Phosphorylation of the human leukemia inhibitory factor (LIF) receptor by mitogen-activated protein kinase and the regulation of LIF receptor function by heterologous receptor activation.

We used a bacterially expressed fusion protein containing the entire cytoplasmic domain of the human leukemia inhibitory factor (LIF) receptor to study its phosphorylation in response to LIF stimulation. The dose- and time-dependent relationships for phosphorylation of this construct in extracts of LIF-stimulated 3T3-L1 cells were superimposable with those for the stimulation of mitogen-activated protein kinase (MAPK). Indeed, phosphorylation of the cytoplasmic domain of the low-affinity LIF receptor alpha-subunit (LIFR) in Mono Q-fractionated, LIF-stimulated 3T3-L1 extracts occurred only in those fractions containing activated MAPK; Ser-1044 served as the major phosphorylation site in the human LIFR for MAPK both in agonist-stimulated 3T3-L1 lysates and by recombinant extracellular signal-regulated kinase 2 in vitro. Expression in rat H-35 hepatoma cells of LIFR or chimeric granulocyte-colony-stimulating factor receptor (G-CSFR)-LIFR mutants lacking Ser-1044 failed to affect cytokine-stimulated expression of a reporter gene under the control of the beta-fibrinogen gene promoter but eliminated the insulin-induced attenuation of cytokine-stimulated gene expression. Thus, our results identify the human LIFR as a substrate for MAPK and suggest a mechanism of heterologous receptor regulation of LIFR signaling occurring at Ser-1044.

Full-length myotonin protein kinase (72 kDa) displays serine kinase activity.

We describe the full-length (72 kDa) myotonin protein kinase (Mt-PK) and demonstrate its kinase activity. The 72-kDa protein corresponds to the translation product from the first in-frame AUG codon. This protein was found in the cytoplasmic fraction, whereas the previously reported 55-kDa protein was observed in nuclear extracts. Only the 72-kDa protein was phosphorylated by [32P]phosphate in normal human fibroblasts. To investigate the putative kinase activity of Mt-PK, a construct containing the full-length open reading frame of Mt-PK was expressed in bacterial cells. The recombinant Mt-PK autophosphorylates a Ser residue and phosphorylates the synthetic peptide Gly-Arg-Gly-Leu-Ser-Leu-Ser-Arg, which contains a Ser residue in the phosphorylation site. We examined phosphorylation of the voltage-dependent Ca(2+)-release channel, or dihydropyridine receptor (DHPR), by recombinant Mt-PK. We observed that the beta subunit of DHPR was phosphorylated in vitro by Mt-PK. A beta-subunit DHPR peptide containing some of the Ser residues predicted to be phosphorylated was synthesized and found to be a substrate for Mt-PK in vitro. We conclude that the 72-kDa Mt-PK has a protein kinase activity specific for Ser residues.

Chimeric plant calcium/calmodulin-dependent protein kinase gene with a neural visinin-like calcium-binding domain.

Calcium, a universal second messenger, regulates diverse cellular processes in eukaryotes. Ca2+ and Ca2+/calmodulin-regulated protein phosphorylation play a pivotal role in amplifying and diversifying the action of Ca(2+)-binding domain was cloned and characterized from lily. The cDNA clone contains an open reading frame coding for a protein of 520 amino acids. The predicted structure of CCaMK contains a catalytic domain followed by two regulatory domains, a calmodulin-binding domain and a visinin-like Ca(2+)-binding domain. The amino-terminal region of CCaMK contains all 11 conserved subdomains characteristic of serine/threonine protein kinases. The calmodulin-binding region of CCaMK has high homology (79%) to alpha subunit of mammalian Ca2+/calmodulin-dependent protein kinase. The calmodulin-binding region is fused to a neural visinin-like domain that contains three Ca(2+)-binding EF-hand motifs and a biotin-binding site. The Escherichia coli-expressed protein (approximately 56 kDa) binds calmodulin in a Ca(2+)-dependent manner. Furthermore, 45Ca-binding assays revealed that CCaMK directly binds Ca2+. The CCaMK gene is preferentially expressed in developing anthers. Southern blot analysis revealed that CCaMK is encoded by a single gene. The structural features of the gene suggest that it has multiple regulatory controls and could play a unique role in Ca2+ signaling in plants.

Molecular cloning and characterization of a conserved nuclear serine(threonine) protein kinase.

Human, Drosophila melanogaster, and Caenorhabditis elegans cDNA clones encoding homologues of a serine(threonine) protein kinase (EC 2.7.1.37) (designated Ndr protein kinase) have been isolated and sequenced. The human and Drosophila cDNAs predict polypeptides of 54 kDa and 52 kDa, respectively, which share approximately 80% amino acid similarity. Northern analysis of human tissues revealed a ubiquitously expressed 3.9-kb transcript. Recombinant GST-Ndr underwent intramolecular autophosphorylation on serine and threonine residues in vitro but failed to transphosphorylate several standard protein kinase substrates. Transfection of the human cDNA into COS-1 cells resulted in the appearance of an intense nuclear staining in cells analyzed by indirect immunofluorescence; deletion mutagenesis identified a short basic peptide, KRKAETWKRNRR, responsible for the nuclear accumulation of Ndr. Thus, Ndr is a conserved and widely expressed nuclear protein kinase. The closest known relative of this previously uncharacterized kinase is Dbf2, a budding yeast protein kinase required for the completion of nuclear division.

Isolation of a yeast protein kinase that is activated by the protein encoded by SRP1 (Srp1p) and phosphorylates Srp1p complexed with nuclear localization signal peptides.

Srp1p, the protein encoded by SRP1 of Saccharomyces cerevisiae, is a nuclear-pore-associated protein. Its Xenopus homolog, importin, was recently shown to be an essential component required for nuclear localization signal (NLS)-dependent binding of karyophilic proteins to the nuclear envelope [Gorlich, D., Prehn, S., Laskey, R. A. & Hartman, E. (1994) Cell 79, 767-778]. We have discovered a protein kinase whose activity is stimulated by Srp1p (Srp1p fused to glutathione S-transferase and expressed in Escherichia coli) and is detected by phosphorylation of Srp1p and of a 36-kDa protein, a component of the protein kinase complex. The enzyme, called Srp1p kinase, is a protein-serine kinase and was found in extracts in two related complexes of approximately 180 kDa and 220 kDa. The second complex, when purified, contained four protein components including the 36-kDa protein. We observed that, upon purification of the kinase, phosphorylation of Srp1p became very weak, while activation of phosphorylation of the 36-kDa protein by Srp1p remained unaltered. Significantly, NLS peptides and the nuclear proteins we have tested greatly stimulated phosphorylation of Srp1p, suggesting that Srp1p, complexed with karyophilic proteins carrying an NLS, is the in vivo substrate of this protein kinase.

Cellular protein kinase C isoform zeta regulates human parainfluenza virus type 3 replication.

Phosphorylation of the P proteins of nonsegmented negative-strand RNA viruses is critical for their function as transactivators of the viral RNA polymerases. Using unphosphorylated P protein of human parainfluenza virus type 3 (HPIV3) expressed in Escherichia coli, we have shown that the cellular protein kinase that phosphorylates P in vitro is biochemically and immunologically indistinguishable from cellular protein kinase C isoform zeta (PKC-zeta). Further, PKC-zeta is specifically packaged within the progeny HPIV3 virions and remains tightly associated with the ribonucleoprotein complex. The P protein seems also to be phosphorylated intracellularly by PKC-zeta, as shown by the similar protease digestion pattern of the in vitro and in vivo phosphorylated P proteins. The growth of HPIV3 in CV-1 cells is completely abrogated when a PKC-zeta-specific inhibitor pseudosubstrate peptide was delivered into cells. These data indicate that PKC-zeta plays an important role in HPIV3 gene expression by phosphorylating P protein, thus providing an opportunity to develop antiviral agents against an important human pathogen.

Evidence for a role of endothelin 1 and protein kinase C in nitroglycerin tolerance.

We sought to examine mechanisms responsible for increased vasoconstriction that occurs during development of nitroglycerin tolerance. Rabbits were treated for 3 days with nitroglycerin patches (0.4 mg/hr), and their aortic segments were studied in organ chambers. This treatment resulted in attenuated in vitro relaxations to nitroglycerin and increased contractile sensitivity to angiotensin II, serotonin, phenylephrine, KCl, and a direct activator of protein kinase C, the phorbol ester phorbol 12,13-dibutyrate. The protein kinase C antagonists calphostin C (100 nM) and staurosporine (10 nM) corrected the hypersensitivity to constrictors in tolerant vessels, yet had minimal effects on constrictions in control vessels. Paradoxically, constrictions caused by endothelin 1 were decreased in nitrate-tolerant vessels. Immunocytochemical analysis revealed intense endothelin 1-like and big endothelin 1-like immunoreactivity in the media of nitroglycerin-tolerant but not of control aortas. The enhanced vasoconstriction to angiotensin II, serotonin, KCl, and phenylephrine could be mimicked in normal vessels by addition of subthreshold concentrations of endothelin 1, and this effect was prevented by calphostin C. We propose that increased autocrine production of endothelin 1 in nitrate tolerance sensitizes vascular smooth muscle to a variety of vasoconstrictors through a protein kinase C-mediated mechanism.

Activation of the Lck tyrosine protein kinase by hydrogen peroxide requires the phosphorylation of Tyr-394.

Exposure of cells to H2O2 mimics many of the effects of treatment of cells with extracellular ligands. Among these is the stimulation of tyrosine phosphorylation. In this study, we show that exposure of cells to H2O2 increases the catalytic activity of the lymphocyte-specific tyrosine protein kinase p56lck (Lck) and induces tyrosine phosphorylation of Lck at Tyr-394, the autophosphorylation site. Using mutant forms of Lck, we found that Tyr-394 is required for H2O2-induced activation of Lck, suggesting that phosphorylation of this site may activate Lck. In addition, H2O2 treatment induced phosphorylation at Tyr-394 in a catalytically inactive mutant of Lck in cells that do not express endogenous Lck. This demonstrates that a kinase other than Lck itself is capable of phosphorylating Lck at the so-called autophosphorylation site and raises the possibility that this as yet unidentified tyrosine protein kinase functions as an activator of Lck. Such an activating enzyme could play an important role in signal transduction in T cells.

Cdc37 is required for association of the protein kinase Cdc28 with G1 and mitotic cyclins.

Studies of the temperature-sensitive cdc37-1 mutant of Saccharomyces cerevisiae suggest that Cdc37 is required for passage through the G1 phase of the cell cycle, but its precise function is not known. We have investigated the role of Cdc37 in the regulation of the cyclin-dependent protein kinase Cdc28. We find that G1 arrest in the cdc37-1 mutant is accompanied by a decrease in the Cdc28 activity associated with the G1 cyclin Cln2. This defect appears to be caused by a decrease in the binding of Cdc28 and Cln2. cdc37-1 mutants also exhibit a defect in the binding and activation of Cdc28 by the mitotic cyclin Clb2. Thus Cdc37 may be a regulator that is required for the association of Cdc28 with multiple cyclins.

Mutant mice lacking the gamma isoform of protein kinase C show decreased behavioral actions of ethanol and altered function of gamma-aminobutyrate type A receptors.

Calcium/phospholipid-dependent protein kinase (protein kinase C, PKC) has been suggested to play a role in the sensitivity of gamma-aminobutyrate type A (GABAA) receptors to ethanol. We tested a line of null mutant mice that lacks the gamma isoform of PKC (PKC gamma) to determine the role of this brain-specific isoenzyme in ethanol sensitivity. We found that the mutation reduced the amount of PKC gamma immunoreactivity in cerebellum to undetectable levels without altering the levels of the alpha, beta I, or beta II isoforms of PKC. The mutant mice display reduced sensitivity to the effects of ethanol on loss of righting reflex and hypothermia but show normal responses to flunitrazepam or pentobarbital. Likewise, GABAA receptor function of isolated brain membranes showed that the mutation abolished the action of ethanol but did not alter actions of flunitrazepam or pentobarbital. These studies show the unique interactions of ethanol with GABAA receptors and suggest protein kinase isoenzymes as possible determinants of genetic differences in response to ethanol.

Cyclin-dependent protein kinase and cyclin homologs SSN3 and SSN8 contribute to transcriptional control in yeast.

The SSN3 and SSN8 genes of Saccharomyces cerevisiae were identified by mutations that suppress a defect in SNF1, a protein kinase required for release from glucose repression. Mutations in SSN3 and SSN8 also act synergistically with a mutation of the MIG1 repressor protein to relieve glucose repression. We have cloned the SSN3 and SSN8 genes. SSN3 encodes a cyclin-dependent protein kinase (cdk) homolog and is identical to UME5. SSN8 encodes a cyclin homolog 35% identical to human cyclin C. SSN3 and SSN8 fusion proteins interact in the two-hybrid system and coimmunoprecipitate from yeast cell extracts. Using an immune complex assay, we detected protein kinase activity that depends on both SSN3 and SSN8. Thus, the two SSN proteins are likely to function as a cdk-cyclin pair. Genetic analysis indicates that the SSN3-SSN8 complex contributes to transcriptional repression of diversely regulated genes and also affects induction of the GAL1 promoter.

Loss of the catalytic subunit of the DNA-dependent protein kinase in DNA double-strand-break-repair mutant mammalian cells.

The DNA-dependent protein kinase (DNA-PK) consists of three polypeptide components: Ku-70, Ku-80, and an approximately 350-kDa catalytic subunit (p350). The gene encoding the Ku-80 subunit is identical to the x-ray-sensitive group 5 complementing gene XRCC5. Expression of the Ku-80 cDNA rescues both DNA double-strand break (DSB) repair and V(D)J recombination in group 5 mutant cells. The involvement of Ku-80 in these processes suggests that the underlying defect in these mutant cells may be disruption of the DNA-PK holoenzyme. In this report we show that the p350 kinase subunit is deleted in cells derived from the severe combined immunodeficiency mouse and in the Chinese hamster ovary cell line V-3, both of which are defective in DSB repair and V(D)J recombination. A centromeric fragment of human chromosome 8 that complements the scid defect also restores p350 protein expression and rescues in vitro DNA-PK activity. These data suggest the scid gene may encode the p350 protein or regulate its expression and are consistent with a model whereby DNA-PK is a critical component of the DSB-repair pathway.

Activation of YRP kinase by v-Src and protein kinase C-mediated signal transduction pathways.

We have previously reported that a serine(threonine) protein kinase that phosphorylates histone H1 in vitro is activated by tyrosine phosphorylation in v-Src-transformed rat 3Y1 fibroblasts. We now refer to this kinase as YRP kinase, for tyrosine-regulated protein kinase. Since YRP kinase may play a role in mediating the growth-stimulatory and morphology-altering effects of v-Src, we have further examined the signal transduction involved in the activation of YRP kinase. Although YRP kinase is constitutively activated in fibroblasts transformed by v-Src, activation of protein kinase C was also found to lead to activation of YRP kinase. Activation of YRP kinase by protein kinase C was found to be potentiated by vanadate treatment or overexpression of c-Src. The activation of YRP kinase by v-Src, however, does not appear to be mediated by protein kinase C, suggesting that YRP kinase can be activated by two separate signal transduction pathways. Transformation of fibroblasts by v-Ras or v-Mil did not result in activation of YRP kinase, indicating that the MAP kinase pathway does not mediate the activation of YRP kinase by v-Src or protein kinase C.

Neospora caninum calcium-dependent protein kinase 1 is an effective drug target for neosporosis therapy.

Despite the enormous economic importance of Neospora caninum related veterinary diseases, the number of effective therapeutic agents is relatively small. Development of new therapeutic strategies to combat the economic impact of neosporosis remains an important scientific endeavor. This study demonstrates molecular, structural and phenotypic evidence that N. caninum calcium-dependent protein kinase 1 (NcCDPK1) is a promising molecular target for neosporosis drug development. Recombinant NcCDPK1 was expressed, purified and screened against a select group of bumped kinase inhibitors (BKIs) previously shown to have low IC50s against Toxoplasma gondii CDPK1 and T. gondii tachyzoites. NcCDPK1 was inhibited by low concentrations of BKIs. The three-dimensional structure of NcCDPK1 in complex with BKIs was studied crystallographically. The BKI-NcCDPK1 structures demonstrated the structural basis for potency and selectivity. Calcium-dependent conformational changes in solution as characterized by small-angle X-ray scattering are consistent with previous structures in low Calcium-state but different in the Calcium-bound active state than predicted by X-ray crystallography. BKIs effectively inhibited N. caninum tachyzoite proliferation in vitro. Electron microscopic analysis of N. caninum cells revealed ultra-structural changes in the presence of BKI compound 1294. BKI compound 1294 interfered with an early step in Neospora tachyzoite host cell invasion and egress. Prolonged incubation in the presence of 1294 interfered produced observable interference with viability and replication. Oral dosing of BKI compound 1294 at 50 mg/kg for 5 days in established murine neosporosis resulted in a 10-fold reduced cerebral parasite burden compared to untreated control. Further experiments are needed to determine the PK, optimal dosage, and duration for effective treatment in cattle and dogs, but these data demonstrate proof-of-concept for BKIs, and 1294 specifically, for therapy of bovine and canine neosporosis.