes radius of 39 A, suggesting a molecular weight of about 122000. Polyacrylamide gel electrophoresis of the enzyme in the presence of sodium dodecyl sulfate suggests the enzyme is composed of subunits of molecular weights 44 000, 40 000, and 26 000 present in a molar ratio of 1:1:2. Incubation of the enzyme alone in the presence of [gamma-32P]ATP results in the phosphorylation of the 26 000-dalton subunit. Protein kinase II actively phosphorylates phosvitin, casein, and nonhistone chromosomal proteins but does not phosphorylate basic proteins such as histones or protamine to an appreciable extent. Km values of 3.6 micron for ATP and 6.5 micronM for GTP were determined in the presence of 4mM Mg2+. The enzyme is neither stimulated by cyclic adenosine 3',5'-monophosphate or cyclic guanosine 3', 5'-monophosphate nor inhibited by the regulatory subunit of rabbit muscle protein kinase. Its activity is stimulated by KCl at concentrations below 0.2 M and inhibited by higher concentrations.

Cyclic AMP↗

PubMed1975

Relationship between RNA polymerase and protein kinase activities in rat mammary gland nuclei.

1. Extracts from rat mammary gland nuclei contain cyclic AMP -independent protein kinases which phosphorylate casein rather than histone. 2. A major increase in nuclear protein kinase activity occurred during late pregnancy and was maintained with the onset of lactation. 3. Two major peaks of activity were resolved by chomatography of nuclear extracts on DEAE-Sephadex; the first (NI) appeared in the void volume and the second (NII) was eluted by 0.05-0.12 M ammonium sulfate. Several other regions of lesser activity were also present. 4. Protein kinases in the cytosol 105,000 times g supernatant, precipitated by 70 percent ammonium sulfate, dialyzed against buffer, and chromatographed on DEAE-Sephadex, yielded a major components phosphorylated histone in preference to casein, and this was stimulated by cyclic AMP if histone was the substrate, but only the first (void volume) fraction was cyclic AMP-dependent when casein was used. 5. Most of RNA polymerases Ib and II, derived from the nucleolus and nucleoplasm, respectively, appeared in column fractions distinct from those containing the major NI and NII protein kinases. 6. Cyclic AMP altered the amount of RNA product synthesized by polymerases Ib and II, but the explanation for this is unknown. Due to their elution profiles and cyclic AMP-independence, protein kinases NI and NII are excluded from playing a catalytic role in these effects; participation of quantitatively minor protein kinases which co-elute with polymerase Ib and II is not yet excluded.

Animals↗

PubMed1976

Protein kinases from liver mitochondria of tumour-bearing rats.

The mitochondria of liver of Yoshida ascites tumour-bearing rats contained two forms of protein kinase distinguishable on the basis of their kinetic properties, substrate specificity and responses to cyclic adenosine 3',5'-monophosphate (cAMP). One of these (kinase I) was activated 2-3 fold by cAMP while the other form (kinase II) was insensitive to the action of cAMP. Kinase I which was selective towards histone F1 as substrate was obtained as a homogeneous preparation and was observed to have a molecular weight of 170 000 by Sephadex G-150 gel filtration. Protein kinase II appeared to be a smaller protein with molecular weight of 54 000 and was specific towards acidic proteins namely casein and phosvitin. Protein kinases isolated from liver mitochondria of normal rats showed variations in respect to elution profile of DEAE-cellulose and electrophoretic mobility. The preparation corresponding to kinase I did not show stimulatory responses to cAMP.

Animals↗

PubMed1977

Endogenous protein kinase inhibitors. Purification, characterization, and distribution in different tissues.

A thermostable inhibition of ATP-protein phosphotransferase (EC 2.7.1.37) (protein kinase) which is present in crude tissue extracts has been resolved by gel chromatography (Sephadex G-100) into two molecular forms. These two forms will be referred to as type I and type II inhibitor. The type I inhibitor (Mr approximately or equal to 24,000) is specific for cAMP-dependent protein kinase and corresponds to the inhibitor described earlier (Walsh, D. A., Ashby, C. D., Gonzalez, C., Calkins, D., Fisher, E. H., and Krebs, E. G. (1971) J. Biol. Chem. 246, 1977-1985). The type II inhibitor (Mr approximately or equal to 15,000) competes for the enzyme with various substrate proteins (histone, alpha-casein, and Leu-Arg-Arg-Ala-Ser-Leu-Gly (kemptide). The type II inhibitor blocks protein phosphorylation catalyzed by several types of protein kinases (cAMP- and cGMP-dependent or cyclic nucleotide-independent protein kinases). The type II inhibitor from rat brain has been purified 1500-fold; this protein is thermostable, has acidic characteristics, and does not require Ca2+ ions for its activity. Different ratios and concentrations of type I and type II inhibitors of protein kinase are found in rat skeletal muscle, pancreas, cerebellum and corpus striatum, and in lobster tail muscle.

Animals↗

PubMed1977

Resolution and general properties of different types of ribosomal protein kinases in mouse plasmocytoma.

Three different types of protein kinases (ATP: protein phosphotransferase, EC 2.7.1.37) were isolated and partially purified from a mouse plasmacytoma microsomal KCl wash fraction, then chromatographed on DEAE cellulose and phosphocellulose. The three protein kinase activities designated by protein kinase I, II and III were characterized with respect to their capacity to utilize [gamma-32P]ATP and [gamma-32P]GTP, to interact with cyclic AMP, stimulation by cyclic AMP, substrate specificity and sedimentation behaviour on glycerol gradient centrifugation. Protein kinase I was found to be cyclic AMP dependent and preferentially phosphorylated histones. Protein kinase II and III were insensitive to cyclic AMP, protein kinase II preferentially phosphorylated histones and the protein(s) of a ribosomal KCl wash fraction eluted from DEAE cellulose between 0.2 and 0.35 M KCl and termed "PPx". Protein kinase III phosphorylated casein and ribosomal proteins to a great extent. Studies with glycerol density gradient centrifugation indicated that protein kinase I sediments as a component of about 4.4 S, protein kinase II of 4.3 S and protein kinase III of 3 S. Chromatography on phosphocellulose of the protein kinases isolated from purified free polysomes showed the same type of protein kinases as those from microsomes. So it appears unlikely that protein kinase I and II were contaminants from the cytosol.

Animals↗

PubMed1979

Cyclic AMP-dependent and -independent protein kinases of the water mold, Blastocladiella emersonii.

Protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) and cyclic adenosine 3',5'-monophosphate binding activities have been identified in zoospore extracts of the water mold Blastocladiella emersonii. More than 75% of these activities is found in the soluble fraction. Soluble protein kinase activity is resolved in three peaks(I, II and III) by DEAE-cellulose chromatography. Peak I is casein dependent and insensitive to cyclic AMP. Peak II is histone dependent and cyclic AMP independent; this enzyme is inhibited by the heat-stable inhibitor from bovine muscle. Peak III utilizes histone as substrate and is activated by cyclic AMP.

Blastocladiella↗

PubMed1975

Changes in cyclic AMP-dependent protein dinase activity in Tetrahymena pyriformis during the growth cycle.

An adenosine 3':5'-monophosphate-dependent protein kinase II (ATP:protein phosphotransferase, EC 2.7.1.37) was partially purified from the cytosol fraction of an exponentially growing culture of Tetrahymena pyriformis. Protein kinase II represented approximately 90% of the cytosolic protein kinase activity. The enzyme had a high degree of substrate specificity for calf thymus and Tetrahymena histones as compared to casein, protamine and phosvitin. The enzyme incorporated the terminal phosphate of ATP into serine and threonine residues of all the histone fractions. The apparent Km of the enzyme for adenosine 3':5'-monophosphate (cyclic AMP) was 1-10-minus 8 M. Protein kinase II was also activated by other cyclic nucleotides with apparent Km values in the range 2.k-10-minus 6 M. Ther specific activity of the cyclic AMP-dependent protein kinase of Tetrahymena decreases markedly from initial high values during the transition from the lag to early log phase of growth. This is followed by a shrp increase in the activity of the enzyme as the log phase of growth progresses. The specific activity of the enzyme increases rapidly during the heat-induced synchronization of Tetrahymena cells. The capacity for rapid phosphorylation of multiple classed of organelle-specific phosphoproteins and the level of cyclic AMP were maximal in Tetrahymena during the earliest phase of growth. These results demonstrate that the cell cycle of Tetrahymena may be coordinated by marked variations in the level of cyclic AMP which in turn regulate the cyclic AMP-dependent protein kinase.

Animals↗

PubMed1979

Human platelet myosin light chain kinase requires the calcium-binding protein calmodulin for activity.

In an actomyosin fraction isolated from human platelets, phosphorylation of the 20,000-dalton light chain of myosin is stimulated by calcium and the calcium-binding protein calmodulin. The enzyme catalyzing this phosphorylation has been isolated by using calmodulin-affinity chromatography. Platelet myosin light chain kinase activity was monitored throughout the isolation procedures by using the 20,000-dalton smooth muscle myosin light chain purified from turkey gizzards as substrate. The partially purified myosin kinase requires both calcium and calmodulin for activity and has a specific activity of 3.1 mumol of phosphate transferred to the 20,000-dalton light chain per mg of kinase per min under optimal assay conditions. Km values determined for ATP and myosin light chains are 121 microM and 18 microM, respectively. Of several substrates surveyed as phosphate acceptors (alpha-casein, histone II-A, phosphorylase b, protamine, histone V-S, and phosvitin), only the 20,000-dalton myosin light chain is phosphorylated at a significant rate. These results suggest that platelet myosin light chain kinase is a calcium-dependent enzyme and that the requirement for calcium is mediated by the calcium-binding protein calmodulin.

Actomyosin↗

PubMed1977

Effect of Mg2+ concentration on the cAMP-dependent protein kinase-catalyzed activation of rabbit skeletal muscle phosphorylase kinase.

Phosphorylase kinase was found to be activated and phosphorylated at 10mM Mg2+ by the cAMP-dependent protein kinase-catalyzed reaction ot much higher levels than observed previously when reactions were carried out in 1 to 2 mM Mg2+ (Cohen, P. (1973) Eur. J. Biochem. 34, 1; Hayakawa, T., Perkin, J.P., and Krebs, E.G. (1973) Biochemistry 12, 574). That the reaction at 10 mM Mg2+ is protein kinase-catalyzed is supported by several observations: (a) the reaction is facilitated by the addition of protein kinase; (b) the reaction depends on cAMP when protein kinase holoenzyme is uded; (c) the reaction is not inhibited by 1 mM ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetate which is known to inhibit autoactivation and autophosphorylation of phosphorylase kinase; and (d) the protein inhibitor of protein kinase inhibits this reaction. The phosphorylation and activation of phosphorylase kinase seem to occur in two phases. At low Mg2+ only the first phase is manifested and involves the incorporation of 2 mol of phosphate, 1 mol into each of Subunits A and B. At high Mg2+ additional sites are phosphorylated almost exclusively on Subunit A, with phosphate incorporation approaching the final level of 7 to 9 mol. Enzyme activity at high Mg2+ is 2 to 3 times higher than that observed when activation is studied at low Mg2+. The observation that both casein and type II histone are phosphorylated to the same extent at 1 mM and 10 mM Mg2+ suggested that high Mg2+ may be altering the conformation of phosphorylase kinase thus rendering more phosphorylation sites accessible to protein kinase. Since the phosphorylation of phosphorylase kinase by either the protein kinase-catalyzed or autocatalytic reaction can result in the incorporation of 7 to 9 mol of phosphate, the finding that only about seven sites become phosphorylated by both mechanisms acting together suggest that activation by these two mechanisms may involve common phosphorylation sites.

Animals↗

PubMed1979

Purification and properies of cAMP dependent and independent histone kinases from human leukocytes.

Histone kinase activity was purified from human polymorphonuclear leukocytes by ammonium sulphate precipitation of a 180 000 x g supernatant, followed by DEAE-cellulose chromatography and gelfiltration. On DEAE-cellulose cAMP dependent kinase activity eluted in two peaks, I and III, at 1.2 mmho and 6.5 mmho, respectively. Catalytic subunit (C) from both peaks had Mr 33 000, 3.0S. Regulatory subunit (R) from peak I and III both had Mr 33 000 upon gelfiltration, but sedimented at 2.8--3.0S and 3.0--3.2S, respectively. R2 and R4 subunits were identified. The R-C dimer from peak I and III sedimented at 4.8S and (4.8)--5.1S, respectively. The holoenzyme from peak I had Mr 165 000, 6.7S, which suggest a R2C2 structure, while that of peak III sedimented at 6.7S, but eluted at Mr 330 000 (2R2C2) by gelfiltration. The Kmapp for peak I and III enzymes were, respectively: histone IIA 0.5 mg/ml (both forms), ATP 18 microM and 23 microM, and cAMP 5 X 10(-8) M and 6.3 x 10(-8) M. Both enzymes had pH optimum 6.7--6.9 and were equally sensitive to Ca2+, temperature and protein kinase inhibitor. The substrate specificity was histone VS greater than histone IIA = histone VIS greater than casein greater than phosvitin. Peak I enzyme, but not peak III enzyme, was dissociated by histone and high ionic strength and reassociation of R and C subunits were facilitated by ATP-Mg. It is concluded that peak I and III enzymes represent type I and II cAMP dependent protein kinases, respectively. Type I comprises 20--30% of cAMP dependent protein kinase activity and is absent from the 180 000 x g supernatant of gently disrupted cells. Purified catalytic subunit had Kmapp (ATP) 20 microM with rabbit muscle glycogen synthease I as substrates. Synthase I from rabbit muscle and human leukocytes were phosphorylated by catalytic subunit to synthase D (ratio of independence less than 0.07). cAMP independent histone kinase activity eluted in one peak (Peak II) at3 mmho. The enzymatic activity sedimented at 3.4S and eluted from gelfiltration with Mr 78 000. Kmapp for ATP was 78 microM and for histone IIA 0.5 mg/ml. The enzyme was sensitive to temperature, but less sensitive than cAMP dependent protein kinase to Ca2+, and insensitive to protein kinase inhibitor. The substrate specificity was histone IIA greater than histone VS = histone VIS, while casein and phosvitin were poor substrates. Glycogen synthase I was not phosphorylated. The cAMP independent histone kinase activity comprised 15% of the total histone kinase activity in a crude homogenate of leukocytes. Its physiological substrate is unknown.

Chromatography, DEAE-Cellulose↗

PubMed1976

Characterization of the protein kinases in a transplantable islet cell tumor of the Syrian hamster.

The protein kinase activities of a transplantable, insulin-producing hamster islet cell tumor were characterized using gel filtration, sucrose density gradient centrifugation and acrylamide gel electrophoresis. The post-microsomal supernatant fluid contains 70-80% of the protein kinase activity present in crude homogenates. A cAMP-dependent protein kinase, PK I (Mr 170,000), represents 25% of the soluble protein kinase activity assayed with protamine as substrate. It dissociates in the presence of cAMP into a cAMP-binding protein, R2 (Mr 90,000) and a catalytic subunit C (Mr 33,000). The dissociation induced by cAMP seems to be facilitated by the addition of Mg2+ and ATP. The regulatory subunit, R2, changes its gel filtration pattern in the presence of 0.5 M NaCl suggesting dissociation into a smaller subunit, R1 (Mr 44,000). By analogy with purified beef heart protein kinase (Erlichman et al., 1973) and skeletal muscle protein kinase, PK I. The presence in crude homogenates of a free cAMP-binding protein indistinguishable from the R2 derived by dissociation of PK I, suggests that PK I is partially dissociated in vivo. A cAMP-independent (casein) kinase (Mr 210,000) elutes with PK I on columns of Sepharose 6B. Another cAMP-independent protein kinase, PK II (Mr 88,000), is the predominatn form of soluble protein kinase accounting for approximately 75% of the soluble protein kinase activity detected using protaimine as substrate. This cAMP-independent protein kinase changes its gel filtration pattern in the presence of 0.5 M NaCl giving rise to a form which appears to have the same Mr (33,000) as the catalytic subunit of PK I. Studies comparing the catalytic subunit C of PK I with PK II and its salt-induced smaller molecular form demonstrate facile association of C with the cAMP-binding protein of purified bovine heart protein kinase to yield a hybrid holoenzyme, whereas PK II and its smaller form fail to recombine in this fashion. The 33,000 dalton forms derived from PK I (by cAMP) and PK II (by salt) also show different substrate specificities. It would appear, therefore, that pK II is a cAMP-independent protein kinase unrelated to PK I.

Adenoma, Islet Cell↗

PubMed1975

Isolation of a glycogen synthase I kinase that is independent of adenosine 3':5'-monophosphate.

Three protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) were detected when the soluble fraction of rabbit kidney medulla was chromatographed on DEAE-cellulose with a linear NaC1 gradient. The first two kinases eluted (Peak 1 and Peak II) were cyclic-AMP-dependent, wheras Peak III was cyclic-AMP-independent. A procedure was developed to separate the catalytic subunit of Peak II cyclic-AMP-dependent protein kinase (representing the bulk of the histone kinase activity) from Peak III protein kinase. In contrast to the catalytic subunit, Peak III protein kinase phosphorylated casein more rapidly than histone. Peak III was insensitive to the heat-stable protein inhibitor of cyclic-AMP-dependent protein kinases and appeared to have a higher requirement for ATP than did the catalytic subunit. Peak III catalyzed the conversion of glycogen synthase (UDPglucose:glycogen alpha-4-glucosyltransferase, EC 2.4.1.11) from the I (glucose-6-phosphate-independent) to the D (glucose-6-phosphate-dependent) form. This conversion was dependent on Mg-2+ and ATP and was unaffected by cyclic AMP, cyclic GMP, or the protein inhibitor. Glycogen synthase I in the soluble fraction of kidney medulla could be converted to the D form by endogenous glycogen synthase I kinase if Mg-2+ and ATP were added. Most of this glycogen synthase I kinase activity was unaffected by cyclic AMP or by the protein inhibitor, suggesting that Peak III may be of major importance in the regulation of glycogen synthase in vivo.

Ammonium Sulfate↗