p38α mitogen-activated protein kinase (MAPK) is activated in cancer cells in response to environmental factors, oncogenic stress, radiation, and chemotherapy. p38α MAPK phosphorylates a number of ...substrates, including MAPKAP-K2 (MK2), and regulates the production of cytokines in the tumor microenvironment, such as TNF-α, interleukin-1β (IL-1β), IL-6, and CXCL8 (IL-8). p38α MAPK is highly expressed in human cancers and may play a role in tumor growth, invasion, metastasis, and drug resistance. LY2228820 dimesylate (hereafter LY2228820), a trisubstituted imidazole derivative, is a potent and selective, ATP-competitive inhibitor of the α- and β-isoforms of p38 MAPK in vitro (IC(50) = 5.3 and 3.2 nmol/L, respectively). In cell-based assays, LY2228820 potently and selectively inhibited phosphorylation of MK2 (Thr334) in anisomycin-stimulated HeLa cells (at 9.8 nmol/L by Western blot analysis) and anisomycin-induced mouse RAW264.7 macrophages (IC(50) = 35.3 nmol/L) with no changes in phosphorylation of p38α MAPK, JNK, ERK1/2, c-Jun, ATF2, or c-Myc ≤ 10 μmol/L. LY2228820 also reduced TNF-α secretion by lipopolysaccharide/IFN-γ-stimulated macrophages (IC(50) = 6.3 nmol/L). In mice transplanted with B16-F10 melanoma, tumor phospho-MK2 (p-MK2) was inhibited by LY2228820 in a dose-dependent manner threshold effective dose (TED)(70) = 11.2 mg/kg. Significant target inhibition (>40% reduction in p-MK2) was maintained for 4 to 8 hours following a single 10 mg/kg oral dose. LY2228820 produced significant tumor growth delay in multiple in vivo cancer models (melanoma, non-small cell lung cancer, ovarian, glioma, myeloma, breast). In summary, LY2228820 is a p38 MAPK inhibitor, which has been optimized for potency, selectivity, drug-like properties (such as oral bioavailability), and efficacy in animal models of human cancer.
Owing to the prevalence of the JAK2V617F mutation in myeloproliferative neoplasms (MPNs), its constitutive activity, and ability to recapitulate the MPN phenotype in mouse models, JAK2V617F kinase is ...an attractive therapeutic target. We report the discovery and initial characterization of the orally bioavailable imidazopyridazine, LY2784544, a potent, selective and ATP-competitive inhibitor of janus kinase 2 (JAK2) tyrosine kinase. LY2784544 was discovered and characterized using a JAK2-inhibition screening assay in tandem with biochemical and cell-based assays. LY2784544 in vitro selectivity for JAK2 was found to be equal or superior to known JAK2 inhibitors. Further studies showed that LY2784544 effectively inhibited JAK2V617F-driven signaling and cell proliferation in Ba/F3 cells (IC50=20 and 55 nM, respectively). In comparison, LY2784544 was much less potent at inhibiting interleukin-3-stimulated wild-type JAK2-mediated signaling and cell proliferation (IC50=1183 and 1309 nM, respectively). In vivo, LY2784544 effectively inhibited STAT5 phosphorylation in Ba/F3-JAK2V617F-GFP (green fluorescent protein) ascitic tumor cells (TED50=12.7 mg/kg) and significantly reduced (P<0.05) Ba/F3-JAK2V617F-GFP tumor burden in the JAK2V617F-induced MPN model (TED50=13.7 mg/kg, twice daily). In contrast, LY2784544 showed no effect on erythroid progenitors, reticulocytes or platelets. These data suggest that LY2784544 has potential for development as a targeted agent against JAK2V617F and may have properties that allow suppression of JAK2V617F-induced MPN pathogenesis while minimizing effects on hematopoietic progenitor cells.
Signal transduction through both cytokine and lymphocyte antigen receptors shares some common pathways by which they initiate cellular responses, such as activation of mitogen-activated protein ...kinase(s),. However, other signalling components appear to be uniquely coupled to each receptor. For example, the interferon receptors transduce regulatory signals through the JAK/STAT pathway, resulting in an inhibition of growth and of antiviral effects, whereas this pathway apparently plays no role in T-cell-receptor (TCR)-dependent gene expression,. Conversely, signal transduction through the TCR requires the tyrosine kinases Lck and ZAP-70 and the tyrosine phosphatase CD45 (ref. 5). Here we show that, unexpectedly, transmission of growth-inhibitory signals by interferon-α (IFN-α) in T cells requires the expression and association of CD45, Lck and ZAP-70 with the IFN-α-receptor signalling complex.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Interleukin-12 (IL-12) is a key immunoregulatory cytokine that promotes Th1 differentiation and cell-mediated immune responses. The transcription factor STAT4 (signal transducer and activator of ...transcription 4) is an important element in mediating IL-12 signals, as evidenced by the fact that STAT4−/− mice display impaired responsiveness to IL-12 and deficient Th1 differentiation. STAT4 is inducibly phosphorylated on tyrosine and serine in response to IL-12, but the kinase(s) responsible for the latter event is unknown. Here we show that IL-12 induces STAT4 phosphorylation on serine 721 and that mutation of serine 721 interferes with STAT4 transcriptional activity. In addition, we show that mutation of tyrosine 693 abrogates IL-12–induced STAT4 tyrosine phosphorylation and transcriptional activity. Although the site surrounding serine 721 is an optimum consensus sequence for mitogen-activated family of protein kinases (MAPKs)-mediated phosphorylation, we demonstrate that IL-12 does not induce extracellular signal-regulated kinase (ERK) or c-Jun N-terminal kinase (JNK) activation in T and natural killer (NK) cells and that IL-12–induced STAT4 transcriptional activity is not affected by these kinases. Rather, we show that IL-12 induces p38 activation. Moreover, we demonstrate that p38α and its upstream activator, MKK6, phosphorylate STAT4 on serine 721, and are required for STAT4 full transcriptional activity induced by IL-12, establishing the MKK6/p38α/STAT4 pathway as an important mediator of IL-12 actions.
Recently, we have demonstrated that the tyrosine kinase pp60v-src can undergo cell-free assembly into a heterocomplex with
rabbit hsp90 and p50 when the immunoadsorbed protein is incubated with ...rabbit reticulocyte lysate (Hutchison, K. A., Brott,
B. K., De Leon, J. H., Perdew, G. H., Jove, R., and Pratt, W. B. (1992) J. Biol. Chem 267, 2902-2908). Using a baculovirus
system to express a high level of human c-Raf serine/threonine kinase in Sf9 insect cells, we show here that immunoadsorbed
c-Raf undergoes similar lysate-mediated assembly into a heterocomplex with hsp90 and p50. As with pp60v-src and steroid receptors,
binding of c-Raf to hsp90 occurs in an ATP-dependent and K(+)-dependent manner and the resulting heterocomplex is stabilized
by molybdate. With a very rapid and gentle procedure of Sf9 cell cytosol preparation and c-Raf immunoadsorption, we show coimmunoadsorption
of the insect homologue of hsp90. The same procedures permit detection of a native complex of v-Raf with rat hsp90 and p50
in stably transfected rat 3Y1 fibroblasts, and v-Raf is also assembled into a heterocomplex with rabbit hsp90 and p50 by reticulocyte
lysate. Using the 22W mutant of c-Raf in which the NH2-terminal half has been deleted, we show that the catalytic domain of
the kinase is sufficient for both formation of the native heterocomplex in mouse NIH 3T3 cells and cell-free reconstitution
of the heterocomplex by rabbit reticulocyte lysate. Although the native Raf-heat shock protein heterocomplex is less stable
than native pp60v-src and glucocorticoid receptor heterocomplexes, by analogy with these proteins its detection may have important
implications regarding the mechanism of Raf trafficking through the cytoplasm.
We have expressed the mitogenic signaling proteins Src, Ras, Raf-1, Mek (MAP kinase kinase), and Erk (MAP kinase) in baculovirus-infected Sf9 insect cells in order to study a potential role for the ...chaperone hsp90 in formation of multiprotein complexes. One such complex obtained by immunoadsorption with anti-Ras antibody of cytosol prepared from cells simultaneously expressing Ras, Raf, Mek, and Erk contained Ras, Raf, and Erk. To detect directly the protein-protein interactions involved in forming multiprotein complexes, we combined cytosols from single infections in vitro in all possible combinations of protein pairs. We detected complexes between Ras·Raf, Ras·Src, Raf·Mek, and Raf·Src, but no complex containing Erk was obtained by mixing cytosols. Thus, cellular factors appear to be required for assembly of the Erk-containing multiprotein complex. One cellular factor thought to be involved in signaling protein complex formation is the chaperone hsp90, and we show that Src, Raf, and Mek are each complexed with insect hsp90. Treatment of Sf9 cells with geldanamycin, a benzoquinone ansamycin that binds to hsp90 and disrupts its function, did not decrease coadsorption of either Raf or Erk with Ras, although it did decrease the level of cytosolic Raf. To study geldanamycin action, we treated rat 3Y1 fibroblasts expressing v-Raf and showed that the antibiotic blocked assembly of Raf·hsp90 complexes at an intermediate stage of assembly where Raf is still bound to the p60 and hsp70 components of the assembly mechanism. As in Sf9 cells, Raf levels decline with geldanamycin treatment of 3Y1 cells. To determine if geldanamycin affects mitogenic response, we treated HeLa cells with epidermal growth factor (EGF) and showed that geldanamycin treatment decreased EGF signaling and decreased the level of Raf protein without affecting the EGF-mediated increase in Raf kinase activity. We conclude that hsp90 is not required for forming complexes between the mitogenic signaling proteins or for Raf kinase activity and that EGF signaling is decreased indirectly by geldanamycin because the antibiotic increases degradation of Raf and perhaps other components of the signaling pathway.
Checkpoint kinase 1 (CHK1) is a key regulator of the DNA damage response and a mediator of replication stress through modulation of replication fork licensing and activation of S and G
-M cell-cycle ...checkpoints. We evaluated prexasertib (LY2606368), a small-molecule CHK1 inhibitor currently in clinical testing, in multiple preclinical models of pediatric cancer. Following an initial assessment of prexasertib activity, this study focused on the preclinical models of neuroblastoma.
We evaluated the antiproliferative activity of prexasertib in a panel of cancer cell lines; neuroblastoma cell lines were among the most sensitive. Subsequent Western blot and immunofluorescence analyses measured DNA damage and DNA repair protein activation. Prexasertib was investigated in several cell line-derived xenograft mouse models of neuroblastoma.
Within 24 hours, single-agent prexasertib promoted γH2AX-positive double-strand DNA breaks and phosphorylation of DNA damage sensors ATM and DNA-PKcs, leading to neuroblastoma cell death. Knockdown of CHK1 and/or CHK2 by siRNA verified that the double-strand DNA breaks and cell death elicited by prexasertib were due to specific CHK1 inhibition. Neuroblastoma xenografts rapidly regressed following prexasertib administration, independent of starting tumor volume. Decreased Ki67 and increased immunostaining of endothelial and pericyte markers were observed in xenografts after only 6 days of exposure to prexasertib, potentially indicating a swift reduction in tumor volume and/or a direct effect on tumor vasculature.
Overall, these data demonstrate that prexasertib is a specific inhibitor of CHK1 in neuroblastoma and leads to DNA damage and cell death in preclinical models of this devastating pediatric malignancy.
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Virtually all known cellular processes involve modulation of cellular signaling pathways via changes in protein phosphorylation. With genomics efforts more than doubling the number of proteins ...available for analysis, a major challenge will be to identify unknown phosphoproteins as they exist in the normal or diseased intracellular environment. Recent advances in proteomic technology have made it possible to examine changes in protein expression with much greater resolution than was previously possible. In this report, we describe a rapid and reproducible method for identifying phosphoproteins upregulated in response to activation of cell surface receptors. Phosphotyrosine‐containing proteins were immunoprecipitated from IFNα‐ or IL2‐treated primary human lymphocyte extracts using a novel anti‐phosphotyrosine immunoprecipitation technique. This technique takes advantage of differing antibody affinities for epitopes on native versus denatured proteins. Following separation from the immunopellets, phosphoproteins are resolved by two‐dimensional polyacrylamide gel electrophoresis. With this method, we identified known proteins phosphorylated in response to IL2 or IFNα using both silver staining and Western blotting for protein detection/identification. The silver‐stained immunoprecipitation profile serves as a fingerprint for phosphorylation events that occur in response to cytokine treatment. By merging these techniques with mass spectrometric microsequencing, new capabilities are achieved. It will then be possible to identify novel signaling proteins that are activated in response to a variety of stimuli, including receptor activation, disease progression, etc.
The heat shock proteins hsp90 and hsp70 have been immunopurified from rabbit reticulocyte lysate in a multiprotein complex that acts as a self-sufficient protein folding machine. This immunopurified ...“foldosome” directs the assembly of the glucocorticoid receptor-hsp90 complex and refolds the receptor to the steroid binding state (Hutchison, K. A., Dittmar, K. D., and Pratt, W. B.(1994) J. Biol. Chem. 269, 27894-27899). Extensive washing of the immunoadsorbed foldosome eliminates a weakly bound component required for receptor heterocomplex assembly and folding. This protein factor is contained in a Centricon C-100 filtrate of lysate which reconstitutes the receptor activating activity of the washed foldosome. This hsp90-associated protein folding system is present in both animal and plant cells, and the Centricon C-100 fraction of rabbit reticulocyte lysate potentiates receptor folding directed by wheat germ lysate. We have used this ability to stimulate wheat germ lysate-directed folding of the glucocorticoid receptor as a rapid assay for the factor. We demonstrate that the activity segregates with the 23-kDa acidic protein component of the hsp90 foldosome when rabbit reticulocyte lysate is fractionated by ammonium sulfate precipitation and ion exchange chromatography. Immunoadsorption of the Centricon C-100 filtrate with a monoclonal antibody against p23 eliminates its ability to stimulate the wheat germ heterocomplex assembly/receptor folding system, and the activity is replaced by purified, bacterially expressed p23. Immunodepletion of p23 also eliminates the ability of the Centricon C-100 filtrate to reconstitute receptor activating activity of the washed foldosome and addition of purified, bacterially expressed p23 restores its activity, confirming that p23 is the weakly bound component of the foldosome complex required for refolding of the receptor to the steroid binding conformation.
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