High tyrosine-specific protein kinase activity in normal human peripheral blood lymphocytes

Tyrosine-specific protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) activity was measured in normal human nonadherent peripheral blood lymphocytes using synthetic peptide substrates having sequence homologies with either pp60^src or c-myc. A high level of tyrosine-specific protein kinase activity was found associated with the cell particulate fraction (100 000 × g pellet). High-pressure liquid chromatography and phosphoamino acid analysis of the synthetic peptide substrates substantiated the phosphorylation of tyrosine residues by the particulate fraction enzyme. The human enzyme was also capable of phosphorylating a synthetic random polymer of 80% glutamic acid and 20% tyrosine. Enzyme activity was half-maximal with 22 μM Mg·ATP and had apparent K_m values for the synthetic peptides from 1.9 to 7.1 mM. The enzyme preferred Mg²⁺ to Mn²⁺ for optimal activity and was stimulated 2–5-fold by low levels (0.05%) of some ionic as well as non-ionic detergents including deoxycholate, Nonidet P-40 and Triton X-100. The enzyme activity was not stimulated by N⁶;O^2′-dibutyryl cyclic AMP (100 μM), N⁶;O^2′-dibutyryl cyclic GMP (100 μM), Ca²⁺ (200 μM), insulin (1 μg/ml) or homogeneous human T-cell growth factor (3 μg/ml) under the conditions used. Alkaline-resistant phosphorylation of particulate proteins in vitro revealed protein bands with M_r 59 000 and 54 000 suggesting that there are endogenous substrates for the human lymphocyte tyrosine protein kinase.     

Acute cytotoxicity of arabinofuranosyl nucleoside analogs is not dependent on mitochondrial DNA

The nucleoside analogs 9-β-D-arabinofuranosylguanine (araG) and 1-β-d-arabinofuranosylthymine (araT) are substrates of mitochondrial nucleoside kinases and have previously been shown to be predominantly incorporated into mtDNA of cells, but the pharmacological importance of their accumulation in mtDNA is not known. Here, we examined the role of mtDNA in the response to araG, araT and other anti-cancer and anti-viral agents in a MOLT-4 wild-type (wt) T-lymphoblastoid cell line and its petite mutant MOLT-4 ρ⁰ cells (lacking mtDNA). The mRNA levels and activities of deoxyguanosine kinase (dGK), deoxycytidine kinase (dCK), thymidine kinase 1 (TK1) and thymidine kinase 2 (TK2) were determined in the two cell lines. Compared to that in the MOLT-4 wt cells the mRNA level of the constitutively expressed TK2 was higher (p < 0.01) in the ρ⁰ cells, whereas the TK1 mRNA level was lower (p < 0.05). The enzyme activity of the S-phase restricted TK1 was also lower (p < 0.05) in the MOLT-4 ρ⁰ cells, whereas the activities of dGK, dCK and TK2 were similar in MOLT-4 wt and ρ⁰ cell lines. The sensitivities to different cytotoxic nucleoside analogs were determined and compared between the two cell lines. Interestingly, we found that the acute cytotoxicity of araG, araT and other anti-viral and anti-cancer agents is independent of the presence of mtDNA in MOLT-4 T-lymphoblastoid cells.     

An expanded adenylate kinase gene family in the protozoan parasite Trypanosoma cruzi

Adenylate kinases supply energy routes in cellular energetic homeostasis. In this work, we identified and characterized the adenylate kinase activity in extracts from the flagellated parasite Trypanosoma cruzi, the causative agent of Chagas' disease. Adenylate kinase activity was detected in different subcellular fractions and the cytosolic isoform was biochemically characterized. Cytosolic adenylate kinase specific activity increases continuously during the epimastigote growth and is down-regulated when other soluble phosphotransferase, arginine kinase, is overexpressed. Six different genes of adenylate kinase isoforms were identified and the mRNA expression was confirmed by RT-PCR and Northern Blot. Three open reading frames coding for different enzyme isoforms named TzADK1, TzADK2 and TzADK5 were cloned and functionally expressed in E. coli. This work reports an unusually large number of genes of adenylate kinases and suggests a coordinated regulation of phosphotransferase-mediated ATP regenerating pathways in the unicellular parasite Trypanosoma cruzi.     

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.     

Thymidine phosphorylating enzymes in the gonads of marine invertebrates

The activities of enzymes involved in the consecutive phosphorylation of thymidine were revealed in the gonad extracts of marine invertebrates. Along with thymidine kinase activity, thymidilate kinase activity was revealed in all the studied species; however, the specific activities of nucleoside and nucleotide kinases varied in different species of mollusks, sea stars and sea urchins. Thymidine and thymidilate kinases were isolated from the gonads of the scallop Mizuhopecten yessoensis and some of their enzymat properties were studied. The thymidine kinase of M. yessoensis catalyzed the phosphorylation of thymidine and deoxycytidine at a lesser rate, but didn’s use purine ribo-and deoxyribonucleosides or pyrimidine ribonucleosides as phosphate acceptors. The thymidilate kinase carried out both TMP and dCMP phosphorylation. As well as ATP, the enzymes of M. yessoensis were also able to use dATP, dGTP, GTP, UTP and CTP as donors of phosphate groups. The thymidine kinase activity was inhibited by TMP, TTP and dCTP.     

Substrate specificity of vaccinia virus thymidylate kinase

Anti-poxvirus therapies are currently limited to cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine], but drug-resistant strains have already been characterized. In the aim of finding a new target, the thymidylate (TMP) kinase from vaccinia virus, the prototype of Orthopoxvirus, has been overexpressed in Escherichia coli after cloning the gene (A48R). Specific inhibitors and alternative substrates of pox TMP kinase should contribute to virus replication inhibition. Biochemical characterization of the enzyme revealed distinct catalytic features when compared to its human counterpart. Sharing 42% identity with human TMP kinase, the vaccinia virus enzyme was assumed to adopt the common fold of nucleoside monophosphate kinases. The enzyme was purified to homogeneity and behaves as a homodimer, like all known TMP kinases. Initial velocity studies showed that the Km for ATP-Mg2+ and dTMP were 0.15 mm and 20 microM, respectively. Vaccinia virus TMP kinase was found to phosphorylate dTMP, dUMP and also dGMP from any purine and pyrimidine nucleoside triphosphate. 5-Halogenated dUMP such as 5-iodo-2'-deoxyuridine 5'-monophosphate (5I-dUMP) and 5-bromo-2'-deoxyuridine 5'-monophosphate (5Br-dUMP) were also efficient alternative substrates. Using thymidine-5'-(4-N'-methylanthraniloyl-aminobutyl)phosphoramidate as a fluorescent probe of the dTMP binding site, we detected an ADP-induced conformational change enhancing the binding affinity of dTMP and analogues. Several thymidine and dTMP derivatives were found to bind the enzyme with micromolar affinities. The present study provides the basis for the design of specific inhibitors or substrates for poxvirus TMP kinase.     

Molecular characterization, heterologous expression and kinetic analysis of recombinant Plasmodium falciparum thymidylate kinase

The gene encoding for thymidylate kinase from Plasmodium falciparum was obtained by PCR and expressed in Escherichia coli and the enzyme was investigated as a possible new drug target. The enzyme is a homodimer exhibiting maximal kinase activity over a wide pH range of 7-9 and is characterized by marked stability. Compared with the human enzyme, the recombinant P. falciparum TMP kinase showed a broader spectrum of substrate specificity. The enzyme not only phosphorylates dTMP and dUMP but can also tolerate the bulkier purines dGMP, GMP and dIMP. Initial velocity studies showed that the Km values for TMP and dGMP are 22 and 30 microM, respectively. The turnover number kcat(TMP) was found to be 3.4 s(-1), a value indicating the higher catalytic efficiency of the plasmodium enzyme. From the present study, we suggest that the design of appropriate inhibitors especially purine based compounds could have a selective inhibitory effect on the parasite enzyme.     

Synthesis and Enantioselectivity of Cyclopropavir Phosphates for Cellular GMP Kinase

Enantiomeric cyclopropavir phosphates (+)-9 and (?)-9 were synthesized and investigated as substrates for GMP kinase. N²-Isobutyryl-di-O-acetylcyclopropavir (11) was converted to (+)-monoacetate 12 using hydrolysis catalyzed by porcine liver esterase. Phosphorylation via phosphite 13 gave after deacylation, phosphate (+)-9. Acid-catalyzed tetrahydropyranylation of (+)-monoacetate 12 gave, after deacylation, tetrahydropyranyl derivative 14. Phosphorylation via phosphite 15 furnished, after deprotection, enantiomeric phosphate (-)-9. Racemic diphosphate 16 was also synthesized. The phosphate (+)-9 is a relatively good substrate for GMP kinase with a K_M value of 57 μM that is similar to that of the natural substrates GMP (61 μM) and dGMP (82 μM). In contrast, the enantiomer (?)-9 is not a good substrate (K_M 1200 μM) indicating a significant enantioselectivity for the GMP kinase catalyzed reaction of monophosphate to diphosphate.     

Adrenocortical cyclic GMP-dependent protein kinase: purification, characterization, and modification of its activity by calmodulin, and its relationship with steroidogenesis

Cyclic GMP-dependent protein kinase has been purified to apparent homogeneity from bovine adrenal cortex and its presence in the rat adrenal cortex has been demonstrated. Sucrose density sedimentation studies indicated that the M_r of the enzyme was 145,000. This protein was composed to two identical subunits each with M_r of 75,000. The enzyme molecule was asymmetric with a frictional coefficient of 1.54, Stokes radius of 53.5 Å and a sedimentation coefficient of 6.5. The enzyme self-phosphorylated and the stoichiometry of cyclic GMP binding was two molecules per holoenzyme. Calmodulin or troponin C markedly stimulated the apparent maximal velocity of cyclic GMP-dependent protein kinase without affecting its basal activity. This effect of protein modulators was independent of calcium. Sucrose density gradient studies indicated that the stimulatory effect of calmodulin was due to its interaction with histones. An interaction of calmodulin with the enzyme was not observed. The steroidogenic potential of cyclic GMP and its analogs correlated closely with their ability to stimulate cyclic GMP-dependent protein kinase; the order of potency for both activities was 8-bromocylic GMP > cyclic GMP > N²-monobutyryl cyclic GMP > N², O²-dibutyryl cyclic GMP. In each case, calmodulin enhanced the cyclic GMP-dependent protein kinase activity for histone phosphorylation. These results indicate that although cyclic GMP is the primary regulator of cyclic GMP-dependent protein kinase, other modulator proteins such as calmodulin could act as additional regulators of the phosphorylation of substrate proteins. In addition, the demonstration of cyclic GMP-dependent protein kinase in rat adrenal glands, and the results with cyclic GMP and its analogs relating to their activation of protein kinase and steroidogenesis are consistant with the concept that cyclic GMP is one of the mediators of adrenal steroidogenesis.     

An ATP analog-sensitive version of the tomato cell death suppressor protein kinase Adi3 for use in substrate identification

Adi3 is a protein kinase from tomato that functions as a cell death suppressor and its substrates are not well defined. As a step toward identifying Adi3 substrates we developed an ATP analog-sensitive version of Adi3 in which the ATP-binding pocket is mutated to allow use of bulky ATP analogs. Met385 was identified as the “gatekeeper” residue and the M385G mutation allows for the use of two bulky ATP analogs. Adi3^M385G can also specifically utilize N⁶-benzyl-ATP to phosphorylate a known substrate and provides a tool for identifying Adi3 substrates.     

N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist,also inhibits phospholipid sensitive calcium-dependent protein kinase

N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), commonly regared as a calmodulin antagonist, inhibted phospholipid-sensitive Ca²⁺-dependent protein kinase and to a lesser extent cyclic GMP- and cyclic AMP-dependent protein kinases. Kinetic studies of the inhibition of the homogenous spleen phospholipid-sensitive Ca²⁺-dependent protein kinase indicated that W-7 inhibited the enzyme activity competitively with respect to phospholipid (K_i = 60 μM). N-(6-Aminohexyl)-1-naphthalenesulfonamide (W-5) was found to be musch less potent than W-7. The findings indicate that W-6 was able to inhibit a variety of protein kinases, in addition to those requiring calmodulin previously reported.     

A human kinase yeast array for the identification of kinases modulating phosphorylation‐dependent protein–protein interactions

Protein kinases play an important role in cellular signaling pathways and their dysregulation leads to multiple diseases, making kinases prime drug targets. While more than 500 human protein kinases are known to collectively mediate phosphorylation of over 290,000 S/T/Y sites, the activities have been characterized only for a minor, intensively studied subset. To systematically address this discrepancy, we developed a human kinase array in Saccharomyces cerevisiae as a simple readout tool to systematically assess kinase activities. For this array, we expressed 266 human kinases in four different S. cerevisiae strains and profiled ectopic growth as a proxy for kinase activity across 33 conditions. More than half of the kinases showed an activity‐dependent phenotype across many conditions and in more than one strain. We then employed the kinase array to identify the kinase(s) that can modulate protein–protein interactions (PPIs). Two characterized, phosphorylation‐dependent PPIs with unknown kinase–substrate relationships were analyzed in a phospho‐yeast two‐hybrid assay. CK2α1 and SGK2 kinases can abrogate the interaction between the spliceosomal proteins AAR2 and PRPF8, and NEK6 kinase was found to mediate the estrogen receptor (ERα) interaction with 14‐3‐3 proteins. The human kinase yeast array can thus be used for a variety of kinase activity‐dependent readouts.     

Slow Myosin ATP Turnover in the Super-Relaxed State in Tarantula Muscle

We measured the nucleotide turnover rate of myosin in tarantula leg muscle fibers by observing single turnovers of the fluorescent nucleotide analog 2′-/3′-O-(N′-methylanthraniloyl)adenosine-5′-O-triphosphate, as monitored by the decrease in fluorescence when 2′-/3′-O-(N′-methylanthraniloyl)adenosine-5′-O-triphosphate (mantATP) is replaced by ATP in a chase experiment. We find a multiexponential process with approximately two-thirds of the myosin showing a very slow nucleotide turnover time constant (∼ 30 min). This slow-turnover state is termed the super-relaxed state (SRX). If fibers are incubated in 2′-/3′-O-(N′-methylanthraniloyl)adenosine-5′-O-diphosphate and chased with ADP, the SRX is not seen, indicating that trinucleotide-relaxed myosins are responsible for the SRX. Phosphorylation of the myosin regulatory light chain eliminates the fraction of myosin with a very long lifetime. The data imply that the very long-lived SRX in tarantula fibers is a highly novel adaptation for energy conservation in an animal that spends extremely long periods of time in a quiescent state employing a lie-in-wait hunting strategy. The presence of the SRX measured here correlates well with the binding of myosin heads to the core of the thick filament in a structure known as the “interacting-heads motif,” observed previously by electron microscopy. Both the structural array and the long-lived SRX require relaxed filaments or relaxed fibers, both are lost upon myosin phosphorylation, and both appear to be more stable in tarantula than in vertebrate skeletal or vertebrate cardiac preparations.     

The phosphorylation site of Ca2+-dependent protein kinase from alfalfa

A 50 kDa, calcium-dependent protein kinase (CDPK) was purified about 1000-fold from cultured cells of alfalfa (Medicago varia) on the basis of its histone H1 phosphorylation activity. The major polypeptide from bovine histone H1 phosphorylated by either animal protein kinase C (PK-C) or by the alfalfa CDPK gave an identical phosphopeptide pattern. The phosphoamino acid determination showed phosphorylation of serine residues in histone H1 by the plant enzyme. Histone-related oligopeptides known to be substrates for animal histone kinases also served as substrates for the alfalfa kinase. Both of the studied peptides (GKKRKRSRKA; AAASFKAKK) inhibited phosphorylation of H1 histones by bovine and alfalfa kinases. The results of competition studies with the nonapeptide (AAASFKAKK), which is a PK-C specific substrate, suggest common features in target recognition between the plant Ca²⁺-dependent kinase and animal protein kinase C. We also propose that synthetic peptides like AAASFKAKK can be used as a tool to study substrates of plant kinases in crude cell extracts.     

Tyrosine kinase activity of a Ca2+/calmodulin-dependent protein kinase II catalytic fragment

A 30-kDa fragment of Ca²⁺/calmodulin-dependent protein kinase II (30K-CaMKII) is a constitutively active protein Ser/Thr kinase devoid of autophosphorylation activity. We have produced a chimeric enzyme of 30K-CaMKII (designated CX₄₀-30K-CaMKII), in which the N-terminal 40 amino acids of Xenopus Ca²⁺/calmodulin-dependent protein kinase I (CX₄₀) were fused to the N-terminal end of 30K-CaMKII. Although CX₄₀-30K-CaMKII exhibited essentially the same substrate specificity as 30K-CaMKII, it underwent significant autophosphorylation. Surprisingly, its autophosphorylation site was found to be Tyr-18 within the N-terminal CX₄₀ region of the fusion protein, although it did not show any Tyr kinase activity toward exogenous substrates. Several lines of evidence suggested that the autophosphorylation occurred via an intramolecular mechanism. These data suggest that even typical Ser/Thr kinases such as 30K-CaMKII can phosphorylate Tyr residues under certain conditions. The possible mechanism of the Tyr residue autophosphorylation is discussed.     

The mouse guanylate kinase double mutant E72Q/D103N is a functional adenylate kinase.

Guanylate kinase catalyzes the phosphorylation of either GMP to GDP or dGMP to dGDP and is an important enzyme in nucleotide metabolic pathways. Because of its essential intracellular role, guanylate kinase is a target for a number of cancer chemotherapeutic agents such as 6-thioguanine and 8-azaguanine and is involved in antiviral drug activation. Guanylate kinase shares a similarity in function and structure to other nucleoside monophosphate kinases especially with that of the well-studied adenylate kinase. Amino acid substitutions were made within the GMP binding site of mouse guanylate kinase to alter the polarity of the side chains that interact with GMP as a means of evaluating the role that these residues play on substrate interaction. One of these mutants, E72Q/D103N, was shown by functional complementation and enzyme assays to embody both guanylate kinase activity and a novel adenylate kinase activity.     

Identification and characterization of bi-thiazole-2,2′-diamines as kinase inhibitory scaffolds

Based on bioinformatics interrogation of the genome, > 500 mammalian protein kinases can be clustered within seven different groups. Of these kinases, the mitogen-activated protein kinase (MAPK) family forms part of the CMGC group of serine/threonine kinases that includes extracellular signal regulated kinases (ERKs), cJun N-terminal kinases (JNKs), and p38 MAPKs. With the JNKs considered attractive targets in the treatment of pathologies including diabetes and stroke, efforts have been directed to the discovery of new JNK inhibitory molecules that can be further developed as new therapeutics. Capitalizing on our biochemical understanding of JNK, we performed in silico screens of commercially available chemical databases to identify JNK1-interacting compounds and tested their in vitro JNK inhibitory activity. With in vitro and cell culture studies, we showed that the compound, 4′-methyl-N²-3-pyridinyl-4,5′-bi-1,3-thiazole-2,2′-diamine (JNK Docking (JD) compound 123, but not the related compound (4′-methyl-N ~ 2 ~ -(6-methyl-2-pyridinyl)-4,5′-bi-1,3-thiazole-2,2′-diamine (JD124), inhibited JNK1 activity towards a range of substrates. Molecular docking, saturation transfer difference NMR experiments and enzyme kinetic analyses revealed both ATP- and substrate-competitive inhibition of JNK by JD123. In characterizing JD123 further, we noted its ATP-competitive inhibition of the related p38-γ MAPK, but not ERK1, ERK2, or p38-α, p38-β or p38-δ. Further screening of a broad panel of kinases using 10 μM JD123, identified inhibition of kinases including protein kinase Bβ (PKBβ/Aktβ). Appropriately modified thiazole diamines, as typified by JD123, thus provide a new chemical scaffold for development of inhibitors for the JNK and p38-γ MAPKs as well as other kinases that are also potential therapeutic targets such as PKBβ/Aktβ.     

Novel compounds, ‘1,3-selenazine derivatives’ as specific inhibitors of eukaryotic elongation factor-2 kinase

The inhibitory activities of 5,6-dihydro-4H-1,3-selenazine derivatives on protein kinases were investigated. In a multiple protein kinase assay using a postnuclear fraction of v-src-transformed NIH3T3 cells, 4-ethyl-4-hydroxy-2-p-tolyl-5,6-dihydro-4H-1,3-selenazine (TS-2) and 4-hydroxy-6-isopropyl-4-methyl-2-p-tolyl-5,6-dihydro-4H-1,3-selenazine (TS-4) exhibited selective inhibitory activity against eukaryotic elongation factor-2 kinase (eEF-2K) over protein kinase A (PKA), protein kinase C (PKC) and protein tyrosine kinase (PTK). In further experiments using purified kinases, TS-2 (IC₅₀=0.36 μM) and TS-4 (IC₅₀=0.31 μM) inhibited eEF-2K about 25-fold more effectively than calmodulin-dependent protein kinase-I (CaMK-I), and about 6-fold (TS-2) or 33-fold (TS-4) more effectively than calmodulin-dependent protein kinase-II (CaMK-II), respectively. TS-2 and TS-4 showed much weaker inhibitory activity toward PKA and PKC, while TS-4, but not TS-2, moderately inhibited immunoprecipitated v-src kinase. TS-2 (10.7-fold) and TS-4 (12.5-fold) demonstrated more potent and more specific eEF-2K inhibitory activity than rottlerin, a previously identified eEF-2K inhibitor. TS-2 inhibited ATP or eEF-2 binding to eEF-2K in a competitive or non-competitive manner, respectively. In cultured v-src-transformed NIH3T3 cells, TS-2 also decreased phospho-eEF-2 protein level (IC₅₀=4.7 μM) without changing the total eEF-2 protein level. Taken together, these results suggest that TS-2 and TS-4 are the first identified selective eEF-2K inhibitors and should be useful tools for studying the function of eEF-2K.     

Novel Ca/calmodulin-dependent protein kinase expressed in actively growing mycelia of the basidiomycetous mushroom Coprinus cinereus

We isolated cDNA clones for novel protein kinases by expression screening of a cDNA library from the basidiomycetous mushroom Coprinus cinereus. One of the isolated clones was found to encode a calmodulin (CaM)-binding protein consisting of 488 amino acid residues with a predicted molecular weight of 53,906, which we designated CoPK12. The amino acid sequence of the catalytic domain of CoPK12 showed 46% identity with those of rat Ca²⁺/CaM-dependent protein kinase (CaMK) I and CaMKIV. However, a striking difference between these kinases is that the critical Thr residue in the activating phosphorylation site of CaMKI/IV is replaced by a Glu residue at the identical position in CoPK12. As predicted from its primary sequence, CoPK12 was found to behave like an activated form of CaMKI phosphorylated by an upstream CaMK kinase, indicating that CoPK12 is a unique CaMK with different properties from those of the well-characterized CaMKI, II, and IV. CoPK12 was abundantly expressed in actively growing mycelia and phosphorylated various proteins, including endogenous substrates, in the presence of Ca²⁺/CaM. Treatment of mycelia of C. cinereus with KN-93, which was found to inhibit CoPK12, resulted in a significant reduction in growth rate of mycelia. These results suggest that CoPK12 is a new type of multifunctional CaMK expressed in C. cinereus, and that it may play an important role in the mycelial growth.     

Identification and activation of mitogen-activated protein (MAP) kinase in normal human osteoblastic and bone marrow stromal cells: Attenuation of MAP kinase activation by cAMP,parathyroid hormone and forskolin

The mitogen-activated protein (MAP) kinases (p44^mapk and p42^mapk), also known as extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), are activated in response to a variety of extracellular signals, including growth factors, hormones and, neurotransmitters. We have investigated MAP kinase signal transduction pathways in normal human osteoblastic cells. Normal human bone marrow stromal (HBMS), osteoblastic (HOB), and human (TE85, MG-63, SaOS-2), rat (ROS 17/2.8, UMR-106) and mouse (MC3T3-E1) osteoblastic cell lines contained immunodetectable p44^mapk/ERK1 and p42^mapk/ERK2. MAP kinase activity was measured by 'in-gel' assay myelin basic protein as the substrate. Mainly ERK2 was rapidly activated (within 10 min) by bFGF, IGF-I and PDGF-BB in normal HOB, HBMS and human osteosarcoma cells, whereas both ERK1 and ERK2 were activated by growth factors in rat osteoblast-like cell lines, ROS 17/2.8 and UMR-106. The ERK1 activation was greater than the ERK2 in ROS 17/2.8 cells. Furthermore, ERK2 was also activated by bFGF and PDGF-BB in the mouse osteoblastic cell line, MC3T3-E1. This is the first demonstration of inter-species differences in the activation of MAP kinases in osteoblastic cells. Cyclic AMP derivatives or cAMP generating agents such as PTH and forskolin inhibited ERK2 activation by bFGF and PDGF-BB suggesting a 'cross-talk' between the two different signalling pathways activated by receptor tyrosine kinases and cAMP-dependent protein kinase. The accumulated results also suggest that the MAP kinases may be involved in mediating mitogenic and other biological actions of bFGF, IGF-I and PDGF-BB in normal human osteoblastic and bone marrow stromal cells.