TRAF2 Ser-11 Phosphorylation Promotes Cytosolic Translocation of the CD40 Complex To Regulate Downstream Signaling Pathways.

CD40 plays an important role in immune responses by activating the c-Jun N-terminal protein kinase (JNK) and NF-κB pathways; however, the precise mechanisms governing the spatiotemporal activation of these two signaling pathways are not fully understood. Here, using four different TRAF2-deficient cell lines (A20.2J, CH12.LX, HAP1, and mouse embryonic fibroblasts [MEFs]) reconstituted with wild-type or phosphorylation mutant forms of TRAF2, along with immunoprecipitation, immunoblotting, gene expression, and immunofluorescence analyses, we report that CD40 ligation elicits TANK-binding kinase 1 (TBK1)-mediated phosphorylation of TRAF2 at Ser-11. This phosphorylation interfered with the interaction between TRAF2's RING domain and membrane phospholipids and enabled translocation of the TRAF2 complex from CD40 to the cytoplasm. We also observed that this cytoplasmic translocation is required for full activation of the JNK pathway and the secondary phase of the NF-κB pathway. Moreover, we found that in the absence of Ser-11 phosphorylation, the TRAF2 RING domain interacts with phospholipids, leading to the translocation of the TRAF2 complex to lipid rafts, resulting in its degradation and activation of the noncanonical NF-κB pathway. Thus, our results provide new insights into the CD40 signaling mechanisms whereby Ser-11 phosphorylation controls RING domain-dependent subcellular localization of TRAF2 to modulate the spatiotemporal activation of the JNK and NF-κB pathways.

TRAF2 exerts opposing effects on basal and TNFα-induced activation of the classic IKK complex in hematopoietic cells in mice.

The role of TRAF2 and TRAF5 in TNFα-induced NF-κB activation has become complicated owing to the accumulation of conflicting data. Here, we report that 7-day-old TRAF2-knockout (KO) and TRAF2 TRAF5 double KO (TRAF2/5-DKO) mice exhibit enhanced canonical IκB kinase (IKK) and caspase-8 activation in spleen and liver, and that subsequent knockout of TNFα suppresses the basal activity of caspase-8, but not of IKK. In primary TRAF2 KO and TRAF2/5-DKO cells, TNFα-induced immediate IKK activation is impaired, whereas delayed IKK activation occurs normally; as such, owing to elevated basal and TNFα-induced delayed IKK activation, TNFα stimulation leads to significantly increased induction of a subset of NF-κB-dependent genes in these cells. In line with this, both TRAF2 KO and TRAF2/5-DKO mice succumb to a sublethal dose of TNFα owing to increased expression of NF-κB target genes, diarrhea and bradypnea. Notably, depletion of IAP1 and IAP2 (also known as BIRC2 and BIRC3, respectively) also results in elevated basal IKK activation that is independent of autocrine TNFα production and that impairs TNFα-induced immediate IKK activation. These data reveal that TRAF2, IAP1 and IAP2, but not TRAF5, cooperatively regulate basal and TNFα-induced immediate IKK activation.

RIP1 Cleavage in the Kinase Domain Regulates TRAIL-Induced NF-κB Activation and Lymphoma Survival.

Although TRAIL is considered a potential anticancer agent, it enhances tumor progression by activating NF-κB in apoptosis-resistant cells. Cellular FLICE-like inhibitory protein (cFLIP) overexpression and caspase-8 activation have been implicated in TRAIL-induced NF-κB activation; however, the underlying mechanisms are unknown. Here, we report that caspase-8-dependent cleavage of RIP1 in the kinase domain (KD) and intermediate domain (ID) determines the activation state of the NF-κB pathway in response to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment. In apoptosis-sensitive cells, caspase-8 cleaves RIP1 in the KD and ID immediately after the recruitment of RIP1 to the receptor complex, impairing IκB kinase (IKK) recruitment and NF-κB activation. In apoptosis-resistant cells, cFLIP restricts caspase-8 activity, resulting in limited RIP1 cleavage and generation of a KD-cleaved fragment capable of activating NF-κB but not apoptosis. Notably, depletion of the cytoplasmic pool of TRAF2 and cIAP1 in lymphomas by CD40 ligation inhibits basal RIP1 ubiquitination but does not prompt cell death, due to CD40L-induced cFLIP expression and limited RIP1 cleavage. Inhibition of RIP1 cleavage at the KD suppresses NF-κB activation and cell survival even in cFLIP-overexpressing lymphomas. Importantly, RIP1 is constitutively cleaved in human and mouse lymphomas, suggesting that cFLIP-mediated and caspase-8-dependent limited cleavage of RIP1 is a new layer of mechanism that promotes NF-κB activation and lymphoma survival.

TRAIL activates JNK and NF-κB through RIP1-dependent and -independent pathways.

The death receptor (DR) ligand TRAIL is being evaluated in clinical trials as an anti-cancer agent; however, many studies have found that TRAIL also enhances tumor progression by activating the NF-κB pathway in apoptosis-resistant cells. Although RIP1, cFLIP and caspase-8 have been implicated in TRAIL-induced JNK and NF-κB activation, underlying mechanisms are unclear. By examining the kinetics of pathway activation in TRAIL-sensitive lymphoma cells wild-type or deficient for RIP1, TRAF2, cIAP1/2 or HOIP, we report here that TRAIL induces two phases of JNK and NF-κB activation. The early phase is activated by TRAF2- and cIAP1-mediated ubiquitination of RIP1, whereas the delayed phase is induced by caspase-dependent activation of MEKK1 independent of RIP1 and TRAF2 expression. cFLIP overexpression promotes the early phase but completely suppresses the delayed phase of pathway activation in lymphoma cells, whereas Bcl-2 overexpression promotes both the early and delayed phases of the pathways. In addition, stable overexpression of cFLIP in RIP1- or TRAF2-deficient cells confers resistance to apoptosis, but fails to mediate NF-κB activation. HOIP is not essential for, but contributes to, TRAIL-induced NF-κB activation in cFLIP-overexpressing cells. These findings not only elucidate details of the mechanisms underlying TRAIL-induced JNK and NF-κB activation, but also clarify conflicting reports in the field.

Two coordinated mechanisms underlie tumor necrosis factor alpha-induced immediate and delayed IκB kinase activation.

Tumor necrosis factor alpha (TNF-α)-induced NF-κB activation has been believed to depend on TRAF2- and cIAP1-mediated RIP1 ubiquitination. However, recent findings have challenged the notion that these proteins play essential roles in NF-κB activation. Here, by assessing the kinetics and amplitude of IκB kinase (IKK) activation, we report that TNF-α-induced immediate and robust activation of IKK requires K63-linked and linearly linked ubiquitination of RIP1 and that in the absence of RIP1 expression, TRAF2 and cIAP1 cooperatively induce delayed IKK activation by recruiting LUBAC to TNFR1. Knockdown of HOIP (a component of LUBAC) in RIP1-deficient cells completely impairs the recruitment and activation of IKK but does not affect K63-linked ubiquitination of TRAF2 and recruitment of TAK1 to TNFR1, suggesting that the K63-linked ubiquitin chain is not capable of recruiting IKK in vivo. We also demonstrate that TRAF2 and cIAP1 together, but not either one alone, directly catalyze linearly linked ubiquitination of RIP1. Importantly, in embryonic hepatocytes, TNF-α activates NF-κB through a RIP1-independent pathway. Thus, our findings clarify molecular details of this important signaling mechanism by providing evidence for the existence of two phases of IKK activation: the immediate phase, induced by TRAF2/cIAP1-mediated ubiquitination of RIP1, and the delayed phase, activated by TRAF2/cIAP1-dependent recruitment of LUBAC.

TRAF2 phosphorylation promotes NF-κB-dependent gene expression and inhibits oxidative stress-induced cell death.

Tumor necrosis factor α (TNF-α) receptor-associated factor 2 (TRAF2) regulates activation of the c-Jun N-terminal kinase (JNK)/c-Jun and the inhibitor of κB kinase (IKK)/nuclear factor κB (NF-κB) signaling cascades in response to TNF-α stimulation. Gene knockout studies have revealed that TRAF2 inhibits TNF-α-induced cell death but promotes oxidative stress-induced apoptosis. Here we report that TNF-α and oxidative stress both induce TRAF2 phosphorylation at serines 11 and 55 and that this dual phosphorylation promotes the prolonged phase of IKK activation while inhibiting the prolonged phase of JNK activation. Prolonged IKK activation trigged by TNF-α plays an essential role in efficient expression of a subset of NF-κB target genes but has no substantial role in TNF-α-induced cell death. On the other hand, TRAF2 phosphorylation in response to oxidative stress significantly promotes cell survival by inducing prolonged IKK activation and by inhibiting the prolonged phase of JNK activation. Notably, stable expression of phospho-null mutant TRAF2 in cancer cells leads to an increase in the basal and inducible JNK activation and B-cell lymphoma 2 (Bcl-2) phosphorylation. In addition, exposure of cells expressing phospho-null mutant TRAF2 to sublethal oxidative stress results in a rapid degradation of Bcl-2 and cellular inhibitor of apoptosis 1 as well as significantly increased cell death. These results suggest that TRAF2 phosphorylation is essential for cell survival under conditions of oxidative stress.

The RING domain of TRAF2 plays an essential role in the inhibition of TNFalpha-induced cell death but not in the activation of NF-kappaB.

Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) and receptor-interacting protein 1 (RIP1) play critical roles in activating c-Jun N-terminal kinase (JNK) and inhibitor of kappaB kinase (IKK), as well as in inhibiting apoptosis induced by TNFalpha. The TRAF2 RING domain-mediated polyubiquitination of RIP1 is believed to be essential for TNFalpha-induced IKK activation, and the RING-domain-deleted TRAF2 (TRAF2-DeltaR) has been widely used as a dominant negative in transient overexpression systems to block TNFalpha-induced JNK and IKK activation. Here, we report that stable expression of TRAF2-DeltaR at a physiological level in TRAF2 and TRAF5 double knockout (TRAF2/5 DKO) cells almost completely restores normal TNFalpha-induced IKK activation, but not RIP1 polyubiquitination. In addition, stable expression of TRAF2-DeltaR in TRAF2/5 DKO cells efficiently inhibited the TNFalpha-induced later phase of prolonged JNK activation, yet failed to inhibit TNFalpha-induced cell death. Although the basal and inducible expression of anti-apoptotic proteins in TRAF2-DeltaR-expressing TRAF2/5 DKO cells was normal, the cells remained sensitive to TNFalpha-induced cell death because anti-apoptotic proteins were not recruited to the TNFR1 complex efficiently. Moreover, stable expression of TRAF2-DeltaR in TRAF2/5 DKO cells failed to suppress constitutive p100 processing in these cells. These data suggest that (i) the TRAF2 RING domain plays a critical role in inhibiting cell death induced by TNFalpha and is essential for suppressing the noncanonical nuclear factor kappaB pathway in unstimulated cells; (ii) RIP1 polyubiquitination is not essential for TNFalpha-induced IKK activation; and (iii) prolonged JNK activation has no obligate role in TNFalpha-induced cell death.

TRAF2 suppresses basal IKK activity in resting cells and TNFalpha can activate IKK in TRAF2 and TRAF5 double knockout cells.

Tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2) and TRAF5 are adapter proteins involved in TNFalpha-induced activation of the c-Jun N-terminal kinase and nuclear factor kappaB (NF-kappaB) pathways. Currently, TNFalpha-induced NF-kappaB activation is believed to be impaired in TRAF2 and TRAF5 double knockout (T2/5 DKO) cells. Here, we report instead that T2/5 DKO cells exhibit high basal IkappaB kinase (IKK) activity and elevated expression of NF-kappaB-dependent genes in unstimulated conditions. Although TNFalpha-induced receptor-interacting protein 1 ubiquitination is indeed impaired in T2/5 DKO cells, TNFalpha stimulation further increases IKK activity in these cells, resulting in significantly elevated expression of NF-kappaB target genes to a level higher than that in wild-type cells. Inhibition of NIK in T2/5 DKO cells attenuates basal IKK activity and restores robust TNFalpha-induced IKK activation to a level comparable with that seen in wild-type cells. This suggests that TNFalpha can activate IKK in the absence of TRAF2 and TRAF5 expression and receptor-interacting protein 1 ubiquitination. In addition, both the basal and TNFalpha-induced expression of anti-apoptotic proteins are normal in T2/5 DKO cells, yet these DKO cells remain sensitive to TNFalpha-induced cell death, due to the impaired recruitment of anti-apoptotic proteins to the TNFR1 complex in the absence of TRAF2. Thus, our data demonstrate that TRAF2 negatively regulates basal IKK activity in resting cells and inhibits TNFalpha-induced cell death by recruiting anti-apoptotic proteins to the TNFR1 complex rather than by activating the NF-kappaB pathway.

Phosphorylation of TRAF2 within its RING domain inhibits stress-induced cell death by promoting IKK and suppressing JNK activation.

Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) is an adaptor protein that modulates the activation of the c-Jun NH(2) terminal kinase (JNK)/c-Jun and IkappaB kinase (IKK)/nuclear factor-kappaB (NF-kappaB) signaling cascades in response to TNFalpha stimulation. Although many serine/threonine kinases have been implicated in TNFalpha-induced IKK activation and NF-kappaB-dependent gene expression, most of them do not directly activate IKK. Here, we report that protein kinase Czeta phosphorylates TRAF2 at Ser(55), within the RING domain of the protein, after TNFalpha stimulation. Although this phosphorylation event has a minimal effect on induction of the immediate/transient phase of IKK and JNK activation by TNFalpha, it promotes the secondary/prolonged phase of IKK activation and inhibits that of JNK. Importantly, constitutive TRAF2 phosphorylation increased both basal and inducible NF-kappaB activation and rendered Ha-Ras-V12-transformed cells resistant to stress-induced apoptosis. Moreover, TRAF2 was found to be constitutively phosphorylated in some malignant cancer cell lines and Hodgkin's lymphoma. These results reveal a new level of complexity in TNFalpha-induced IKK activation modulated by TRAF2 phosphorylation and suggest that TRAF2 phosphorylation is one of the events that are responsible for elevated basal NF-kappaB activity in certain human cancers.

TRAF2 phosphorylation modulates tumor necrosis factor alpha-induced gene expression and cell resistance to apoptosis.

TRAF2 is an adaptor protein that regulates the activation of the c-Jun N-terminal kinase (JNK) and IkappaB kinase (IKK) signaling cascades in response to tumor necrosis factor alpha (TNF-alpha) stimulation. Although the downstream events in TNF-alpha signaling are better understood, the membrane-proximal events are still elusive. Here, we demonstrate that TNF-alpha and cellular stresses induce TRAF2 phosphorylation at serine 11 and that this phosphorylation is required for the expression of a subset of NF-kappaB target genes. Although TRAF2 phosphorylation had a minimal effect on the TNF-alpha-induced rapid and transient IKK activation, it was essential for secondary and prolonged IKK activation. Consistent with this, TRAF2 phosphorylation is not required for its recruitment to the TNFR1 complex in response to TNF-alpha stimulation but is required for its association with a cytoplasmic complex containing RIP1 and IKK. In addition, TRAF2 phosphorylation was essential for the full TNF-alpha-induced activation of JNK. Notably, TRAF2 phosphorylation increased both basal and inducible c-Jun and NF-kappaB activities and rendered cells resistant to stress-induced apoptosis. Moreover, TRAF2 was found to be constitutively phosphorylated in some lymphomas. These results unveil a new, finely tuned mechanism for TNF-alpha-induced IKK activation modulated by TRAF2 phosphorylation and suggest that TRAF2 phosphorylation contributes to elevated levels of basal NF-kappaB activity in certain human cancers.

Tau binds both subunits of calcineurin, and binding is impaired by calmodulin.

Calcineurin, an important protein Ser/Thr phosphatase which acts on tau in vivo, is a heterodimer of a catalytic subunit, calcineurin A, and a regulatory subunit, calcineurin B, and is unique in being regulated by calmodulin. Here, we find that both subunits of calcineurin bind tau, and calmodulin interferes with the association between calcineurin and tau. The domains of both subunits of calcineurin and tau involved in binding are mapped. We also investigate the functional consequences of the interactions between both subunits of calcineurin, tau and calmodulin, and reveal the interactions affect dephosphorylation of tau by calcineurin and contribute to the balance of phosphorylation and dephosphorylation of tau in vivo. Our findings may be of potential significance in neuronal physiology and also in neurodegenerative disorders. They shed some light on how the interactions might control the phosphorylation state of tau under physiological conditions, and provide new insights into the treatment of tauopathies such as Alzheimer's disease.

Inhibition of calcineurin by infusion of CsA causes hyperphosphorylation of tau and is accompanied by abnormal behavior in mice.

Calcineurin is a Ca2+/calmodulin-dependent phosphatase that dephosphorylates numerous substrates in different neuronal compartments. Genetic and pharmacological studies have provided insight into its involvement in the brain. Cyclosporin A (CsA) is used as a specific calcineurin inhibitor in many pharmacological experiments. However, the calcineurin activity of CsA-treated brain has not been reported. To examine the relationship between calcineurin activity and brain function, we injected CsA into the left lateral ventricle of the mouse brain and assayed calcineurin activity. CsA reduced calcineurin activity in a dose-dependent manner, without affecting the amount of calcineurin protein. Assays of the effect of protein phosphatase inhibitors on CsA-injected mouse brain extracts and kinetic analysis revealed that CsA inhibited calcineurin activity in a non-competitive manner in vivo, in agreement with in vitro results. Injection of CsA led to enhanced phosphorylation of tau at Ser-262 (12E8 site), Ser-198, Ser-199, and/or Ser-202 (Tau-1 site) and Ser-396 and/or Ser-404 (PHF-1 site), as well as to impaired spatial memory, which are two characteristic features of Alzheimer's disease. We propose that inhibition of calcineurin may play an important role in Alzheimer's disease.