Lipopolysaccharide activation of the TPL-2/MEK/extracellular signal-regulated kinase mitogen-activated protein kinase cascade is regulated by IkappaB kinase-induced proteolysis of NF-kappaB1 p105

The MEK kinase TPL-2 (also known as Cot) is required for lipopolysaccharide (LPS) activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase cascade in macrophages and consequent upregulation of genes involved in innate immune responses. In resting cells, TPL-2 forms a stoichiometric complex with NF-kappaB1 p105, which negatively regulates its MEK kinase activity. Here, it is shown that lipopolysaccharide (LPS) stimulation of primary macrophages causes the release of both long and short forms of TPL-2 from p105 and that TPL-2 MEK kinase activity is restricted to this p105-free pool. Activation of TPL-2, MEK, and ERK by LPS is also demonstrated to require proteasome-mediated proteolysis. p105 is known to be proteolysed by the proteasome following stimulus-induced phosphorylation of two serines in its PEST region by the IkappaB kinase (IKK) complex. Expression of a p105 point mutant, which is not susceptible to signal-induced proteolysis, in RAW264.7 macrophages impairs LPS-induced release of TPL-2 from p105 and its subsequent activation of MEK. Furthermore, expression of wild-type but not mutant p105 reconstitutes LPS stimulation of MEK and ERK phosphorylation in primary NF-kappaB1-deficient macrophages. Consistently, pharmacological blockade of IKK inhibits LPS-induced release of TPL-2 from p105 and TPL-2 activation. These data show that IKK-induced p105 proteolysis is essential for LPS activation of TPL-2, thus revealing a novel function of IKK in the regulation of the ERK MAP kinase cascade.     

Phosphorylation of TPL-2 on serine 400 is essential for lipopolysaccharide activation of extracellular signal-regulated kinase in macrophages

Tumor progression locus 2 (TPL-2) kinase is essential for Toll-like receptor 4 activation of the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) and for upregulation of the inflammatory cytokine tumor necrosis factor (TNF) in lipopolysaccharide (LPS)-stimulated macrophages. LPS activation of ERK requires TPL-2 release from associated NF-kappaB1 p105, which blocks TPL-2 access to its substrate, the ERK kinase MEK. Here we demonstrate that TPL-2 activity is also regulated independently of p105, since LPS stimulation was still needed for TPL-2-dependent activation of ERK in Nfkb1(-/-) macrophages. In wild-type macrophages, LPS induced the rapid phosphorylation of serine (S) 400 in the TPL-2 C-terminal tail. Mutation of this conserved residue to alanine (A) blocked the ability of retrovirally expressed TPL-2 to induce the activation of ERK in LPS-stimulated Nfkb1(-/-) macrophages. TPL-2(S400A) expression also failed to reconstitute LPS activation of ERK and induction of TNF in Map3k8(-/-) macrophages, which lack endogenous TPL-2. Consistently, the S400A mutation was found to block LPS stimulation of TPL-2 MEK kinase activity. Thus, induction of TPL-2 MEK kinase activity by LPS stimulation of macrophages requires TPL-2 phosphorylation on S400, in addition to its release from NF-kappaB1 p105. Oncogenic C-terminal truncations of TPL-2 that remove S400 could promote its transforming potential by eliminating this critical control step.     

ABIN-2 forms a ternary complex with TPL-2 and NF-kappa B1 p105 and is essential for TPL-2 protein stability

NF-kappa B1 p105 forms a high-affinity, stoichiometric interaction with TPL-2, a MEK kinase essential for TLR4 activation of the ERK mitogen-activated protein kinase cascade in lipopolysaccharide (LPS)-stimulated macrophages. Interaction with p105 is required to maintain TPL-2 metabolic stability and also negatively regulates TPL-2 MEK kinase activity. Here, affinity purification identified A20-binding inhibitor of NF-kappa B 2 (ABIN-2) as a novel p105-associated protein. Cotransfection experiments demonstrated that ABIN-2 could interact with TPL-2 in addition to p105 but preferentially formed a ternary complex with both proteins. Consistently, in unstimulated bone marrow-derived macrophages (BMDMs), a substantial fraction of endogenous ABIN-2 was associated with both p105 and TPL-2. Although the majority of TPL-2 in these cells was complexed with ABIN-2, the pool of TPL-2 which could activate MEK after LPS stimulation was not, and LPS activation of TPL-2 was found to correlate with its release from ABIN-2. Depletion of ABIN-2 by RNA interference dramatically reduced steady-state levels of TPL-2 protein without affecting levels of TPL-2 mRNA or p105 protein. In addition, ABIN-2 increased the half-life of cotransfected TPL-2. Thus, optimal TPL-2 stability in vivo requires interaction with ABIN-2 as well as p105. Together, these data raise the possibility that ABIN-2 functions in the TLR4 signaling pathway which regulates TPL-2 activation.     

Coordinate Regulation of TPL-2 and NF-κB Signaling in Macrophages by NF-κB1 p105

The role of IκB kinase (IKK)-induced proteolysis of NF-κB1 p105 in innate immune signaling was investigated using macrophages from Nfkb1(SSAA/SSAA) mice, in which the IKK target serines on p105 are mutated to alanines. We found that the IKK/p105 signaling pathway was essential for TPL-2 kinase activation of extracellular signal-regulated kinase (ERK) mitogen-activate protein (MAP) kinase and modulated the activation of NF-κB. The Nfkb1(SSAA) mutation prevented the agonist-induced release of TPL-2 from its inhibitor p105, which blocked activation of ERK by lipopolysaccharide (LPS), tumor necrosis factor (TNF), CpG, tripalmitoyl-Cys-Ser-Lys (Pam(3)CSK), poly(I · C), flagellin, and R848. The Nfkb1(SSAA) mutation also prevented LPS-induced processing of p105 to p50 and reduced p50 levels, in addition to decreasing the nuclear translocation of RelA and cRel. Reduced p50 in Nfkb1(SSAA/SSAA) macrophages significantly decreased LPS induction of the IκBζ-regulated Il6 and Csf2 genes. LPS upregulation of Il12a and Il12b mRNAs was also impaired although specific blockade of TPL-2 signaling increased expression of these genes at late time points. Activation of TPL-2/ERK signaling by IKK-induced p105 proteolysis, therefore, induced a negative feedback loop to downregulate NF-κB-dependent expression of the proinflammatory cytokine interleukin-12 (IL-12). Unexpectedly, TPL-2 promoted soluble TNF production independently of IKK-induced p105 phosphorylation and its ability to activate ERK, which has important implications for the development of anti-inflammatory drugs targeting TPL-2.     

NF-κB1 inhibits TLR-induced IFN-β production in macrophages through TPL-2-dependent ERK activation

Although NF-κB1 p50/p105 has critical roles in immunity, the mechanism by which NF-κB1 regulates inflammatory responses is unclear. In this study, we analyzed the gene expression profile of LPS-stimulated Nfkb1(-/-) macrophages that lack both p50 and p105. Deficiency of p50/p105 selectively increased the expression of IFN-responsive genes, which correlated with increased IFN-β expression and STAT1 phosphorylation. IFN Ab-blocking experiments indicated that increased STAT1 phosphorylation and expression of IFN-responsive genes observed in the absence of p50/p105 depended upon autocrine IFN-β production. Markedly higher serum levels of IFN-β were observed in Nfkb1(-/-) mice than in wild-type mice following LPS injection, demonstrating that Nfkb1 inhibits IFN-β production under physiological conditions. TPL-2, a mitogen-activated protein kinase kinase kinase stabilized by association with the C-terminal ankyrin repeat domain of p105, negatively regulates LPS-induced IFN-β production by macrophages via activation of ERK MAPK. Retroviral expression of TPL-2 in Nfkb1(-/-) macrophages, which are deficient in endogenous TPL-2, reduced LPS-induced IFN-β secretion. Expression of the C-terminal ankyrin repeat domain of p105 in Nfkb1(-/-) macrophages, which rescued LPS activation of ERK, also inhibited IFN-β expression. These data indicate that p50/p105 negatively regulates LPS-induced IFN signaling in macrophages by stabilizing TPL-2, thereby facilitating activation of ERK.     

The C terminus of p53 family proteins is a cell fate determinant

The p53 tumor suppressor is the most commonly mutated gene in human cancers. The ability of p53 to induce cell cycle arrest, apoptosis, DNA repair, and other p53-dependent activities is well known; however, the mechanism by which p53 induces a specific activity over another is unclear. Here, we showed that stringent regulation of and by p53 family isoforms facilitates differential target gene expression and thus determines cell fate. Through the use of engineered deletion mutants, we found that activation domain 2 is required for induction of the proapoptotic target gene insulin-like growth factor binding protein 3 (IGFBP3) by p53 and that the basic domain inhibits induction of this gene by p53. Thus, for the first time we provide evidence that the basic domain of p53 is inhibitory in vivo as has been determined in vitro. We also showed that the in vivo inhibitory activity of the basic domain depends upon activation domain 1, such that combined deletion of activation domain 1 and the basic domain was required to alleviate the inhibition by the basic domain. Importantly, deletion of the inhibitory functional domains, namely N-terminal activation domain 1 and the C-terminal basic domain, is paralleled in nature. We found that the IGFBP3 promoter was activated by p53(DeltaNDeltaBD), which mimics a naturally occurring N- and C-terminally truncated human p53 isoform, and by p53AS, a C-terminally truncated murine p53 isoform generated through alternative splicing, but not by full-length human or murine p53. In addition, we found that the C termini of p63 and p73 inhibit the induction of IGFBP3, such that C-terminally truncated p63 and p73 isoforms induce the expression of IGFBP3, whereas full-length ones cannot. We also demonstrated that IGFBP3 is an important effector of the apoptosis induced by N- and C-terminally truncated p53, such that knockdown of IGFBP3 by using an IGFBP3 neutralizing antibody or IGFBP3 small interfering RNA partially rescues the cell death induced by N- and C-terminally truncated p53. In addition, we identified that histone deacetylase activity, not p53 DNA binding ability, governs the regulation of IGFBP3 by full-length p53 family proteins, as inhibition of histone deacetylases restores the induction of IGFBP3 by exogenous full-length p53, p63, and p73 proteins. Furthermore, we found that activation of p53 or inhibition of histone deacetylases alone was not sufficient to induce IGFBP3; however, combined treatment endowed endogenous p53 with this activity. To better understand the significance of this regulation, we performed a microarray study and identified several target genes differentially regulated by full-length p53 and p53 lacking the N-terminal activation domain 1 and the C-terminal basic domain. Taken together, our data suggest a novel mechanism by which p53 family proteins differentially regulate gene expression and provide an insight for designing a combined therapy for cancer treatment.     

Activation of the Abl tyrosine kinase in vivo by Src homology 3 domains from the Src homology 2/Src homology 3 adaptor Nck.

The nonreceptor tyrosine kinase c-Abl is tightly regulated in vivo, but the mechanisms that normally repress its activity are not well understood. We find that a construct encoding the first two Src homology 3 (SH3) domains of the Src homology 2/SH3 adaptor protein Nck can activate c-Abl in human 293T cells. A myristoylated Nck SH3 domain construct, which is expected to localize to membranes, potently activated Abl when expressed at low levels. An unmyristoylated Nck SH3 domain construct, which localizes to the cytosol and nucleus, also activated Abl but only at high levels of expression. Activation by both myristoylated and unmyristoylated Nck constructs required the C terminus of Abl; a C-terminally truncated form of Abl was not activated, although this construct could still be activated by deletion of its SH3 domain. Activation did not require the major binding sites in the Abl C terminus for Nck SH3 domains, however, suggesting that the mechanism of activation does not require direct binding to the C terminus. Activation of c-Abl by Nck SH3 domains provides a robust experimental system for analyzing the mechanisms that normally repress Abl activity and how that normal regulation can be perturbed.     

Mouse Receptor Interacting Protein 3 Does Not Contain a Caspase-Recruiting or a Death Domain but Induces Apoptosis and Activates NF-κB

The death domain-containing receptor superfamily and their respective downstream mediators control whether or not cells initiate apoptosis or activate NF-kappaB, events critical for proper immune system function. A screen for upstream activators of NF-kappaB identified a novel serine-threonine kinase capable of activating NF-kappaB and inducing apoptosis. Based upon domain organization and sequence similarity, this novel kinase, named mRIP3 (mouse receptor interacting protein 3), appears to be a new RIP family member. RIP, RIP2, and mRIP3 contain an N-terminal kinase domain that share 30 to 40% homology. In contrast to the C-terminal death domain found in RIP or the C-terminal caspase-recruiting domain found in RIP2, the C-terminal tail of mRIP3 contains neither motif and is unique. Despite this feature, overexpression of the mRIP3 C terminus is sufficient to induce apoptosis, suggesting that mRIP3 uses a novel mechanism to induce death. mRIP3 also induced NF-kappaB activity which was inhibited by overexpression of either dominant-negative NIK or dominant-negative TRAF2. In vitro kinase assays demonstrate that mRIP3 is catalytically active and has autophosphorylation site(s) in the C-terminal domain, but the mRIP3 catalytic activity is not required for mRIP3 induced apoptosis and NF-kappaB activation. Unlike RIP and RIP2, mRIP3 mRNA is expressed in a subset of adult tissues and is thus likely to be a tissue-specific regulator of apoptosis and NF-kappaB activity. While the lack of a dominant-negative mutant precludes linking mRIP3 to a known upstream regulator, characterizing the expression pattern and the in vitro functions of mRIP3 provides insight into the mechanism(s) by which cells modulate the balance between survival and death in a cell-type-specific manner.     

Isolation of the protein kinase TAO2 and identification of its mitogen-activated protein kinase/extracellular signal-regulated kinase kinase binding domain.

We previously reported the cloning of the thousand and one-amino acid protein kinase 1 (TAO1), a rat homolog of the Saccharomyces cerevisiae protein kinase sterile 20 protein. Here we report the complete sequence and properties of a related rat protein kinase TAO2. Like TAO1, recombinant TAO2 selectively activated mitogen-activated protein/extracellular signal-regulated kinase kinases (MEKs) 3, 4, and 6 of the stress-responsive mitogen-activated protein kinase pathways in vitro and copurified with MEK3 endogenous to Sf9 cells. To examine TAO2 interactions with MEKs, the MEK binding domain of TAO2 was localized to an approximately 135-residue sequence just C-terminal to the TAO2 catalytic domain. In vitro this MEK binding domain associated with MEKs 3 and 6 but not MEKs 1, 2, or 4. Using chimeric MEK proteins, we found that the MEK N terminus was sufficient for binding to TAO2. Catalytic activity of full-length TAO2 enhanced its binding to MEKs. However, neither the autophosphorylation of the MEK binding domain of TAO2 nor the activity of MEK itself was required for MEK binding. These results suggest that TAO proteins lie in stress-sensitive kinase cascades and define a mechanism by which these kinases may organize downstream targets.     

p53 deficiency increases transformation by v-Abl and rescues the ability of a C-terminally truncated v-Abl mutant to induce pre-B lymphoma in vivo

Abelson murine leukemia virus (A-MuLV) is an acute transforming retrovirus that preferentially transforms early B-lineage cells both in vivo and in vitro. Its transforming protein, v-Abl, is a tyrosine kinase related to v-Src but containing an extended C-terminal domain. Many mutations affecting the C-terminal portion of the molecule block the pre-B-transforming activity of v-Abl without affecting the fibroblast-transforming ability. In this study we have determined the abilities of both wild-type and C-terminally truncated (p90) forms of v-Abl to transform cells from p53(-/-) mice. Lack of p53 increases the susceptibility of bone marrow cells to transformation by v-Abl by a factor of more than 7 but does not alter v-Abl's preference for B220(+) IgM(-) pre-B cells. p53-deficient mice have earlier tumor onset, more rapid tumor progression, and decreased survival time following A-MuLV infection, but all of the tumors are pre-B lymphomas. Thus, p53-dependent pathways inhibit v-Abl transformation but play no role in conferring preferential transformation of pre-B cells. Surprisingly, the C-terminally truncated form of v-Abl (p90) transforms pre-B cells very efficiently in mice lacking p53, thus demonstrating that the C terminus of v-Abl does not determine preB tropism but is necessary to overcome p53-dependent inhibition of transformation.     

p105.Ikappa Bgamma and prototypical Ikappa Bs use a similar mechanism to bind but a different mechanism to regulate the subcellular localization of NF-kappa B

p105, also known as NF-kappaB1, is an atypical IkappaB molecule with a multi-domain organization distinct from other prototypical IkappaBs, like IkappaBalpha and IkappaBbeta. To understand the mechanism by which p105 binds and inhibits NF-kappaB, we have used both p105 and its C-terminal inhibitory segment known as IkappaBgamma for our study. We show here that one IkappaBgamma molecule binds to NF-kappaB dimers wherein at least one NF-kappaB subunit is p50. We suggest that the obligatory p50 subunit in IkappaBgamma.NF-kappaB complexes is equivalent to the N-terminal p50 segment in all p105.NF-kappaB complexes. The nuclear localization signal (NLS) of the obligatory p50 subunit is masked by IkappaBgamma, whereas the NLS of the nonobligatory NF-kappaB subunit is exposed. Thus, the global binding mode of all IkappaB.NF-kappaB complexes seems to be similar where one obligatory (or specific) NF-kappaB subunit makes intimate contact with IkappaB and the nonobligatory (or nonspecific) subunit is bound primarily through its ability to dimerize. In the case of IkappaBalpha and IkappaBbeta, the specific NF-kappaB subunit in the complex is p65. In contrast to IkappaBalpha.NF-kappaB complexes, where the exposed NLS of the nonspecific subunit imports the complex to the nucleus, p105.NF-kappaB and IkappaBgamma.NF-kappaB complexes are cytoplasmic. We show that the death domain of p105 (also of IkappaBgamma) is essential for the cytoplasmic sequestration of NF-kappaB by p105 and IkappaBgamma. However, the death domain does not mask the exposed NLS of the complex. We also demonstrate that the death domain alone is not sufficient for cytoplasmic retention and instead functions only in conjunction with other parts in the three-dimensional scaffold formed by the association of the ankyrin repeat domain (ARD) and NF-kappaB dimer. We speculate that additional cytoplasmic protein(s) may sequester the entire p105.NF-kappaB complex by binding through the death domain and other segments, including the exposed NLS.     

Identification of a novel homotypic interaction motif required for the phosphorylation of receptor-interacting protein (RIP) by RIP3.

Receptor-interacting protein (RIP), a Ser/Thr kinase component of the tumor necrosis factor (TNF) receptor-1 signaling complex, mediates activation of the nuclear factor kappaB (NF-kappaB) pathway. RIP2 and RIP3 are related kinases that share extensive sequence homology with the kinase domain of RIP. Unlike RIP, which has a C-terminal death domain, and RIP2, which has a C-terminal caspase activation and recruitment domain, RIP3 possesses a unique C terminus. RIP3 binds RIP through this unique C-terminal segment to inhibit RIP- and TNF receptor-1-mediated NF-kappaB activation. We have identified a unique homotypic interaction motif at the C terminus of both RIP and RIP3 that is required for their association. Sixty-four amino acids within RIP3 and 88 residues within RIP are sufficient for interaction of the two proteins. This interaction is a prerequisite for RIP3-mediated phosphorylation of RIP and subsequent attenuation of TNF-induced NF-kappaB activation.