The mode of action of adenosine 3′:5′-cyclic monophosphate in mammalian islets of Langerhans. Preparation and properties of islet-cell protein phosphokinase

1. A protein was demonstrated in mammalian islets of Langerhans that after purification appeared as a single component possessing both cyclic-AMP (adenosine 3':5'-cyclic monophosphate)-binding and cyclic-AMP-dependent protein phosphokinase activities. 2. The protein had an intrinsic association constant for cyclic AMP of 1.15x10(-8)m, which was similar to the K(m) for cyclic AMP (1.11x10(-8)m) of the protein phosphokinase activity. 3. Incubation of the protein in the presence of cyclic AMP resulted in its dissociation into cyclic-AMP-independent protein phosphokinase (catalytic) and cyclic-AMP-binding (receptor) subunits, which could be separated on Sephadex G-200. 4. The cyclic-AMP-dependent protein phosphokinase was capable of phosphorylating a variety of proteins, the most readily phosphorylated being histone, casein and protein components of sub-cellular fractions prepared from islets of Langerhans. 5. The cyclic-AMP-dependent phosphorylation of histone had a K(m) for ATP of 1.1x10(-5)m. 6. The endogenous protein phosphokinase activity in rat islets incubated with agents that are known to alter the intracellular concentration of cyclic AMP was investigated. Theophylline and 3-isobutyl-1-methylxanthine, agents that raise cyclic AMP concentrations in islets, increased the activity of the protein phosphokinase, whereas adrenaline, which lowers islet cyclic AMP concentrations, decreased its activity. 7. It is suggested that cyclic AMP may exert its effects on insulin release by increasing the activity of a protein phosphokinase and may thereby promote the phosphorylation and activity of a rate-determining component of the secretory mechanism.     

Nucleoside phosphorylation by phosphate minerals

In the presence of formamide, crystal phosphate minerals may act as phosphate donors to nucleosides, yielding both 5'- and, to a lesser extent, 3'-phosphorylated forms. With the mineral Libethenite the formation of 5'-AMP can be as high as 6% of the adenosine input and last for at least 10(3) h. At high concentrations, soluble non-mineral phosphate donors (KH(2)PO(4) or 5'-CMP) afford 2'- and 2':3'-cyclic AMP in addition to 5'-and 3'-AMP. The phosphate minerals analyzed were Herderite Ca[BePO(4)F], Hureaulite Mn(2+)(5)(PO(3)(OH)(2)(PO(4))(2)(H(2)O)(4), Libethenite Cu(2+)(2)(PO(4))(OH), Pyromorphite Pb(5)(PO(4))(3)Cl, Turquoise Cu(2+)Al(6)(PO(4))(4)(OH)(8)(H(2)O)(4), Fluorapatite Ca(5)(PO(4))(3)F, Hydroxylapatite Ca(5)(PO(4))(3)OH, Vivianite Fe(2+)(3)(PO(4))(2)(H(2)O)(8), Cornetite Cu(2+)(3)(PO(4))(OH)(3), Pseudomalachite Cu(2+)(5)(PO(4))(2)(OH)(4), Reichenbachite Cu(2+)(5)(PO(4))(2)(OH)(4), and Ludjibaite Cu(2+)(5)(PO(4))(2)(OH)(4)). Based on their behavior in the formamide-driven nucleoside phosphorylation reaction, these minerals can be characterized as: 1) inactive, 2) low level phosphorylating agents, or 3) active phosphorylating agents. Instances were detected (Libethenite and Hydroxylapatite) in which phosphorylation occurs on the mineral surface, followed by release of the phosphorylated compounds. Libethenite and Cornetite markedly protect the beta-glycosidic bond. Thus, activated nucleic monomers can form in a liquid non-aqueous environment in conditions compatible with the thermodynamics of polymerization, providing a solution to the standard-state Gibbs free energy change (DeltaG degrees ') problem, the major obstacle for polymerizations in the liquid phase in plausible prebiotic scenarios.     

Purification and characterization of a phosvitin kinase from the thyroid gland

1. Two cyclic AMP independent protein kinases phosphorylating preferentially acidic substrates have been identified in soluble extract from human, rat and pig thyroid glands. Both enzymes were retained on DEAE-cellulose. The first enzyme activity eluted between 60 and 100 mM phosphate (depending on the species), phosphorylated both casein and phosvitin and was retained on phosphocellulose; this enzyme likely corresponds to a casein kinase already described in many tissues. The second enzyme activity eluted from DEAE-cellulose at phosphate concentrations higher than 300 mM, phosphorylated only phosvitin and was not retained on phosphocellulose. These enzymes were neither stimulated by cyclic AMP, cyclic GMP and calcium, nor inhibited by the inhibitor of the cyclic AMP dependent protein kinases. 2. The second enzyme activity was purified from pig thyroid gland by the association of affinity chromatography on insolubilized phosvitin and DEAE-cellulose chromatography. Its specific activity was increased by 8400. 3. The purified enzyme (phosvitin kinase) was analyzed for biochemical and enzymatic properties. Phosvitin kinase phosphorylated phosvitin with an apparent Km of 100 micrograms/ml; casein, histone, protamine and bovine serum albumin were not phosphorylated. The enzyme utilized ATP as well as GTP as phosphate donor with an apparent Km of 25 and 28 microM, respectively. It had an absolute requirement for Mg2+ with a maximal activity at 4 mM and exhibited an optimal activity at pH 7.0. The molecular weight of the native enzyme was 110 000 as determined by Sephacryl S300 gel filtration. The analysis by SDS-polyacrylamide gel electrophoresis revealed a major band with a molecular weight of 35000 suggesting a polymeric structure of the enzyme.     

Stereoselective phosphorylation of branched cyclodextrins with inorganic cyclo-triphosphate

The phosphorylation by inorganic sodium cyclo-triphosphate (P(3m)) having a six-membered ring was examined for cyclomaltohexaose (alpha-cyclodextrin) and branched cyclodextrins (mono-6-O-alpha-D-glucopyranosylcyclomaltohexaose, mono-6-O-alpha-D-maltosylcyclomaltohexaose, mono-6-O-alpha-D-glucopyranosylcyclomaltoheptaose, and mono-6-O-alpha-D-maltosylcyclomaltoheptaose) in aqueous solution. For all cyclomaltooligosaccharides (cyclodextrins) studied, the 2-OH group was stereoselectively phosphorylated. In the reaction of branched cyclodextrins and P(3m), only the 2-OH on the alpha-D-glucopyranosyl group of the cyclodextrin rings was phosphorylated with maximum yields of more than 27%. The phosphorylation mechanism of branched cyclodextrins with P(3m) is also discussed.     

In Vitro Phosphorylation of Bovine Adrenal Chromaffin Cell Tyrosine Hydroxylase by Endogenous Protein Kinases

Under phosphorylating conditions, addition of Ca2+ or cyclic AMP to the 100,000 g supernatant of purified bovine adrenal chromaffin cells increases both the incorporation of 32P into tyrosine hydroxylase and the activity of the enzyme. Combining maximally effective concentrations of each of these stimulating agents produces an additive increase in both the level of 32P incorporation into tyrosine hydroxylase and the degree of activation of the enzyme. The increased phosphorylation by Ca2+ is due to stimulation of endogenous Ca2+-dependent protein kinase activity and not inhibition of phosphoprotein phosphatases. When the chromaffin cell supernatant is subjected to diethylaminoethyl (DEAE) chromatography to remove calmodulin and phospholipids, tyrosine hydroxylase is no longer phosphorylated or activated by Ca2+; on the other hand, phosphorylation and activation of tyrosine hydroxylase by cyclic AMP are not affected. Subsequent replacement of either Ca2+ plus calmodulin or Ca2+ plus phosphatidylserine to the DEAE-fractionated cell supernatant restores the phosphorylation, but not activation of the enzyme. Reverse-phase HPLC peptide mapping of tryptic digests of tyrosine hydroxylase from the 100,000 g supernatant shows that the Ca2+-dependent phosphorylation occurs on three phosphopeptides, whereas the cyclic AMP-dependent phosphorylation occurs on one of these peptides. In the DEAE preparation, either cyclic AMP alone or Ca2+ in the presence of phosphatidylserine stimulates the phosphorylation of only a single phosphopeptide peak, the same peptide phosphorylated by cyclic AMP in the crude supernatant. In contrast, Ca2+ in the presence of calmodulin stimulates the phosphorylation of three peptides having reverse-phase HPLC retention times that are identical to peptides phosphorylated by Ca2+ addition to the crude unfractionated 100,000 g supernatant. Rechromatography of the peaks from each of the in vitro phosphorylations, either in combination with each other or in combination with each of the seven peaks generated from phosphorylation of tyrosine hydroxylase in situ, established that cyclic AMP, Ca2+/phosphatidylserine, and Ca2+/calmodulin all stimulate the phosphorylation of the same reverse-phase HPLC peptide: in situ peptide 6. Ca2+/calmodulin stimulates the phosphorylation of in situ peptides 3 and 5 as well. Thus, tyrosine hydroxylase can be phosphorylated in vitro by protein kinases endogenous to the chromaffin cell. Phosphorylation occurs on a maximum of three of the seven in situ phosphorylated sites, and all three of these sites can be phosphorylated by a Ca2+/calmodulin-dependent protein kinase.     

Purification and characterization fo a phosvitin kinase from the thyroid gland

1. Two cyclic AMP independent protein kinases phosphorylating preferentially acidic substrates have been identified in soluble extract from human, rat and pig thyroid glands/ Both enzymes were retained on DEAE-cellulose. The first enzyme activity eluted between 60 and 100 mM phosphate (depending on the species), phosphorylated both casein and phosvitin and was retained on phosphocellulose; this enzyme likely corresponds to a casein kinase already described in many tissues. The second enzyme activity eluted from DEAE-cellulose at phosphate concentrations higher than 3000 mM, phosphorylated only phosvitin and was not retained on phophocellulose. These enzymes were neither stimulated by cyclic AMP, cyclic GMP and calcium, nor inhbiited by the inhibitor of the cyclic AMP dependent protein kinases. 2. The second enzyme activity was purified from pig thyroid gland by the association of affinity chromatography on insolubilized phosvitin and DEAE-cellulose chromatography. Its specific activity was increased by 8400. 3. The purified enzyme (phosvitin kinase) was analyzed for biochemical and enzymatic properties. Phosvitin kinase phosphorylated phosvitin with an apparent K_m of 100 μg/ml; casein, histone, protamine and bovine serum albumin were not phosphorylated. The enzyme utilized ATP as well as GTP as phosphate donor with an apparent K_m of 25 and 28 μM, respectively. It had an absolute requirement for Mg²⁺ with a maximal activity at 4 mM and exhibited an optimal activity at pH 7.0. The molecular weight of the native enzyme was 110 000 as determined by Sephacryl S300 gel filtration. The analysis by SDS-polyacrylamide gel electrophoresis revealed a major band with a molecualr weight of 35 000 suggesting a polymeric structure of the enzyme.     

Turnover of protein-bound serine phosphate in respiring slices of guinea-pig cerebral cortex. Effects of putative transmitters, tetrodotoxin and other agents

1. The effect of various agents on the turnover of protein-bound phosphorus in respiring slices of cerebral cortex was studied. 2. Confirming previous work turnover was increased by the application of electrical pulses for 10s to the tissue. 3. Turnover was also increased by exposure of the slices for 10min to noradrenaline (0.5mm), 5-hydroxytryptamine (1mum) and histamine (0.1mm). 4. When slices were stimulated by electrical pulses in the presence of histamine the increase in turnover was the sum of the responses given by each agent above, suggesting that different phosphorylating systems were involved. 5. Tetrodotoxin (0.5mum) blocked the increased turnover due to electrical pulses, but not that due to histamine. Tetrodotoxin also prevented the increase in tissue cyclic AMP content caused by the application of electrical pulses. 6. Phosphoprotein turnover was not affected by adenosine, despite the increase in tissue cyclic AMP content given by this agent. 7. Adenosine blocked the phosphoprotein response to histamine, but did not affect the response to electrical pulses. 8. The results are discussed in relation to the hypothesis that the stimulation of protein phosphorus turnover by electrical pulses is secondary to the release of cyclic AMP in the tissue.     

Nuclear events during early chondrogenesis: phosphorylation of the precartilage 35.5-kDa domain-specific chromatin protein and its regulation by cyclic AMP

During chondrogenesis in vivo and in vitro, a family of nonhistone proteins (Mr 35,500), designated PCP 35.5, is lost from the nuclei of precartilage mesenchyme cells. A basic subcomponent of this family, designated PCP 35.5b, is phosphorylated during the first few hours of chondrogenesis in vitro by a phosphorylating system whose activity is enhanced 12- to 15-fold by exposure of differentiating precartilage cells to dibutyryl cyclic AMP. This phosphorylating system is present in isolated precartilage cell nuclei, where it retains its dependence on cyclic AMP and its specificity for PCP 35.5b. Assays for nuclear cyclic AMP inhibitable protein phosphatase activity capable of dephosphorylating PCP 35.5b were negative, indicating that the system responsible for phosphorylating this protein is a cyclic AMP-dependent protein kinase. Chromatin fractionation studies indicate that PCP 35.5b is localized at sites previously shown to be closely associated with DNase I-sensitive domains of precartilage cell chromatin. These studies define PCP 35.5b as a strategically located component of precartilage cell chromatin which is the major or sole chromatin target of cyclic AMP-dependent phosphorylation during chondrogenesis. This chromatin modification occurs prior to overt cartilage differentiation and may therefore play a regulatory role in the acquisition of the cartilage cell phenotype.     

Rapid enhancement of protein phosphorylation in A-431 cell membrane preparations by epidermal growth factor.

Membranes prepared from A-431 human epidermoid carcinoma cells retained the ability to bind 125I-labeled epidermal growth factor (EGF) in a specific manner. In the presence of [gamma-32P]ATP and Mn2+ or Mg2+, this membrane preparation was capable of phosphorylating endogenous membrane components, including membrane-associated proteins; the major phosphorylated amino acid residue detected in partial acid hydrolysates was phosphothreonine. The binding of EGF to these membranes in vitro resulted in a severalfold stimulation of the phosphorylation reaction; again, the major phosphorylated amino acid residue detected in partial acid hydrolysates was phosphothreonine. Membrane-associated dephosphorylation reactions did not appear to be affected by EGF. The phosphorylation reaction was not stimulated by cyclic AMP or cyclic GMP in the absence or presence of EGF. The phosphorylation system of the membrane was able to utilize [gamma-32P]GTP in both the basal and EGF-stimulated reactions. The enhanced membrane phosphorylation was specific for EGF and its derivatives; a wide variety of other peptide hormones were ineffective. The A-431 membrane preparation also was capable of phosphorylating exogenous proteins, such as histone, phosvitin, and ribonuclease, by a process which was stimulated by EGF. These findings suggest that one of the biochemical consequences of the binding of EGF to membranes is a rapid activation of a cyclic AMP-independent phosphorylating system.     

Adenosine 3':5'-monophosphate-regulated phosphorylation of erythrocyte membrane proteins. Separation of membrane-associated cyclic adenosine 3':5'-monophosphate-dependent protein kinase from its endogenous substrates.

An adenosine 3':5'-monophosphate (cyclic AMP)-binding protein in the human erythrocyte plasma membrane was isotopically labeled using a photoaffinity analog of cyclic AMP, N6-(ethyl 2-diazomalonyl) cyclic [3H]AMP. The cyclic AMP-binding site is located in a polypeptide chain having a molecular weight of 48,000. Cyclic AMP-binding protein and cyclic AMP-dependent protein kinase were solubilized with 0.5% Triton X-100 in 56 mM sodium borate, pH 8, but 32P-labeled membrane phosphoproteins were retained in the Triton-insoluble fraction, suggesting that the membrane-associated binding protein is not a primary substrate for protein kinase. Triton-solubilized and membrane-associated protein kinase activities were stimulated 15- and 17-fold by cyclic AMP, suggesting that the degree of association between the catalytic anc cyclic AMP-binding components was very similar in both preparations. Fractionation and characterization of membrane phosphoproteins have shown that protein III and a co-migrating minor protein are substrates for protein kinase but membrane sialoglycoproteins are not phosphorylated.     

Evidence of an intact N-terminal translocation sequence of human mitochondrial adenylate kinase 4

Adenylate kinases are abundant nucleoside monophosphate kinases, which catalyze the phosphorylation of AMP by using ATP or GTP as phosphate donors. A previously cloned cDNA was named adenylate kinase 4 (AK4) based on its sequence similarity with known AKs but with no confirmed AK enzyme activity. In the present study the AK4 cDNA was expressed in Escherichia coli and the substrate specificity and kinetic properties of the recombinant protein were characterized. The enzyme catalyzed the phosphorylation of AMP, dAMP, CMP and dCMP with ATP or GTP as phosphate donors and AK4 also phosphorylated AMP with UTP as phosphate donor. The kinetic parameters of the enzyme were determined for AMP and dAMP with ATP as phosphate donor and for AMP with GTP as phosphate donor. AK4 showed its highest efficiency when phosphorylating AMP with GTP and a slightly lower efficiency for the phosphorylation of AMP with ATP. Among the three reactions for which kinetics were performed, dAMP was the poorest substrate. The AK4 mitochondrial localization was confirmed by expression of AK4 as a fusion protein with GFP in HeLa cells. The mitochondrial import sequence was shown to be located within the first N-terminal 11 amino acid residues, very close to the ATP-binding region of the enzyme. Import analysis suggested that the mitochondrial import sequence was not cleaved and thus the enzyme retained its activity upon entering the mitochondria. Site directed mutagenesis of amino acids Lys 4 and Arg 7 showed that these two residues were essential for mitochondrial import.     

Metformin and phenformin activate AMP-activated protein kinase in the heart by increasing cytosolic AMP concentration

AMP-activated protein kinase (AMPK) acts as a cellular energy sensor: it responds to an increase in AMP concentration ([AMP]) or the AMP-to-ATP ratio (AMP/ATP). Metformin and phenformin, which are biguanides, have been reported to increase AMPK activity without increasing AMP/ATP. This study tests the hypothesis that these biguanides increase AMPK activity in the heart by increasing cytosolic [AMP]. Groups of isolated rat hearts (n = 5-7 each) were perfused with Krebs-Henseleit buffer with or without 0.2 mM phenformin or 10 mM metformin, and (31)P-NMR-measured phosphocreatine, ATP, and intracellular pH were used to calculate cytosolic [AMP]. At various times, hearts were freeze-clamped and assayed for AMPK activity, phosphorylation of Thr(172) on AMPK-alpha, and phosphorylation of Ser(79) on acetyl-CoA carboxylase, an AMPK target. In hearts treated with phenformin for 18 min and then perfused for 20 min with Krebs-Henseleit buffer, [AMP] began to increase at 26 min and AMPK activity was elevated at 36 min. In hearts treated with metformin, [AMP] was increased at 50 min and AMPK activity, phosphorylated AMPK, and phosphorylated acetyl-CoA carboxylase were elevated at 61 min. In metformin-treated hearts, HPLC-measured total AMP content and total AMP/ATP did not increase. In summary, phenformin and metformin increase AMPK activity and phosphorylation in the isolated heart. The increase in AMPK activity was always preceded by and correlated with increased cytosolic [AMP]. Total AMP content and total AMP/ATP did not change. Cytosolic [AMP] reported metabolically active AMP, which triggered increased AMPK activity, but measures of total AMP did not.     

The effects of aluminum loading on the renal response to parathyroid hormone in the vitamin D-replete rat

Aluminum (Al) may cause vitamin D-resistant osteomalacia and depress the serum levels of immunoreactive parathyroid hormone (iPTH) in patients treated with maintenance dialysis and those on total parental nutrition (TPN). Both conditions have been associated with low serum levels of 1,25(OH)2-vitamin D (1,25(OH)2D). Al may inhibit PTH secretion in vitro; however, induction of hypocalcemia can enhance endogenous PTH secretion in Al-loaded dogs and TPN patients. Despite hypocalcemia and/or increased endogenous iPTH levels, Al-loaded TPN patients fail to show the expected rise in serum 1,25(OH)2D levels. Such observations suggest that Al may impair the renal response to PTH. We studied vitamin D-replete rats given Al or saline vehicle IP for 5 days. Al and control rats then received a saline infusion with an IV bolus of PTH 1-34. Urinary cyclic AMP and P excretion rose in Al and control rats by 1 hr post-PTH, without differences between the groups. Serum P and ionized Ca levels were not different between Al and control rats. In other Al and control rats, serum 1,25(OH)2D levels were measured after saline without PTH. Serum 1,25(OH)2D levels were higher in controls given PTH than in those without, but 1,25(OH)2D levels were not different between Al rats given PTH and those with none. Thus, aluminum does not affect cyclic AMP or P excretion but may impair 25(OH)D-1 alpha-hydroxylase activity in response to PTH.     

Pattern of protein phosphorylation in intact stimulated cells: thyrotropin and dog thyroid

Two-dimensional, high-resolution electrophoretic technique of O'Farrell has been adapted to the analysis of thyroid phosphorylated proteins. Proteins were extracted from dog thyroid slices which had been incubated in the presence of [32P]phosphate with thyrotropin or with different agents which enhance the intracellular accumulation of cyclic AMP. About 350 phosphorylated polypeptides have been separated. Thyrotropin stimulates the phosphorylation of at least eight of these polypeptides. An increase in the phosphorylation of the same polypeptides was observed was observed when dog thyroid slices were incubated with dibutyryl adenosine 3':5'-monophosphate, cholera toxin or prostaglandin E1 instead of thyrotropin. Our results confirm that most of dog thyroid protein phosphorylation is independent of cyclic AMP. They offer a first link between the action of cyclic AMP on protein kinase and the physiological effects of thyrotropin. They strongly substantiate the hypothesis that most thyrotropin effects are mediated by cyclic AMP.     

Phosphorylation of pyrimidine deoxynucleoside analog diphosphates: selective phosphorylation of L-nucleoside analog diphosphates by 3-phosphoglycerate kinase.

D-Nucleoside analogs, which are in the natural configuration, as well as the L-nucleoside analogs, are clinically relevant antiviral and anticancer agents. Metabolism of L-nucleoside analog diphosphates to the triphosphates, however, remains unexplored. Studies with recombinant nm23-H1 and -H2 isoforms indicated that L-nucleoside analog diphosphates were not phosphorylated by their nucleoside diphosphate kinase (NDPK) activity. Therefore, roles of creatine kinase, 3-phosphoglycerate kinase, and pyruvate kinase were evaluated using preparations from commercial sources and human HepG2 cells. Phosphorylation of L-OddC, L-SddC, L-Fd4C, L-FMAU, and L-ddC were compared with D-deoxynucleoside analogs, AraC, dFdC, and D-FMAU, and D-dideoxynucleoside analogs, ddC and d4T. Results based on preparations from HepG2 cells showed that L-nucleoside analog diphosphates were selectively phosphorylated by 3-phosphoglycerate kinase, whereas, D-deoxynucleoside analog diphosphates were phosphorylated by NDPK. Interestingly, ddCDP and d4TDP were substrates for creatine kinase, but were not phosphorylated by NDPK. In conclusion, it is proposed that specificity of the phosphorylating enzymes toward the nucleoside analog diphosphates is dependent on the configuration of the analog (L or D) and the presence or absence of 3'-hydroxyl group in the sugar moiety. The enzymatic process of phosphorylation of L- and D-nucleoside analog diphosphates is different in cells.     

Cell cycle regulation of the p34cdc2 inhibitory kinases.

In cells of higher eukaryotic organisms the activity of the p34cdc2/cyclin B complex is inhibited by phosphorylation of p34cdc2 at two sites within its amino-terminus (threonine 14 and tyrosine 15). In this study, the cell cycle regulation of the kinases responsible for phosphorylating p34cdc2 on Thr14 and Tyr15 was examined in extracts prepared from both HeLa cells and Xenopus eggs. Both Thr14- and Tyr15- specific kinase activities were regulated in a cell cycle-dependent manner. The kinase activities were high throughout interphase and diminished coincident with entry of cells into mitosis. In HeLa cells delayed in G2 by the DNA-binding dye Hoechst 33342, Thr14- and Tyr15-specific kinase activities remained high, suggesting that a decrease in Thr14- and Tyr15- kinase activities may be required for entry of cells into mitosis. Similar cell cycle regulation was observed for the Thr14/Tyr15 kinase(s) in Xenopus egg extracts. These results indicate that activation of CDC2 and entry of cells into mitosis is not triggered solely by activation of the Cdc25 phosphatase but by the balance between Thr14/Tyr15 kinase and phosphatase activities. Finally, we have detected two activities capable of phosphorylating p34cdc2 on Thr14 and/or Tyr15 in interphase extracts prepared from Xenopus eggs. An activity capable of phosphorylating Tyr15 remained soluble after ultracentrifugation of interphase extracts whereas a second activity capable of phosphorylating both Thr14 and Tyr15 pelleted. The pelleted fraction contained activities that were detergent extractable and that phosphorylated p34cdc2 on both Thr14 and Tyr15. The Thr14- and Tyr15-specific kinase activities co-purified through three successive chromatographic steps indicating the presence of a dual-specificity protein kinase capable of acting on p34cdc2.     

Serine and tyrosine protein kinase activities in Streptococcus pyogenes. Phosphorylation of native and synthetic peptides of streptococcal M proteins

Two forms of protein kinase activity were isolated from crude extracts of Streptococcus pyogenes and partially purified by ion exchange chromatography and affinity chromatography. The phosphorylation activities were shown to be insensitive to cAMP, required the presence of divalent cations, and eluted from a Sephadex G-200 column with approximate molecular masses of 60 and 45 kDa, respectively. Both enzymes were capable of phosphorylating eukaryotic proteins and synthetic polypeptides in addition to endogenous and heterologous prokaryotic proteins at serine and tyrosine residues. Firm evidence for tyrosine kinase activity was obtained by the use of a tyrosine kinase-specific substrate, a 4:1 glutamate:tyrosine copolymer. Both protein kinases phosphorylated HPr, a phosphocarrier protein of the phosphotransferase system isolated from S. pyogenes and Bacillus stearothermophilus, but failed to phosphorylate HPr isolated from Escherichia coli. Both also phosphorylated a native polypeptide fragment (pep M24) as well as synthetic peptide copies of M protein, the major virulence determinant of group A streptococci. These results indicate that prokaryotic protein kinases are capable of phosphorylating eukaryotic proteins and suggest that the protein kinases of streptococci may play an important role not only in the phosphotransferase system but also in the virulence properties of these organisms.     

The role of cyclic 3',5'-AMP in the regulation of aminoacyl-tRNA synthetase activities in mouse uterus and liver following 17beta-oestradiol treatment. Activation of a phosphoaminoacyl-tRNA synthetase phosphatase by phosphorylation with cyclic 3',5'-AMP dependent protein kinase.

The changes in the activities of 17 aminoacyl-tRNA synthetases induced by phosphorylation [1] were reversed by the action of cyclic AMP in preparations from both uterus and liver. Cyclic AMP also inhibited the phosphorylation of aminoacyl-tRNA synthetase protein by endogenous non-cyclic AMP-dependent protein kinase and [gamma-32P]ATP. The effect was not due to a stimulation of phosphoaminoacyl-tRNA synthetase phosphatase or to an influence of cyclic AMP on aminoacyl-tRNA synthetases. The activity of phosphoaminoacyl-tRNA synthetase phosphatase was increased by treatment with endogenous cyclic AMP-dependent protein kinase, ATP and cyclic AMP. Affinity chromatography of the 32P-labeled phosphorylated phosphosynthetase phosphatase protein followed by gel electrophoresis showed that the activated phosphatase was phosphorylated. In the uterus, the changes in 17 aminoacyl-tRNA synthetase activities observed 5 min after dibutyryl cyclic AMP administration to ovariectomized mice were similar to those observed after 17beta-oestradiol treatment, whereas in the liver the changes in these activities were the opposite to those found after treatment with 17beta-oestradiol. A mechanism for the regulation of the 17 aminoacyl-tRNA synthetase activities is proposed, which suggests that the synthetase activities inhibited (group I) or stimulated (group II) by phosphorylation with a non-cyclic AMP-dependent aminoacyl-tRNA synthetase kinase are reactivated (group I) or inhibited (group II), respectively, by the action of a cyclic AMP-dependent phosphatase kinase through the increased activity of phosphorylated phosphoaminoacyl-tRNA synthetase phosphatase.     

Phosphorylation of ankyrin decreases its affinity for spectrin tetramer

The effects of phosphorylation on the interaction between spectrin and ankyrin were investigated. Spectrin and ankyrin were phosphorylated using purified human erythrocyte membrane and cytosolic (casein kinase A) kinases. These two kinases have similar properties as well as activities toward spectrin and ankyrin. Both kinases catalyzed the incorporation of about 2 mol of phosphate/mol of spectrin and about 7 mol of phosphate/mol of ankyrin. These phosphates were incorporated primarily into seryl and threonyl residues of the proteins. The phosphopeptide maps of ankyrin phosphorylated by the membrane kinase and casein kinase A were identical. Binding studies indicate that ankyrin exhibits different affinities for spectrin dimers (KD = 2.5 +/- 0.9 X 10(-6) M) and tetramers (KD = 2.7 +/- 0.8 X 10(-7) M). These dissociation constants were not appreciably affected by the phosphorylation of spectrin. On the other hand, phosphorylation of ankyrin was found to significantly reduce its affinity for either phosphorylated or unphosphorylated spectrin tetramers (KD = 1.2 +/- 0.1 X 10(-6) M) but not spectrin dimers (KD = 2.5 +/- 0.4 X 10(-6) M). The same results were obtained using either the membrane kinase or casein kinase A as the phosphorylating enzyme. The above observation suggests that ankyrin phosphorylation may provide an important mechanism for the regulation of the erythrocyte membrane cytoskeletal network.     

Failure of 1, 25 dihydroxycholecalciferol (1, 25-(OH) 2-D3) to modify cyclic AMP levels in parathyroid hormone-treated and untreated bone cells.

Treatment with 1, 25-(OH) 2-D3 (1 ng/ml-25 ng/ml) for periods ranging from 2.5 min. to 60 min. did not alter cyclic AMP levels in bone cells isolated from periosteum-free rat calvaria, or in cells isolated from rat periosteal tissues. 1, 25-(OH) 2-D3 failed to modify the acute increases in cyclic AMP elicited by PTH (10 ng/ml-1 ug/ml). Two separate 1, 25-(OH) 2-D3 preparations, biologically active in other systems, were ineffective under a wide variety of experimental conditions. These results suggest that 1, 25-(OH) 2-D3 is not an acute modulator of cyclic AMP metabolism in PTH-treated and untreated bone cells.