Tpl2 Kinase Research Articles

Identification of a novel class of selective Tpl2 kinase inhibitors: 4-Alkylamino-[1,7]naphthyridine-3-carbonitriles

We have previously reported the discovery and initial SAR of the [1,7]naphthyridine-3-carbonitriles and quinoline-3-carbonitriles as Tumor Progression Loci-2 (Tpl2) kinase inhibitors. In this paper, we report new SAR efforts which have led to the identification of 4-alkylamino-[1,7]naphthyridine-3-carbonitriles. These compounds show good in vitro and in vivo activity against Tpl2 and improved pharmacokinetic properties. In addition they are highly selective for Tpl2 kinase over other kinases, for example, EGFR, MEK, MK2, and p38. Lead compound 4-cycloheptylamino-6-[(pyridin-3-ylmethyl)-amino]-[1,7]naphthyridine-3-carbonitrile ( 30) was efficacious in a rat model of LPS-induced TNF-α production.

Inhibition of Tpl2 Kinase and TNFα Production with Quinoline‐3‐carbonitriles for the Treatment of Rheumatoid Arthritis.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

Inhibition of Tpl2 kinase and TNFα production with quinoline-3-carbonitriles for the treatment of rheumatoid arthritis

The synthesis and structure–activity studies of a series of quinoline-3-carbonitriles as inhibitors of Tpl2 kinase are described. Potent inhibitors of Tpl2 kinase with selectivity against a panel of selected kinases in enzymatic assays and specificity in cell-based phosphorylation assays in LPS-treated human monocytes were identified. Selected inhibitors with moderate activity in human whole blood assay effectively inhibited LPS/D-Gal induced TNFα release when administered intraperitoneally in mice.

Inhibition of Tpl2 Kinase and TNF‐α Production with 1,7‐Naphthyridine‐3‐carbonitriles: Synthesis and Structure—Activity Relationships.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

The Tyrosine Kinase Syk Regulates TPL2 Activation Signals

Tpl2/Cot is a serine/threonine kinase that plays a key physiological role in the regulation of immune responses to pro-inflammatory stimuli, including tumor necrosis factor-alpha (TNF-alpha). TNF-alpha stimulates the JNK, ERK, and p38 mitogen-activated protein kinases and the NF-kappaB pathway by recruiting RIP1 and TRAF2 to the TNF receptor 1. Here we showed that Tpl2 activation by TNF-alpha signals depends on the integrity of the Tpl2-interacting proteins RIP1 and TRAF2, which are required for the engagement of the ERK mitogen-activated protein kinase pathway. However, neither RIP1 nor TRAF2 overexpression was sufficient to activate Tpl2 and ERK. We also showed that Tpl2 activation by TNF-alpha depends on a tyrosine kinase activity that is detected in TNF-alpha-stimulated cells. Based on both genetic and biochemical evidence, we concluded that in a variety of cell types, Syk is the tyrosine kinase that plays an important role in the activation of Tpl2 upstream of ERK. These data therefore dissect the TNF receptor 1 proximal events that regulate Tpl2 and ERK and highlight a role for RIP1, TRAF2, and Syk in this pathway.

Inhibition of Tpl2 kinase and TNF-α production with 1,7-naphthyridine-3-carbonitriles: Synthesis and structure–activity relationships

The synthesis and structure–activity studies of a series of 6-substituted-4-anilino-[1,7]-naphthyridine-3-carbonitriles as inhibitors of Tpl2 kinase are described. The early exploratory work described here may lead to the discovery of compounds with significant therapeutic potential for treating rheumatoid arthritis and other inflammatory diseases.

Tpl2/Cot Signals Activate ERK, JNK, and NF-κB in a Cell-type and Stimulus-specific Manner

Macrophages and B-cells from Tpl2 knock-out mice exhibit a restricted defect in lipopolysaccharide and death receptor signaling that is limited to the activation of ERK. Here we show that Tpl2-/- MEFs exhibit defects in ERK, JNK, and NF-kappaB activation, or ERK activation only when stimulated with tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta, respectively. In addition, we show that the activation of Tpl2 by TNF-alpha depends on signals transduced by both TRAF2 and RIP1. Activated Tpl2 phosphorylates MKK4/SEK1 upstream of JNK and stimulates NF-kappaB DNA binding and transcriptional activity by mechanisms that are independent of the nuclear translocation of p50 and p65. Tpl2-transduced TNF-alpha signals instead promote the phosphorylation of p65 at Ser276 and modulate the spectrum of proteins associated with p65. Phosphorylation stimulates the transcriptional activity of NF-kappaB but does not affect its ability to bind DNA, which may be affected by the composition of the nuclear NF-kappaB complexes. These data confirm that defects caused by a single mutation may be cell-type and signal-specific and delineate the role of Tpl2 in the transduction of TNF-alpha signals that activate JNK and NF-kappaB in MEFs.

Phosphorylation at Thr-290 regulates Tpl2 binding to NF-κB1/p105 and Tpl2 activation and degradation by lipopolysaccharide

The serine-threonine protein kinase encoded by the Tpl2 protooncogene transduces Toll-like and death receptor signals in a variety of cell types and plays an important role in innate immunity and inflammation. Differential translational initiation of the Tpl2 mRNA gives rise to 58-kDa (p58) and 52-kDa (p52) isoforms. In unstimulated cells, both isoforms are stabilized and inactivated by stoichiometric binding to NF-kappaB1/p105. After lipopolysaccharide or TNF-alpha stimulation, p58 is released from p105 preferentially relative to p52. The released p58 is active but unstable and undergoes rapid degradation via the proteasome. Recent studies revealed that Tpl2 undergoes phosphorylation at Thr-290 and that phosphorylation at this site is required for activation. Here, we present evidence showing that it is the p58 isoform that is preferentially phosphorylated at Thr-290 and that phosphorylation is more efficient when p58 is complexed to p52. Because p58 is preferentially released from p105 after stimulation, we examined whether Tpl2 phosphorylation at this site controls the dissociation of the two proteins in response to external signals and the subsequent events leading to the activation of Tpl2. The results showed that lipopolysaccharide-induced Tpl2 phosphorylation at Thr-290 in macrophages promotes the release of Tpl2 from p105, contributes to the enzymatic activation of the Tpl2 kinase, and is required for the degradation of Tpl2 via the proteasome.

IkappaB kinase is an essential component of the Tpl2 signaling pathway.

IkappaB kinase (IKK), a key regulator of immune and inflammatory responses, is known as an effector kinase mediating activation of the transcription factor NF-kappaB. Whether IKK also participates in other signaling events is not known. Here we show that IKK serves as an essential component of a signaling pathway that involves activation of the Tpl2 kinase and its downstream targets, MEK1 and ERK. Inhibition of IKKbeta in macrophages eliminates Tpl2 activation and ERK phosphorylation induced by lipopolysaccharide and tumor necrosis factor alpha. Using IKK-deficient murine fibroblasts, we further demonstrate that IKKbeta, but not IKKalpha, is required for Tpl2 activation. Moreover, this novel function of IKKbeta appears to involve phosphorylation and degradation of the Tpl2 inhibitor NF-kappaB1/p105. These findings suggest that IKKbeta exerts its immune-regulatory functions by targeting different downstream signaling pathways.

Activation of NF-κB nuclear transcription factor by flow in human endothelial cells

The tractive force generated by blood flow, called fluid shear stress, is an important regulator of endothelial cell gene expression. Several transcription factors are activated by shear stress, including members of the NF-κB/Rel family. The nature of the upstream-signaling components involved in the activation of NF-κB by flow has been studied in human endothelial cells. Flow rapidly increased endogenous IKK1/2 activity and transiently degraded IκBα and IκBβ1, but not p105/p50. Nuclear translocation of the p65 subunit was induced by flow in wild-type (w/t) cells and in cells overexpressing w/t NIK, IKK1 or IKK2, but not in cells transiently transfected with kinase-inactive mutants of these enzymes. Nuclear translocation of p65 in response to flow was not affected by overexpressing a dominant-negative mutant of a MAPKKK related to NIK, called TPL2 kinase, nor by pretreating cells with the selective PKC inhibitor bisindoylmaleimide-1. Gel shift assays showed that the binding of p50/p65 heterodimer to radiolabeled oligonucleotide containing a shear-stress response element was increased by flow. The activity of a 3κB conA-luciferase reporter was also increased, confirming that NF-κB activated by flow was transcriptionally active. We conclude that shear stress induces gene transactivation by NF-κB (p50/p65) via the NIK-IKK1/2 pathway and proteosome-dependent degradation of IκB and that induction by flow does not involve TPL-2 kinase or PKC.

NF-kappaB1 p105 negatively regulates TPL-2 MEK kinase activity.

Activation of the oncogenic potential of the MEK kinase TPL-2 (Cot) requires deletion of its C terminus. This mutation also weakens the interaction of TPL-2 with NF-kappaB1 p105 in vitro, although it is unclear whether this is important for the activation of TPL-2 oncogenicity. It is demonstrated here that TPL-2 stability in vivo relies on its high-affinity, stoichiometric association with NF-kappaB1 p105. Formation of this complex occurs as a result of two distinct interactions. The TPL-2 C terminus binds to a region encompassing residues 497 to 534 of p105, whereas the TPL-2 kinase domain interacts with the p105 death domain. Binding to the p105 death domain inhibits TPL-2 MEK kinase activity in vitro, and this inhibition is significantly augmented by concomitant interaction of the TPL-2 C terminus with p105. In cotransfected cells, both interactions are required for inhibition of TPL-2 MEK kinase activity and, consequently, the catalytic activity of a C-terminally truncated oncogenic mutant of TPL-2 is not affected by p105. Thus, in addition to its role as a precursor for p50 and cytoplasmic inhibitor of NF-kappaB, p105 is a negative regulator of TPL-2. Insensitivity of C-terminally truncated TPL-2 to this regulatory mechanism is likely to contribute to its ability to transform cells.

New Regulatory Role for NF-κB Precursor

The NF-kappa B (NF-κB) transcription factors, which have critical roles in immune function, are translated as large precursor proteins that are proteolytically processed to form the mature transcription factor. However, expression of p50, the mature form of NF-κB1, does not fully compensate for loss of the p105 precursor form, which suggests that the precursor may have a distinct function of its own. Waterfield et al. now propose that one such function of p105 is to interact directly with and to inhibit the MAP (mitogen-activated protein) kinase kinase kinase Tpl2. Toll-like receptors recognize infection by binding bacterially derived molecules like lipopolysaccharide (LPS). The Tpl2 protein kinase participates in signaling from Toll-like receptors to the nucleus. Waterfield et al. report that LPS-induced activation of Tpl2 is lost in macrophages from mice lacking p105. The authors confirmed previous reports that p105 interacts with the Tpl2 protein in vivo. Expression of both proteins in 293 cells indicated that the interaction stabilizes the Tpl2 protein, but also inhibits the kinase activity of Tpl2. Tpl2 protein isolated from wild-type macrophages in a complex with p105 lacked kinase activity, and stimulation of cells with LPS caused release of the kinase. The authors' model expands the role of NF-κB1 in LPS signaling from the traditional one of activating target genes (mediated by the mature form) to one in which the precursor form of the transcription factor modulates upstream signals in a distinct gene-regulatory pathway mediated by MAP kinases. M. R. Waterfield, M. Zhang, L. P. Norman, S.-C. Sun. NF-κB1/p105 regulates lipopolysaccharide-stimulated MAP kinase signaling by governing the stability and function of the Tpl2 kinase. Mol. Cell 11 , 685-694 (2003). [Online Journal]

NF-κB1/p105 Regulates Lipopolysaccharide-Stimulated MAP Kinase Signaling by Governing the Stability and Function of the Tpl2 Kinase

NF-kappaB family of transcription factors plays a pivotal role in regulation of immune and inflammatory responses. NF-kappaB is known to function by binding to the kappaB enhancer and directly activating target gene transcription. Here we demonstrate another function of NF-kappaB, in which the nfkappab1 gene product p105 regulates MAP kinase signaling triggered by the bacterial component lipopolysaccharide. p105 exerts this signaling function by controlling the stability and function of an upstream kinase, Tpl2. In macrophages, Tpl2 forms a stable and inactive complex with p105, and activation of Tpl2 involves its dissociation from p105 and subsequent degradation. Thus, p105 functions as a physiological partner and inhibitor of Tpl2, which provides an example of how a transcription factor component regulates upstream signaling events.

Tpl-2 induces IL-2 expression in T-cell lines by triggering multiple signaling pathways that activate NFAT and NF-kappaB.

The Tpl-2 kinase activates the nuclear factor of activated T cells (NFAT) and induces IL-2 expression in T-cell lines. Here we show that the activation of the IL-2 promoter by Tpl-2 is inhibited by mutant signaling molecules that inhibit the mitogen-activated protein kinase (MAPK) or the calcineurin/NFAT pathways and is promoted by combinations of signaling molecules that activate these pathways. We, therefore, conclude that signals generated by the convergence of the MAPK and the calcineurin/NFAT pathway are necessary and sufficient for the activation of the IL-2 promoter by Tpl-2. The activation of both the IL-2 promoter and an NFAT-driven minimal promoter were shown to depend on signals transduced by Raf1. However, it was only the IL-2 promoter whose activation by Tpl-2 was fully blocked by the dominant negative mutant MEK1S218/222A and the MEK1/MEK2 inhibitor PD098059. Since the activation of NFAT is MAPK-dependent these findings suggested that the activation of MAPK by Tpl-2 is either independent or only partially dependent on MEK1 and MEK2. In addition, they suggested that the activation of the IL-2 promoter is under the control of not only NFAT but also a second factor whose activation is MEK-dependent. Experiments in COS-1 and EL-4 cells confirmed both hypotheses and revealed that the second factor activated by Tpl-2 is NF-kappaB. While the activation of the IL-2 promoter and an NFAT-driven minimal promoter by Tpl-2 was fully blocked by the dominant negative mutant NFAT delta418, it was only partially blocked by the calcineurin inhibitor cyclosporin A suggesting that the Tpl-2-mediated NFAT activation is under the control of a combination of calcineurin-dependent and independent pathways. Both pathways were fully blocked by Bcl-2 or Bcl-X(L).

Tpl-2 is an oncogenic kinase that is activated by carboxy-terminal truncation.

Provirus insertion in the last intron of the Tpl-2 gene in retrovirus-induced rat T-cell lymphomas results in the enhanced expression of a carboxy-terminally truncated Tpl-2 kinase. Here we show that the truncated protein exhibits an approximately sevenfold higher catalytic activity and is two- to threefold more efficient in activating the MAPK and SAPK pathways relative to the wild-type protein. The truncated Tpl-2 protein and a GST fusion of the Tpl-2 carboxy-terminal tail interact when coexpressed in Sf9 cells. Their interaction down-regulates the kinase activity of the truncated protein suggesting that tail-directed intramolecular interactions regulate the Tpl-2 kinase. Tpl-2 transgenic mice expressing the wild-type protein from the proximal Lck promoter fail to show a biological phenotype, whereas mice expressing the truncated protein develop large-cell lymphoblastic lymphomas of T-cell origin. These results show that Tpl-2 is an oncogenic kinase that is activated by carboxy-terminal truncation.