Sequence-specific antitumor activity of a phosphorothioate oligodeoxyribonucleotide targeted to human C-raf kinase supports an antisense mechanism of action in vivo

To determine the mechanism of action responsible for the in vivo antitumor activity of a phosphorothioate antisense inhibitor targeted against human C-raf kinase (ISIS 5132, also known as CGP69846A), a series of mismatched phosphorothioate analogs of ISIS 5132 or CGP69846A were synthesized and characterized with respect to hybridization affinity, inhibitory effects on C-raf gene expression in vitro, and antitumor activity in vivo. Incorporation of a single mismatch into the sequence of ISIS 5132 or CGP69846A resulted in reduced hybridization affinity toward C-raf RNA sequences and reduced inhibitory activity against C-raf expression in vitro and tumor growth in vivo. Moreover, incorporation of additional mismatches resulted in further loss of in vitro and in vivo activity in a manner that correlated well with a hybridization-based (i.e., antisense) mechanism of action. These results provide important experimental evidence supporting an antisense mechanism of action underlying the in vivo antitumor activity displayed by ISIS 5132 or CGP69846A.

A functional genetic screen identifies regions at the C-terminal tail and death-domain of death-associated protein kinase that are critical for its proapoptotic activity

Death-associated protein kinase (DAP-kinase) is a Ca+2/calmodulin-regulated serine/threonine kinase with a multidomain structure that participates in apoptosis induced by a variety of signals. To identify regions in this protein that are critical for its proapoptotic activity, we performed a genetic screen on the basis of functional selection of short DAP-kinase-derived fragments that could protect cells from apoptosis by acting in a dominant-negative manner. We expressed a library of randomly fragmented DAP-kinase cDNA in HeLa cells and treated these cells with IFN-γ to induce apoptosis. Functional cDNA fragments were recovered from cells that survived the selection, and those in the sense orientation were examined further in a secondary screen for their ability to protect cells from DAP-kinase-dependent tumor necrosis factor-α-induced apoptosis. We isolated four biologically active peptides that mapped to the ankyrin repeats, the “linker” region, the death domain, and the C-terminal tail of DAP-kinase. Molecular modeling of the complete death domain provided a structural basis for the function of the death-domain-derived fragment by suggesting that the protective fragment constitutes a distinct substructure. The last fragment, spanning the C-terminal serine-rich tail, defined a new regulatory region. Ectopic expression of the tail peptide (17 amino acids) inhibited the function of DAP-kinase, whereas removal of this region from the complete protein caused enhancement of the killing activity, indicating that the C-terminal tail normally plays a negative regulatory role. Altogether, this unbiased screen highlighted functionally important regions in the protein and revealed an additional level of regulation of DAP-kinase apoptotic function that does not affect the catalytic activity.

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.

Jun N-terminal kinase 2 modulates thymocyte apoptosis and T cell activation through c-Jun and nuclear factor of activated T cell (NF-AT)

The Jun N-terminal kinases (JNKs) recently have been shown to be required for thymocyte apoptosis and T cell differentiation and/or proliferation. To investigate the molecular targets of JNK signaling in lymphoid cells, we used mice in which the serines phosphorylated by JNK in c-Jun were replaced by homologous recombination with alanines (junAA mice). Lymphocytes from these mice showed no phosphorylation of c-Jun in response to activation stimuli, whereas c-Jun was rapidly phosphorylated in wild-type cells. Despite the fact that c-jun is essential for early development, junAA mice develop normally; however, c-Jun N-terminal phosphorylation was required for efficient T cell receptor-induced and tumor necrosis factor-α-induced thymocyte apoptosis. In contrast, c-Jun phosphorylation by JNK is not required for T cell proliferation or differentiation. Because jnk2−/− T cells display a proliferation defect, we concluded that JNK2 must have other substrates required for lymphocyte function. Surprisingly, jnk2−/− T cells showed reduced NF-AT DNA-binding activity after activation. Furthermore, overexpression of JNK2 in Jurkat T cells strongly enhanced NF-AT-dependent transcription. These results demonstrate that JNK signaling differentially uses c-Jun and NF-AT as molecular effectors during thymocyte apoptosis and T cell proliferation.

IL-1-induced NFκB and c-Jun N-terminal kinase (JNK) activation diverge at IL-1 receptor-associated kinase (IRAK)

Mutant I1A cells, lacking IL-1 receptor-associated kinase (IRAK) mRNA and protein, have been used to study the involvement of IRAK in NFκB and c-Jun N-terminal kinase (JNK) activation. A series of IRAK deletion constructs were expressed in I1A cells, which were then tested for their ability to respond to IL-1. Both the N-terminal death domain and the C-terminal region of IRAK are required for IL-1-induced NFκB and JNK activation, whereas the N-proximal undetermined domain is required for the activation of NFκB but not JNK. The phosphorylation and ubiquitination of IRAK deletion mutants correlate tightly with their ability to activate NFκB in response to IL-1, but IRAK can mediate IL-1-induced JNK activation without being phosphorylated. These studies reveal that the IL-1-induced signaling pathways leading to NFκB and JNK activation diverge either at IRAK or at a point nearer to the receptor.

Role of MEKK2-MEK5 in the regulation of TNF-α gene expression and MEKK2-MKK7 in the activation of c-Jun N-terminal kinase in mast cells

Cross-linking of the high-affinity IgE receptor (FcɛRI) on mast cells with IgE and multivalent antigen triggers mitogen-activated protein (MAP) kinase activation and cytokine gene expression. We report here that MAP kinase kinase 4 (MKK4) gene disruption does not affect either MAP kinase activation or cytokine gene expression in response to cross-linking of FcɛRI in embryonic stem cell-derived mast cells. MKK7 is activated in response to cross-linking of FcɛRI, and this activation is inhibited by MAP/ERK kinase (MEK) kinase 2 (MEKK2) gene disruption. In addition, expression of kinase-inactive MKK7 in the murine mast cell line MC/9 inhibits c-Jun NH2-terminal kinase (JNK) activation in response to cross-linking of FcɛRI, whereas expression of kinase-inactive MKK4 does not affect JNK activation by this stimulus. However, FcɛRI-induced activation of the tumor necrosis factor-α (TNF-α) gene promoter is not affected by expression of kinase-inactive MKK7. We describe an alternative pathway by which MEKK2 activates MEK5 and big MAP kinase1/extracellular signal-regulated kinase 5 in addition to MKK7 and JNK, and interruption of this pathway inhibits TNF-α promoter activation. These findings suggest that JNK activation by antigen cross-linking is dependent on the MEKK2-MKK7 pathway, and cytokine production in mast cells is regulated in part by the signaling complex MEKK2-MEK5-ERK5.

Mutational analysis of NM23-H2/NDP kinase identifies the structural domains critical to recognition of a c-myc regulatory element.

NM23-H2, a presumed regulator of tumor metastasis in humans, is a hexameric protein with both enzymatic (NDP kinase) and regulatory (transcriptional activation) activity. While the structure and catalytic mechanisms have been well characterized, the mode of DNA binding is not known. We examined this latter function in a site-directed mutational study and identified residues and domains essential for the recognition of a c-myc regulatory sequence. Three amino acids, Arg-34, Asn-69, and Lys-135, were found among 30 possibilities to be critical for DNA binding. Two of these, Asn-69 and Lys-135, are not conserved between NM23 variants differing in DNA-binding potential, suggesting that DNA recognition resides partly in nonconserved amino acids. All three DNA-binding defective mutant proteins are active enzymatically and appear to be stable hexamers, suggesting that they perform at the level of DNA recognition and that separate functional domains exist for enzyme catalysis and DNA binding. In the context of the known crystal structure of NM23-H2, the DNA-binding residues are located within distinct structural motifs in the monomer, which are exposed to the surface near the 2-fold axis of adjacent subunits in the hexamer. These findings are explained by a model in which NM23-H2 binds DNA with a combinatorial surface consisting of the "outer" face of the dimer. Chemical crosslinking data support a dimeric DNA-binding mode by NM23-H2.

Activation of c-Jun N-terminal kinase in bacterial lipopolysaccharide-stimulated macrophages.

Activation of macrophages by bacterial lipopolysaccharide (LPS) induces transcription of genes that encode for proinflammatory regulators of the immune response. Previous work has suggested that activation of the transcription factor activator protein 1 (AP-1) is one LPS-induced event that mediates this response. Consistent with this notion, we found that LPS stimulated AP-1-mediated transcription of a transfected reporter gene in the murine macrophage cell line RAW 264.7. As AP-1 activity is regulated in part by activation of the c-Jun N-terminal kinase (JNK), which phosphorylates and subsequently increases the transcriptional activity of c-Jun, we examined whether LPS treatment of macrophages resulted in activation of this kinase. LPS treatment of RAW 264.7 cells, murine bone marrow-derived macrophages, and the human monocyte cell line THP-1 resulted in rapid activation of the p46 and p54 isoforms of JNK. Treatment with wild-type and rough mutant forms of LPS and synthetic lipid A resulted in JNK activation, while pretreatment with the tyrosine kinase inhibitor herbimycin A inhibited this response. Binding of LPS-LPS binding protein (LBP) complexes to CD14, a surface receptor that mediates many LPS responses, was found to be crucial, as pretreatment of THP-1 cells with the monoclonal antibody 60b, which blocks this binding, inhibited JNK activation. These results suggest that LPS activation of JNK in monocyte/macrophage cells is a CD14- and protein tyrosine phosphorylation-dependent event that may mediate the early activation of AP-1 in regulating LPS-triggered gene induction.

Cloning and characterization of Lnk, a signal transduction protein that links T-cell receptor activation signal to phospholipase C gamma 1, Grb2, and phosphatidylinositol 3-kinase.

A cDNA encoding a signal transduction protein with a Src homology 2 (SH2) domain and a tyrosine phosphorylation site was cloned from a rat lymph node cDNA library. This protein, which we designate Lnk, has a calculated molecular weight of 33,988. When T lymphocytes were activated by antibody-mediated crosslinking of the T-cell receptor and CD4, Lnk became tyrosine phosphorylated. In activated T lymphocytes, phospholipase C gamma 1, phosphatidylinositol 3-kinase, and Grb-2 coimmunoprecipitated with Lnk. Our results suggest that Lnk becomes tyrosine phosphorylated and links the immediate tyrosine phosphorylation signals of the TCR to the distal phosphatidylinositol 3-kinase, phospholipase C gamma 1 and Ras signaling pathways through its multifunctional tyrosine phosphorylation site.

Identification of human cyclin-dependent kinase 8, a putative protein kinase partner for cyclin C.

Metazoan cyclin C was originally isolated by virtue of its ability to rescue Saccharomyces cerevisiae cells deficient in G1 cyclin function. This suggested that cyclin C might play a role in cell cycle control, but progress toward understanding the function of this cyclin has been hampered by the lack of information on a potential kinase partner. Here we report the identification of a human protein kinase, K35 [cyclin-dependent kinase 8 (CDK8)], that is likely to be a physiological partner of cyclin C. A specific interaction between K35 and cyclin C could be demonstrated after translation of CDKs and cyclins in vitro. Furthermore, cyclin C could be detected in K35 immunoprecipitates prepared from HeLa cells, indicating that the two proteins form a complex also in vivo. The K35-cyclin C complex is structurally related to SRB10-SRB11, a CDK-cyclin pair recently shown to be part of the RNA polymerase II holoenzyme of S. cerevisiae. Hence, we propose that human K35(CDK8)-cyclin C might be functionally associated with the mammalian transcription apparatus, perhaps involved in relaying growth-regulatory signals.

The inositol polyphosphate 4-phosphatase forms a complex with phosphatidylinositol 3-kinase in human platelet cytosol

Inositol polyphosphate 4-phosphatase (4-phosphatase) is an enzyme that catalyses the hydrolysis of the 4-position phosphate from phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2]. In human platelets the formation of this phosphatidylinositol, by the actions of phosphatidylinositol 3-kinase (PI 3-kinase), correlates with irreversible platelet aggregation. We have shown previously that a phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase forms a complex with the p85 subunit of PI 3-kinase. In this study we investigated whether PI 3-kinase also forms a complex with the 4-phosphatase in human platelets. Immunoprecipitates of the p85 subunit of PI 3-kinase from human platelet cytosol contained 4-phosphatase enzyme activity and a 104-kDa polypeptide recognized by specific 4-phosphatase antibodies. Similarly, immunoprecipitates made using 4-phosphatase-specific antibodies contained PI 3-kinase enzyme activity and an 85-kDa polypeptide recognized by antibodies to the p85 adapter subunit of PI 3-kinase. After thrombin activation, the 4-phosphatase translocated to the actin cytoskeleton along with PI 3-kinase in an integrin- and aggregation-dependent manner. The majority of the PI 3-kinase/4-phosphatase complex (75%) remained in the cytosolic fraction. We propose that the complex formed between the two enzymes serves to localize the 4-phosphatase to sites of PtdIns(3,4)P2 production.

Disruption of cellular translational control by a viral truncated eukaryotic translation initiation factor 2α kinase homolog

Phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) is a common cellular mechanism to limit protein synthesis in stress conditions. Baculovirus PK2, which resembles the C-terminal half of a protein kinase domain, was found to inhibit both human and yeast eIF2α kinases. Insect cells infected with wild-type, but not pk2-deleted, baculovirus exhibited reduced eIF2α phosphorylation and increased translational activity. The negative regulatory effect of human protein kinase RNA-regulated (PKR), an eIF2α kinase, on virus production was counteracted by PK2, indicating that baculoviruses have evolved a unique strategy for disrupting a host stress response. PK2 was found in complex with PKR and blocked kinase autophosphorylation in vivo, suggesting a mechanism of kinase inhibition mediated by interaction between truncated and intact kinase domains.

The RIIβ regulatory subunit of protein kinase A binds to cAMP response element: An alternative cAMP signaling pathway

cAMP, through the activation of cAMP-dependent protein kinase (PKA), is involved in transcriptional regulation. In eukaryotic cells, cAMP is not considered to alter the binding affinity of CREB/ATF to cAMP-responsive element (CRE) but to induce serine phosphorylation and consequent increase in transcriptional activity. In contrast, in prokaryotic cells, cAMP enhances the DNA binding of the catabolite repressor protein to regulate the transcription of several operons. The structural similarity of the cAMP binding sites in catabolite repressor protein and regulatory subunit of PKA type II (RII) suggested the possibility of a similar role for RII in eukaryotic gene regulation. Herein we report that RIIβ subunit of PKA is a transcription factor capable of interacting physically and functionally with a CRE. In contrast to CREB/ATF, the binding of RIIβ to a CRE was enhanced by cAMP, and in addition, RIIβ exhibited transcriptional activity as a Gal4-RIIβ fusion protein. These experiments identify RIIβ as a component of an alternative pathway for regulation of CRE-directed transcription in eukaryotic cells.

Two binding modes reveal flexibility in kinase/response regulator interactions in the bacterial chemotaxis pathway

The crystal structure at 2.0-Å resolution of the complex of the Escherichia coli chemotaxis response regulator CheY and the phosphoacceptor-binding domain (P2) of the kinase CheA is presented. The binding interface involves the fourth and fifth helices and fifth β-strand of CheY and both helices of P2. Surprisingly, the two heterodimers in the asymmetric unit have two different binding modes involving the same interface, suggesting some flexibility in the binding regions. Significant conformational changes have occurred in CheY compared with previously determined unbound structures. The active site of CheY is exposed by the binding of the kinase domain, possibly to enhance phosphotransfer from CheA to CheY. The conformational changes upon complex formation as well as the observation that there are two different binding modes suggest that the plasticity of CheY is an essential feature of response regulator function.