Shk1, a homolog of the Saccharomyces cerevisiae Ste20 and mammalian p65PAK protein kinases, is a component of a Ras/Cdc42 signaling module in the fission yeast Schizosaccharomyces pombe.

We describe a protein kinase, Shk1, from the fission yeast Schizosaccharomyces pombe, which is structurally related to the Saccharomyces cerevisiae Ste20 and mammalian p65PAK protein kinases. We provide genetic evidence for physical and functional interaction between Shk1 and the Cdc42 GTP-binding protein required for normal cell morphology and mating in S. pombe. We further show that expression of the STE20 gene complements the shk1 null mutation and that Shk1 is capable of signaling to the pheromone-responsive mitogen-activated protein kinase cascade in S. cerevisiae. Our results lead us to propose that signaling modules composed of small GTP-binding proteins and protein kinases related to Shk1, Ste20, and p65PAK, are highly conserved in evolution and participate in both cytoskeletal functions and mitogen-activated protein kinase signaling pathways.

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.

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.

Deletion of the loop region of Bcl-2 completely blocks paclitaxel-induced apoptosis

At high concentrations, the tubule poison paclitaxel is able to kill cancer cells that express Bcl-2; it inhibits the antiapoptotic activity of Bcl-2 by inducing its phosphorylation. To localize the site on Bcl-2 regulated by phosphorylation, mutant forms of Bcl-2 were constructed. Mutant forms of Bcl-2 with an alteration in serine at amino acid 70 (S70A) or with deletion of a 60-aa loop region between the α1 and α2 helices (Δloop Bcl-2, which also deletes amino acid 70) were unable to be phosphorylated by paclitaxel treatment of MDA-MB-231 cells into which the genes for the mutant proteins were transfected. The Δloop mutant completely inhibited paclitaxel-induced apoptosis. In cells expressing the S70A mutant, paclitaxel induced about one-third the level of apoptosis seen with wild-type Bcl-2. To evaluate the role of mitogen-activated protein kinases (MAPKs) in Bcl-2 phosphorylation, the activation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 was examined. Paclitaxel-induced apoptosis was associated with phosphorylation of Bcl-2 and activation of ERK and JNK MAPKs. If JNK activation was blocked by transfections with either a stress-activated protein kinase kinase dominant-negative (K→R) gene (which prevents the activation of a kinase upstream of JNK) or MAPK phosphatase-1 gene (which dephosphorylates and inactivates JNK), Bcl-2 phosphorylation did not occur, and the cells were not killed by paclitaxel. By contrast, neither an ERK inhibitor (PD098059) nor p38 inhibitors (SB203580 and SB202190) had an effect on Bcl-2 phosphorylation. Thus, our data show that the antiapoptotic effects of Bcl-2 can be overcome by phosphorylation of Ser-70; forms of Bcl-2 lacking the loop region are much more effective at preventing apoptosis than wild-type Bcl-2 because they cannot be phosphorylated. JNK, but not ERK or p38 MAPK, appear to be involved in the phosphorylation of Bcl-2 induced by paclitaxel.

Dissociation of cytokine-induced phosphorylation of Bad and activation of PKB/akt: Involvement of MEK upstream of Bad phosphorylation

The phosphatidylinositol 3-kinase (PI3K)-signaling pathway has emerged as an important component of cytokine-mediated survival of hemopoietic cells. Recently, the protein kinase PKB/akt (referred to here as PKB) has been identified as a downstream target of PI3K necessary for survival. PKB has also been implicated in the phosphorylation of Bad, potentially linking the survival effects of cytokines with the Bcl-2 family. We have shown that granulocyte/macrophage colony-stimulating factor (GM-CSF) maintains survival in the absence of PI3K activity, and we now show that when PKB activation is also completely blocked, GM-CSF is still able to stimulate phosphorylation of Bad. Interleukin 3 (IL-3), on the other hand, requires PI3K for survival, and blocking PI3K partially inhibited Bad phosphorylation. IL-4, unique among the cytokines in that it lacks the ability to activate the p21ras–mitogen-activated protein kinase (MAPK) cascade, was found to activate PKB and promote cell survival, but it did not stimulate Bad phosphorylation. Finally, although our data suggest that the MAPK pathway is not required for inhibition of apoptosis, we provide evidence that phosphorylation of Bad may be occurring via a MAPK/ERK kinase (MEK)-dependent pathway. Together, these results demonstrate that although PI3K may contribute to phosphorylation of Bad in some instances, there is at least one other PI3K-independent pathway involved, possibly via activation of MEK. Our data also suggest that although phosphorylation of Bad may be one means by which cytokines can inhibit apoptosis, it may be neither sufficient nor necessary for the survival effect.

ErbB2 expression increases the spectrum and potency of ligand-mediated signal transduction through ErbB4

Interleukin 3-dependent murine 32D cells do not detectably express members of the ErbB receptor family and do not proliferate in response to known ligands for these receptors. 32D transfectants were generated expressing human ErbB4 alone (32D.E4) or with ErbB2 (32D.E2/E4). Epidermal growth factor (EGF), neuregulin 1-β (NRG1-β), betacellulin (BTC), transforming growth factor-α (TGF-α), heparin binding-EGF (HB-EGF), and amphiregulin were analyzed for their ability to mediate mitogenesis in these transfectants. 32D.E4 responded mitogenically to NRG1-β and BTC. Surprisingly, EGF also induced significant DNA synthesis and TGF-α was negligibly mitogenic on 32D.E4 cells, whereas HB-EGF and amphiregulin were inactive. Although coexpression of ErbB2 with ErbB4 in 32D.E2/E4 cells did not significantly alter DNA synthesis in response to NRG1-β or BTC, it greatly enhanced mitogenesis elicited by EGF and TGF-α and unmasked the ability of HB-EGF to induce proliferation. EGF-related ligands that exhibited potent mitogenic activity on 32D.E2/E4 cells at low concentrations induced adherence, morphological alterations, and up-regulation of the Mac-1 integrin and FcγRII/III at higher concentrations. While 125I-EGF could be specifically crosslinked to both 32D.E4 and 32D.E2/E4 cells, its crosslinking capacity was greatly enhanced in the cotransfected cells. The ability of the various ligands to mediate proliferation and/or adhesion in the two transfectants correlated with their capacity to induce substrate tyrosine phosphorylation and to initiate and sustain activation of mitogen-activated protein kinase. We conclude that the ability of ErbB4 to mediate signal transduction through EGF-like ligands is broader than previously assumed and can be profoundly altered by the concomitant expression of ErbB2.