ILPIP,a novel anti-apoptotic protein that enhances XIAP-mediated activation of JNK1 and protection against apoptosis

We have previously described a new aspect of the Inhibitor of Apoptosis (IAP) family of proteins anti-apoptotic activity that involves the TAK1/JNK1 signal transduction pathway (1,2). Our findings suggest the existence of a novel mechanism that regulates the anti-apoptotic activity of IAPs that is separate from caspase inhibition but instead involves TAK1-mediated activation of JNK1. In a search for proteins involved in the XIAP/TAK1/JNK1 signaling pathway we isolated by yeast two-hybrid screening a novel X chromosome-linked IAP (XIAP)-interacting protein that we called ILPIP (hILP-Interacting Protein). Whereas ILPIP moderately activates JNK family members when expressed alone, it strongly enhances XIAP-mediated activation of JNK1, JNK2, and JNK3. The expression of a catalytically inactive mutant of TAK1 blocked XIAP/ILPIP synergistic activation of JNK1 thereby implicating TAK1 in this signaling pathway. ILPIP moderately protects against interleukin-1beta converting enzyme- or Fas-induced apoptosis and significantly potentiates the anti-apoptotic activity of XIAP. In vivo co-precipitation experiments show that both ILPIP and XIAP interact with TAK1 and tumor necrosis factor receptor-associated factor 6. Finally, expression of ILPIP did not affect the ability of XIAP to inhibit caspase activation, further supporting the idea that XIAP protection against apoptosis is achieved by two separate mechanisms: one requiring JNK1 activation and a second involving caspase inhibition.     

Restoration of NF-kappaB activation by tumor necrosis factor alpha receptor complex-targeted MEKK3 in receptor-interacting protein-deficient cells

Receptor-interacting protein (RIP) plays a critical role in tumor necrosis factor alpha (TNF-alpha)-induced NF-kappaB activation. However, the mechanism by which RIP mediates TNF-alpha-induced signal transduction is not fully understood. In this study, we reconstituted RIP-deficient Jurkat T cells with a fusion protein composed of full-length MEKK3 and the death domain of RIP (MEKK3-DD). In these cells, MEKK3-DD substitutes for RIP and directly associates with TRADD in TNF receptor complexes following TNF-alpha stimulation. We found that TNF-alpha-induced NF-kappaB activation was fully restored by MEKK3-DD in these cells. In contrast, expression of a fusion protein composed of NEMO, a component of the IkappaB kinase complex, and the death domain of RIP (NEMO-DD) cannot restore TNF-alpha-induced NF-kappaB activation in RIP-deficient cells. These results indicate that the role of RIP is to specifically recruit MEKK3 to the TNF-alpha receptor complex, whereas the forced recruitment of NEMO to the TNF-alpha receptor complex is insufficient for TNF-alpha-induced NF-kappaB activation. Although MEKK2 has a high degree of homology with MEKK3, MEKK2-DD, unlike MEKK3-DD, also fails to restore TNF-alpha-induced NF-kappaB activation in RIP-deficient cells, indicating that RIP-dependent recruitment of MEKK3 plays a specific role in TNF-alpha signaling.     

hKSR-2 inhibits MEKK3-activated MAP kinase and NF-kappaB pathways in inflammation

Kinase suppressor of ras (KSR) and MEKK3 (MAP kinase kinase kinase) are integral members of the MAP kinase pathway. We have recently identified a new isoform of the KSR family named human kinase suppressor of ras-2 (hKSR-2), and demonstrated that hKSR-2 negatively regulates Cot, a MAP3K family member which is important in inflammation and oncogenesis [P.L. Channavajhala, L. Wu, J.W. Cuozzo, J.P. Hall, W. Liu, L.L. Lin, Y. Zhang, J. Biol. Chem. 278 (2003) 47089-47097]. In this report, we provide evidence that hKSR-2 also regulates the activity of MEKK3 (another MAP3K family member) in HEK-293T cells. We demonstrate that hKSR-2 is a negative regulator of MEKK3-mediated activation of MAP kinase (specifically ERK and JNK) and NF-kappaB pathways, and concurrently inhibits MEKK3-mediated interleukin-8 production. We find that while hKSR-2 blocks MEKK3 activation, it has little to no effect on other members of the MAP3K family, including MEKK4, TAK1, and Ras-Raf, suggesting that its effects are selective.     

Cyr61 expression confers resistance to apoptosis in breast cancer MCF-7 cells by a mechanism of NF-kappaB-dependent XIAP up-regulation

The aggressiveness of a tumor is partly attributed to its resistance to chemotherapeutic agent-induced apoptosis. Cysteine-rich 61 (Cyr61), from the CCN gene family, is a secreted and matrix-associated protein, which is involved in many cellular activities such as growth and differentiation. Here we established a cell model system to examine whether stable expression of Cyr61 in MCF-7 cells can confer resistance to apoptosis and identify possible participating mechanisms. We showed that stable cell lines overexpressing Cyr61 had acquired a remarkable resistance to apoptosis induced by paclitaxel, adriamycin, and beta-lapachone. Most interesting, gel shift and reporter assays showed that the Cyr61-overexpressing cells had significantly increased NF-kappaB activity compared with neo control cells. Blockage of NF-kappaB activity in Cyr61-expressing cells by transfecting with a dominant negative (DN)-IkappaB or with an NF-kappaB decoy rendered them more susceptible to anti-cancer drugs-induced apoptosis. In addition, several NF-kappaB-regulated anti-apoptotic genes were examined, and we found that only XIAP showed a significant 3-4-fold increase in mRNA and protein in Cyr61-overexpressing cells but not in neo control cells. Treatment with inhibitor of apoptosis protein (XIAP)-specific antisense, but not sense, oligonucleotides abolished the apoptosis resistance of the Cyr61-overexpressing cells. At the same time, transfection of these stable cells with DN-IkappaB to block NF-kappaB activity also effectively reduced the elevated XIAP level. Function-neutralizing antibodies to alpha(v)beta(3) and alpha(v)beta(5) could inhibit Cyr61-mediated NF-kappaB activation as well as XIAP expression. Taken together, our data suggested that Cyr61 plays an important role in resistance to chemotherapeutic agent-induced apoptosis in human breast cancer MCF-7 cells by a mechanism involving the activation of the integrins/NF-kappaB/XIAP signaling pathway.     

Uncoupling of the signaling and caspase-inhibitory properties of X-linked inhibitor of apoptosis

In addition to its well described function as an enzymatic inhibitor of specific caspases, X-linked inhibitor of apoptosis (X-linked IAP or XIAP) can function as a cofactor in Smad, NF-kappaB, and JNK signaling pathways. However, caspases themselves have been shown to regulate the activity of a number of signaling cascades, raising the possibility that the effect of XIAP in these pathways is indirect. Here we examine this question by introducing point mutations in XIAP predicted to disrupt the ability of the molecule to bind to and inhibit caspases. We show that whereas these mutant variants of XIAP lost caspase-inhibitory activity, they maintained their ability to activate Smad, NF-kappaB, and JNK signaling pathways. Indeed, the signaling properties of the molecule were mapped to domains not directly involved in caspase binding and inhibition. The activation of NF-kappaB by XIAP was dependent on the E3 ubiquitin ligase activity of the RING domain. On the other hand, the ability of XIAP to activate Smad-dependent signaling was mapped to the third baculoviral IAP repeat (BIR) and loop regions of the molecule. Thus, the anti-apoptotic and signaling properties of XIAP can be uncoupled.     

Nuclear factor-kappa B activation by the CXC chemokine melanoma growth-stimulatory activity/growth-regulated protein involves the MEKK1/p38 mitogen-activated protein kinase pathway

Melanoma growth stimulatory activity/growth-regulated protein (MGSA/GRO), a CXC chemokine, plays an important role in inflammation, wound healing, growth regulation, angiogenesis, and tumorigenesis. Constitutive expression of MGSA/GROalpha in melanoma tumors is associated with constitutive nuclear factor (NF)-kappaB activity. We show here that either exogenous addition or continuous expression of MGSA/GROalpha in immortalized melanocytes enhances NF-kappaB activation, as well as mitogen-activated protein (MAP) kinase kinase kinase (MEKK) 1, MAP kinase kinase (MEK) 3/6, and p38 MAP kinase activation. Expression of dominant negative M-Ras (S27N), dominant negative MEKK1 (K432M), or specific chemical inhibitors for p38 MAP kinase (SB202190 and SB203580) block MGSA/GROalpha-induced NF-kappaB transactivation, demonstrating that Ras, MEKK1, and p38 are involved in the signal pathways of MGSA/GROalpha activation of NF-kappaB. Expression of dominant active Ras or dominant active MEKK1 alone can also stimulate NF-kappaB activation. The expression of dominant negative MEKK1 inhibits the Ras-induced NF-kappaB activation, suggesting that MEKK1 is a downstream target of Ras. Moreover, MGSA/GROalpha induction of NF-kappaB is independent of the MEK1/ERK cascade, because MGSA/GROalpha failed to increase ERK and ELK activation, and specific chemical inhibitors for MEK1 (PD98059) had no effect on MGSA/GROalpha-enhanced NF-kappaB activation. These data demonstrate that NF-kappaB activation is required for MGSA/GROalpha-induced melanocyte transformation through a Ras/MEKK1/p38 cascade in melanocytes.     

XIAP regulates cytosol-specific innate immunity to Listeria infection

The inhibitor of apoptosis protein (IAP) family has been implicated in immune regulation, but the mechanisms by which IAP proteins contribute to immunity are incompletely understood. We show here that X-linked IAP (XIAP) is required for innate immune control of Listeria monocytogenes infection. Mice deficient in XIAP had a higher bacterial burden 48 h after infection than wild-type littermates, and exhibited substantially decreased survival. XIAP enhanced NF-kappaB activation upon L. monocytogenes infection of activated macrophages, and prolonged phosphorylation of Jun N-terminal kinase (JNK) specifically in response to cytosolic bacteria. Additionally, XIAP promoted maximal production of pro-inflammatory cytokines upon bacterial infection in vitro or in vivo, or in response to combined treatment with NOD2 and TLR2 ligands. Together, our data suggest that XIAP regulates innate immune responses to L. monocytogenes infection by potentiating synergy between Toll-like receptors (TLRs) and Nod-like receptors (NLRs) through activation of JNK- and NF-kappaB-dependent signaling.     

X-linked inhibitor of apoptosis protein functions as a cofactor in transforming growth factor-beta signaling

X-linked inhibitor of apoptosis protein (XIAP) is a potent suppressor of apoptotic cell death, which functions by directly inhibiting caspases, the principal effectors of apoptosis. Here we report that XIAP can also function as a cofactor in the regulation of gene expression by transforming growth factor-beta (TGF-beta). XIAP, but not the related proteins c-IAP1 or c-IAP2, associated with several members of the type I class of the TGF-beta receptor superfamily and potentiated TGF-beta-induced signaling. Although XIAP-mediated activation of c-Jun N-terminal kinase and nuclear factor kappa B was found to require the TGF-beta signaling intermediate Smad4, the ability of XIAP to suppress apoptosis was found to be Smad4-independent. These data implicate a role for XIAP in TGF-beta-mediated signaling that is distinct from its anti-apoptotic functions.     

Ubiquitin‐dependent regulation of MEKK2/3‐MEK5‐ERK5 signaling module by XIAP and cIAP1

Mitogen‐activated protein kinases (MAPKs) are highly conserved protein kinase modules, and they control fundamental cellular processes. While the activation of MAPKs has been well studied, little is known on the mechanisms driving their inactivation. Here we uncover a role for ubiquitination in the inactivation of a MAPK module. Extracellular‐signal‐regulated kinase 5 (ERK5) is a unique, conserved member of the MAPK family and is activated in response to various stimuli through a three‐tier cascade constituting MEK5 and MEKK2/3. We reveal an unexpected role for Inhibitors of Apoptosis Proteins (IAPs) in the inactivation of ERK5 pathway in a bimodal manner involving direct interaction and ubiquitination. XIAP directly interacts with MEKK2/3 and competes with PB1 domain‐mediated binding to MEK5. XIAP and cIAP1 conjugate predominantly K63‐linked ubiquitin chains to MEKK2 and MEKK3 which directly impede MEK5–ERK5 interaction in a trimeric complex leading to ERK5 inactivation. Consistently, loss of XIAP or cIAP1 by various strategies leads to hyperactivation of ERK5 in normal and tumorigenic cells. Loss of XIAP promotes differentiation of human primary skeletal myoblasts to myocytes in a MEKK2/3‐ERK5‐dependent manner. Our results reveal a novel, obligatory role for IAPs and ubiquitination in the physical and functional disassembly of ERK5‐MAPK module and human muscle cell differentiation.     

X-linked inhibitor of apoptosis (XIAP) inhibits c-Jun N-terminal kinase 1 (JNK1) activation by transforming growth factor beta1 (TGF-beta1) through ubiquitin-mediated proteosomal degradation of the TGF-beta1-activated kinase 1 (TAK1)

Active NF-kappaB renders malignant hepatocytes refractory to the growth inhibitory and pro-apoptotic properties of transforming growth factorbeta1 (TGF-beta1). NF-kappaB counteracts TGF-beta1-induced apoptosis through up-regulation of downstream target genes, such as XIAP and Bcl-X(L), which in turn inhibit the intrinsic pathway of apoptosis. In addition, induction of NF-kappaB by TGF-beta1 inhibits JNK signaling, thereby attenuating TGF-beta1-induced cell death of normal hepatocytes. However, the mechanism involved in the negative cross-talk between the NF-kappaB and JNK pathways during TGF-beta1 signaling has not been determined. In this study, we have identified the XIAP gene as one of the critical mediators of NF-kappaB-mediated suppression of JNK signaling. We show that NF-kappaB plays a role in the up-regulation of XIAP gene expression in response to TGF-beta1 treatment and forms a TGF-beta1-inducible complex with TAK1. Furthermore, we show that the RING domain of XIAP mediates TAK1 polyubiquitination, which then targets this molecule for proteosomal degradation. Down-regulation of TAK1 protein expression inhibits TGF-beta1-mediated activation of JNK and apoptosis. Conversely, silencing of XIAP promotes persistent JNK activation and potentiates TGF-beta1-induced apoptosis. Collectively, our findings identify a novel mechanism for the regulation of JNK activity by NF-kappaB during TGF-beta1 signaling and raise the possibility that pharmacologic inhibition of the NF-kappaB/XIAP signaling pathway might selectively abolish the pro-oncogenic activity of TGF-beta1 in advanced hepatocellular carcinomas (HCCs) without affecting the pro-apoptotic effects of TGF-beta1 involved in normal liver homeostasis.     

Casein kinase 1alpha interacts with RIP1 and regulates NF-kappaB activation

Tumor necrosis factor alpha (TNFalpha) triggers a signaling pathway converging on the activation of NF-kappaB, which forms the basis for many physiological and pathological processes. In a kinase gene screen using a NF-kappaB reporter, we observed that overexpression of casein kinase 1alpha (CK1alpha) enhanced TNFalpha-induced NF-kappaB activation, and a CK1alpha kinase dead mutant, CK1alpha (K46A), reduced NF-kappaB activation induced by TNFalpha. We subsequently demonstrated that CK1alpha interacted with receptor interacting protein 1 (RIP1) but not with TRADD, TRAF2, MEKK3, IKKalpha, IKKbeta, or IKKgamma in mammalian cells. RIP1 is an indispensable molecule in TNFalpha/NF-kappaB signaling. We demonstrated that CK1alpha interacted with and phosphorylated RIP1 at the intermediate domain. Finally, we showed that CK1alpha enhanced RIP1-mediated NF-kappaB activation. Taken together, our studies suggest that CK1alpha is another kinase that regulates RIP1 function in NF-kappaB activation.