A Pilot Phase II Study of Erlotinib for the Treatment of Patients with Relapsed/Refractory Acute Myeloid Leukemia.

Erlotinib, an epidermal growth factor receptor (EGFR) inhibitor, may have off-target activity inducing acute myeloid leukemia (AML) differentiation, possibly through SYK inhibition. We investigated erlotinib in a pilot phase II study for efficacy in relapsed/refractory AML patients at a dose of 150 mg once daily in 28-day cycles. Twenty-nine patients were treated for a median of 29 days (range 12-142 days). Seven patients (24%) received > 1 cycle of therapy and 12 (41%) discontinued treatment before day 28 due to disease progression. One patient (3%) achieved complete remission and 2 (7%) a > 50% reduction in blasts. The most common toxicities associated with erlotinib were fatigue in 10 patients (34%), diarrhea in 10 (34%), nausea in 8 (28%), and rash in 7 (24%). Only 2 patients (7%) had study drug-related adverse events requiring dose reductions and eventual discontinuation. The main reason for treatment discontinuation was disease progression in 26 patients (90%). All patients had died by the time of the last follow-up. Progression of disease was the primary cause of death in all patients. Median overall survival was 14 weeks (range 2.3-96.9 weeks) and median event-free survival was 5 weeks (range 1.7-21.0 weeks). Erlotinib as a single agent has limited clinical efficacy in patients with relapsed/refractory AML.

Critical role of monoubiquitination of histone H2AX protein in histone H2AX phosphorylation and DNA damage response.

DNA damage response is an important surveillance mechanism used to maintain the integrity of the human genome in response to genotoxic stress. Histone variant H2AX is a critical sensor that undergoes phosphorylation at serine 139 upon genotoxic stress, which provides a docking site to recruit the mediator of DNA damage checkpoint protein 1 (MDC1) and DNA repair protein complex to sites of DNA breaks for DNA repair. Here, we show that monoubiquitination of H2AX is induced upon DNA double strand breaks and plays a critical role in H2AX Ser-139 phosphorylation (γ-H2AX), in turn facilitating the recruitment of MDC1 to DNA damage foci. Mechanistically, we show that monoubiquitination of H2AX induced by RING finger protein 2 (RNF2) is required for the recruitment of active ataxia telangiectasia mutated to DNA damage foci, thus affecting the formation of γ-H2AX. Importantly, a defect in monoubiquitination of H2AX profoundly enhances ionizing radiation sensitivity. Our study therefore suggests that monoubiquitination of H2AX is an important step for DNA damage response and may have important clinical implications for the treatment of cancers.

Structural basis for the lack of E2 interaction in the RING domain of TRAF2.

TRAF proteins are intracellular signal transducers for a number of immune receptor superfamilies. Specifically, TRAF2 interacts with members of the TNF receptor superfamily and connects the receptors to downstream signaling proteins. It has been assumed that TRAF2 is a ubiquitin ligase like TRAF6 and mediates K63-linked polyubiquitination of RIP1, a kinase pivotal in TNFalpha-induced NF-kappaB activation. Here we report the crystal structure of the RING and the first zinc finger domains of TRAF2. We show that the TRAF2 RING structure is very different from the known TRAF6 RING structure. The differences are multifaceted, including amino acid differences at the critical Ubc13-interacting site, local conformational differences, and a unique nine-residue insertion between the RING domain and the first zinc finger in TRAF2. These structural differences prevent TRAF2 from interacting with Ubc13 and other related E2s via steric clash and unfavorable interfaces. Our structural observation should prompt a re-evaluation of the role of TRAF2 in TNFalpha signaling and may indicate that TRAF2-associated proteins such as cIAPs may be the ubiquitin ligases for NF-kappaB signaling.

The docking protein Gab1 is an essential component of an indirect mechanism for fibroblast growth factor stimulation of the phosphatidylinositol 3-kinase/Akt antiapoptotic pathway.

The docking protein Gab1 has been implicated as a mediator of multiple signaling pathways that are activated by a variety of receptor tyrosine kinases and cytokines. We have previously proposed that fibroblast growth factor 1 (FGF1) stimulation of tyrosine phosphorylation of Gab1 and recruitment of phosphatidylinositol (PI) 3-kinase are mediated by an indirect mechanism in which the docking protein fibroblast receptor substrate 2alpha (FRS2alpha) plays a critical role. In this report, we explore the role of Gab1 in FGF1 signaling by using mouse embryo fibroblasts (MEFs) derived from Gab1(-/-) or FRS2alpha(-/-) mice. We demonstrate that Gab1 is essential for FGF1 stimulation of both PI 3-kinase and the antiapoptotic protein kinase Akt, while FGF1-induced mitogen-activated protein kinase (MAPK) stimulation is not affected by Gab1 deficiency. To test the indirect mechanism for FGF1 stimulation of PI 3-kinase and Akt, we use a chimeric docking protein composed of the membrane targeting signal and the phosphotyrosine-binding domain of FRS2alpha fused to the C-terminal portion of Gab1, the region including the binding sites for the complement of signaling proteins that are recruited by Gab1. We demonstrate that expression of the chimeric docking protein in Gab1(-/-) MEFs rescues PI 3-kinase and the Akt responses, while expression of the chimeric docking protein in FRS2alpha(-/-) MEFs rescues stimulation of both Akt and MAPK. These experiments underscore the essential role of Gab1 in FGF1 stimulation of the PI 3-kinase/Akt signaling pathway and provide further support for the indirect mechanism for FGF1 stimulation of PI 3-kinase involving regulated assembly of a multiprotein complex.

TRAFs in RANK signaling.

Members of the tumor necrosis factor (TNF) family govern many diverse physiological and cellular responses including cellular proliferation, differentiation, and apoptosis. Ligands of this family interact through a distinct set of specific receptors that lack enzymatic activity and therefore are dependent on the association of adaptor molecules. One receptor/ligand pair known as receptor activator of nuclear factor-kappa B (RANK) and RANK ligand (RANKL) regulates bone remodeling, mammary gland development, and lymph node organogenesis. RANK interacts with five members of the TNF receptor-associated factor (TRAF) family, of which TRAF6 is indispensable for its signaling capability. An accumulation of evidence from various research laboratories indicates TRAFs, but more importantly TRAF6, is the key to understanding how RANKL links cytoplasmic signaling to the nuclear transcriptional program.

Carfilzomib Triggers Cell Death in Chronic Lymphocytic Leukemia by Inducing Proapoptotic and Endoplasmic Reticulum Stress Responses.

PURPOSE: Carfilzomib, while active in B-cell neoplasms, displayed heterogeneous response in chronic lymphocytic leukemia (CLL) samples from patients and showed interpatient variability to carfilzomib-induced cell death. To understand this variability and predict patients who would respond to carfilzomib, we investigated the mechanism by which carfilzomib induces CLL cell death. EXPERIMENTAL DESIGN: Using CLL patient samples and cell lines, complementary knockdown and knockout cells, and carfilzomib-resistant cell lines, we evaluated changes in intracellular networks to identify molecules responsible for carfilzomib's cytotoxic activity. Lysates from carfilzomib-treated cells were immunoblotted for molecules involved in ubiquitin, apoptotic, and endoplasmic reticulum (ER) stress response pathways and results correlated with carfilzomib cytotoxic activity. Coimmunoprecipitation and pull-down assays were performed to identify complex interactions among MCL-1, Noxa, and Bak. RESULTS: Carfilzomib triggered ER stress and activation of both the intrinsic and extrinsic apoptotic pathways through alteration of the ubiquitin proteasome pathway. Consequently, the transcription factor CCAAT/enhancer-binding protein homology protein (CHOP) accumulated in response to carfilzomib, and CHOP depletion conferred protection against cytotoxicity. Carfilzomib also induced accumulation of MCL-1 and Noxa, whereby MCL-1 preferentially formed a complex with Noxa and consequently relieved MCL-1's protective effect on sequestering Bak. Accordingly, depletion of Noxa or both Bak and Bax conferred protection against carfilzomib-induced cell death. CONCLUSIONS: Collectively, carfilzomib induced ER stress culminating in activation of intrinsic and extrinsic caspase pathways, and we identified the CHOP protein level as a biomarker that could predict sensitivity to carfilzomib in CLL. Clin Cancer Res; 22(18); 4712-26. ©2016 AACR.

The docking protein Gab1 is the primary mediator of EGF-stimulated activation of the PI-3K/Akt cell survival pathway.

BACKGROUND: Gab1 is a docking protein that recruits phosphatidylinositol-3 kinase (PI-3 kinase) and other effector proteins in response to the activation of many receptor tyrosine kinases (RTKs). As the autophosphorylation sites on EGF-receptor (EGFR) do not include canonical PI-3 kinase binding sites, it is thought that EGF stimulation of PI-3 kinase and its downstream effector Akt is mediated by an indirect mechanism. RESULTS: We used fibroblasts isolated from Gab1-/- mouse embryos to explore the mechanism of EGF stimulation of the PI-3 kinase/Akt anti-apoptotic cell signaling pathway. We demonstrate that Gab1 is essential for EGF stimulation of PI-3 kinase and Akt in these cells and that these responses are mediated by complex formation between p85, the regulatory subunit of PI-3 kinase, and three canonical tyrosine phosphorylation sites on Gab1. Furthermore, complex formation between Gab1 and the protein tyrosine phosphatase Shp2 negatively regulates Gab1 mediated PI-3 kinase and Akt activation following EGF-receptor stimulation. We also demonstrate that tyrosine phosphorylation of ErbB3 may lead to recruitment and activation of PI-3 kinase and Akt in Gab1-/- MEFs. CONCLUSIONS: The primary mechanism of EGF-induced stimulation of the PI-3 kinase/Akt anti-apoptotic pathway occurs via the docking protein Gab1. However, in cells expressing ErbB3, EGF and neuroregulin can stimulate PI-3 kinase and Akt activation in a Gab1-dependent or Gab1-independent manner.

Pharmacological and Protein Profiling Suggests Venetoclax (ABT-199) as Optimal Partner with Ibrutinib in Chronic Lymphocytic Leukemia.

PURPOSE: Bruton's tyrosine kinase (BTK) is a critical enzyme in the B-cell receptor pathway and is inhibited by ibrutinib due to covalent binding to the kinase domain. Though ibrutinib results in impressive clinical activity in chronic lymphocytic leukemia (CLL), most patients achieve only partial remission due to residual disease. We performed a pharmacologic profiling of residual circulating CLL cells from patients receiving ibrutinib to identify optimal agents that could induce cell death of these lymphocytes. EXPERIMENTAL DESIGN: Ex vivo serial samples of CLL cells from patients on ibrutinib were obtained prior and after (weeks 2, 4, and 12) the start of treatment. These cells were incubated with PI3K inhibitors (idelalisib or IPI-145), bendamustine, additional ibrutinib, or BCL-2 antagonists (ABT-737 or ABT-199), and cell death was measured. In vitro investigations complemented ex vivo studies. Immunoblots for BTK signaling pathway and antiapoptotic proteins were performed. RESULTS: The BCL-2 antagonists, especially ABT-199, induced high cell death during ex vivo incubations. In concert with the ex vivo data, in vitro combinations also resulted in high cytotoxicity. Serial samples of CLL cells obtained before and 2, 4, 12, or 36 weeks after the start of ibrutinib showed inhibition of BTK activity and sensitivity to ABTs. Among the three BCL-2 family antiapoptotic proteins that are overexpressed in CLL, levels of MCL-1 and BCL-XL were decreased after ibrutinib while ABT-199 selectively antagonizes BCL-2. CONCLUSIONS: Our biologic and molecular results suggest that ibrutinib and ABT-199 combination should be tested clinically against CLL.

Partial rescue of insulin receptor-deficient mice by transgenic complementation with an activated insulin receptor in the liver.

Insulin receptor (IR)-deficient mice develop severe diabetes mellitus, diabetic ketoacidosis (DKA) and liver steatosis and die within 1 week after birth. We examined in this work whether the metabolic phenotype of IR(-/-) mutants could be improved by transgenic complementation with IR selectively in the liver. We first generated transgenic mice expressing a human DNA complementary to RNA encoding a truncated constitutively activated form of IR (IRdelta) under the control of liver-specific phenylalanine hydroxylase (PAH) gene promoter. These mice presented more pronounced fasting hypoglycemia and showed slightly improved glucose tolerance as compared to controls. The transgenic mice were crossed with IR(+/-) mutants to generate IR(-/-) mice carrying the PAH-IRDelta transgene. Although such mutants developed glycosuria, DKA was delayed by more than 1 week and survival was prolonged to 8-20 days in approximately 10% of mice. In these partially rescued pups, serum glucose and triglyceride levels were lowered, hepatic glycogen stores were reconstituted and liver steatosis was absent as compared with pups which developed strong DKA and died earlier. Thus, lack of insulin action in the liver is responsible in large part for the metabolic disorders seen in IR(+/-) mice. This study should stimulate interest in therapeutic strategies aimed at improving hepatic function in diabetes.

Ectopic expression of Bcl-2 and Bcl-xL inhibits apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL) through suppression of caspases-8, 7, and 3 and BID cleavage in human acute myelogenous leukemia cell line HL-60.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is one of the latest members of the TNF superfamily known to induce apoptosis in a wide variety of tumor cells. Some cell types, however, are quite resistant to TRAIL. We investigated the effect of ectopic expression of Bcl-2 and Bcl-xL on TRAIL-induced apoptosis in human acute myelogenous leukemia HL-60 cells. We found that HL-60 cells, which express TRAIL receptors (also called death receptor, DR) DR4, DR5, and Dc (decoy) R2, are highly sensitive to TRAIL-induced cytotoxicity. Greater than 90% killing occurred within 24 h of TRAIL treatment. The expression of Bcl-2 and Bcl-xL, however, completely abolished the TRAIL-induced cytotoxic effects. Treatment of HL-60 cells with TRAIL induced caspase-8 activation within 2-4 h, but no activation could be seen in Bcl-2-expressing or Bcl-xL-expressing cells. TRAIL also induced cleavage of BID, which was also abolished by Bcl-2 and Bcl-xL. Similarly, TRAIL activated caspase-3 and caspase-7 in control cells but not in cells expressing Bcl-2 or Bcl-xL. Cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase (PARP), was abrogated by ectopic expression of Bcl-2 and Bcl-xL. Inhibition of caspases by the pan-caspase inhibitor, benzyloxycarbonyl-valine-alanine-aspartate-fluoromethylketone (zVAD-fmk) abolished the TRAIL-induced apoptosis. Overall, these results indicate that TRAIL-induced apoptosis involves activation of caspase-8, caspase-7, caspase-3, and BID cleavage, and Bcl-2 and Bcl-xL prevents TRAIL-induced apoptosis by abrogating caspase activation and BID cleavage.

TAK1 is essential for osteoclast differentiation and is an important modulator of cell death by apoptosis and necroptosis.

Transforming growth factor ß (TGF-ß)-activated kinase 1 (TAK1), a mitogen-activated protein 3 (MAP3) kinase, plays an essential role in inflammation by activating the IκB kinase (IKK)/nuclear factor κB (NF-κB) and stress kinase (p38 and c-Jun N-terminal kinase [JNK]) pathways in response to many stimuli. The tumor necrosis factor (TNF) superfamily member receptor activator of NF-κB ligand (RANKL) regulates osteoclastogenesis through its receptor, RANK, and the signaling adaptor TRAF6. Because TAK1 activation is mediated through TRAF6 in the interleukin 1 receptor (IL-1R) and toll-like receptor (TLR) pathways, we sought to investigate the consequence of TAK1 deletion in RANKL-mediated osteoclastogenesis. We generated macrophage colony-stimulating factor (M-CSF)-derived monocytes from the bone marrow of mice with TAK1 deletion in the myeloid lineage. Unexpectedly, TAK1-deficient monocytes in culture died rapidly but could be rescued by retroviral expression of TAK1, inhibition of receptor-interacting protein 1 (RIP1) kinase activity with necrostatin-1, or simultaneous genetic deletion of TNF receptor 1 (TNFR1). Further investigation using TAK1-deficient mouse embryonic fibroblasts revealed that TNF-α-induced cell death was abrogated by the simultaneous inhibition of caspases and knockdown of RIP3, suggesting that TAK1 is an important modulator of both apoptosis and necroptosis. Moreover, TAK1-deficient monocytes rescued from programmed cell death did not form mature osteoclasts in response to RANKL, indicating that TAK1 is indispensable to RANKL-induced osteoclastogenesis. To our knowledge, we are the first to report that mice in which TAK1 has been conditionally deleted in osteoclasts develop osteopetrosis.

Deletion of TAK1 in the myeloid lineage results in the spontaneous development of myelomonocytic leukemia in mice.

Previous studies of the conditional ablation of TGF-ß activated kinase 1 (TAK1) in mice indicate that TAK1 has an obligatory role in the survival and/or development of hematopoietic stem cells, B cells, T cells, hepatocytes, intestinal epithelial cells, keratinocytes, and various tissues, primarily because of these cells' increased apoptotic sensitivity, and have implicated TAK1 as a critical regulator of the NF-κB and stress kinase pathways and thus a key intermediary in cellular survival. Contrary to this understanding of TAK1's role, we report a mouse model in which TAK1 deletion in the myeloid compartment that evoked a clonal myelomonocytic cell expansion, splenomegaly, multi-organ infiltration, genomic instability, and aggressive, fatal myelomonocytic leukemia. Unlike in previous reports, simultaneous deletion of TNF receptor 1 (TNFR1) failed to rescue this severe phenotype. We found that the features of the disease in our mouse model resemble those of human chronic myelomonocytic leukemia (CMML) in its transformation to acute myeloid leukemia (AML). Consequently, we found TAK1 deletion in 13 of 30 AML patients (43%), thus providing direct genetic evidence of TAK1's role in leukemogenesis.

The E3 ligase TRAF6 regulates Akt ubiquitination and activation.

Akt signaling plays a central role in many biological functions, such as cell proliferation and apoptosis. Because Akt (also known as protein kinase B) resides primarily in the cytosol, it is not known how these signaling molecules are recruited to the plasma membrane and subsequently activated by growth factor stimuli. We found that the protein kinase Akt undergoes lysine-63 chain ubiquitination, which is important for Akt membrane localization and phosphorylation. TRAF6 was found to be a direct E3 ligase for Akt and was essential for Akt ubiquitination, membrane recruitment, and phosphorylation upon growth-factor stimulation. The human cancer-associated Akt mutant displayed an increase in Akt ubiquitination, in turn contributing to the enhancement of Akt membrane localization and phosphorylation. Thus, Akt ubiquitination is an important step for oncogenic Akt activation.

E2 interaction and dimerization in the crystal structure of TRAF6.

Tumor necrosis factor (TNF) receptor-associated factor (TRAF)-6 mediates Lys63-linked polyubiquitination for NF-kappaB activation via its N-terminal RING and zinc finger domains. Here we report the crystal structures of TRAF6 and its complex with the ubiquitin-conjugating enzyme (E2) Ubc13. The RING and zinc fingers of TRAF6 assume a rigid, elongated structure. Interaction of TRAF6 with Ubc13 involves direct contacts of the RING and the preceding residues, and the first zinc finger has a structural role. Unexpectedly, this region of TRAF6 is dimeric both in the crystal and in solution, different from the trimeric C-terminal TRAF domain. Structure-based mutagenesis reveals that TRAF6 dimerization is crucial for polyubiquitin synthesis and autoubiquitination. Fluorescence resonance energy transfer analysis shows that TRAF6 dimerization induces higher-order oligomerization of full-length TRAF6. The mismatch of dimeric and trimeric symmetry may provide a mode of infinite oligomerization that facilitates ligand-dependent signal transduction of many immune receptors.

The RING domain and first zinc finger of TRAF6 coordinate signaling by interleukin-1, lipopolysaccharide, and RANKL.

TRAF6, a crucial adaptor molecule in innate and adaptive immunity, contains three distinct functional domains. The C-terminal TRAF domain facilitates oligomerization and sequence-specific interaction with receptors or other adaptor proteins. In conjunction with the dimeric E2 enzyme Ubc13-Uev1A, the N-terminal RING domain of TRAF6 functions as an E3 ubiquitin (Ub) ligase that facilitates its own site-specific ubiquitination through the generation of a Lys-63-linked poly-Ub chain. This modification does not cause its proteasomal degradation but rather serves as a scaffold to activate both the IKK and stress kinase pathways. Connecting the N-and C-terminal regions, the four internal zinc finger (ZF) motifs have yet to be functionally defined. In this study, we examined the role of the ZF domains in interleukin-1, lipopolysaccharide, and RANKL signaling by reconstitution of TRAF6-deficient cells with point mutations or deletions of these ZF motifs. Although ZF domains 2-4 are dispensable for activating IKK, p38, and JNK by interleukin-1 and lipopolysaccharide, the first ZF domain together with an intact RING domain of TRAF6 is essential for activating these pathways. Furthermore, TRAF6 autoubiquitination and its interaction with Ubc13 are dependent on ZF1 and an intact RING domain. Additionally, expression of TRAF6 lacking ZF2-4 in TRAF6-deficient monocytes rescues RANKL-mediated osteoclast differentiation and LPS-stimulated interleukin-6 production. These data provide evidence for the critical role of the Ub ligase activity of TRAF6, which is coordinated via the RING domain and ZF1 to supply the necessary elements in signaling by cytokines dependent upon TRAF6.

Molecular basis for the unique deubiquitinating activity of the NF-kappaB inhibitor A20.

Nuclear factor kappaB (NF-kappaB) activation in tumor necrosis factor, interleukin-1, and Toll-like receptor pathways requires Lys63-linked nondegradative polyubiquitination. A20 is a specific feedback inhibitor of NF-kappaB activation in these pathways that possesses dual ubiquitin-editing functions. While the N-terminal domain of A20 is a deubiquitinating enzyme (DUB) for Lys63-linked polyubiquitinated signaling mediators such as TRAF6 and RIP, its C-terminal domain is a ubiquitin ligase (E3) for Lys48-linked degradative polyubiquitination of the same substrates. To elucidate the molecular basis for the DUB activity of A20, we determined its crystal structure and performed a series of biochemical and cell biological studies. The structure reveals the potential catalytic mechanism of A20, which may be significantly different from papain-like cysteine proteases. Ubiquitin can be docked onto a conserved A20 surface; this interaction exhibits charge complementarity and no steric clash. Surprisingly, A20 does not have specificity for Lys63-linked polyubiquitin chains. Instead, it effectively removes Lys63-linked polyubiquitin chains from TRAF6 without dissembling the chains themselves. Our studies suggest that A20 does not act as a general DUB but has the specificity for particular polyubiquitinated substrates to assure its fidelity in regulating NF-kappaB activation in the tumor necrosis factor, interleukin-1, and Toll-like receptor pathways.

Association between imatinib-resistant BCR-ABL mutation-negative leukemia and persistent activation of LYN kinase.

BACKGROUND: Imatinib is a tyrosine kinase inhibitor that is used to treat chronic myelogenous leukemia (CML). BCR-ABL mutations are associated with failure of imatinib treatment in many CML patients. LYN kinase regulates survival and responsiveness of CML cells to inhibition of BCR-ABL kinase, and differences in LYN regulation have been found between imatinib-sensitive and -resistant CML cell lines. METHODS: We evaluated cells from 12 imatinib-resistant CML patients with mutation-negative BCR-ABL and from six imatinib-sensitive patients who discontinued therapy because of imatinib intolerance. Phosphorylation of BCR-ABL and LYN was assessed in patient cells and cell lines by immunoblotting with activation state-specific antibodies, co-immunoprecipitation studies, and mass spectroscopy analysis of phosphopeptides. Cell viability, caspase activation, and apoptosis were also measured. Mutations were analyzed by sequencing. The effect of silencing LYN with short interfering RNAs (siRNAs) or reducing activation by treatment with tyrosine kinase inhibitors was evaluated in cell lines and patient cells. RESULTS: Imatinib treatment suppressed LYN phosphorylation in cells from imatinib-sensitive CML patients and imatinib-sensitive cell lines. Imatinib treatment blocked BCR-ABL signaling but did not suppress LYN phosphorylation in cells from imatinib-resistant patients, and persistent activation of LYN kinase was not associated with mutations in LYN kinase or its carboxyl-terminal regulatory domains. Unique LYN phosphorylation sites (tyrosine-193 and tyrosine-459) and associated proteins (c-Cbl and p80) were identified in cells from imatinib-resistant patients. Reducing LYN expression (siRNA) or activation (dasatinib) was associated with loss of cell survival and cytogenetic or complete hematologic responses in imatinib-resistant disease. CONCLUSIONS: LYN activation was independent of BCR-ABL in cells from imatinib-resistant patients. Thus, LYN kinase may be involved in imatinib resistance in CML patients with mutation-negative BCR-ABL and its direct inhibition is consistent with clinical responses in these patients.

Site-specific Lys-63-linked tumor necrosis factor receptor-associated factor 6 auto-ubiquitination is a critical determinant of I kappa B kinase activation.

Tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) is a key mediator in proximal signaling of the interleukin-1/Toll-like receptor and the TNF receptor superfamily. Analysis of TRAF6-deficient mice revealed a fundamental role of TRAF6 in osteoclastogenesis; however, the molecular mechanism underlying TRAF6 signaling in this biological process is not understood. Recent biochemical evidence has indicated that TRAF6 possesses ubiquitin ligase activity that controls the activation of IKK and NF-kappaB. Because these studies are primarily based on cell-free systems, the role of the ubiquitin ligase activity of TRAF6 and its auto-ubiquitination to initiate the NF-kappaB pathway in vivo remain elusive. Here we show that an intact RING domain of TRAF6 in conjunction with the E2 enzyme Ubc13/Uev1A is necessary for Lys-63-linked auto-ubiquitination of TRAF6 and for its ability to activate IKK and NF-kappaB. Furthermore, a RING mutant of TRAF6 abolishes its ability to induce receptor activator of NF-kappaB-independent osteoclast differentiation and nuclear accumulation of the transcription factor NFATc1. Notably, we map the auto-ubiquitination site of TRAF6 to a single Lys residue, which if mutated renders TRAF6 unable to activate transforming growth factor-beta-activated kinase 1 and IKK and to cause spontaneous osteoclast differentiation. Additionally, we provide biochemical and in vivo evidence that TRAF6 serves as an E3 to directly ubiquitinate NEMO. Reconstituting TRAF6-deficent cells with various TRAF6 mutants, we clearly demonstrate the requirement for the TRAF6 RING domain and site-specific auto-ubiquitination of TRAF6 to activate IKK in response to interleukin-1. These data establish a signaling cascade in which regulated site-specific Lys-63-linked TRAF6 auto-ubiquitination is the critical upstream mediator of IKK.

TRAF6 ubiquitin ligase is essential for RANKL signaling and osteoclast differentiation.

Tumor necrosis factor receptor-associated factor 6 (TRAF6), the crucial adaptor molecule of receptor activator of NF-kappaB (RANK), plays an essential role in governing the formation of multi-nucleated osteoclasts. TRAF6 is a RING-dependent ubiquitin (Ub) ligase that in conjunction with Ubc13/Uev1A catalyzes its own auto-ubiquitination via Lys63-linked poly-Ub chains. While the receptor-adaptor function of TRAF6 in RANK signaling is well understood, the significance of its Ub ligase activity in this process remains largely unknown. In this study, we show that retroviral expression of TRAF6, but not a RING mutant of TRAF6 was able to rescue TRAF6-deficient monocytes for the activation of IKK and osteoclast differentiation by RANKL. Furthermore, a catalytically inactive Ubc13 or stable knockdown of Ubc13 significantly prevents RANK-mediated TRAF6 ubiquitination and NF-kappaB and JNK activation. These data establish a signaling cascade in which regulated Lys63-linked TRAF6 auto-ubiquitination is the critical upstream mediator of osteoclast differentiation.