TAK1-binding protein 1, TAB1, mediates osmotic stress-induced TAK1 activation but is dispensable for TAK1-mediated cytokine signaling

TAK1 kinase is an indispensable intermediate in several cytokine signaling pathways including tumor necrosis factor, interleukin-1, and transforming growth factor-beta signaling pathways. TAK1 also participates in stress-activated intracellular signaling pathways such as osmotic stress signaling pathway. TAK1-binding protein 1 (TAB1) is constitutively associated with TAK1 through its C-terminal region. Although TAB1 is known to augment TAK1 catalytic activity when it is overexpressed, the role of TAB1 under physiological conditions has not yet been identified. In this study, we determined the role of TAB1 in TAK1 signaling by analyzing TAB1-deficient mouse embryonic fibroblasts (MEFs). Tumor necrosis factor- and interleukin-1-induced activation of TAK1 was entirely normal in Tab1-deficient MEFs and could activate both mitogen-activated protein kinases and NF-kappaB. In contrast, we found that osmotic stress-induced activation of TAK1 was largely impaired in Tab1-deficient MEFs. Furthermore, we showed that the C-terminal 68 amino acids of TAB1 were sufficient to mediate osmotic stress-induced TAK1 activation. Finally, we attempted to determine the mechanism by which TAB1 activates TAK1. We found that TAK1 is spontaneously activated when the concentration is increased and that it is totally dependent on TAB1. Cell shrinkage under the osmotic stress condition increases the concentration of TAB1-TAK1 and may oligomerize and activate TAK1 in a TAB1-dependent manner. These results demonstrate that TAB1 mediates TAK1 activation only in a subset of TAK1 pathways that are mediated through spontaneous oligomerization of TAB1-TAK1.     

TAB1beta (transforming growth factor-beta-activated protein kinase 1-binding protein 1beta ), a novel splicing variant of TAB1 that interacts with p38alpha but not TAK1

The mitogen-activated protein kinases (MAPKs) play an important role in a variety of biological processes. Activation of MAPKs is mediated by phosphorylation on specific regulatory tyrosine and threonine sites. We have recently found that activation of p38alpha MAPK can be carried out not only by its upstream MAPK kinases (MKKs) but also by p38alpha autophosphorylation. p38alpha autoactivation requires an interaction of p38alpha with TAB1 (transforming growth factor-beta-activated protein kinase 1-binding protein 1). The autoactivation mechanism of p38alpha has been found to be important in cellular responses to a number of physiologically relevant stimuli. Here, we report the characterization of a splicing variant of TAB1, TAB1beta. TAB1 and TAB1beta share the first 10 exons. The 11th and 12th exons of TAB1 were spliced out in TAB1beta, and an extra exon, termed exon beta, downstream of exons 11 and 12 in the genome was used as the last exon in TAB1beta. The mRNA of TAB1beta was expressed in all cell lines examined. The TAB1beta mRNA encodes a protein with an identical sequence to TAB1 except the C-terminal 69 amino acids were replaced with an unrelated 27-amino acid sequence. Similar to TAB1, TAB1beta interacts with p38alpha but not other MAPKs and stimulates p38alpha autoactivation. Different from TAB1, TAB1beta does not bind or activate TAK1. Inhibition of TAB1beta expression with RNA interference in MDA231 breast cancer cells resulted in the reduction of basal activity of p38alpha and invasiveness of MDA231 cells, suggesting that TauAlphaBeta1beta is involved in regulating p38alpha activity in physiological conditions.     

Heat shock protein 27 functions in inflammatory gene expression and transforming growth factor-beta-activated kinase-1 (TAK1)-mediated signaling

Heat shock protein (HSP) 27 has long been known to be a component of the p38 mitogen-activated protein kinase (MAPK) signaling pathway. p38 MAPK has important functions in the inflammatory response, but the role of HSP27 in inflammation has remained unknown. We have used small interfering RNAs to suppress HSP27 expression in HeLa cells and fibroblasts and found that it is required for pro-inflammatory cell signaling and the expression of pro-inflammatory genes. HSP27 is needed for the activation by interleukin (IL)-1 of TAK1 and downstream signaling by p38 MAPK, JNK, and their activators (MKK-3, -4, -6, -7) and IKKbeta. IL-1-induced ERK activation appears to be independent of HSP27. HSP27 is required for both IL-1 and TNF-induced signaling pathways for which the most upstream common signaling protein is TAK1. HSP27 is also required for IL-1-induced expression of the pro-inflammatory mediators, cyclooxygenase-2, IL-6, and IL-8. HSP27 functions to drive cyclooxygenase-2 and IL-6 expression by augmenting the activation of the kinase downstream of p38 MAPK, MK2, resulting in stabilization of cyclooxygenase-2 and IL-6 mRNAs. The mechanism may not occur in cells of myeloid lineage because HSP27 protein was undetectable in human monocytes and murine macrophages.     

Interleukin-1 (IL-1) receptor-associated kinase leads to activation of TAK1 by inducing TAB2 translocation in the IL-1 signaling pathway

Interleukin-1 (IL-1) is a proinflammatory cytokine that recognizes a surface receptor complex and generates multiple cellular responses. IL-1 stimulation activates the mitogen-activated protein kinase kinase kinase TAK1, which in turn mediates activation of c-Jun N-terminal kinase and NF-kappaB. TAB2 has previously been shown to interact with both TAK1 and TRAF6 and promote their association, thereby triggering subsequent IL-1 signaling events. The serine/threonine kinase IL-1 receptor-associated kinase (IRAK) also plays a role in IL-1 signaling, being recruited to the IL-1 receptor complex early in the signal cascade. In this report, we investigate the role of IRAK in the activation of TAK1. Genetic analysis reveals that IRAK is required for IL-1-induced activation of TAK1. We show that IL-1 stimulation induces the rapid but transient association of IRAK, TRAF6, TAB2, and TAK1. TAB2 is recruited to this complex following translocation from the membrane to the cytosol upon IL-1 stimulation. In IRAK-deficient cells, TAB2 translocation and its association with TRAF6 are abolished. These results suggest that IRAK regulates the redistribution of TAB2 upon IL-1 stimulation and facilitates the formation of a TRAF6-TAB2-TAK1 complex. Formation of this complex is an essential step in the activation of TAK1 in the IL-1 signaling pathway.     

Mitogen-activated protein kinase feedback phosphorylation regulates MEK1 complex formation and activation during cellular adhesion

Cell adhesion and spreading depend on activation of mitogen-activated kinase, which in turn is regulated both by growth factor and integrin signaling. Growth factors, such as epidermal growth factor, are capable of activating Ras and Raf, but integrin signaling is required to couple Raf to MEK and MEK to extracellular signal-regulated protein kinase (ERK). It was previously shown that Rac-p21-activated kinase (PAK) signaling regulated the physical association of MEK1 with ERK2 through phosphorylation sites in the proline-rich sequence (PRS) of MEK1. It was also shown that activation of MEK1 and ERK by integrins depends on PAK phosphorylation of S298 in the PRS. Here we report a novel MEK1-specific regulatory feedback mechanism that provides a means by which activated ERK can terminate continued PAK phosphorylation of MEK1. Activated ERK can phosphorylate T292 in the PRS, and this blocks the ability of PAK to phosphorylate S298 and of Rac-PAK signaling to enhance MEK1-ERK complex formation. Preventing ERK feedback phosphorylation on T292 during cellular adhesion prolonged phosphorylation of S298 by PAK and phosphorylation of S218 and S222, the MEK1 activating sites. We propose that activation of ERK during adhesion creates a feedback system in which ERK phosphorylates MEK1 on T292, and this in turn blocks additional S298 phosphorylation in response to integrin signaling.     

Roles for TAB1 in regulating the IL-1-dependent phosphorylation of the TAB3 regulatory subunit and activity of the TAK1 complex

The protein kinase TAK1 (transforming growth factor-beta-activated kinase 1), which has been implicated in the activation of MAPK (mitogen-activated protein kinase) cascades and the production of inflammatory mediators by LPS (lipopolysaccharide), IL-1 (interleukin 1) and TNF (tumour necrosis factor), comprises the catalytic subunit complexed to the regulatory subunits, termed TAB (TAK1-binding subunit) 1 and either TAB2 or TAB3. We have previously identified a feedback-control mechanism by which p38alpha MAPK down-regulates TAK1 and showed that p38alpha MAPK phosphorylates TAB1 at Ser(423) and Thr(431). In the present study, we identified two IL-1-stimulated phosphorylation sites on TAB2 (Ser(372) and Ser(524)) and three on TAB3 (Ser(60), Thr(404) and Ser(506)) in human IL-1R cells [HEK-293 (human embryonic kidney) cells that stably express the IL-1 receptor] and MEFs (mouse embryonic fibroblasts). Ser(372) and Ser(524) of TAB2 are not phosphorylated by pathways dependent on p38alpha/beta MAPKs, ERK1/2 (extracellular-signal-regulated kinase 1/2) and JNK1/2 (c-Jun N-terminal kinase 1/2). In contrast, Ser(60) and Thr(404) of TAB3 appear to be phosphorylated directly by p38alpha MAPK, whereas Ser(506) is phosphorylated by MAPKAP-K2/MAPKAP-K3 (MAPK-activated protein kinase 2 and 3), which are protein kinases activated by p38alpha MAPK. Studies using TAB1(-/-) MEFs indicate important roles for TAB1 in recruiting p38alpha MAPK to the TAK1 complex for the phosphorylation of TAB3 at Ser(60) and Thr(404) and in inhibiting the dephosphorylation of TAB3 at Ser(506). TAB1 is also required to induce TAK1 catalytic activity, since neither IL-1 nor TNFalpha was able to stimulate detectable TAK1 activity in TAB1(-/-) MEFs. Surprisingly, the IL-1 and TNFalpha-stimulated activation of MAPK cascades and IkappaB (inhibitor of nuclear factor kappaB) kinases were similar in TAB1(-/-), MEKK3(-/-) [MAPK/ERK (extracellular-signal-regulated kinase) kinase kinase 3] and wild-type MEFs, suggesting that another MAP3K (MAPK kinase kinase) may mediate the IL-1/TNFalpha-induced activation of these signalling pathways in TAB1(-/-) and MEKK3(-/-) MEFs.     

Role of the TAB2-related protein TAB3 in IL-1 and TNF signaling

The cytokines IL-1 and TNF induce expression of a series of genes that regulate inflammation through activation of NF-kappaB signal transduction pathways. TAK1, a MAPKKK, is critical for both IL-1- and TNF-induced activation of the NF-kappaB pathway. TAB2, a TAK1-binding protein, is involved in IL-1-induced NF-kappaB activation by physically linking TAK1 to TRAF6. However, IL-1-induced activation of NF-kappaB is not impaired in TAB2-deficient embryonic fibroblasts. Here we report the identification and characterization of a novel protein designated TAB3, a TAB2-like molecule that associates with TAK1 and can activate NF-kappaB similar to TAB2. Endogenous TAB3 interacts with TRAF6 and TRAF2 in an IL-1- and a TNF-dependent manner, respectively. Further more, IL-1 signaling leads to the ubiquitination of TAB2 and TAB3 through TRAF6. Cotransfection of siRNAs directed against both TAB2 and TAB3 inhibit both IL-1- and TNF-induced activation of TAK1 and NF-kappaB. These results suggest that TAB2 and TAB3 function redundantly as mediators of TAK1 activation in IL-1 and TNF signal transduction.     

TAB2, a novel adaptor protein, mediates activation of TAK1 MAPKKK by linking TAK1 to TRAF6 in the IL-1 signal transduction pathway

The TAK1 MAPKKK mediates activation of JNK and NF-KB in the IL-1-activated signaling pathway. Here we report the identification of TAB2, a novel intermediate in the IL-1 pathway that functionally links TAK1 to TRAF6. Expression of TAB2 induces JNK and NF-kappaB activation, whereas a dominant-negative mutant TAB2 impairs their activation by IL-1. IL-1 stimulates translocation of TAB2 from the membrane to the cytosol where it mediates the IL-1-dependent association of TAK1 with TRAF6. These results define TAB2 as an adaptor linking TAK1 and TRAF6 and as a mediator of TAK1 activation in the IL-1 signaling pathway.     

Interleukin-1 (IL-1) receptor-associated kinase-dependent IL-1-induced signaling complexes phosphorylate TAK1 and TAB2 at the plasma membrane and activate TAK1 in the cytosol

Interleukin-1 (IL-1) receptor-associated kinase (IRAK) plays an important role in the sequential formation and activation of IL-1-induced signaling complexes. Previous studies showed that IRAK is recruited to the IL-1-receptor complex, where it is hyperphosphorylated. We now find that the phosphorylated IRAK in turn recruits TRAF6 to the receptor complex (complex I), which differs from the previous concept that IRAK interacts with TRAF6 after it leaves the receptor. IRAK then brings TRAF6 to TAK1, TAB1, and TAB2, which are preassociated on the membrane before stimulation to form the membrane-associated complex II. The formation of complex II leads to the phosphorylation of TAK1 and TAB2 on the membrane by an unknown kinase, followed by the dissociation of TRAF6-TAK1-TAB1-TAB2 (complex III) from IRAK and consequent translocation of complex III to the cytosol. The formation of complex III and its interaction with additional cytosolic factors lead to the activation of TAK1, resulting in NF-kappaB and JNK activation. Phosphorylated IRAK remains on the membrane and eventually is ubiquitinated and degraded. Taken together, the new data reveal that IRAK plays a critical role in mediating the association and dissociation of IL-1-induced signaling complexes, functioning as an organizer and transporter in IL-1-dependent signaling.     

Effect of inositol hexakisphosphate kinase 2 on transforming growth factor beta-activated kinase 1 and NF-kappaB activation

We previously showed that inositol hexakisphosphate kinase 2 (IHPK2) functions as a growth-suppressive and apoptosis-enhancing kinase during cell stress. Overexpression of IHPK2 sensitized ovarian carcinoma cell lines to the growth-suppressive and apoptotic effects of interferon beta (IFN-beta), IFN-alpha2, and gamma-irradiation. Expression of a kinase-dead mutant abrogated 50% of the apoptosis induced by IFN-beta. Because the kinase-dead mutant retained significant response to cell stressors, we hypothesized that a portion of the death-promoting function of IHPK2 was independent of its kinase activity. We now demonstrate that IHPK2 binds to tumor necrosis factor (TNF) receptor-associated factor (TRAF) 2 and interferes with phosphorylation of transforming growth factor beta-activated kinase 1 (TAK1), thereby inhibiting NF-kappaB signaling. IHPK2 contains two sites required for TRAF2 binding, Ser-347 and Ser-359. Compared with wild type IHPK2-transfected cells, cells expressing S347A and S359A mutations displayed 3.5-fold greater TAK1 activation following TNF-alpha. This mutant demonstrated a 6-10-fold increase in NF-kappaB DNA binding following TNF-alpha compared with wild type IHPK2-expressing cells in which NF-kappaB DNA binding was inhibited. Cells transfected with wild type IHPK2 or IHPK2 mutants that lacked S347A and S359A mutations displayed enhanced terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling staining following TNF-alpha. We believe that IHPK2-TRAF2 binding leads to attenuation of TAK1- and NF-kappaB-mediated signaling and is partially responsible for the apoptotic activity of IHPK2.     

Serine/threonine protein phosphatases type 2A and their roles in stress signaling

Serine/threonine protein phosphatases are ubiquitous enzymes in all eukaryotes but many of their physiological roles in plants remain unknown. The available results have demonstrated critical functions for these enzymes in the regulation of adaptive stress responses, and recent studies have directed attention to the functional roles of Ser/Thr phosphatases type 2A (PP2A) as components of stress signaling pathways. This review is focused primarily on plant PP2As and their participation in the control of biotic and abiotic stress responses.Key words: protein phosphatases type 2A, PP2A, biotic stress, abiotic stress, signaling, okadaic acid     

NF-kappaB activation by a signaling complex containing TRAF2, TANK and TBK1, a novel IKK-related kinase

The activation of NF-kappaB by receptors in the tumor necrosis factor (TNF) receptor and Toll/interleukin-1 (IL-1) receptor families requires the TRAF family of adaptor proteins. Receptor oligomerization causes the recruitment of TRAFs to the receptor complex, followed by the activation of a kinase cascade that results in the phosphorylation of IkappaB. TANK is a TRAF-binding protein that can inhibit the binding of TRAFs to receptor tails and can also inhibit NF-kappaB activation by these receptors. However, TANK also displays the ability to stimulate TRAF-mediated NF-kappaB activation. In this report, we investigate the mechanism of the stimulatory activity of TANK. We find that TANK interacts with TBK1 (TANK-binding kinase 1), a novel IKK-related kinase that can activate NF-kappaB in a kinase-dependent manner. TBK1, TANK and TRAF2 can form a ternary complex, and complex formation appears to be required for TBK1 activity. Kinase-inactive TBK1 inhibits TANK-mediated NF-kappaB activation but does not block the activation mediated by TNF-alpha, IL-1 or CD40. The TBK1-TANK-TRAF2 signaling complex functions upstream of NIK and the IKK complex and represents an alternative to the receptor signaling complex for TRAF-mediated activation of NF-kappaB.     

In vivo activation of protein kinase A in Schizosaccharomyces pombe requires threonine phosphorylation at its activation loop and is dependent on PDK1

Phosphoinositide-dependent protein kinase 1 (PDK1) plays a central role in cellular signaling by phosphorylating members of the AGC family of kinases. This family includes protein kinase C (PKC), protein kinase B (PKB), p70/p90 ribosomal S6 kinases (RSK and S6K), and the catalytic subunit of cAMP-dependent protein kinase (PKA). Although PDK1 phosphorylates and activates PKC, PKB, and RSK in vivo, PDK1 regulation of PKA remains controversial. We isolated ksg1, the fission yeast ortholog of mammalian PDK1, as a suppressor of growth defects caused by loss of the stress-activated MAP kinase, Spc1. Here, we demonstrate that Ksg1 is required for activation of PKA. Cells containing the ksg1.12 thermolabile allele exhibit pleiotropic phenotypes, including the failure to arrest in G(1) and an inability to conjugate. The ksg1.12 allele strongly suppresses defects associated with unregulated PKA. Pka1, the catalytic subunit of cAMP-dependent protein kinase, is phosphorylated in vivo at Thr-356, which is located in the activation loop of the kinase and corresponds to Thr-197 in mammalian PKA. Phosphorylation of Thr-356 is required for in vivo activation of Pka1 and is dependent upon Ksg1. These data provide experimental evidence that PKA is a physiological substrate for PDK1.     

Protein phosphatase 2A is a negative regulator of transforming growth factor-beta1-induced TAK1 activation in mesangial cells

TAK1 (transforming growth factor (TGF)-beta-activated kinase 1) is a serine/threonine kinase that is rapidly activated by TGF-beta1 and plays a vital function in its signal transduction. Once TAK1 is activated, efficient down-regulation of TAK1 activity is important to prevent excessive TGF-beta1 responses. The regulatory mechanism of TAK1 inactivation following TGF-beta1 stimulation has not been elucidated. Here we demonstrate that protein phosphatase 2A (PP2A) plays a pivotal role as a negative regulator of TAK1 activation in response to TGF-beta1 in mesangial cells. Treatment with okadaic acid (OA) induces autophosphorylation of Thr-187 in the activation loop of TAK1. In vitro dephosphorylation assay suggests that Thr-187 in TAK1 is a major dephosphorylation target of PP2A. TGF-beta1 stimulation rapidly activates TAK1 in a biphasic manner, indicating that TGF-beta1-induced TAK1 activation is tightly regulated. The association of PP2A(C) with TAK1 is enhanced in response to TGF-beta1 stimulation and closely parallels TGF-beta1-induced TAK1 activity. Attenuation of PP2A activity by OA treatment or targeted knockdown of PP2A(C) with small interfering RNA enhances TGF-beta1-induced phosphorylation of TAK1 at Thr-187 and MKK3 (MAPK kinase 3). Endogenous TAK1 co-precipitates with PP2A(C) but not PP6(C), another OA-sensitive protein phosphatase, and knockdown of PP6(C) by small interfering RNA does not affect TGF-beta1-induced phosphorylation of TAK1 at Thr-187 and MKK3. Moreover, ectopic expression of phosphatase-deficient PP2A(C) enhances TAK1-mediated MKK3 phosphorylation by TGF-beta1 stimulation, whereas the expression of wild-type PP2A(C) suppresses the MKK3 phosphorylation. Taken together, our data indicate that PP2A functions as a negative regulator in TGF-beta1-induced TAK1 activation.