Spermine specifically inhibits the phosphorylation of an 11,000-dalton nuclear protein in various cultured mammalian cell lines

The effect of polyamines (putrescine, spermidine and spermine) on endogenous protein phosphorylation in mouse neuroblastoma cells was investigated by using techniques of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The results indicated that spermine at 1mM completely inhibited the phosphorylation of the 11,000-dalton and 120,000-dalton proteins in nuclear fractions. The inhibition of the phosphorylation of the 11,000-dalton but not the 120,000-dalton protein by spermine was also observed in five other cell lines examined and appeared to be a general phenomenon. The inhibitory effect of spermine on the phosphorylation of the 11,000-dalton protein was specific, other cations such as ammonium chloride, arginine, putrescine, cyclen and trien were ineffective at equal molar or much higher concentrations.     

Rat liver nuclear protein kinases NI and NII. Purification, subunit composition, substrate specificity, possible levels of regulation

Rat liver nuclear protein kinases NI and NII have been purified to homogeneity by an improved method. This method includes a casein-phosvitin-Sepharose column step, which separates the enzymes from the other chromosomal non-histone proteins, and a gel filtration at high ionic strength in the presence of a high concentration of protease inhibitors to separate the two enzymes from each other. NI has an apparent molecular mass of approximately 50 kDa and is composed of a single subunit. NII has an apparent molecular mass of 133 kDa and is composed of two subunits of identical molecular mass. The V and the Km of the two enzymes were determined for several substrates. Both enzymes phosphorylate chromosomal non-histone proteins with partly different specificities as shown by two-dimensional electrophoreses. When incubated in the absence of protease inhibitors, the enzymes were degraded into discrete polypeptides. Autophosphorylation of a polypeptide derived from NII was observed after incubation of the enzyme with ATP. This phosphorylation stimulated the enzyme activity. Several chromosomal proteins coeluted with NII from the casein-phosvitin-Sepharose column. They remained associated with the enzyme in sucrose gradients, during gel filtration performed at physiological ionic strength, and are dissociated at high ionic strength. These proteins were highly phosphorylated when the protein-NII complex was incubated with ATP.     

Purification of nuclear cAMP-independent protein kinases from rat ventral prostate

Two nuclear cAMP-independent protein kinases (designated PK-N1 and PK-N2) were purified from rat ventral-prostate and liver. The yield of enzyme units was 4-5% and 7-9% for each enzyme from the prostatic nuclei and liver nuclei, respectively. The average fold purification for prostatic nuclear protein kinase N1 and N2 was 1360 and 1833, respectively. The respective average specific activity of the two enzymes towards casein was 81,585 and 110,000 nmol 32P incorporated/hr/mg of enzyme. Protein kinase N1 comprised one polypeptide of Mr 35,000 which underwent phosphorylation in the presence of Mg2+ + ATP. Protein kinase N2 comprised two polypeptides Mr 40,000 and 30,000 of which only the Mr 30,000 polypeptide was autophosphorylated. Both enzymes were active towards casein, phosvitin, dephosphophosvitin, spermine-binding protein, and non-histone proteins in vitro. Little activity was detected towards histones. Both enzymes were stimulated by 150-200 mM NaCl. MgCl2 requirement varied with the protein substrate but was between 2-4 mM for both enzymes. With dephosphophosvitin as substrate, the apparent Km for ATP for N1 protein kinase was 0.01 mM. GTP did not replace ATP in this reaction. Protein kinase N2 was active in the presence of ATP or GTP. The apparent Km was 0.01 mM for ATP, but 0.1 mM for GTP.     

Purification and characterization of a nuclear protein kinase from rat liver and a hepatoma that is capable of activating poly(A) polymerase

The cyclic nucleotide-independent protein kinase which is separated from poly(A) polymerase during its purification from nuclei of rat liver and Morris hepatoma 3924A was purified essentially to homogeneity. Liver nuclear poly(A) polymerase was dissociated from protein kinase by phosphocellulose column chromatography. In contrast, protein kinase copurified with the hepatoma poly(A) polymerase on the phosphocellulose column. Neither liver nor hepatoma kinase was stimulated by spermine or inhibited by heparin. These enzymes did not utilize GTP as phosphoryl donor, or histones or tyrosine-containing [Val5]-angiotensin II as phosphoryl acceptors. The apparent Km with respect to ATP was similar for the liver (4.7 microM) and hepatoma (11 microM) kinases, and the apparent Km with respect to casein was identical (0.6 microgram/microliter) for these enzymes. Both enzymes were capable of phosphorylating poly(A) polymerase and stimulating both tumor and liver poly(A) polymerase activity. However, in addition to their different chromatographic properties, the two kinases differed in molecular weight (liver, 37,000; hepatoma, 56,000), in their response to various divalent metal ions, and in their ability to phosphorylate hepatoma poly(A) polymerase (Km 7.9 and 30 microgram/microliter for liver and hepatoma enzymes, respectively). These latter characteristics distinguished the liver and hepatoma protein kinases from each other as well as from the previously described NI protein kinase.     

Major 56,000-dalton, soluble phosphoprotein present in bovine sperm is the regulatory subunit of a type II cAMP-dependent protein kinase

It has been shown that cAMP-dependent phosphorylation of a soluble sperm protein is important for the initiation of flagellar motion. The suggestion has been made that this motility initiation protein, named axokinin, is the major 56,000-dalton phosphoprotein present in both dog sperm and in other cells containing axokinin-like activity. Since the regulatory subunit of a type II cAMP-dependent protein kinase is a ubiquitous cAMP-dependent phosphoprotein of similar subunit molecular weight as reported for axokinin, we have addressed the question of how many soluble 56,000-dalton cAMP-dependent phosphoproteins are present in mammalian sperm. We report that in bovine sperm cytosol, the ratio of the type I to type II cAMP-dependent protein kinase is approximately 1:1. The type II regulatory subunit is related to the non-neural form of the enzyme and undergoes a phosphorylation-dependent electrophoretic mobility shift. The apparent subunit molecular weights of the phospho and dephospho forms are 56,000 and 54,000 daltons, respectively. When bovine sperm cytosol or detergent extracts are phosphorylated in the presence of catalytic subunits, two major proteins are phosphorylated and have subunit molecular weights of 56,000 and 40,000 daltons. If, however, the type II regulatory subunit (RII) is quantitatively removed from these extracts using either immobilized cAMP or an anti-RII monoclonal affinity column, the ability to phosphorylate the 56,000- but not 40,000-dalton polypeptide is lost. These data suggest that the major 56,000 dalton cAMP-dependent phosphoprotein present in bovine sperm is the regulatory subunit of a type II cAMP-dependent protein kinase and not the motility initiator protein, axokinin.     

Spermine Stimulation of a Nuclear NII Kinase from Pea Plumules and Its Role in the Phosphorylation of a Nuclear Polypeptide

We have previously demonstrated that spermine stimulates the phosphorylation of a 47 kilodalton nuclear polypeptide from pea plumules (N Datta, LK Hardison, SJ Roux 1986 Plant Physiol 82: 681-684) In this paper we report that spermine stimulates the activity of a cyclic AMP independent casein kinase, partially purified from a chromatin fraction of pea plumule nuclei. This effect of spermine was substrate specific; i.e. with casein as substrate, spermine stimulated the kinase activity, and with phosvitin as substrate, spermine completely inhibited the activity. The stimulation by spermine of the casein kinase was, in part, due to the lowering of the Mg²⁺ requirement of the kinase. Heparin could partially inhibit this casein kinase activity and spermine completely overcame this inhibition. By further purification of the casein kinase extract on high performance liquid chromatography, we fractionated it into an NI and an NII kinase. Spermine stimulated the NII kinase by 5- to 6-fold but had no effect on the NI kinase. Using [γ-³²P]GTP, we have shown that spermine promotes the phosphorylation of the 47 kilodalton polypeptide(s) in isolated nuclei, at least in part by stimulating an NII kinase.     

Identification of tyrosine-phosphorylated proteins associated with the nuclear envelope.

The nuclear envelope separates the nucleoplasm from the rest of the cell. Throughout the cell cycle, its structural integrity is controlled by reversible protein phosphorylation. Whereas its phosphorylation-dependent disassembly during mitosis is well characterized, little is known about phosphorylation events at this structure during interphase. The few characterized examples cover protein phosphorylation at serine and threonine residues, but not tyrosine phosphorylation at the nuclear envelope. Here, we demonstrate that tyrosine phosphorylation and dephosphorylation occur at the nuclear envelope of intact Neuro2a mouse neuroblastoma cells. Tyrosine kinase and phosphatase activities remain associated with purified nuclear envelopes. A similar pattern of tyrosine-phosphorylated nuclear envelope proteins suggests that the same tyrosine kinases act at the nuclear envelope of intact cells and at the purified nuclear envelope. We have also identified eight tyrosine-phosphorylated nuclear envelope proteins by 2D BAC/SDS/PAGE, immunoblotting with phosphotyrosine-specific antibodies, tryptic in-gel digestion, and MS analysis of tryptic peptides. These proteins are the lamina proteins lamin A, lamin B1, and lamin B2, the inner nuclear membrane protein LAP2beta, the heat shock protein hsc70, and the DNA/RNA-binding proteins PSF, hypothetical 16-kDa protein, and NonO, which copurify with the nuclear envelope.     

The role of protein kinases in anoxia tolerance in facultative anaerobes: purification and characterization of a protein kinase that phosphorylates pyruvate kinase

A protein kinase which phosphorylates pyruvate kinase (PK) in vitro was purified and characterized from the foot muscle of the anoxia-tolerant gastropod mollusc Busycon canaliculatum. Purification involved four steps: poly(ethylene glycol) fractionation, affinity chromatography on Blue agarose, ion-exchange chromatography on phosphocellulose and preparative isoelectric focusing (pI = 5.5). The activity was monitored by following changes in pyruvate kinase I50 values for L-alanine which have previously been linked to changes in the degree of enzyme phosphorylation. The correlation between enzyme phosphorylation and changes in the L-alanine inhibition constant was also directly demonstrated in the present paper by radioactively labelling PK with [tau-32P]ATP. The final purified protein kinase solution gave a single band on SDS-gel electrophoresis with a molecular weight of 37,000 +/- 2000. Kinetic analysis of the purified protein kinase (PK-kinase) showed a pH optimum of 7.0, an absolute requirement for magnesium ions (Km = 1.29 mM), a relatively high affinity for MgATP (Km = 57 microM), and inhibition by increasing salt concentrations (I50 = 55 mM KCl). The protein kinase activity was not affected by either spermine, heparin, cAMP, cGMP or concentrations of CaCl2 less than 10 mM. The enzyme did not phosphorylate either phosphofructokinase or glycogen phosphorylase, two enzymes that are also phosphorylated during anoxia in whelks. The purified enzyme is different from the catalytic subunit of cAMP-dependent protein kinase as shown by the inability of cAMP to stimulate the protein kinase at all stages of the preparation; cAMP did not activate either crude enzyme, the 7% poly(ethylene glycol) supernatant, or any of the column eluant peak fractions when measured by changes in pyruvate kinase kinetic parameters.     

Stimulation of CK2-dependent Grp94 phosphorylation by the nuclear localization signal peptide

The nuclear localization signal sequence (NLS) of SV40 Large T antigen is essential and sufficient for the nuclear translocation of the protein. Phosphorylation often modulates the intracellular distribution of signaling proteins. In this study, we investigated effects of the NLS-peptide of Large T antigen on protein phosphorylation. When crude cell lysates were incubated with [γ-³²P]ATP, phosphorylation of several endogenous substrates with molecular masses of 100, 80, 50, and 45 kDa by an endogenous kinase was stimulated by the addition of the wild type NLS-peptide (CPKKKRKVEDP). The mutated NLS-peptide (CPKTKRKVEDP) and the reversed NLS-peptide (PDEVKRKKKPC) are weak in the nuclear localization activity, and they only weakly stimulated phosphorylation of these substrates. The mobility of the 100 kDa phosphoprotein was indistinguishable with that of an endoplasmic reticulum (ER)-resident molecular chaperone glucose-regulated protein 94 (Grp94) belonging to the Hsp90 family, and purified Grp94 was phosphorylated by a kinase in cell lysates in an NLS-dependent fashion. The 100 kDa protein was identified as Grp94 by immunoprecipitation and reconstitution experiments. Purification of the NLS-dependent Grp94 kinase by sequential biochemical column chromatography steps resulted in isolation of two polypeptides with molecular masses of 42 and 27 kDa, which were identified as α and β subunit of protein kinase CK2, respectively, by western blotting analysis and biochemical characterization. Moreover, effect of an excess amount of GTP and V8 peptide mapping showed that the NLS-dependent Grp94 kinase in the cell lysate is identical with CK2. Surprisingly purified CK2 did phosphorylate Grp94 even without the NLS-peptide, suggesting that an additional suppressive factor is required for NLS-dependent phosphorylation of Grp94 by CK2. We suggest a possible general role for CK2-catalyzed phosphorylation in the regulation of NLS-dependent protein nuclear translocation.     

Purification of rat liver nuclear protein kinase NI.

Rat liver nuclear protein kinase NI, which appears in the flowthrough of DEAE-Sephadex columns, has been purified approximately 15,000-fold from soluble nuclear protein with yields of up to 10%. The method of purification involved chromatography of the DEAE-flowthrough protein successively on phosvitin-Sepharose and casein-Sepharose followed by rechromatography on phosvitin-Sepharose. The purified enzyme has an s20,w and molecular weight of 3.7 and 47,000, respectively, as determined by sucrose density gradient centrifugation in 0.4 M NaCl. A similar molecular weight of 42,000 was determined by gel filtration using Sephadex G-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed a single polypeptide with a molecular weight of 25,000. Protein kinase NI therefore consists of a dimer of two identical subunits. Protein kinase NI exhibits maximal activity on casein substrate and is not stimulated by 10(-5) to 10(-4) M cAMP or cGMP when either casein or histone H2b is used as a substrate.     

Biochemical characterization of a casein kinase I-like actin kinase responsible for the actin-induced suppression of casein kinase II activity in vitro

By combination of column chromatographies (heparin-agarose, HiTrap heparin and HiTrap SP columns) and gel filtration on a Superdex 200-pg HPLC column, an actin kinase was partially purified from a 1. 5 M NaCl extract of porcine liver. The actin kinase was finally purified, by actin-Sepharose column chromatography (HPLC), as an actin-binding protein kinase. The biochemical properties, such as (1) requirements of divalent cations (10 mM Mg(2+) and 3 mM Mn(2+)) and effective phosphate acceptors (actin and alpha-casein), (2) phosphorylation of both Ser- and Thr-residues on these two phosphate acceptors, (3) autophosphorylation of the catalytic subunit (approximately 37 kDa), and (4) inhibition kinetics by CK-I-7 (a CK-I specific inhibitor), of the purified actin kinase were similar to those reported for CK-I purified from various mammalian cells, but it was distinguishable from three cellular actin kinases (A-kinase, C-kinase and actin-fragmin kinase (approximately 80 kDa)). The 37 kDa actin kinase-mediated phosphorylation of actin did not relate to its polymerizability. Inhibition of CK-II-mediated phosphorylation of functional cellular proteins, including calmodulin (CaM), by actin was significantly stimulated after its full phosphorylation by the purified 37 kDa actin kinase or rCK-I in vitro. These results suggest that: (1) the 37 kDa Ser/Thr actin-binding kinase may be classified as a member of the CK-I family; and (2) specific phosphorylation of actin by the actin kinase may be involved in the suppression mechanism of CK-II-mediated signal transduction at the cellular level.     

DNA binding by cyclic adenosine 3',5'-monophosphate dependent protein kinase from calf thymus nuclei.

Cyclic adenosine 3',5'-monophosphate (cAMP) dependent protein kinase and proteins specifically binding cAMP have been extracted from calf thymus nuclei and analyzed for their abilities to bind to DNA. Approximately 70% of the cAMP-binding activity in the nucleus can be ascribed to a nuclear acidic protein with physical and biochemical characteristics of the regulatory (R) subunit of cAMP-dependent protein kinase. Several peaks of protein kinase activity and of cAMP-binding activity are resolved by affinity chromatography of nuclear acidic proteins on calf thymus DNA covalently linked to aminoethyl Sephrarose 4B. When an extensively purified protein kinase is subjected to chromatography on the DNA column in the presence of 10(-7) M cAMP, the R subunit of the kinase is eluted from the column at 0.05 M NaCl while the catalytic (C) subunit of the enzyme is eluted at 0.1-0.2 M NaCl. When chromatographed in the presence of histones, the R subunit is retained on the column and is eluted at 0.6-0.9 M NaCl. In the presence of cAMP, association of the C subunit with DNA is enhanced, as determined by sucrose density gradient centrifugation of DNA-protein kinase complexes. cAMP increases the capacity of the calf thymus cAMP-dependent protein kinase preparation to bind labeled calf thymus DNA, as determined by a technique employing filter retention of DNA-protein complexes. This protein kinase preparation binds calf thymus DNA in preference to salmon DNA, Escherichia coli DNA, or yeast RNA. Binding of protein kinases to DNA may be part of a mechanism for localizing cyclic nucleotide stimulated protein phosphorylation at specific sites in the chromatin.     

Polyamine-like effects of aminoglycosides on various messenger-independent protein kinase reactions

Gentamicin and several other aminoglycoside antibiotics in millimolar concentrations directly stimulate the phosphorylation of casein by purified preparations of cAMP- and Ca2+-independent protein kinases PK-C2 (equivalent to cytosolic casein kinase II) and its nuclear counterpart PK-N2 from rat liver and ventral prostate. These stimulatory effects of aminoglycoside antibiotics were similar to those exerted by the aliphatic polyamine spermine. Phosphorylation of casein by purified preparations of messenger-independent protein kinases PK-C1 (equivalent to cytosolic casein kinase I) and its nuclear counterpart PK-N1 was much less enhanced by spermine and the aminoglycoside antibiotics tested. Stimulations of PK-N2 reactions evoked by gentamicin or spermine (at 0.5 and 1.0 mM) were not additive. Several amino sugars tested were without effect on these protein kinases. Methylglyoxal bis(guanylhydrazone) which is known to block the stimulatory effects of polyamines on certain other enzymes did not alter spermine-stimulated phosphorylation of casein catalyzed by PK-N2 preparations.     

Head Proteins from T-Even Bacteriophages II. Physical and Chemical Characterization

Three classes of structural head proteins, having molecular weights of 43,000, 18,000, and 11,000 daltons, were isolated from the T-even bacteriophages and were characterized. Based on electrophoretic studies, the 43,000-dalton class contained one major protein and one (or two) minor components, the 18,000-dalton class contained two protein components, and the 11,000-dalton class contained one major component. The N-terminal residues for the 43,000- and the 11,000-dalton classes were alanine, and the N-terminal residues for the 18,000-dalton class were methionine and alanine. Of the three classes of proteins, the 18,000-dalton proteins were the most acidic, whereas the 11,000-dalton proteins were the most basic. The amino acid composition of the 11,000-dalton class revealed that methionine and cysteine were absent and lysine, histidine, and tryptophan content was higher in the 11,000-dalton class than in the other two classes of proteins. Estimates of the relative number of the three classes of structural proteins were made and indicated that there were between 1,600 and 2,000 subunits of the 43,000-dalton proteins, 100 to 200 of the 18,000-dalton proteins, and 1,000 to 1,500 of the 11,000-dalton proteins. Evidence was presented that the 43,000-dalton proteins and the 11,000-dalton proteins readily formed aggregates with themselves but not with each other. The significance of these interactions to the structure of the T-even phage head was discussed.     

Phosphorylation of skeletal and cardiac muscle C-proteins by the catalytic subunit of cAMP-dependent protein kinase

Catecholamines are known to influence the contractility of cardiac and skeletal muscles, presumably via cAMP-dependent phosphorylation of specific proteins. We have investigated the in vitro phosphorylation of myofibrillar proteins by the catalytic subunit of cAMP-dependent protein kinase of fast- and slow-twitch skeletal muscles and cardiac muscle with a view to gaining a better understanding of the biochemical basis of catecholamine effects on striated muscles. Incubation of canine red skeletal myofibrils with the isolated catalytic subunit of cAMP-dependent protein kinase and Mg-[gamma-32P]ATP led to the rapid incorporation of [32P]phosphate into five major protein substrates of subunit molecular weights (MWs) 143,000, 60,000, 42,000, 33,000, and 11,000. The 143,000 MW substrate was identified as C-protein; the 42,000 MW substrate is probably actin; the 33,000 MW substrate was shown not to be a subunit of tropomyosin and, like the 60,000 and 11,000 MW substrates, is an unidentified myofibrillar protein. Isolated canine red skeletal muscle C-protein as phosphorylated to the extent of approximately 0.5 mol Pi/mol C-protein. Rabbit white skeletal muscle and bovine cardiac muscle C-proteins were also phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, both in myofibrils and in the isolated state. Cardiac C-protein was phosphorylated to the extent of 5-6 mol Pi/mol C-protein, whereas rabbit white skeletal muscle C-protein was phosphorylated at the level of approximately 0.5 mol Pi/mol C-protein. As demonstrated earlier by others, C-protein of skeletal and cardiac muscles inhibited the actin-activated myosin Mg2+-ATPase activity at low ionic strength in a system reconstituted from the purified skeletal muscle contractile proteins (actin and myosin).(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of rat liver heterogeneous nuclear ribonucleoproteins A2 and C can be modulated by calmodulin.

It was previously reported that the phosphorylation of three proteins of 36, 40 to 42, and 50 kDa by casein kinase 2 is inhibited by calmodulin in nuclear extracts from rat liver cells (R. Bosser, R. Aligué, D. Guerini, N. Agell, E. Carafoli, and O. Bachs, J. Biol. Chem. 268:15477-15483, 1993). By immunoblotting, peptide mapping, and endogenous phosphorylation experiments, the 36- and 40- to 42-kDa proteins have been identified as the A2 and C proteins, respectively, of the heterogeneous nuclear ribonucleoprotein particles. To better understand the mechanism by which calmodulin inhibits the phosphorylation of these proteins, they were purified by using single-stranded DNA chromatography, and the effect of calmodulin on their phosphorylation by casein kinase 2 was analyzed. Results revealed that whereas calmodulin inhibited the phosphorylation of purified A2 and C proteins in a Ca(2+)-dependent manner, it did not affect the casein kinase 2 phosphorylation of a different protein substrate, i.e., beta-casein. These results indicate that the effect of calmodulin was not on casein kinase 2 activity but on specific protein substrates. The finding that the A2 and C proteins can bind to a calmodulin-Sepharose column in a Ca(2+)-dependent manner suggests that this association could prevent the phosphorylation of the proteins by casein kinase 2. Immunoelectron microscopy studies have revealed that such interactions could also occur in vivo, since calmodulin and A2 and C proteins colocalize on the ribonucleoprotein particles in rat liver cell nuclei.     

Properties of rat liver nuclear protein kinases.

Two cyclic nucleotide-independent protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) have been purified to homogeneity from rat liver nuclei. While these enzymes have many similar catalytic properties (preference for acid rather than basic proteins), they differ in molecular weight and subunit composition. Protein kinase NII will utilize ATP and GTP as phosphate donors while protein kinase NI will only effectively use ATP. Both enzymes reveal an unusual activation by Fe2+.     

Stimulation of ascites tumor RNA polymerase II by protein kinase.

The activity of purified RNA polymerase II from Novikoff ascites tumor cells is stimulated 5-7-fold by a purified protein factor. This protein factor, designated HLF2, has extensive protein kinase activity and catalyzed the incorporation of gamma-32G from ATP into protein under normal RNA polymerase assay conditions. Protein phosphorylation is totally dependent on the presence of HLF2 and is stimulated 2-3-fold by the presence of highly purified RNA polymerase II. The purification procedure developed for the isolation of the polymerase stimulatory factor resulted in a 4000-fold purification of a protein kinase. Chromatography on carboxymethylcellulose, phosphocellulose, and Sephadex G-100 did not resolve polymerase stimulatory activity from protein kinase activity. Adenylimidodiphosphate (AMP-PNP), an inhibitor of protein kinases, inhibited the stimulatory activity of purified factor by 80%. The heat denaturation profile of protein kinase was paralleled by the loss of polymerase stimulatory activity. Concentrations of (NH4)2SO4 which are known to inhibit polymerase stimulation (Lee and Dahmus, 1973) also inhibit protein kinase activity. The protein kinase activity associated with stimulatory factor catalyzes the phosphorylation of basic proteins such as protamine or histone. The protein kinase is not stimulated by cyclic 3', 5'-AMP or -GMP over a concentration range of 10(-6)-10(-4)M. Furthermore, protein kinase activity is not inhibited by either the regulatory subunit of rabbit muscle protein kinase or by the heat-stable inhibitor of cyclic 3', 5'-AMP-dependent protein kinases. Protein kinase activity is stimulated by KCl or NH4Cl and is inhibited by MnCl2. The apparent Km values, determined in the presence of 4 mM Mg2+, are 0.02 mM for ATP, and 4.1 mM for GTP.     

Polyamines stimulate endogenous protein phosphorylation in thyroid cytosol

The polyamine, spermine (1-5 mM), when added to rat thyroid cytosol, increases the phosphorylation of a 107 kDa protein 4-fold as analyzed by sodium dodecyl sulfate polyacrylamide gradient gel electrophoresis (SDS-PAGE) and autoradiography; spermidine was less effective and putrescine was without effect. Sodium chloride, when tested at equivalent ionic strengths (4-40 mM), did not reproduce the effects of spermine. In addition to stimulating the phosphorylation of a 107 kDa protein, spermine had an apparent biphasic effect on the phosphorylation of 88 and 65 kDa proteins; maximum stimulation of approximately 60-70% was observed at 0.5-2 mM. Both basal and spermine-stimulated protein phosphorylation patterns were identical whether [gamma-32P] ATP or [gamma-32P] GTP was used as phosphate donors. Heparin (1 microgram/ml) reduced spermine-stimulated phosphorylation of the 107 kDa protein by 64%. Phosphorylation of a 107 kDa protein was not restricted to rat thyroid as spermine was found to augment the phosphorylation of 107 kDa protein(s) in mouse and beef thyroid cytosol preparations.     

Effect of spermine on tyrosine hydroxylase activity before and after phosphorylation by cyclic AMP-dependent protein kinase

The effect of spermine on tyrosine hydroxylase (TH) activity purified from bovine adrenal medulla was examined before and after phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase (A-kinase). Before phosphorylation, spermine (less than 1 mM) inhibited the enzymatic activity, and negative cooperative effect of spermine on TH (Hill coefficient = 0.7) was observed from the kinetic analysis concerning 6-methyl-5,6,7,8-tetrahydropterin (6MPH4). Spermine interacted noncompetitively toward tyrosine and the Ki for spermine was calculated to be 68 microM. Phosphorylation abolished the ability of spermine to inhibit TH activity in a negative cooperative manner against the pterin cofactor, and also increased four-fold the Ki value against the substrate. These results suggest that spermine may inhibit TH activity by interacting with the pterin binding site of the enzyme molecule in a manner of negative cooperativity, and that this inhibition is reversed by the conformational change of regulatory domain of TH after phosphorylation by A-kinase.