Purification and characterization of nucleoside diphosphate kinase from the brain of Bombyx mori

Nucleoside diphosphate kinase in the brain of Bombyx mori was purified by ammonium sulfate fractionation, and a sequence of chromatographies on DEAE-Cellulofine, hydroxyapatite, Mono-S, and Mono-Q column. The purified enzyme preparation was found to be electrophoretically homogeneous on SDS-PAGE, and its molecular mass was determined to be 18 kDa. The purified protein was digested and the amino acid sequences of resulting peptides were determined. The enzyme showed high similarity to the amino acid sequences of the Drosophila NDP kinase. The enzyme showed NDP kinase activity and mediated the phosphorylation of myelin basic protein. Gel filtration and Hill plot analysis indicate that the purified NDP kinase forms a tetramer and shows little interaction among substrates. Dephosphorylation of NDP kinase by bacterial alkaline phosphatase increased NDP kinase activity. This result indicates that phosphorylation of NDP kinase represses NDP kinase activity.     

Putative functions of nucleoside diphosphate kinase in plants and fungi

The putative functions of NDP (nucleoside diaphosphate) kinases from various organisms focusing to fungi and plants are described. The biochemical reactions catalyzed by NDP kinase are as follows. (i) Phosphotransferring activity from mainly ATP to cognate NDPs generating nucleoside triphosphates (NTPs). (ii) Autophosphorylation activity from ATP and GTP. (iii) Protein kinase (phosphotransferring) activity phosphorylating such as myelin basic protein. NDP kinase could function to provide NTPs as a housekeeping enzyme. However, recent works proved possible functions of the NDP kinases in the processes of signal transduction in various organisms, as described below. By use of the extracts of the mycelia of a filamentous fungus Neurospora crassa blue-light irradiation could increase the phosphorylation of a 15-kDa protein, which was purified and identified to be NDP kinase (NDK-1). By use of the etiolated seedlings of Pisum sativum cv Alaska and Oryza sativa red-light irradiation of intact plants increased the phosphorylation of NDP kinase. However, successive irradiation by red–far-red reversed the reaction, indicating that phytochrome-mediated light signals are transduced to the phosphorylation of NDP kinase. NDP kinase localizing in mitochondria is encoded by nuclear genome and different from those localized in cytoplasm. NDP kinase in mitochondria formed a complex with succinyl CoA synthetase. In Spinicia oleraceae two different NDP kinases were detected in the chloroplast, and in Pisum sativum two forms of NDP kinase originated from single species of mRNA could be detected in the choloroplast. However, the function of NDP kinases in the choloroplast is not yet known. In Neurospora crassa a Pro72His mutation in NDP kinase (ndk-1 ^Pro72His ) deficient in the autophosphorylation and protein kinase activity resulted in lacking the light-induced polarity of perithecia. In wild-type directional light irradiation parallel to the solid medium resulted in the formation of the perithecial beak at the top of perithecia, which was designated as light-induced polarity of perithecia. In wild-type in darkness the beak was formed at random places on perithecia, and in ndk ^Pro72His mutant the perithecial beak was formed at random places even under directional light illumination. The introduction of genomic DNA and cDNA for ndk-1 demonstrated that the wild-type DNAs suppressed the mutant phenotype. With all these results except for the demonstration in Neurospora, most of the phenomena are elusive and should be solved in the molecular levels concerning with NDP kinases.     

Distribution subcellulaire des protéine-kinases stimulables par l'AMP cyclique et des protéines réceptrices de l'AMP cyclique dans la thyroïde de porc

The distribution of cyclic AMP-dependent protein kinase activity in porcine thyroid glands has been studied. Enzyme activity catalyzing phosphorylation of exogenous substrate (protamine) from ATP, and cyclic AMP binding were determined in parallel in subcellular fractions purified by differential centrifugation and flotation on sucrose density layers. Both activities were found in all the studied fractions; they were quantitatively the highest in the cytosol but particles showed the highest specific activities.Latent protein-kinase activity was unmasked by action of detergents on microsomes (× 5–10 fold) and solubilized (85 to 99 p. cent of the initial total activity). Cyclic AMP binding capacity was also recovered in detergent-treated microsomal extracts in spite of reduced cyclic AMP binding in the presence of detergent.Protein kinase activity and cyclic AMP-binding proteins were less represented in purified nuclei than in microsomes. Again both activities were unmasked by detergent.Preparations highly enriched in Golgi membranes were compared to rough microsomal preparations. Higher protein kinase activity was detected in rough microsomes as compared to Golgi membranes, whereas the reverse was true for cyclic AMP binding. Both activities were equalized after detergent treatment. Since unmasking of protein kinase activity was the highest in Golgi membranes, this fraction contains more enzyme activity and cyclic AMP binding capacity than rough microsomes.The localization of endogeneous protein substrates of cyclic AMP-dependent protein kinases was investigated using purified soluble protein kinase subcellular fractions. The better endogeneous substrates seemed to be localized in the rough microsomal and in the nuclear fractions.     

Purification and properties of a protamine kinase from bovine kidney microsomes.

About an eightfold increase in protamine kinase activity was detected following extraction of highly purified microsomes from bovine kidney with 1% Triton X-100. Relative to the soluble fraction, the microsomes contained about 30% protamine kinase activity. The microsomal protamine kinase was purified to apparent homogeneity. The purified enzyme exhibited an apparent M(r) approximately 45,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel permeation chromatography on Sephacryl S-200. Relative to protamine, the purified kinase exhibited about 100% activity with the synthetic peptide RRLSSLRA and about 5, 8, and less than 0.1% activity with casein, histone H2B, and histone H1, respectively. The purified kinase phosphorylated several 40 S ribosome polypeptides. One of these polypeptides was identified as ribosomal protein S6 by N-terminal sequencing. About 2.5 mol of phosphoryl groups was incorporated per mole of ribosomal protein S6 following incubation of the 40 S ribosomes with the purified kinase. Following incubation with protein phosphatase 2A2, purified preparations of the protamine kinase were inactivated. These properties were identical to those of purified preparations of a protamine kinase from extracts of bovine kidney cytosol (Z. Damuni, G.D. Amick, and T.R. Sneed, 1989, J. Biol. Chem. 264, 6412-6418). Near identical peptide patterns were obtained following incubation of purified preparations of the microsomal and cytosolic protamine kinases with Staphylococcus aureus V8 proteinase. The results indicate that a form of the cytosolic protamine kinase is present in microsomes.     

Protein Tyrosine Kinase Activity and Its Endogenous Substrates in Rat Brain: A Subcellular and Regional Survey

The rat CNS contains high levels of tyrosine-specific protein kinases that specifically phosphorylate the tyrosine-containing synthetic peptide poly(Glu80,Tyr20). The phosphorylation of this peptide is rapid and occurs with normal Michaelis-Menten kinetics. Using this peptide to assay for enzyme activity, we have measured the protein tyrosine kinase activity in homogenates from various regions of rat CNS. A marked regional distribution pattern was observed, with high activity present in cerebellum, hippocampus, olfactory bulb, and pyriform cortex, and low activity in the pons/medulla and spinal cord. The distribution of protein tyrosine kinase activity was examined in various subcellular fractions of rat forebrain. The majority of the activity was associated with the particulate fractions, with enrichment in the crude microsomal (P3) and crude synaptic vesicle (LP2) fractions. Moreover, the subcellular distribution of pp60csrc, a well-characterized protein tyrosine kinase, was examined by immunoblot analysis using an affinity-purified antibody specific for pp60csrc. The subcellular distribution of pp60csrc paralleled the overall protein tyrosine kinase activity. In addition, using an antibody specific for phosphotyrosine, endogenous substrates for protein tyrosine kinases were demonstrated on immunoblots of homogenates from the various regions and the subcellular fractions. The immunoblots revealed numerous phosphotyrosine-containing proteins that were present in many of the CNS regions examined and were associated with specific subcellular fractions. The differences in tyrosine-specific protein kinase activity, and in phosphotyrosine-containing proteins, observed in various regional areas and subcellular fractions may reflect specific functional roles for protein tyrosine kinase activity in mammalian brain.     

Purification and characterization of the 65-kDa protein phosphorylated in murine macrophages by stimulation with bacterial lipopolysaccharide

Modification of cellular proteins via phosphorylation is known to be a major regulatory mechanism whereby external stimuli control intracellular events. We demonstrated that bacterial LPS induced a distinct set of phosphorylated protein (pp) in murine peritoneal macrophages, and that the LPS-induced pp were specifically located in cytosol and/or membrane fractions. One of the most heavily phosphorylated substrate proteins with a molecular mass of 65 kDa (pp65) was purified to homogeneity via SDS-PAGE analysis and autoradiography by sequential chromatography on Sephacryl S-200, HPLC anion exchange, and hydroxyapatite HPLC. Our pp65 is apparently the first purified LPS-induced pp, and is thought to be a novel protein. Serine residues on pp65 were found to be exclusively phosphorylated, indicating a contribution by LPS-inducible serine kinase. Interestingly, LPS-induced phosphorylation of pp65 was not observed in macrophages from a LPS-nonresponsive C3H/HeJ strain of mice, although their macrophages had about the same amounts of unphosphorylated p65 as normal macrophages when detected under Western blot analysis by using polyclonal anti-pp65 antibodies. This suggests that the functional defect of C3H/HeJ macrophages exists somewhere in the process before the pp65 phosphorylation. Moreover, the degree of the pp65 phosphorylation in macrophages stimulated with LPS or lipd A correlated well to that of cellular responses such as IL-1 production in the same macrophages. Considering these observations, the pp65 seems to play a crucial role in macrophage activation, and the studies on the structure and function of the pp65 should lead to progress in our understanding of the mechanisms of macrophage activation by LPS.     

Redistribution of a 120 kDa Phosphoprotein in the Parietal Cell Associated with Stimulation

When rabbit isolated gastric glands were stimulated via the cyclic AMP pathway, a phosphorylated protein band of about 120 kDa (pp120) was markedly increased in the apical membrane-rich fraction, concomitant with an increase in the amount of H,K-ATPase and the phosphorylation of the cytoskeletal protein ezrin in the same fraction. The cytosolic fraction, but not other membrane fractions, also contained a protein with common features to the membrane-bound pp120, i.e., comigration in two-dimensional gels with a pI of ∼4.5, anomalous mobility in SDS-PAGE, reactivity to antibodies, and phosphorylation, indicating that these two proteins were identical. The possibility that pp120 is vinculin was completely excluded. Using antibody against pp120, this protein was found to be almost exclusively in the gastric parietal cell. Biochemical and immunohistochemical analyses suggest that pp120 exists mainly in the cytosol, and that a small part of the protein binds to the apical membrane when the parietal cell is stimulated via the cyclic AMP pathway. In the presence of histone, purified pp120 produced phosphorylation on pp120 as well as histone. The inhibitor profile of this kinase activity is not consistent with any known kinase. We conclude that pp120 is closely associated with a new type of kinase, and translocates from cytosol to the apical membrane when the parietal cell is stimulated. Received: 9 September 1998/Revised: 29 December 1998     

A Nucleoside Diphosphate Kinase from Paramecium tetraurelia with Protein Kinase Activity

Nucleoside diphosphate kinase (NDP kinase) from Paramecium was purified to homogeneity. The native enzyme was 80 kDa (by gel filtration), with subunits of 18 and 20 kDa. Near the amino terminus, 15 of 20 residues were identical with those in human NDP kinase, and 17 of 20 with the awd gene product from Drosophila. NDP kinase bound α-labeled ATP and GTP, and a photoreactive GTP analog labeled both subunits. Purified NDP kinase underwent autophosphorylation on a histidine and a serine residue using either ATP or GTP as a substrate. The enzyme also catalyzed acid-stable phosphorylation of casein and phosvitin. This protein kinase activity is distinct from the histidine phosphorylation that is part of the NDP kinase catalytic cycle. Antiserum against the purified protein from Paramecium cross-reacted with 16- to 20-kDa proteins in most species tested, and with a larger protein (44 kDa) in Paramecium, Xenopus, and two human lines. The multiple forms (20 and 44 kDa) of the NDP kinase in Paramecium and its protein kinase activity, suggest that the protein is more than a housekeeping enzyme; it may have regulatory roles such as those of the NDP kinase-like awd protein of Drosophila and Nm23 protein of humans.     

Characterization of an activated ribosomal S6 kinase variant from maturing sea star oocytes: association with phosphatase 2A and substrate specificity.

Two ribosomal protein S6 kinases (i.e., pp52(S6K) and pp70(S6K)) of the p70 S6 kinase family were markedly activated during meiotic maturation of Pisaster ochraceus sea star oocytes. A rapid protocol was developed for the purification from the oocyte cytosol of pp52(S6K) by approximately 50,000-fold with a specific enzyme activity of 1.6 micromol per min per mg. The purified enzyme apparently featured the N- and C-terminal regions of pp70(S6K) as it immunoreacted with antibodies directed to peptides patterned after these amino acid sequences in mammalian pp70(S6K). pp52(S6K) was inhibited by fluoride (IC(50) approximately 60 mM), but was relatively insensitive to beta-glycerolphosphate, EGTA, dithiothreitol, spermine, heparin, NaCl, and metal ions such as Mn(2+), Zn(2+), and Ca(2+). The consensus sequence for substrate phosphorylation was determined to be RXXSXR, which was partially distinct from mammalian p70(S6K) in its requirement for an amino-terminal arginine. Phosphorylation of ribosomal protein S6 by p52(S6K) occurred exclusively on serine on at least five tryptic peptides. Inhibition of sea star p52(S6K) phosphotransferase activity after treatment with protein serine/threonine phosphatases confirmed that p52(S6K) was still regulated by phosphorylation. The sea star S6 kinase was purified to near homogeneity with the regulatory and catalytic subunits of protein-serine phosphatase 2A and the heat shock protein 60. The association of an S6 kinase with phosphatase 2A was confirmed by coimmunoprecipitation of S6 kinase activity with phosphatase 2A-specific antibodies. The purified S6 kinase and the sea star oocyte system will be useful for analysis of upstream and downstream signaling events that lead to phosphorylation of the S6 protein and other targets.     

Rapid cycling of autophosphorylation of a ca-calmodulin regulated plasma membrane located protein kinase from pea

Plasma membrane vesicles from pea (Pisum sativum L.) seedlings contain an autophosphorylating calcium-activated protein kinase of relative molecular weight 18,000 (phosphoprotein 18, pp 18). Pulse chase analysis revealed that pp 18 autophosphorylation exhibited very rapid turnover. pp 18 was the only detectable plasma membrane protein to have this property. pp 18 has been highly purified by affinity chromatography and the final preparation contains peptides of relative molecular weight 67,000, 48,000, and 18,000. Highly purified pp 18 still showed rapid cycling of autophosphorylated phosphate attached to pp 18. Turnover of pp 18 autophosphorylation was accelerated by ADP, but this effect did not appear to be the back reaction of the kinase since inorganic phosphate accumulation is increased by ADP. A rapid cycling of phosphorylation is an ideal control point in signal transduction.     

Phosphorylation of nitric oxide synthase by protein kinase A.

Nitric oxide synthase was purified to apparent homogeneity from the cytosolic fractions obtained from rat and porcine cerebellum. Enzyme activity--measured as [3H]citrulline formation after incubation with [3H]arginine--was dependent on Ca2+/calmodulin, NADPH, and tetrahydro-L-biopterin. Specific activity varied between 450 to 780 nmol/min/mg protein. Purified nitric oxide synthases showed a single band on 8% SDS/PAGE gels and had an apparent molecular mass of 150,000 Da. The purified proteins were used as substrate for phosphorylation with different protein kinases. In the assays using two Ca2+/calmodulin-dependent protein kinases, CaM kinase II and CaM kinase-Gr, protein kinase C, and the catalytic subunit of protein kinase A, nitric oxide synthase was exclusively phosphorylated by protein kinase A. Such phosphorylation was linear over time for at least 60 min and resulted in nearly stoichiometric phosphate/protein incorporation. The serine in the protein kinase A-consensus sequence KRFGS is probably the site of phosphorylation in nitric oxide synthase. Kemptide, a known protein kinase A substrate, inhibited phosphorylation of nitric oxide synthase in a dose-dependent manner. No changes in nitric oxide synthase activity were observed upon phosphorylation by protein kinase A.     

Phosphorylation of the p220 subunit of eIF-4F by cAMP dependent protein kinase and protein kinase C in vitro

Changes in the extent of phosphorylation of the 25 kDa subunit of eIF-4F occur during several major biological events including mitosis and heat shock in mammalian cells and shortly after fertilization of sea urchin (Lytechinus pictus) eggs. In vitro phosphorylation studies using highly purified protein kinases demonstrated that the 220 kDa subunit of eIF-4F was phosphorylated by cAMP dependent protein kinase, protein kinase C and probably to a lesser extent by cGMP dependent protein kinase. In addition, eIF-4A was readily phosphorylated by cAMP and cGMP dependent protein kinases whereas p48 of eIF-4F was not. The effect of these phosphorylation events on eIF-4F function, its assembly or disassembly, susceptibility to viral initiated proteolysis or the ability of p25 to be phosphorylated at serine-53 remain to be investigated.     

Protein phosphorylation substrates in normal and neoplastic squamous epithelia of the human upper aero-digestive tract

Protein phosphorylation was studied in crude and in protein kinase C (Pk-C)-enriched preparations from squamous cell carcinomas and normal mucosa of the human upper aero-digestive tract. In crude soluble preparations from neoplastic mucosa we found a 5-fold higher basal endogenous phosphorylation when compared to normal mucosa. In particulate fractions the increase was 3-fold. SDS-PAGE and autoradiography of phosphorylated proteins in crude soluble tumor extracts showed bands corresponding to proteins with apparent molecular weights of 18, 37, 40-42, 52, 60, 62 and 90 kDa. In normal mucosa the phosphorylation of these proteins was very low or absent, except for the proteins with molecular weights of 40-42 and 52-55 kDa. Addition of Ca2+ or Ca2+/phospholipids to the reaction mixture caused phosphorylation of additional proteins with apparent molecular weight of 45-50 kDa in soluble preparations of tumors. Cyclic AMP or cGMP had no significant effect on the phosphorylation of endogenous proteins. In the partially purified, Pk-C-enriched fractions the phosphorylation in the presence of Ca2+/phospholipids was distinctly higher in tumors when compared to the phosphorylation observed in normal mucosa, and some phosphorylation substrates were detected only in tumor tissue. In order to find out whether the elevated basal phosphorylation was due to an endogenous activation of protein kinases, different inhibitors of serine/threonine protein kinases were tested. These inhibitors included: heat-stable cyclic AMP-dependent protein kinase (Pk-A) inhibitor, Pk-A inhibitor peptide (Wiptide), heparin and the Pk-C inhibitors peptide 19-36 and H-7. None of these inhibitors had any significant effect on the basal phosphorylation. In conclusion, our results show the existence of endogenous phosphorylation substrates in human squamous cell carcinomas from the upper aerodigestive tract, and indicates that there is a significantly higher basal and Pk-C specific phosphorylation of endogenous substrates in tumors compared to normal mucosa. This may be of importance for the transformation and altered growth regulation in epithelial tumors.     

Nucleoside diphosphate kinase from haloalkaliphilic archaeon Natronobacterium magadii: purification and characterization

An ATP-binding protein from the haloalkaliphilic archaeon Natronobacterium magadii was purified and characterized by affinity chromatography on ATP-agarose and by fast protein liquid chromatography (FPLC) on a Mono Q column. The N-terminal 20 amino acid sequence of the kinase showed a strong sequence similarity of this protein with nucleoside diphosphate (NDP) kinases from different organisms and, accordingly, we believe that this protein is a nucleoside diphosphate kinase, an enzyme whose main function is to exchange γ-phosphates between nucleoside triphosphates and diphosphates. Comparison of the molecular weights of the NDP kinase monomer determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (23 000) and of the oligomer determined by sedimentation equilibrium experiments (125 000) indicated that the oligomer is a hexamer. The enzyme was autophosphorylated in the presence of [γ-³²P]ATP, and Mg²⁺ was required for the incorporation of phosphate. The kinase preserved the ability to transfer γ-phosphate from ATP to GDP in the range of NaCl concentration from 90 mM to 3.5 M and in the range of pH from 5 to 12. It was found and confirmed by Western blotting that this kinase is one of the proteins that bind specifically to natronobacterial flagellins. NDP kinase from haloalkaliphiles appeared to be simple to purify and to be a suitable enzyme for studies of structure and stability compared with NDP kinases from mesophilic organisms. Received: December 3, 1997 / Accepted: January 29, 1998     

Towards a molecular description of cell transformation by avian sarcoma virus

The avian sarcoma virus transforming gene product has been identified and partially purified from extracts of transformed cells. It is a phosphoprotein with a relative molecular mass of 60 000 (pp60src) with two major sites of phosphorylation. pp60src appears to be a cyclic-AMP-independent protein kinase as judged by protein phosphorylation with partly purified fractions. The specificity of the phosphorylation observed was judged by inhibition with anti-pp60src IgG but not by normal IgG and by the fact that the protein kinase activity isolated from ts transformation-mutant infected cells was more thermolabile than that from wild-type transformed cells, thus showing more directly the origin of the enzymic activity. A cellular protein substrate of pp60src has been identified as a 34 000 molecular mass protein. These data together suggest that protein phosphorylation by pp60src may be a function of the molecule that plays a major role in transformation.     

Phosphorylation of 130- and 95-kDa substrates associated with tumor necrosis factor-alpha receptor CD120a (p55)

Cross-linking of CD120a (p55), a receptor for tumor necrosis factor alpha (TNFalpha), initiates downstream events, including the activation of protein Ser/Thr kinases. In this report, we have characterized two protein Ser/Thr kinase substrates that are intrinsically associated with CD120a (p55) in mouse macrophages, and we have investigated the mechanism involved in their phosphorylation. pp130 and pp95 were detected by co-immunoprecipitation with CD120a (p55) from lysates of mouse bone marrow-derived macrophages and were phosphorylated on Ser and Thr residues during in vitro kinase assays in the presence of [gamma-(32)P]ATP. The level of phosphorylation of pp130 and pp95 was rapidly and transiently increased in response to TNFalpha in [(32)P]orthophosphate-labeled macrophages, although the level of pp130 protein associated with CD120a (p55) remained unchanged as detected by [(35)S]methionine labeling. In contrast, pp130 and pp95 were efficiently phosphorylated in in vitro kinase assays of CD120a (p55) immunoprecipitates from unstimulated cells, and the level of phosphorylation was rapidly and transiently reduced in response to TNFalpha. Both pp130 and pp95 were sensitive to dephosphorylation with purified protein phosphatase 2A, and okadaic acid, a PP1/PP2A inhibitor, mimicked the ability of TNFalpha to stimulate the phosphorylation of pp130 and pp95 in intact (32)P-labeled macrophages. Collectively, these findings suggest that pp130 and pp95 are constitutively associated with CD120a (p55) and become inducibly phosphorylated in macrophages in response to TNFalpha. We propose that the underlying mechanism of their phosphorylation may involve the inactivation of a cytoplasmic pp130/pp95 Ser/Thr phosphatase.     

Calmodulin-activated protein kinase activity of adipocyte microsomes

A calmodulin-activated phosphorylation activity was identified in microsomal (endoplasmic reticulum) preparations from rat adipocytes. Activity was not detected in mitochondrial or plasma membrane fractions. Although the phosphorylation of several proteins was enhanced by addition of calmodulin, the major calmodulin-sensitive protein had a molecular weight of 54,000. A series of experiments were conducted to determine if the microsomal phosphorylation was either calmodulin-containing phosphorylase kinase or calmodulindependent myosin light chain kinase. The phosphorylation of the 54,000 Dalton band in microsomal preparations was 1) not significantly reduced by potential competing protein substrates, e.g. actomyosin or phosphorylase b, 2) nearly equally well phosphorlyated at pH 8.6 or pH 7.0, unlike actomyosin or phosphorylase b, and 3) not increased by addition of phosphorylase kinase or myosin light chain kinase. The results demonstrate that this microsomal calmodulinactivated phosphorylation is catalysed by a protein kinase distinct from phosphorylase kinase or myosin light chain kinase.     

Protein kinase-mediated regulation of the Na(+)/H(+) exchanger in the rat myocardium by mitogen-activated protein kinase-dependent pathways.

We examined regulation of the Na(+)/H(+) exchanger isoform 1 by phosphorylation in the rat myocardium. We utilized cell extracts from adult rat hearts, adult rat extracts fractionated by fast performance liquid chromatography, and extracts from cultured neonatal cardiac myocytes. The carboxyl-terminal 178 amino acids of the Na(+)/H(+) exchanger were expressed in Escherichia coli fused with glutathione S-transferase. The purified protein was used as a substrate for in vitro phosphorylation and in-gel kinase assays. Unfractionated extracts from neonatal myocytes or adult hearts phosphorylated the COOH-terminal domain of the antiporter. Western blot analysis revealed that mitogen-activated protein (MAP) kinase (44 and 42 kDa) and p90(rsk) (90 kDa) were present in specific fractions of cardiac extracts that phosphorylated the COOH-terminal protein. In-gel kinase assays confirmed that protein kinases of approximately 44 and 90 kDa could phosphorylate this domain. MAP kinase and p90(rsk)-dependent phosphorylation of the antiporter could be demonstrated by immunoprecipitation of these kinases from extracts of neonatal cardiac myocytes. PD98059, a mitogen-activated protein kinase kinase inhibitor, decreased MAP kinase and p90(rsk) phosphorylation of the antiporter and abolished serum and endothelin 1-stimulated increases in steady-state pH(i). These results confirm the presence of MAP kinase-dependent phosphorylation in the regulation of the Na(+)/H(+) exchanger in the rat myocardium and suggest an important role for p90(rsk) phosphorylation in regulation of the protein by endothelin-mediated stimulation of the antiporter.     

A tyrosine kinase related to pp60c-src is associated with membranes of Electrophorus electricus electric organ

A tyrosine-specific protein kinase immunologically related to pp60c-src, the cellular homolog of the Rous sarcoma virus-transforming protein, was expressed at elevated levels in the electric organ of the electric eel Electrophorus electricus. The electric organ kinase phosphorylated antibodies reactive with pp60c-src at tyrosine residues in immune complex protein kinase assays and was associated with electric organ membranes enriched in acetylcholine receptors. The protein recognized by anti-pp60c-src antibodies was phosphorylated in endogenous membrane phosphorylation reactions and was shown to have a relative molecular mass of 57 kDa by two-dimensional gel electrophoresis. In immune complex protein kinase assays the 57-kDa protein was phosphorylated at threonine by a distinct threonine kinase from the electric organ. The tyrosine kinase was purified 844-fold from electric organ membranes by chromatography on omega-aminohexyl agarose, phosphocellulose, and casein-Sepharose. Threonine kinase activity in immunoprecipitates was not observed in the tyrosine kinase fractions after the first step. Incubation of the casein Sepharose fraction with [gamma-32P]ATP-Mn2+ in solution resulted in phosphorylation of only the 57-kDa protein. Phosphorylation occurred solely at tyrosine, suggesting that the kinase is capable of autophosphorylation. The structural and functional properties of the 57-kDa electric organ kinase indicate that the 57-kDa electric organ protein is a member of the src subfamily of tyrosine kinases and is closely related to pp60c-src.     

Protein kinase C phosphorylates pp60src at a novel site

The transforming protein of Rous sarcoma virus (pp60v-src) and its normal cellular homolog (pp60c-src) are demonstrated to be phosphorylated at serine 12 in vivo under certain conditions. We propose that protein kinase C is responsible for this modification based on the following evidence. First, the tumor promoters, 12-O-tetradecanoylphorbol-13-acetate and teleocidin, and synthetic diacylglycerol, known activators of protein kinase C in vivo, cause nearly complete phosphorylation of pp60src at serine 12. Second, among five purified serine/threonine-specific protein kinases tested, only protein kinase C phosphorylates pp60c-src and pp60v-src in vitro at serine 12. Third, purified protein kinase C phosphorylates a synthetic peptide corresponding to the N-terminal 20 amino acids of pp60c-src at serine 12. The physiological significance of this novel phosphorylation is discussed.