Novel yeast protein kinase (YPK1 gene product) is a 40-kilodalton phosphotyrosyl protein associated with protein-tyrosine kinase activity.

Extracts of bakers' yeast (Saccharomyces cerevisiae) contain protein-tyrosine kinase activity that can be detected with a synthetic Glu-Tyr copolymer as substrate (G. Schieven, J. Thorner, and G.S. Martin, Science 231:390-393, 1986). By using this assay in conjunction with ion-exchange and affinity chromatography, a soluble tyrosine kinase activity was purified over 8,000-fold from yeast extracts. The purified activity did not utilize typical substrates for mammalian protein-tyrosine kinases (enolase, casein, and histones). The level of tyrosine kinase activity at all steps of each preparation correlated with the content of a 40-kDa protein (p40). Upon incubation of the most highly purified fractions with Mn-ATP or Mg-ATP, p40 was the only protein phosphorylated on tyrosine. Immunoblotting of purified p40 or total yeast extracts with antiphosphotyrosine antibodies and phosphoamino acid analysis of 32P-labeled yeast proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the 40-kDa protein is normally phosphorylated at tyrosine in vivo. 32P-labeled p40 immunoprecipitated from extracts of metabolically labeled cells by affinity-purified anti-p40 antibodies contained both phosphoserine and phosphotyrosine. The gene encoding p40 (YPK1) was cloned from a yeast genomic library by using oligonucleotide probes designed on the basis of the sequence of purified peptides. As deduced from the nucleotide sequence of YPK1, p40 is homologous to known protein kinases, with features that resemble known protein-serine kinases more than known protein-tyrosine kinases. Thus, p40 is a protein kinase which is phosphorylated in vivo and in vitro at both tyrosine and serine residues; it may be a novel type of autophosphorylating tyrosine kinase, a bifunctional (serine/tyrosine-specific) protein kinase, or a serine kinase that is a substrate for an associated tyrosine kinase.     

Membrane receptors with protein-tyrosine kinase activity

Protein-tyrosine kinase activities have appeared so far to be intrinsic for two classes of proteins: the transforming proteins of certain retroviral oncogenes and the membrane receptors for certain cellular growth factors. In this latter family, the protein-tyrosine kinase is activated upon binding of the growth factor to its receptor and phosphorylates both the receptor itself and other cell target proteins. Growth factor receptors are transmembrane glycoproteins able to undergo not only autophosphorylation but also phosphorylation by other protein kinases (e.g., protein kinase C). Both autophosphorylation and heterologous phosphorylation of the receptor are regulatory events for the ligand binding and protein-tyrosine kinase intrinsic activities of the growth factor receptors.     

The protein-tyrosine kinase substrate, p81, is homologous to a chicken microvillar core protein

p81, a protein-tyrosine kinase substrate previously identified in epidermal growth factor-treated A431 cells, is demonstrated to be homologous to ezrin, an 80-kD component of microvillar core proteins. p81 has been characterized using antiserum raised against purified chicken intestinal ezrin. p81, located by indirect immunofluorescent staining, is concentrated in surface projections of A431 cells such as microvilli and retraction fibers. None of the conditions of biochemical cell fractionation tested completely solubilizes p81; the insoluble p81 partitions as if associated with the cytoskeleton. The soluble form of p81 behaves as a monomer in all extraction procedures studied. EGF-stimulated phosphorylation of p81 does not appear to change its intracellular location. p81 exhibits a wide tissue distribution with highest levels of expression in small intestine, kidney, thymus, and lung. Intermediate levels are found in spleen, thymus, lymph nodes, and bone marrow, with low levels in brain, heart, and testes. p81 is undetectable in muscle and liver. In A431 cells, p81 is phosphorylated on serine and threonine residues. Upon EGF treatment, approximately 10% of p81 becomes phosphorylated on tyrosine, and the phosphorylation of threonine residues increases.     

L-asparaginase of Tetrahymena pyriformis is associated with a kinase activity

Most of L-asparaginase activity of Tetrahymena pyriformis was found to be present in microsomal membranes from which it has been purified to homogeneity (Tsirka, S.A.E. and Kyriakidis, D.A. Mol. Cell. Biochem. 83: 147–155, 1988). The native enzyme has a relative molecular weight of approximately 200 kDa, while under denaturing conditions the enzyme exhibits. a subunit size of 39 kDa. Aminoacid analysis and an oligopeptide from N-terminal sequence have been determined. Dephosphorylation of L-asparaginase by alkaline phosphatase results in an activation of its catalytic activity. This enzyme also exhibits intrinsic phosphorylation activity with a Km value for ATP of 0.5 mM. Autophosphorylation with -^32P ATP of purified L-asparaginase results in the phosphorylation of tyrosine residues as well as in loss of its activity. Mg²⁺ and Ca²⁺ added together act synergistically to stimulate the kinase activity by more than 160%. The polyamines putrescine, spermidine and spermine activate the kinase approximately 100%, while neither cAMP or cGMP have any effect. These results indicate that this membrane protein with dual L-asparaginase/kinase activity must play an important role in regulating the intracellular levels of L-asparagine in Tetrahymena pyriformis.     

A lysine in the ATP-binding site of P130gag-fps is essential for protein-tyrosine kinase activity.

The P130gag-fps transforming protein of Fujinami sarcoma virus (FSV) possesses tyrosine-specific protein kinase activity and autophosphorylates at Tyr-1073. Within the kinase domain of P130gag-fps is a putative ATP-binding site containing a lysine (Lys-950) homologous to lysine residues in cAMP-dependent protein kinase and p60v-src which bind the ATP analogue p-fluorosulfonylbenzoyl-5' adenosine. FSV mutants in which the codon for Lys-950 has been changed to codons for arginine or glycine encode metabolically stable but enzymatically defective proteins which are unable to effect neoplastic transformation. Kinase-defective P130gag-fps containing arginine at residue 950 was normally phosphorylated at serine residues in vivo suggesting that this amino acid substitution has a minimal effect on protein folding and processing. The inability of arginine to substitute for lysine at residue 950 suggests that the side chain of Lys-950 is essential for P130gag-fps catalytic activity, probably by virtue of a specific interaction with ATP at the phosphotransfer active site. Tyr-1073 of the Arg-950 P130gag-fps mutant protein was not significantly autophosphorylated either in vitro or in vivo, but could be phosphorylated in trans by enzymatically active P140gag-fps. These data indicate that Tyr-1073 can be modified by intermolecular autophosphorylation.     

Protein kinase A activity is associated with metacyclogenesis in Leishmania amazonensis

Because of the importance of cell signalling processes in proliferation and differentiation, the adenylate cyclase pathway was studied, specifically the protein kinase A (PKA) in Leishmania amazonensis. The PKAs of soluble (SF) and enriched membrane fractions (MF) from infective/non-infective promastigotes and axenic amastigotes were assayed. In order to purify the PKA molecule, fractions were chromatographed on DEAE-cellulose columns and the phosphorylative activity was evaluated using [gamma(32)P]-ATP as the phosphate source. These experiments were performed in the presence of cyclic adenosine monophosphate (cAMP) and an inhibitor of PKA. Our data demonstrated that the PKA activity was significantly higher (about two times) in SF from promastigotes with a high concentration of metacyclic forms, when compared with the non-infective promastigotes, suggesting an association of this activity and the metacyclogenesis process. A discrete phosphorylative activity in axenic amastigotes was observed. As the adenylate cyclase/cAMP pathway would be involved in the parasite-host interiorization, the PKA activity may constitute a good intracellular target for studies of leishmanicidal drugs.     

Characterization of protein-tyrosine kinase activity in the canine prostate.

Following the measurement of the phosphorylation of the substrate poly(Glu80Na,Tyr20) and the analysis of the alkali-resistant phosphorylation of endogenous proteins, the protein-tyrosine kinase of the canine prostate was partially characterized with regard to its subcellular localization, as well as certain kinetic and molecular properties. This kinase was mainly found in the cytosolic fraction (75%); however, its specific activity was similar to that of the residual enzyme present in the particulate fraction. Conditions for optimal activity of both fractions were determined. Under these conditions, several endogenous phosphoproteins (44-63 kilodaltons upon electrophoresis) were alkali resistant and phosphotyrosine was present in all of the major ones (pp63, pp57, pp52, and pp44). The particulate protein-tyrosine kinase activity was partially solubilized (58%) with 0.5% Triton X-100; this percentage was increased to 85% in the presence of 0.25 M KCl. Upon gel filtration, both cytosolic and particulate kinases showed an apparent molecular mass of 44 kilodaltons; these enzymes also phosphorylated similar major alkali-resistant phosphoproteins. The soluble protein-tyrosine kinase, with a sedimentation coefficient of 4.0S and an isoelectric point of 5.5, could be separated from arginine esterase and prostatic acid phosphatase.     

The human c-fps/fes gene product expressed ectopically in rat fibroblasts is nontransforming and has restrained protein-tyrosine kinase activity.

A 13-kilobase EcoRI genomic restriction fragment containing the human c-fps/fes proto-oncogene locus was expressed transiently in Cos-1 monkey cells and stably in Rat-2 fibroblasts. In both cases, human c-fps/fes directed synthesis of a 92-kilodalton protein-tyrosine kinase (p92c-fes) indistinguishable from a tyrosine kinase previously identified with anti-fps antiserum which is specifically expressed in human myeloid cells. Transfected Rat-2 cells containing approximately 50-fold more human p92c-fes than is found in human leukemic cells remained morphologically normal and failed to grow in soft agar. Synthesis of p92c-fes in this phenotypically normal line exceeded that of the P130gag-fps oncoprotein in a v-fps-transformed Rat-2 line. Despite this elevated expression, human p92c-fes induced no substantial increase in cellular phosphotyrosine and was not itself phosphorylated on tyrosine. In contrast, p92c-fes immunoprecipitated from these Rat-2 cells or expressed as an enzymatically active fragment in Escherichia coli from a c-fps/fes cDNA catalyzed tyrosine phosphorylation with an activity similar to that of v-fps/fes polypeptides. Thus, p92c-fes is not transforming when ectopically overexpressed in Rat-2 fibroblasts. This lack of transforming activity correlates with a restriction imposed on the kinase activity of the normal c-fps/fes product in vivo which is apparently lifted for v-fps/fes oncoproteins, suggesting that regulatory interactions within the host cell modify fps/fes protein function and normally restrain its oncogenic potential.     

Identification of a human gene (HCK) that encodes a protein-tyrosine kinase and is expressed in hemopoietic cells.

We have isolated cDNAs representing a previously unrecognized human gene that apparently encodes a protein-tyrosine kinase. We have designated the gene as HCK (hemopoietic cell kinase) because its expression is prominent in the lymphoid and myeloid lineages of hemopoiesis. Expression in granulocytic and monocytic leukemia cells increases after the cells have been induced to differentiate. The 57-kilodalton protein encoded by HCK resembles the product of the proto-oncogene c-src and is therefore likely to be a peripheral membrane protein. HCK is located on human chromosome 20 at bands q11-12, a region that is affected by interstitial deletions in some acute myeloid leukemias and myeloproliferative disorders. Our findings add to the diversity of protein-tyrosine kinases that may serve specialized functions in hemopoietic cells, and they raise the possibility that damage to HCK may contribute to the pathogenesis of some human leukemias.     

UDP-acetyl-mannosamine dehydrogenase is an endogenous protein substrate of Staphylococcus aureus protein-tyrosine kinase activity

The in silico analysis of the amino acid sequences deduced from the complete genome sequence of Staphylococcus aureus suggested the presence of two protein tyrosine kinase activities, each split into two distinct polypeptides, respectively Cap5A1/Cap5B1 and Cap5A2/Cap5B2, like in some other Gram-positive bacteria. To check this prediction, the corresponding genes were cloned and overexpressed, and the four corresponding proteins were purified by affinity chromatography and assayed for phosphorylating activity in vitro. Individually, none of them was found to autophosphorylate. However, when Cap5B2 was incubated in the presence of Cap5A2 or, with a larger efficiency, in the presence of Cap5A1, this protein exhibited intensive autokinase activity, occurring selectively at tyrosine residues. On the other hand, whatever the protein combination assayed, Cap5B1 did not present any phosphorylating activity. In search of a possible role for the phosphorylation reaction mediated by Cap5B2, an endogenous substrate of this kinase was characterized. This substrate, termed Cap5O, is the enzyme UDP-acetyl-mannosamine dehydrogenase involved in the cascade of reactions leading to the synthesis of the bacterial capsule. It represents the first endogenous substrate for a tyrosine kinase activity so far identified in S. aureus. The analysis of its dehydrogenase activity showed that it was positively controlled by its phosphorylation at tyrosine.     

Characterization of the phosphatase activity of a baculovirus-expressed calcineurin A isoform.

Calcineurin A was purified by calmodulin-Sepharose affinity chromatography from Sf9 cells infected with recombinant baculovirus containing the cDNA of a rat calcineurin A isoform. The Sf9-expressed calcineurin A has a low basal phosphatase activity in the presence of EDTA (0.9 nmol/min/mg) which is stimulated 3-5-fold by Mn2+. Calmodulin increased the Mn2+ stimulated activity 3-5-fold. Bovine brain calcineurin B increased the A subunit activity 10-15-fold, and calmodulin further stimulated the activity of reconstituted A and B subunits 10-15-fold (644 nmol/min/mg). The Km of calcineurin A for 32P-RII pep (a peptide substrate (DLDVPIPGRFDRRVSVAAE) for CaN), was 111 microM with or without calmodulin, and calmodulin increased the Vmax about 4-fold. The Km of reconstituted calcineurin A plus B for 32P-RII pep was 20 microM, and calmodulin increased the Vmax 18-fold without affecting the Km. CaN A467-492, a synthetic autoinhibitory peptide (ITSFEEAKGLDRINERMPPRRDAMP) from calcineurin, inhibited the Mn2+/calmodulin-stimulated activities of the reconstituted enzyme and the A subunit with IC50's of 25 microM and 90 microM, respectively. The reconstitution of the phosphatase activity of an expressed isoform of calcineurin A by purified B subunit and calmodulin may facilitate comparative studies of the regulation of calcineurin A activity by the B subunit and calmodulin.     

Evidence that the Rous sarcoma virus transforming gene product is associated with glycerol kinase activity

This communication provides biochemical, immunological, and genetic evidence that pp60src, the Rous sarcoma virus transforming gene product, is associated with glycerol kinase activity. Our investigations demonstrated that the compound phosphorylated by pp60src or by glycerol kinase (EC 2.7.1.30) from Candida mycoderma share the same electrophoretic and chromatographic mobilities. The glycerol kinase and protein kinase activities of pp60src were inhibited similarly by preincubation with immune IgG. Both activities were reduced 6-9-fold in pp60src preparations derived by immunoaffinity chromatography from cells which were infected with NY68, a temperature-sensitive transformation mutant of Rous sarcoma virus. The thermolability at 41 degrees C of the glycerol kinase activity of pp60src from the mutant virus-infected cells was greater (t/2 = 1.3 min) than the same activity in pp60src preparations from wild type virus-infected cells (t/2 = 4.8 min).     

A Drosophila gene that is homologous to a mammalian gene associated with tumor metastasis codes for a nucleoside diphosphate kinase

The product of the abnormal wing discs (awd) gene of Drosophila is 78% identical to the product of the nm23 gene of mammals, which is differentially expressed in certain metastatic tumors. We present evidence that the awd gene codes for a nucleoside diphosphate kinase (NDP kinase) and that this Awd/NDP kinase is microtubule associated. Neuroblasts in Drosophila larvae homozygous for a null mutation in the awd gene are arrested in metaphase, indicating that microtubule-associated Awd/NDP kinase plays a critical role in spindle microtubule polymerization.     

Kinase activity associated with caldesmon is Ca2+/calmodulin-dependent kinase II.

The relationship of the kinase which co-purifies with caldesmon to Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) was investigated by studying the phosphorylation of bovine brain synapsin I, as well-characterized substrate of CaM-kinase II. Synapsin I is a very good substrate (Km = 90 nM) of the co-purifying kinase, which phosphorylates two sites in synapsin I, both of which are distinct from the single site phosphorylated by cyclic-AMP-dependent protein kinase. Phosphorylation of synapsin I is Ca2(+)- and calmodulin-dependent: half-maximal activation occurs at 0.13 microM-Ca2+ and maximal activity at 0.4 microM-Ca2+. Phosphorylation of the co-purifying kinase slightly enhances the rate, but does not alter the stoichiometry, of subsequent synapsin I phosphorylation; it does, however, circumvent the requirement for Ca2+ and calmodulin. The properties of this kinase therefore closely resemble those of CaM-kinase II, and we conclude that it is probably a smooth-muscle isoenzyme of CaM-kinase II.