Inactivation of pyruvate kinase type M2 from chicken liver by phosphorylation, catalyzed by a cAMP-independent protein kinase.

A cAMP-independent protein kinase from chicken liver phosphorylated and inactivated pyruvate kinase type M2 from the same tissue. Complete inactivation was reached when 4 mol of phosphate were incorporated/mol of tetrameric pyruvate kinase. The protein kinase bound with high affinity to pyruvate kinase type M2 (Km value for pyruvate kinase = 6 X 10(-10)M; it phosphorylated phosvitin and casein but not histones, ATP and GTP were substrates. The differences between the properties of this protein kinase in the interconversion of pyruvate kinase and that described previously are discussed.     

Phosphorylation of the androgen receptor by a nuclear cAMP-independent protein kinase

The androgen receptor was purified from rat ventral prostate. The purified receptor migrated as a single band of mol. wt. 87000 on SDS-polyacrylamide gels, had a kd for R-1881 (17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one) binding as 6 nM, and sedimentation coefficient of 4.5 S. Phosphorylation of the purified receptor was studied by incubating it with [gamma-32P]ATP in the presence of several purified protein kinases including cAMP-dependent protein kinase, and four cAMP-independent protein kinases (which were active towards substrates such as phosvitin and casein). Phosphorylation of the 87000 mol. wt. androgen receptor protein occurred only in the presence of a nuclear cAMP-independent protein kinase (of the N2 type). No auto-phosphorylation of the receptor was detected. The results indicate that the androgen receptor is a phosphoprotein. Further, phosphorylation of the androgen receptor by only a specific nuclear cAMP-independent protein kinase may be important in determining the dynamics of its function.     

A rapid method for determining protein kinase phosphorylation specificity

Selection of target substrates by protein kinases is strongly influenced by the amino acid sequence surrounding the phosphoacceptor site. Identification of the preferred peptide phosphorylation motif for a given kinase permits the production of efficient peptide substrates and greatly simplifies the mapping of phosphorylation sites in protein substrates. Here we describe a combinatorial peptide library method that allows rapid generation of phosphorylation motifs for serine/threonine kinases.     

Cyclic nucleotide-independent protein kinase from rat liver. Purification and characterization of a multifunctional protein kinase

A rat liver cAMP-independent protein kinase that phosphorylates peptide b of ATP-citrate lyase (Ramakrishna, S., Pucci, D. L., and Benjamin, W. B. (1983) J. Biol. Chem. 258, 4950-4956) has been purified to apparent homogeneity. The molecular weight, determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, sucrose density gradient, and by gel filtration, was found to be 36,000. This protein kinase phosphorylates in vitro ATP-citrate lyase, acetyl-CoA carboxylase, and glycogen synthase and does not phosphorylate phosphorylase, phosphorylase kinase, histone, phosvitin, and casein. It has Fa (activity factor) activity stimulating the ATP X Mg-dependent phosphatase and is therefore named a multifunctional protein kinase. This kinase differs from glycogen synthase kinase-3 with regard to substrate specificity, kinetic parameters, and physicochemical properties.     

Role of substrate in determining the phospholipid specificity of protein kinase C activation

The phospholipid selectivity of protein kinase C (PKC) activation was examined by using two substrates, histone and a random copolymer of lysine and serine [poly(lysine, serine)] (PLS), plus phospholipids provided as vesicles or as Triton-mixed micelle preparations. The results indicated that substrate-phospholipid interaction was an essential component of PKC activation and that many in vitro properties of PKC activation are attributable to this interaction. The substrate histone interacted with phospholipid-Triton mixed micelles containing phosphatidylserine (PS), but not with those containing phosphatidylinositol (PI) or phosphatidylglycerol (PG). In direct correlation, only PS-Triton mixed micelles were effective in supporting PKC activity. Also, the minimum PS composition (4 mol % in Triton) required to induce significant histone-PS interaction coincided with the minimum composition required for phosphorylation of histones. Moreover, the PS composition required for maximum activity varied with the histone concentration of the reaction. In contrast to histone, PLS interacted with phospholipid-Triton mixed micelles containing either PS, PI, or PG, and all these mixed micelles supported the phosphorylation of PLS. In fact, by selection of appropriate experimental conditions (e.g., concentration of substrate and phospholipid), any of the three mixed micelles could appear the most effective in supporting PKC activity. Phospholipid vesicles containing PS, PG, or PI were found to interact with both histone and PLS and to support the activity of PKC. Physical properties of the solution and conditions used for preparation of phospholipid vesicles had considerable influence on PKC activation. At high phospholipid concentrations, vesicles containing PS, PI, or PG supported the activity of PKC to essentially the same level, provided that the physical differences among the phospholipid vesicles were minimized.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

cAMP-independent casein kinase mediates phosphorylation of many T3-dependent phosphoproteins in rat liver cytosol

Administration of T3 (20 micrograms/100 g BW) for 3 days increases phosphorylation of several proteins in rat liver cytosol in vitro. To help elucidate the mechanism of T3-induced phosphorylation, we studied which protein kinase(s) mediate phosphorylation of endogenous cytosolic proteins. Five different protein kinases were obtained by DEAE+ cellulose column chromatographic fractionation of liver cytosol. When their ability to phosphorylate heat-inactivated cytosol was investigated, casein kinase, a cAMP independent protein kinase, showed the strongest effect. Casein kinase, purified by phosphocellulose chromatography, phosphorylated more than 10 cytosolic proteins. Several T3-dependent (and cAMP independent) phosphoproteins were included among these. One protein with Mr 39 X 10(3), of which phosphorylation is stimulated by T3 within five hours after injection, was the most active substrate for casein kinase. The results suggest that casein kinase is the enzyme responsible for phosphorylation of many rat liver cytosolic proteins and that several phosphoproteins, apparently under T3-regulation, might be phosphorylated by this enzyme.     

Sphingosine activates protein kinase A type II by a novel cAMP-independent mechanism

Protein kinase A (PKA) has long been recognized as playing a major role in many regulatory processes in cells through its activation by the ubiquitous second messenger cAMP. We show here a novel mode of activation of PKA type II that is independent of cAMP and is, instead, dependent on sphingosine. PKA type II is specifically activated by sphingosine and its analog, dimethylsphingosine, but not by sphingosine-1-phosphate or other lipids. Like cAMP, sphingosine activates PKA holoenzyme but not the catalytic subunit alone, suggesting that the activation is mediated by the regulatory subunits. However, sphingosine-activated PKA, but not cAMP-activated PKA, is inhibited by phosphatidylserine, suggesting a distinct mechanism of activation. Furthermore, unlike cAMP, sphingosine does not induce the dissociation of PKA holoenzyme into catalytic and regulatory subunits. Modulation of sphingosine levels in vivo results in alteration in basal membrane-associated PKA activity consistent with a direct effect of membrane sphingosine on PKA type II. Importantly, sphingosine-dependent but not cAMP-dependent activation of PKA specifically phosphorylates Ser58 of the multifunctional adapter protein 14-3-3zeta, promoting the conversion of dimeric 14-3-3 to a monomeric state, thus potentially modulating several biological functions. These results define a new mode of PKA activation that is sphingosine-dependent and mechanistically different from the classical cAMP-dependent activation of PKA. Furthermore, they suggest that stimuli that induce sphingosine accumulation and modulate phospholipid content at the cell membrane have the potential to activate PKA, thereby inducing the phosphorylation of distinct substrates and biological activities.     

Effect of triiodothyronine on rat liver chromatin protein kinase.

1. Injection of triiodothyronine to rats stimulates protein kinase activity in liver chromatin nonhistone proteins. A significant increase was found after two daily injections. A 4-fold increase was observed with the purified enzyme after eight daily injections of the hormone. No variations were observed in cytosol protein kinase activity. Electrophoretic pattern, effect of heat denaturation, effect of p-hydroxymercuribenzoate seem to indicate that the enzyme present in treated rats is not identical to the enzyme in control animals, which suggests that thyroid hormone has induced nuclear protein kinase. Diiodothyronine, 3, 3', 5'-triiodothyronine have no effect on protein kinase. 2. Chromatin non-histone proteins isolated from rats injected with triiodothyronine incorporated more 32P when incubated with [gamma-32P]ATP than the chromatin proteins from untreated rats. Thyroidectomy reduced the in vitro 32P incorporation. It is suggested that some of the biological activity of thyroid hormone could be mediated through its effect on chromatin non-histone proteins.     

Structural features of pregna-D'-pentaranes determining their interaction with three rat proteins

Competition of a number of progesterone 16alpha,17alpha-cycloalkane derivatives with 3H-labeled ligands for the binding sites of the rat uterine progesterone receptor, uterine pentaranophilin, and blood serum pentaranophilin was studied. We found that the selective ligands for the progesterone receptor are progesterone, 16alpha,17alpha-cyclopropanoprogesterone, and 16alpha,17alpha-cyclopent-3'-enoprogesterone and the selective ligands for serum pentaranophilin are 6alpha-methyl-16alpha,17alpha-cyclohexanopregna-1,4-diene-3,20-dione and 3beta-hydroxy-16alpha,17alpha-cyclohexanopregn-5-en-20-one. No selective ligands for the uterine pentaranophilin were found. The majority of substituents in rings A, B, and D' we studied decreased the affinity of ligands for all the three proteins. The substitution of the delta5-3beta-hydroxy grouping for the delta4-3-keto grouping exerted the strongest negative effect in the case of the progesterone receptor and the uterine pentaranophilin, whereas the introduction of the 3',4'-dimethyl grouping strongly inhibited the ligand affinity for the uterine pentaranophilin. The extent and even the direction of the effect of a substituent on the affinity of ligands for the proteins substantially depended on the presence of other substituents in the steroid molecules. We hypothesized that a certain similarity exists between three proteins studied in respect to the structures of their ligand-binding pockets. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.     

Partial purification of a ribonucleic acid cAMP-independent protein kinase from embryonic chicken muscle

A cAMP-indepedent protein kinase (P38 kinase) from embryonic chicken muscle with ability to phosphorylate a 38,000 molecular weight polypeptide and to bind to RNAs has been further characterized. An approximately 2000-fold purification of this enzyme was achieved by a combination of affinity and ion-exchange chromatography. Our studies indicate that this protein kinase can not phosphorylate the small subunit of rabbit reticulocyte initiation factor eIF-2 in the presence of its normal endogenous substrate, nor is it activated over a wide range of concentrations of double-stranded RNA. This P38 kinase is, therefore, distinct from the hemin-regulated translational inhibitor of protein synthesis in rabbit reticulocytes and from the interferon-induced protein kinase identified In several systems.     

Structural analysis of protein kinase A mutants with Rho-kinase inhibitor specificity

Controlling aberrant kinase-mediated cellular signaling is a major strategy in cancer therapy; successful protein kinase inhibitors such as Tarceva and Gleevec verify this approach. Specificity of inhibitors for the targeted kinase(s), however, is a crucial factor for therapeutic success. Based on homology modeling, we previously identified four amino acids in the active site of Rho-kinase that likely determine inhibitor specificities observed for Rho-kinase relative to protein kinase A (PKA) (in PKA numbering: T183A, L49I, V123M, and E127D), and a fifth (Q181K) that played a surprising role in PKA-PKB hybrid proteins. We have systematically mutated these residues in PKA to their counterparts in Rho-kinase, individually and in combination. Using four Rho-kinase-specific, one PKA-specific, and one pan-kinase-specific inhibitor, we measured the inhibitor-binding properties of the mutated proteins and identify the roles of individual residues as specificity determinants. Two combined mutant proteins, containing the combination of mutations T183A and L49I, closely mimic Rho-kinase. Kinetic results corroborate the hypothesis that side-chain identities form the major determinants of selectivity. An unexpected result of the analysis is the consistent contribution of the individual mutations by simple factors. Crystal structures of the surrogate kinase inhibitor complexes provide a detailed basis for an understanding of these selectivity determinant residues. The ability to obtain kinetic and structural data from these PKA mutants, combined with their Rho-kinase-like selectivity profiles, make them valuable for use as surrogate kinases for structure-based inhibitor design.     

Purification of a novel insulin-stimulated protein kinase from rat liver

We previously described a novel insulin-stimulated protein kinase activity that phosphorylates Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) in cytosolic extracts of adipocytes (Yu, K-T., Khalaf, N., and Czech, M. P. (1987) J. Biol. Chem. 262, 16677-16685). In the present experiments, cytosolic extracts of livers from insulin-treated rats also exhibited a 30-100% increase in this Kemptide kinase activity and served as an abundant source for purification. The Kemptide kinase was purified in parallel from liver extracts of insulin-treated or control rats through five chromatographic steps and one polyethylene glycol precipitation. The chromatographic behavior of the insulin-stimulated Kemptide kinase differed significantly from the control kinase on Mono Q and heparin-Sepharose resins. The purified kinase preparations retain insulin stimulations of 2-10-fold. Analysis of the purified control and insulin-stimulated kinases by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed single bands with similar silver staining intensity and apparent molecular masses of 52 kDa. The insulin-stimulated Kemptide phosphorylating activity also coincided with the major silver-stained band following isoelectric focusing in polyacrylamide gels. The stimulation of kinase activity in response to administration of insulin is due to an increase in Vmax, whereas the Km for Kemptide (0.3 mM) is unchanged. The apparent molecular mass of the native kinase determined by gel filtration is approximately 50 kDa, suggesting that it exists as a monomer. Either Mg2+ or Mn2+ serve as cofactors for the kinase which phosphorylates a variety of basic substrates including a number of peptides and histones. The activity of the Kemptide kinase is not changed by several compounds that have been shown to modulate other kinases. Based on these data, we conclude 1) a novel insulin-sensitive Kemptide kinase in liver cytosol has been purified to near homogeneity, and 2) insulin administration acutely modulates the specific activity of this Kemptide kinase in livers of intact rats.     

Characterization of a 67-kDa S6 protein kinase from rat liver.

Fractionation of rat liver cytosol on DEAE-cellulose resolved two S6 kinases eluting at 25 mM KCl (peak I) and 100 mM KCl (peak II). The apparent molecular weights of the peak I and peak II kinases are 26,300 and 67,000, respectively. The peak II kinase was further purified and characterized. Incubation of the kinase with [gamma-32P] ATP and Mg2+ resulted in the incorporation of 32P predominantly into a 67-kDa band. Optimal activity of the kinase was observed in the presence of 5 mM Mg2+ and in the pH range of 8.0-8.5. The Km for ATP and 40S subunit were 7.3 microM and 1.5 microM, respectively. The Mg(2+)-stimulated kinase activity was inhibited by various divalent metals, NaF, and polyamines. The properties of the peak II S6 kinase are very similar or identical to the previously described mitogen-activated S6 protein kinase and may represent the nonactivated form of this enzyme.     

Identification and solubilization of a cAMP-independent protein kinase from mouse L-cell smooth membranes

1. Smooth membranes have been prepared from mouse L-cells and found to contain an endogenous protein kinase activity. 2. The enzyme(s) responsible for this activity use ATP, but no other nucleoside triphosphates, to phosphorylate endogenous membrane proteins as well as exogenously-added protein substrates such as phosvitin and casein. 3. Mg2+ is required for enzyme activity, maximal activity is observed at pH 7.5-8.0 and the kinase is not dependent on, or stimulated by, cyclic 3'-5' AMP. 4. The kinase activity is not decreased by the Walsh heat-stable inhibitor of cyclic 3'-5' AMP-dependent protein kinases. 5. Fifty percent or more of the membrane-associated kinase activity can be solubilized by extracting membranes with buffer containing 0.6 M NaCl. 6. The solubilized enzyme resembles the membrane-associated activity in its Mg2+ requirement, pH optimum and independence of cyclic 3'-5' AMP. 7. Phosvitin and casein are better exogenous substrates than histones or protamine for phosphorylation by the enzyme in either the membrane-associated or solubilized state.     

Characterization of yeast pyruvate kinase 1 as a protein kinase A substrate, and specificity of the phosphorylation site sequence in the whole protein

Pyk1 (pyruvate kinase 1) from Saccharomyces cerevisiae was characterized as a substrate for PKA (protein kinase A) from bovine heart and yeast. By designing Pyk1 synthetic peptides containing potential PKA sequence targets (Ser22, Thr94 and Thr478) we determined that the peptide S22 was a substrate for PKA in vitro, with a K(sp)* (specificity constant) 10-fold and 3-fold higher than Kemptide for bovine heart and yeast PKA respectively. In vitro phosphorylation of the Pyk1 S22A mutant protein was decreased by as much as 90% when compared with wild-type Pyk1 and the Pyk1 T94A mutant. The K(sp)* values for Pyk1 and Pyk1 T94A were the same, indicating that both proteins are phosphorylated at the same site by PKA. Two-dimensional PAGE of Pyk1 and Pyk1 S22A indicates that in vivo the S22A mutation prevented the formation of one of the Pyk1 isoforms. We conclude that in yeast the major PKA phosphorylation site of Pyk1 is Ser22. Phosphorylation of Ser22 leads to a Pyk1 enzyme that is more active in the absence of FBP (fructose 1,6-bisphosphate). The specificity of yeast and mammalian PKA towards the S22 peptide and towards whole Pyk1 protein was measured and compared. The K(sp)* for the S22 peptide is higher than that for Pyk1, indicating that the peptide modelled on Pyk1 is a much better substrate than Pyk1, regardless of which tissue was used as the source of PKA. However, the K(m) of Pyk1 protein is lower than that of the better substrate, the S22 peptide, indicating that ground-state substrate binding is not the major determinant of substrate specificity for PKA.     

Cyclic AMP-dependent histone-specific nucleoplasmic protein kinase from rat liver.

A nucleoplasmic histone kinase activity was isolated from livers of adult rats and purified 39-fold compared with whole nuclei by ultracentrifugation of the nuclear extract and Sephadex G-200 gel filtration in the presence of cyclic AMP. Analysis by polyacrylamide-gel electrophoresis as well as by gel filtration indicates a mol.wt. of approx. 60,000 for the catalytic subunit and 130000-150000 for the cyclic AMP-binding activity. The purified enzyme displays a 20-fold greater preference for histone fractions 1 and 2b than for any non-histone substrate, including alpha-casein. Endogenous protein in the preparation is not appreciably phosphorylated. The unfractioned enzyme is stimulated significantly by cyclic GMP, cyclic IMP and dibutyryl cyclic AMP as well as by cyclic AMP. The catalytic reaction requires Mg2+ (Km 1.9mM) and ATP (Km 15.4 micron). Half-maximal activity of the enzyme is observed with histone 2b at 12micron and histone 1 at a higher substrate concentration. The pH optima are 6.1 and 6.5 with histones 2b and 1 respectively. This nuclear protein kinase appears to be distinct from other nuclear enzymes that have been reported, on the basis of histone specificity, univalent-salt-sensitivity, pH optima and nuclear location. However, the enzyme possesses many properties similar to those of the cytoplasmic kinases, including cyclic AMP-dependence, Mg2+ and ATP affinities and pH optima. It differs from cytoplasmic protein kinase type I, the major form in the liver, with respect to bivalent-cation effects and response to the heat-stable protein kinase inhibitor protein isolated from ox heart.