DosT and DevS are oxygen-switched kinases in Mycobacterium tuberculosis.

Exposure of Mycobacterium tuberculosis to hypoxia is known to alter the expression of many genes, including ones thought to be involved in latency, via the transcription factor DevR (also called DosR). Two sensory kinases, DosT and DevS (also called DosS), control the activity of DevR. We show that, like DevS, DosT contains a heme cofactor within an N-terminal GAF domain. For full-length DosT and DevS, we determined the ligand-binding parameters and the rates of ATP reaction with the liganded and unliganded states. In both proteins, the heme state was coupled to the kinase such that the unliganded, CO-bound, and NO-bound forms were active, but the O(2)-bound form was inactive. Oxygen-bound DosT was unusually inert to oxidation to the ferric state (half life in air >60 h). Though the kinase activity of DosT was unaffected by NO, this ligand bound 5000 times more avidly than O(2) to DosT (K(d) [NO] approximately 5 nM versus K(d) [O(2)] = 26 microM). These results demonstrate direct and specific O(2) sensing by proteins in M. tuberculosis and identify for the first time a signal ligand for a sensory kinase from this organism. They also explain why exposure of M. tuberculosis to NO donors under aerobic conditions can give results identical to hypoxia, i.e., NO saturates DosT, preventing O(2) binding and yielding an active kinase.

Nucleosomal histone kinase-1 phosphorylates H2A Thr 119 during mitosis in the early Drosophila embryo.

Posttranslational histone modifications are important for the regulation of many biological phenomena. Here, we show the purification and characterization of nucleosomal histone kinase-1 (NHK-1). NHK-1 has a high affinity for chromatin and phosphorylates a novel site, Thr 119, at the C terminus of H2A. Notably, NHK-1 specifically phosphorylates nucleosomal H2A, but not free H2A in solution. In Drosophila embryos, phosphorylated H2A Thr 119 is found in chromatin, but not in the soluble core histone pool. Immunostaining of NHK-1 revealed that it goes to chromatin during mitosis and is excluded from chromatin during S phase. Consistent with the shuttling of NHK-1 between chromatin and cytoplasm, H2A Thr 119 is phosphorylated during mitosis but not in S phase. These studies reveal that NHK-1-catalyzed phosphorylation of a conserved serine/threonine residue in H2A is a new component of the histone code that might be related to cell cycle progression.

Histone phosphorylation: how to proceed.

Among all posttranslational modifications that occur on histone tails, phosphorylation is the one that establishes a direct link between chromatin remodeling and intracellular signaling pathways. Specific, conserved serine residues are present on the N-terminal tails of each histone. These are phosphoacceptor sites for a number of kinases, whose identification is essential to decipher the transduction routes leading to various physiological responses. In the case of histone H3, phosphorylation at the Ser10 residue may lead to either activated gene expression or chromatin condensation during mitosis. In addition, phosphorylation at specific sites may be coupled to other distinct modifications, such as acetylation and methylation, generating the so-called "histone code" which postulates that well defined combinatorial modifications at histone tails correspond to specific physiological responses. Here we describe a number of experimental methodologies that are essential for the study of histone phosphorylation. While chromatin immunoprecipitation is useful in recognizing gene targets, the in-gel kinase assay is a first, essential step in establishing the identity of the kinase(s) that operates in response to a specific signaling pathway. The subsequent use of in vitro kinase assays is helpful in validating the implication of a candidate kinase. These powerful approaches are important as identification of the signaling transduction routes leading to chromatin remodeling is critical to an understanding of all cellular processes.

Purification and characterization of a novel protamine kinase in HL60 cells.

A protamine kinase from HL60 cells was purified to near homogeneity by DEAE-Sephacel, protamine-agarose, Hydroxylapatite, and S-200 chromatography. It was purified by 75.8-fold through four chromatographic steps, and 0.67% of total activity was recovered. The purified enzyme had an apparent molecular mass of 120 kDa and was activated by Mg(2+) or Mn(2+), but inhibited by Ca(2+). Neither phospholipid nor phorbol ester significantly affected the enzyme activity. Staurosporine was the most potent inhibitor of the enzyme among the protein kinase inhibitors tested, K(252a), H(7), heparin, and staurosporine. The purified protamine kinase exhibited a maximum velocity of 5,000 pmol/min/mg and K(m) of 1.3 mM for protamine sulfate as a substrate. Myelin basic protein and protamine sulfate served as the best substrates for the protamine kinase among those tested. The activity of the protamine kinase remained unchanged upon treatment with PMA, retinoic acid, dimethyl sulfoxide, or 1,25 dihydroxy vitamin D(3) for 15 min, while treatment with a differentiating agent, 1,25 dihydroxy vitamin D(3), for one week increased its activity. These results suggest that protamine kinase in HL60 cells is involved in the late stage of the macrophage-monocytic differentiation pathway and may play a role in maintenance of the differentiation after HL60 cells are committed.

Detection of a mammalian histone H4 kinase that has yeast histidine kinase-like enzymic activity.

A well characterized histidine kinase purified from yeast has been shown to phosphorylate histone H4 on a histidine residue. This enzyme is unlike the two-component histidine kinases predominantly found in prokaryotes. Until now, a histidine kinase similar to this yeast enzyme has not been purified from a mammalian source. By using a purification scheme similar to that used to purify the yeast histidine kinase, a protein fraction with histone H4 kinase activity has been isolated from porcine thymus. The yeast histidine kinase was shown to be detectable using an in-gel kinase assay system and using this system, four major bands of histone H4 kinase activity were apparent in the porcine thymus preparation. Through the use of immunoprecipitation, alkaline hydrolysis and subsequent phosphoamino acid analysis it has been demonstrated that this partially purified kinase fraction is capable of phosphorylating histone H4 on histidine. In conclusion, an preparation has been made from porcine thymus that contains histone H4 kinase activity and at least one of the kinases present in this preparation is a histidine kinase.

Cyclic-AMP-dependent activation of an inter-phylum hybrid histone-kinase complex reconstituted from sea urchin sperm-regulatory subunits and bovine heart catalytic subunits.

A cAMP-dependent histone kinase was purified and characterized from spermatozoa of the sea urchin Hemicentrotus pulcherrimus. The molecular mass of the kinase was estimated to be 178 kDa by native PAGE and 400 kDa by gel chromatography on a Superose 6 HR 10/30 column. The enzyme, composed of two 39-kDa catalytic subunits and two 48-kDa regulatory subunits, phosphorylates the lysine-rich histone subspecies (H1 and H2B) isolated from H. pulcherrimus spermatozoa. We isolated cDNA clones encoding a 39-kDa catalytic subunit and a 48-kDa regulatory subunit of the enzyme. The cDNA clone for the 39-kDa subunit was 3881 bp, and the 352-residue deduced amino acid sequence showed 78% similarity with the catalytic subunit of/mammalian cAMP-dependent protein kinase (PKA). The cDNA for the 48-kDa subunit was 4589 bp and the 368-residue deduced amino acid sequence showed 57% similarity with the regulatory subunit of mammalian PKA, although the N-terminal 77 residues showed poor similarity. The mRNAs encoding both the catalytic subunit (7.5 kb) and the regulatory subunit (4.6 kb) were expressed in testis, ovary and egg. An inter-phylum hybrid enzyme, reconstituted from the regulatory subunit of cAMP-dependent histone kinase of sea urchin sperm and the catalytic subunit of bovine heart PKA, has a cAMP-dependent histone kinase activity. Thus, we suggest that the N-terminal 77-amino-acid residues of the regulatory subunit are not essential for inhibition by the regulatory subunit of the catalytic subunit, and that cAMP-dependent inhibitory activity of the regulatory subunit resides in the sequence between the inhibitory site and the C-terminus.

In vitro activation of a 60-70 kDa histone H4 protein kinase from neutrophils by limited proteolysis.

Neutrophils stimulated with the chemotactic peptide fMet-Leu-Phe (fMLP) are known to exhibit a rapid and transient activation of a histone H4 kinase that may function in a stimulatory pathway downstream of phosphatidylinositol 3-kinase. The activity of this histone kinase in unstimulated neutrophils and cells treated with 1.0 microM fMLP for 10 sec was 8.8 +/- 5 and 43 +/- 2 pmol P/min per 10(7) cells, respectively. In this paper, we report that unstimulated neutrophils contain a latent H4 kinase in the 100,000 x g soluble fraction that can be markedly activated by treatment with trypsin. The values for the untreated and trypsin treated enzyme were 5.5 +/- 1.0 and 63.6 +/- 18 pmol P/min per 10(7) cell-equivalents, respectively. This kinase was insensitive to a selective antagonist of protein kinase C (i.e., 50 microM 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7)) but completely blocked by 100 nM staurosporine. Only a single peak of activity was observed for this enzyme when the 100,000 x g supernatant fraction was fractionated on either an exclusion (KW-803) or an anion exchange column (DEAE), or during isoelectric focusing. The molecular weight of the latent kinase was 64 +/- 6 kDa and the isoelectric point was 7.6 +/- 0.1. During all fractionation procedures, the H4 kinase co-chromatographed with a trypsin-activated kinase that catalyzed the phosphorylation of a peptide which corresponds to residues 297-331 of the 47 kDa subunit of the NADPH-oxidase complex (p47-phox). The properties of the trypsin-activated H4 kinase from unstimulated neutrophils are very similar to those reported for this enzyme from fMLP-stimulated cells.

Properties of a resiniferatoxin-stimulated, calcium inhibited but phosphatidylserine-dependent kinase, which is distinct from protein kinase C isotypes alpha, beta 1, gamma, delta, epsilon and eta.

We have separated a resiniferatoxin-stimulated histone-kinase activity from human neutrophils, elicited mouse macrophages and murine alveolar macrophages by hydroxyapatite chromatography. The assay conditions for resiniferatoxin kinase were optimized as part of this study and in the presence of phosphatidylserine but absence of Ca2+ the Ka for histone IIIs phosphorylation by resiniferatoxin was calculated as 16 nM. Using a phosphate gradient of 20-500 mM, peaks of protein kinase C activity could be washed from the hydroxyapatite column in 300 nM phosphate and resiniferatoxin kinase recovered in 500 mM phosphate. At the optimum concentration of 160 nM, the ability of resiniferatoxin to induce enzyme activity was compared with a range of phorbol esters all at the same concentration. These related compounds failed to activate resiniferatoxin kinase although they have previously been shown to activate protein kinase C isotypes. Similarly sn-1,2,-dioleoylglycerol and the potent irritant capsaicin at 30 microM failed to activate the kinase. A Scatchard analysis of [3H] phorbol dibutyrate binding produced a linear plot (Kd 41.6 nM; Bmax 11.6 fmol unit-1) and binding was inhibited by resiniferatoxin and 12-O-tetradecanoylphorbol-13-acetate (TPA), with resiniferatoxin 700 times more potent than TPA in this respect. A radiolabelled resiniferatoxin binding assay was also used to demonstrate specific binding of [3H]resiniferatoxin which could be inhibited by unlabelled compound. Resiniferatoxin kinase activity was shown to be distinct from the protein kinase C isotypes alpha, beta 1, gamma, delta and epsilon by means of immunological analysis and from the eta isotype, because that isotype was not stimulated by resiniferatoxin but was stimulated by TPA when a pseudosubstrate was used. In addition the resiniferatoxin-stimulated activity was inhibited in-vitro by the addition of Ca2+ (Ki 0.1-0.5 nM free Ca2+). Further purification of resiniferatoxin kinase by Superose chromatography indicated a major activity fraction of about 70-90 kDa. Thus resiniferatoxin kinase, isolated from human and mouse inflammatory cells is distinct from the known isotypes of protein kinase C and is a major resiniferatoxin receptor.

Oncogenic ras stimulates a 96-kDa histone H2b kinase activity in activated Xenopus egg extracts. Correlation with the suppression of p34cdc2 kinase.

We previously showed that purified, bacterially expressed oncogenic human rasH protein blocks or delays the progression of the embryonic cell cycle into M-phase in activated Xenopus egg extracts. This block correlates with the suppression of the activation of p34cdc2 kinase (Pan, B.-T., Chen, C.-T., and Lin, S.-M. (1994) J. Biol. Chem. 269, 5968-5975). In an attempt to identify kinases that are involved in mediating the effect of oncogenic Ras on the cell cycle, we assayed aliquots of activated Xenopus egg extracts, which were incubated at 25 degrees C for various times in the absence and presence of oncogenic Ras, for their kinase activities toward a calf thymus histone fraction (Hf1). We find that the suppression of the histone H1 kinase activity of p34cdc2 by oncogenic Ras correlates with a simultaneous stimulation of a histone H2b serine kinase activity. Using a histone H2b in-the-gel kinase assay, we further show that the stimulated histone H2b kinase activity is attributed mainly to a 96-kDa kinase and slightly to p42mapk. Although Xenopus p90rsk is also activated by oncogenic Ras, we demonstrate that activated p90rsk is not responsible for the 96-kDa histone H2b kinase activity. The identity of the 96-kDa kinase remains unclear. Our data suggests that the 96-kDa kinase may be involved in mediating the effect of oncogenic Ras on the embryonic cell cycle of Xenopus.

Ca(2+)-calmodulin-dependent phosphorylation of arginine in histone 3 by a nuclear kinase from mouse leukemia cells.

A Ca(2+)-calmodulin dependent histone 3 kinase was partially purified from a low salt (150 mM NaCl) nuclear extract of mouse leukemia cells by calmodulin-Sepharose affinity chromatography. In vitro, the kinase activity transferred gamma-phosphate from ATP to histone 3 to form an acid-labile and alkaline-stable linkage. Under the assay conditions 1.8 mol of phosphate are incorporated per mol of histone 3. Upon modification of arginine residues with phenylglyoxal prior to phosphorylation, a considerable decrease in the amount of phosphate transferred to histone 3 was observed. Amino acid analysis revealed that H3 was phosphorylated on arginine residues. To identify the phosphorylated peptide(s), histone 3 was cleaved with cyanogen bromide prior to phosphorylation. The phosphorylated mixture was then separated by gel filtration high-performance liquid chromatography under denaturing conditions. Fragments I (N-terminal 10.3-kDa peptide) and III (C-terminal 1.7-kDa peptide) were both phosphorylated. Amino acid sequencing further revealed that the molar yields of 3 of the 4 arginines present in the phosphorylated cyanogen bromide fragment III were reduced by a factor of about 10 compared with the corresponding arginines from the unphosphorylated fragment. In the case of fragment I, 25 cycles of Edman degradation revealed that the recovery of only arginine 2 was reduced by a factor of 20. The putative phosphorylation sites are arginines 2, 128, 129, and 131. The sequence information offered an indirect evidence that these arginines were the sites of phosphorylation. The kinase described in this report represents a first member of a potentially important new class of kinases which are Ca(2+)-calmodulin dependent and which phosphorylate arginine.

Reconstitution of cyclin-dependent cdc2 and cdk2 kinase activities in vitro.

The genes that encode human cdc2 and cdk2 proteins are essential for cell cycle progression. In this report, we describe the purification of cyclin-associated cdc2 and cdk2 kinases as well as cyclin-free cdc2 and cdk2 protein preparations from HeLa cells. The cdc2-cyclin B kinase complex that we have isolated, consisting of two polypeptides of p60 (cyclin B) and p34 (cdc2), phosphorylated both the p34 and p70 subunits of the three-subunit human single-stranded DNA-binding protein (also called RP-A), a DNA replication and repair factor. We also partially purified a histone H1 kinase activity that is associated with the cdk2 and cyclin A proteins. Purified human cyclins A and B1, overproduced in bacteria, complemented a cellular fraction enriched in cdc2 and cdk2 proteins to reconstitute histone H1 kinase activity. Using this complementation system, human cdc2 and cdk2 proteins were purified and separated from one another. Glycerol gradient analyses demonstrated that the purified cdk2 (p33) protein co-sedimented with a cyclin A-dependent H1 kinase activity. Thus, cdk2 and cyclin A proteins are components that assemble to yield a kinase complex that catalyzes the phosphorylation of histone H1.

Mutagenesis of the regulatory domain of rat protein kinase C-eta. A molecular basis for restricted histone kinase activity.

Protein kinase C-theta (PKC-eta) is a member of the protein kinase C family that is characterized by Ca2+ independence and restricted histone kinase activity (Dekker, L. V., Parker, P. J., and McIntyre, P. (1992) FEBS Lett. 312, 195-199). Here we have investigated the molecular basis of this low histone kinase activity by limited proteolysis and site-directed mutagenesis. It is shown that a 46-kDa C-terminal tryptic fragment, representing the catalytic domain of PKC-eta, can phosphorylate histone. The Km value for histone of this catalytic fragment is 25-fold lower than that of intact PKC-eta. Thus, sites in the N-terminal regulatory domain upstream of the trypsin cleavage site (near residue 320) restrict histone kinase activity of intact PKC-eta. Deletion of the "Vo domain" (residues 2-137) generates a PKC-eta mutant that shows the same cofactor dependence and substrate phosphorylation as wild-type PKC-eta, indicating that the relevant sites do not appear to lie in the Vo domain but between amino acid 137 and the start of the catalytic domain. Deletion of the pseudosubstrate region (residue 155-171) generates a cofactor-independent kinase that has high histone kinase activity. A pseudosubstrate site point mutation in which the alanine residue at position 161 is replaced with a glutamic acid residue shows the same properties as the pseudosubstrate site deletion mutant. Km values for histone for both mutants are similar to that observed for the catalytic fragment. Therefore, in addition to its role in conferring cofactor dependence, the pseudosubstrate site also mediates the low histone kinase activity of wild-type PKC-eta. The data are discussed in the light of current models for PKC activation.

Activation of a histone H1 kinase by tyrosine phosphorylation in v-src-transformed fibroblasts.

Using anti-phosphotyrosine immunoaffinity chromatography, we have searched for serine/threonine kinases that are directly regulated by tyrosine phosphorylation in v-src-transformed rat 3Y1 fibroblasts. Tyrosine phosphoprotein preparations from v-src-transformed cells contain a kinase activity that phosphorylates histone H1 in vitro on serine residues and this activity is present at a 20-fold greater level than that in parental cell preparations. This activity elutes from a MonoQ FPLC column as a single peak and gel filtration chromatography suggests that the kinase has a molecular mass of approximately 55 kDa. Tyrosine phosphatase treatment inactivates the histone H1 kinase and this result indicates that the specific activity of the kinase is regulated by tyrosine phosphorylation. Experiments with cells transformed with a temperature-sensitive mutant of the v-src oncogene demonstrate that the tyrosine phosphorylation of the histone H1 kinase is an early event in v-src transformation. The kinase is distinct from known cdc2 family members that contain the PSTAIR motif, because the kinase can be separated almost completely from these proteins by immunoprecipitation with an antibody against p34cdc2. The profile of antibody reactivity and sensitivity to modulators of protein kinases suggests that this activity is distinct from known second messenger-regulated kinases and from previously characterized MAP kinases.

M-phase-specific histone H1 kinase in fish oocytes. Purification, components and biochemical properties.

We demonstrate, for the first time in fish, that a Ca(2+)-independent and cyclic-nucleotide-independent histone H1 kinase activity oscillates according to the cell cycle of the oocyte, peaking at the first and the second meiotic metaphase with a transient drop between them. The kinase, M-phase-specific histone H1 kinase (M-H1K), was purified from mature carp oocytes by using two exogenous substrates for assaying its activity: histone H1 and a synthetic peptide (SP peptide, KKAAKSPKKAKK) containing the sequence KSPKK, which includes the consensus sequence of the site phosphorylated by a serine/threonine-specific protein kinase encoded by the fission yeast cdc2+ gene (cdc 2 kinase). The M-H1K and maturation-promoting factor (MPF) activities coincided closely throughout four steps of purification, strongly suggesting the identity of M-H1K and MPF. The final preparation was purified 5000-fold with a recovery of 4%, when histone H1 was used for the kinase assay, and 10,000-fold with a recovery of 7% when SP peptide was used. The purified molecular mass of the kinase was estimated to be 100 kDa by gel filtration and contained four proteins of 33, 34, 46 and 48 kDa. Anti-PSTAIR antibody recognizing cdc2 kinase cross-reacted with the 33-kDa and 34-kDa proteins, while the 46-kDa and 48-kDa bands cross-reacted with monoclonal antibodies raised against cyclin B. The 33-kDa protein was also recognized by an antibody against a goldfish cdk2 (Eg1) kinase, a cdc2-related kinase which has the PSTAIR sequence and binds to p13suc1 but does not form a complex with cyclin B. M-H1K activity corresponded well to the 34-kDa, 46-kDa and 48-kDa proteins but not to the 33-kDa protein. These results strongly suggest that M-H1K consists of cdc2 kinase forming a complex with cyclin B, and that cdk2 kinase is not a component of M-H1K, although it is found in the highly purified M-H1K. The purified M-H1K utilized Mg2+, Mn2+, ATP and GTP, and had a wide pH optimum ranging over 8.0-10.5. The kinase was thermolabile and sensitive to freezing/thawing.

Cold-sensitive mutants of p34cdc2 that suppress a mitotic catastrophe phenotype in fission yeast.

The p34cdc2 protein kinase plays a central role in the regulation of the eukaryotic cell cycle, being required both in late G1 for the commitment to S-phase and in late G2 for the initiation of mitosis. p34cdc2 also determines the precise timing of entry into mitosis in fission yeast, where a number of gene products that regulate p34cdc2 activity have been identified and characterised. To investigate further the mitotic role of p34cdc2 in this organism we have isolated new cold-sensitive p34cdc2 mutants. These are defective only in their G2 function and are extragenic suppressors of the lethal premature entry into mitosis brought about by mutating the mitotic inhibitor p107wee1 and overproducing the mitotic activator p80cdc25. One of the mutant proteins p34cdc2-E8 is only functional in the absence of p107wee1, and all the mutant strains have reduced histone H1 kinase activity in vitro. Each mutant allele has been cloned and sequenced, and the lesions responsible for the cold-sensitive phenotypes identified. All the mutations were found to map to regions that are conserved between the fission yeast p34cdc2 and functional homologues from higher eukaryotes.

MPM-12: a monoclonal antibody that predominantly stains mitotic cells and recognizes a protein kinase.

The monoclonal antibody MPM-12, raised by using partially purified extract of mitotic HeLa cells as the immunogen, preferentially stains the cytoplasm of mitotic cells by indirect immunofluorescence without exhibiting any species specificity. On immunoblots, MPM-12 recognizes three bands, of 155, 88, and 68 kDa, in mitotic HeLa cell extract but only the 68-kDa band in interphase cell extract. The 68-kDa band seems to be associated with chromatin while the other two are not. All three MPM-12 reactive peptides are phosphorylated, and the phosphorylation seems to be required for MPM-12 reactivity. The MPM-12 immunocomplexes exhibit autophosphorylating and histone H1 kinase activity.

Multiple forms of maturation-promoting factor in unfertilized Xenopus eggs.

Maturation-promoting factor (MPF), which is functionally defined by its ability to induce frog oocyte maturation independent of protein synthesis, is hypothesized to be the mitotic inducer in eukaryotic cells. Previous studies have demonstrated that the cdc2 protein kinase complex (p34cdc2-cyclin) meets the criteria for MPF. In the present study, we show that MPF activity in extracts of unfertilized Xenopus eggs can be resolved into three fractions by Q-Sepharose chromatography. Of the total MPF activity recovered, approximately 20% was in the flow-through fraction that was accounted for by the cdc2 kinase complex, approximately 40% was in the 0.2 M NaCl eluate, and the remaining approximately 40% was in the 0.5 M NaCl eluate. Neither eluate contained cdc2 kinase, but each could activate cdc2 kinase upon microinjection into Xenopus oocytes. The MPF activity in the two eluates, but not in the flow-through fraction, could be depleted by the mitosis-specific monoclonal antibody MPM-2. This antibody has been shown to inhibit Xenopus oocyte maturation and deplete MPF activity from mature oocyte extract but does not recognize the cdc2 kinase complex. The three MPFs differed in apparent molecular size, H1 kinase activity, and stability at 4 degrees C. We propose that MPF activity in unfertilized Xenopus eggs resides in at least three different molecular species, the combined activities of which may be required for autoamplification of MPF.