Biosynthesis of terpenoids: 4-Diphosphocytidyl-2-C-methyl-d-erythritol kinase from tomato

The putative catalytic domain (residues 81–401) of a predicted tomato protein with similarity to 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase of Escherichia coli was expressed in a recombinant E. coli strain. The protein was purified to homogeneity and was shown to catalyze the phosphorylation of the position 2 hydroxy group of 4-diphosphocytidyl-2-C-methyl-d-erythritol at a rate of 33 μmol⋅mg−1⋅min−1. The structure of the reaction product, 4-diphosphocytidyl-2-C-methyl-d-erythritol 2-phosphate, was established by NMR spectroscopy. Divalent metal ions, preferably Mg2+, are required for activity. Neither the tomato enzyme nor the E. coli ortholog catalyzes the phosphorylation of isopentenyl monophosphate.

Sustained activation of Ras/Raf/mitogen-activated protein kinase cascade by the tumor suppressor p53

The p53 tumor suppressor gene can inhibit proliferation transiently, induce permanent cell-cycle arrest/senescence, or cause apoptosis depending on the cellular context. The mitogen-activated protein kinase (MAPK) cascade is known to play a crucial role in cell proliferation and differentiation. Moreover, the duration and intensity of MAPK activation can profoundly influence the biological response observed. We demonstrated that a sustained activation of MAPK cascade could be induced by wild-type p53 expression but not by p21Waf1/Cip1. Furthermore, exposure of normal cells to DNA-damaging agents induced MAPK activation in a p53-dependent manner. Tumor-derived p53 mutants defective in DNA binding failed to activate MAPK, implying that p53 transcriptional activity is essential for this function. Finally, activation of MAPK by p53 was inhibited by expression of dominant-negative Ras (N17Ras) and Raf1 mutants, indicating that MAPK activation by p53 is mediated at a level upstream of Ras. All of these findings establish a biochemical link between p53 signaling and the Ras/Raf/MAPK cascade.

A role for Src kinase in spontaneous epileptiform activity in the CA3 region of the hippocampus

Members of the Src family of nonreceptor protein tyrosine kinases (PTKs) have been implicated in the regulation of cellular excitability and synaptic plasticity. We have investigated the role of these PTKs in in vitro models of epileptiform activity. Spontaneous epileptiform discharges were induced in vitro in the CA3 region of rat hippocampal slices by superfusion with the potassium channel blocker 4-aminopyridine in Mg2+-free medium. In hippocampal slices treated in this fashion, Src kinase activity was increased and the frequency of epileptiform discharges could be greatly reduced by inhibitor of the Src family of PTKs, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), but not by the inactive structural analog 4-amino-7-phenylpyrazol[3,4-d]pyrimidine (PP3). 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine also reduced epileptiform activity induced by either 4-aminopyridine or Mg2+-free medium alone. These observations demonstrate a role for Src family PTKs in the pathophysiology of epilepsy and suggest potential therapeutic targets for antiepileptic therapy.

Essential role for p38α mitogen-activated protein kinase in placental angiogenesis

The p38 family of mitogen-activated protein kinases (MAPKs) mediates signaling in response to environmental stresses and inflammatory cytokines, but the requirements for the p38 MAPK pathway in normal mammalian development have not been elucidated. Here, we show that targeted disruption of the p38α MAPK gene results in homozygous embryonic lethality because of severe defects in placental development. Although chorioallantoic placentation is initiated appropriately in p38α null homozygotes, placental defects are manifest at 10.5 days postcoitum as nearly complete loss of the labyrinth layer and significant reduction of the spongiotrophoblast. In particular, p38α mutant placentas display lack of vascularization of the labyrinth layer as well as increased rates of apoptosis, consistent with a defect in placental angiogenesis. Furthermore, p38α mutants display abnormal angiogenesis in the embryo proper as well as in the visceral yolk sac. Thus, our results indicate a requirement for p38α MAPK in diploid trophoblast development and placental vascularization and suggest a more general role for p38 MAPK signaling in embryonic angiogenesis.

CIKS, a connection to IκB kinase and stress-activated protein kinase

Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-κB and AP-1/ATF families. Activation of NF-κB factors is thought to be mediated primarily via IκB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKα and IKKβ are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-κB essential modulator)/IKKγ. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKγ in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-κB-dependent reporter. Activation of NF-κB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins.

Abnormal integrin-mediated regulation of chronic myelogenous leukemia CD34+ cell proliferation: BCR/ABL up-regulates the cyclin-dependent kinase inhibitor, p27Kip, which is relocated to the cell cytoplasm and incapable of regulating cdk2 activity

β1-integrin engagement on normal (NL) CD34+ cells increases levels of the cyclin-dependent kinase inhibitor (cdki), p27Kip, decreases cdk2 activity, and inhibits G1/S-phase progression. In contrast, β1-integrin engagement on chronic myelogenous leukemia (CML) CD34+ cells does not inhibit G1/S progression. We now show that, in CML, baseline p27Kip levels are significantly higher than in NL CD34+ cells, but adhesion to fibronectin (FN) does not increase p27Kip levels. p27Kip mRNA levels are similar in CML and NL CD34+ cells and remain unchanged after adhesion, suggesting posttranscriptional regulation. Despite the elevated p27Kip levels, cdk2 kinase activity is similar in CML and NL CD34+ cells. In NL CD34+ cells, >90% of p27Kip is located in the nucleus, where it binds to cdk2 after integrin engagement. In CML CD34+ cells, however, >80% of p27Kip is located in the cytoplasm even in FN-adherent cells, and significantly less p27Kip is bound to cdk2. Thus, presence of BCR/ABL induces elevated levels of p27Kip and relocation of p27Kip to the cytoplasm, which contributes to the loss of integrin-mediated proliferation inhibition, characteristic of CML.

Modulation of Akt kinase activity by binding to Hsp90

Serine/threonine kinase Akt/PKB is a downstream effector molecule of phosphoinositide 3-kinase and is thought to mediate many biological actions toward anti-apoptotic responses. We found that Akt formed a complex with a 90-kDa heat-shock protein (Hsp90) in vivo. By constructing deletion mutants, we identified that amino acid residues 229–309 of Akt were involved in the binding to Hsp90 and amino acid residues 327–340 of Hsp90β were involved in the binding to Akt. Inhibition of Akt-Hsp90 binding led to the dephosphorylation and inactivation of Akt, which increased sensitivity of the cells to apoptosis-inducing stimulus. The dephosphorylation of Akt was caused by an increase in protein phosphatase 2A (PP2A)-mediated dephosphorylation and not by a decrease in 3-phosphoinositide-dependent protein kinase-1-mediated phosphorylation. These results indicate that Hsp90 plays an important role in maintaining Akt kinase activity by preventing PP2A-mediated dephosphorylation.

T-cell receptor antagonists induce Vav phosphorylation by selective activation of Fyn kinase

T cell receptor (TCR) antagonists inhibit antigen-induced T cell activation and by themselves fail to induce phenotypic changes associated with T cell activation. However, we have recently shown that TCR antagonists are inducers of antigen-presenting cell (APC)–T cell conjugates. The signaling pathway associated with this cytoskeleton-dependent event appears to involve tyrosine phosphorylation and activation of Vav. In this study, we investigated the role played by the protein tyrosine kinases Fyn, Lck, and ZAP-70 in antagonist-induced signaling pathway. Antagonist stimulation increased tyrosine phosphorylation and kinase activity of Fyn severalfold, whereas little or no increase in Lck and ZAP-70 activity was observed. Second, TCR stimulation of Lck−, Fynhi Jurkat cells induced strong tyrosine phosphorylation of Vav. In contrast, minimal increase in tyrosine phosphorylation of Vav was observed in Lckhi, Fynlo Jurkat cells. Finally, study of T cells from a Fyn-deficient TCR transgenic mouse also showed that Fyn was required for tyrosine phosphorylation and activation of Vav induced by both antagonist and agonist peptides. The deficiency in Vav phosphorylation in Fyn-deficient T cells was associated with a defect in the formation of APC–T cell conjugates when T cells were stimulated with either agonist or antagonist peptide. We conclude from these results that Vav is a selective substrate for Fyn, especially under conditions of low-affinity TCR-mediated signaling, and that this signaling pathway involving Fyn, Vav, and Rac-1 is required for the cytoskeletal reorganization that leads to T cell–APC conjugates and the formation of the immunologic synapse.

Regulation and localization of tyrosine216 phosphorylation of glycogen synthase kinase-3β in cellular and animal models of neuronal degeneration

Inactivation of glycogen synthase kinase-3β (GSK3β) by S9 phosphorylation is implicated in mechanisms of neuronal survival. Phosphorylation of a distinct site, Y216, on GSK3β is necessary for its activity; however, whether this site can be regulated in cells is unknown. Therefore we examined the regulation of Y216 phosphorylation on GSK3β in models of neurodegeneration. Nerve growth factor withdrawal from differentiated PC12 cells and staurosporine treatment of SH-SY5Y cells led to increased phosphorylation at Y216, GSK3β activity, and cell death. Lithium and insulin, agents that lead to inhibition of GSK3β and adenoviral-mediated transduction of dominant negative GSK3β constructs, prevented cell death by the proapoptotic stimuli. Inhibitors induced S9 phosphorylation and inactivation of GSK3β but did not affect Y216 phosphorylation, suggesting that S9 phosphorylation is sufficient to override GSK3β activation by Y216 phosphorylation. Under the conditions examined, increased Y216 phosphorylation on GSK3β was not an autophosphorylation response. In resting cells, Y216 phosphorylation was restricted to GSK3β present at focal adhesion sites. However, after staurosporine, a dramatic alteration in the immunolocalization pattern was observed, and Y216-phosphorylated GSK3β selectively increased within the nucleus. In rats, Y216 phosphorylation was increased in degenerating cortical neurons induced by ischemia. Taken together, these results suggest that Y216 phosphorylation of GSK3β represents an important mechanism by which cellular insults can lead to neuronal death.

Rational design of a global inhibitor of the virulence response in Staphylococcus aureus, based in part on localization of the site of inhibition to the receptor-histidine kinase, AgrC

Two-component signaling systems involving receptor-histidine kinases are ubiquitous in bacteria and have been found in yeast and plants. These systems provide the major means by which bacteria communicate with each other and the outside world. Remarkably, very little is known concerning the extracellular ligands that presumably bind to receptor-histidine kinases to initiate signaling. The two-component agr signaling circuit in Staphylococcus aureus is one system where the ligands are known in chemical detail, thus opening the door for detailed structure–activity relationship studies. These ligands are short (8- to 9-aa) peptides containing a thiolactone structure, in which the α-carboxyl group of the C-terminal amino acid is linked to the sulfhydryl group of a cysteine, which is always the fifth amino acid from the C terminus of the peptide. One unique aspect of the agr system is that peptides that activate virulence expression in one group of S. aureus strains also inhibit virulence expression in other groups of S. aureus strains. Herein, it is demonstrated by switching the receptor-histidine kinase, AgrC, between strains of different agr specificity types, that intragroup activation and intergroup inhibition are both mediated by the same group-specific receptors. These results have facilitated the development of a global inhibitor of virulence in S. aureus, which consists of a truncated version of one of the naturally occurring thiolactone peptides.

A cyclin-dependent kinase-activating kinase regulates differentiation of root initial cells in Arabidopsis

Cell division and differentiation continue throughout the plant life cycle without significant loss of control. However, little is known about the mechanisms that allow the continuous development of meristems. Cell division is controlled by a family of cyclin-dependent kinases (CDKs). CDK-activating kinases (CAKs) are known to phosphorylate and activate almost all CDKs and thus may have a crucial role in controlling CDK activities in each cell of the meristems. Here, we show that overexpression of sense or antisense gene for Cak1At in Arabidopsis by using the glucocorticoid-mediated transcriptional induction system resulted in a reduction of CDK activities. After 14–24 h of glucocorticoid treatment, starch granules appeared in columellar initials in the root meristem, and cortical initials were periclinally divided into cortical and endodermal cells. Accumulation of the cyclin∷β-glucuronidase fusion protein ceased after 72 h of glucocorticoid treatment. Our results indicate that a change of Cak1At activity leads to differentiation of initial cells, followed by cessation of cell division. Therefore, we propose that differentiation of initial cells is controlled by Cak1At but is maintained independent of cell division.

Tyrosine kinase-dependent activation of a chloride channel in CD95-induced apoptosis in T lymphocytes

CD95/Fas/APO-1 mediated apoptosis is an important mechanism in the regulation of the immune response. Here, we show that CD95 receptor triggering activates an outwardly rectifying chloride channel (ORCC) in Jurkat T lymphocytes. Ceramide, a lipid metabolite synthesized upon CD95 receptor triggering, also induces activation of ORCC in cell-attached patch clamp experiments. Activation is mediated by Src-like tyrosine kinases, because it is abolished by the tyrosine kinase inhibitor herbimycin A or by genetic deficiency of p56lck. In vitro incubation of excised patches with purified p56lck results in activation of ORCC, which is partially reversed upon addition of anti-phosphotyrosine antibody. Inhibition of ORCC by four different drugs correlates with a 30–65% inhibition of apoptosis. Intracellular acidification observed upon CD95 triggering is abolished by inhibition of either ORCC or p56lck. The results suggest that tyrosine kinase-mediated activation of ORCC may play a role in CD95-induced cell death in T lymphocytes.

Glucose-regulated interaction of a regulatory subunit of protein phosphatase 1 with the Snf1 protein kinase in Saccharomyces cerevisiae

The Snf1 protein kinase family has been conserved in eukaryotes. In the yeast Saccharomyces cerevisiae, Snf1 is essential for transcription of glucose-repressed genes in response to glucose starvation. The direct interaction between Snf1 and its activating subunit, Snf4, within the kinase complex is regulated by the glucose signal. Glucose inhibition of the Snf1-Snf4 interaction depends on protein phosphatase 1 and its targeting subunit, Reg1. Here we show that Reg1 interacts with the Snf1 catalytic domain in the two-hybrid system. This interaction increases in response to glucose limitation and requires the conserved threonine in the activation loop of the kinase, a putative phosphorylation site. The inhibitory effect of Reg1 appears to require the Snf1 regulatory domain because a reg1Δ mutation no longer relieves glucose repression of transcription when Snf1 function is provided by the isolated catalytic domain. Finally, we show that abolishing the Snf1 catalytic activity by mutation of the ATP-binding site causes elevated, constitutive interaction with Reg1, indicating that Snf1 negatively regulates its own interaction with Reg1. We propose a model in which protein phosphatase 1, targeted by Reg1, facilitates the conformational change of the kinase complex from its active state to the autoinhibited state.

Protein kinase A-catalyzed phosphorylation of heat shock protein 60 chaperone regulates its attachment to histone 2B in the T lymphocyte plasma membrane

Accumulating evidence suggests that the mitochondrial molecular chaperone heat shock protein 60 (hsp60) also can localize in extramitochondrial sites. However, direct evidence that hsp60 functions as a chaperone outside of mitochondria is presently lacking. A 60-kDa protein that is present in the plasma membrane of a human leukemic CD4+ CEM-SS T cell line and is phosphorylated by protein kinase A (PKA) was identified as hsp60. An 18-kDa plasma membrane-associated protein coimmunoprecipitated with hsp60 and was identified as histone 2B (H2B). Hsp60 physically associated with H2B when both molecules were in their dephospho forms. By contrast, PKA-catalyzed phosphorylation of both hsp60 and H2B caused dissociation of H2B from hsp60 and loss of H2B from the plasma membrane of intact T cells. These results suggest that (i) hsp60 and H2B can localize in the T cell plasma membrane; (ii) hsp60 functions as a molecular chaperone for H2B; and (iii) PKA-catalyzed phosphorylation of both hsp60 and H2B appears to regulate the attachment of H2B to hsp60. We propose a model in which phosphorylation/dephosphorylation regulates chaperoning of H2B by hsp60 in the plasma membrane.

Substrate recruitment to cyclin-dependent kinase 2 by a multipurpose docking site on cyclin A

An important question in the cell cycle field is how cyclin-dependent kinases (cdks) target their substrates. We have studied the role of a conserved hydrophobic patch on the surface of cyclin A in substrate recognition by cyclin A-cdk2. This hydrophobic patch is ≈35Å away from the active site of cdk2 and contains the MRAIL sequence conserved among a number of mammalian cyclins. In the x-ray structure of cyclin A-cdk2-p27, this hydrophobic patch contacts the RNLFG sequence in p27 that is common to a number of substrates and inhibitors of mammalian cdks. We find that mutation of this hydrophobic patch on cyclin A eliminates binding to proteins containing RXL motifs without affecting binding to cdk2. This docking site is critical for cyclin A-cdk2 phosphorylation of substrates containing RXL motifs, but not for phosphorylation of histone H1. Impaired substrate binding by the cyclin is the cause of the defect in RXL substrate phosphorylation, because phosphorylation can be rescued by restoring a cyclin A–substrate interaction in a heterologous manner. In addition, the conserved hydrophobic patch is important for cyclin A function in cells, contributing to cyclin A’s ability to drive cells out of the G1 phase of the cell cycle. Thus, we define a mechanism by which cyclins can recruit substrates to cdks, and our results support the notion that a high local concentration of substrate provided by a protein–protein interaction distant from the active site is critical for phosphorylation by cdks.