SUMO化信号途径对JNK信号通路活性的调控

c-Jun氨基末端激酶(the c-Jun N-terminal kinase,JNK)家族是促分裂原活化蛋白激酶(MAPK)超家族成员之一.JNK信号通路对细胞生长、分化和凋亡等生物学活动都有重要作用.而SUMO化是一种重要的生物学修饰,可以调节多种细胞生理活动.最近,黄海等在Development发表文章首次将SUMO化途径与JNK信号通路通过Hipk激酶联系起来,为进一步研究SUMO化的功能及其对JNK通路的调节建立了一个新的模型.     

Sh3rf2/POSHER protein promotes cell survival by ring-mediated proteasomal degradation of the c-Jun N-terminal kinase scaffold POSH (Plenty of SH3s) protein

We report that Sh3rf2, a homologue of the pro-apoptotic scaffold POSH (Plenty of SH3s), acts as an anti-apoptotic regulator for the c-Jun N-terminal kinase (JNK) pathway. siRNA-mediated knockdown of Sh3rf2 promotes apoptosis of neuronal PC12 cells, cultured cortical neurons, and C6 glioma cells. This death appears to result from activation of JNK signaling. Loss of Sh3rf2 triggers activation of JNK and its target c-Jun. Also, apoptosis promoted by Sh3rf2 knockdown is inhibited by dominant-negative c-Jun as well as by a JNK inhibitor. Investigation of the mechanism by which Sh3rf2 regulates cell survival implicates POSH, a scaffold required for activation of pro-apoptotic JNK/c-Jun signaling. In cells lacking POSH, Sh3rf2 knockdown is unable to activate JNK. We further find that Sh3rf2 binds POSH to reduce its levels by a mechanism that requires the RING domains of both proteins and that appears to involve proteasomal POSH degradation. Conversely, knockdown of Sh3rf2 promotes the stabilization of POSH protein and activation of JNK signaling. Finally, we show that endogenous Sh3rf2 protein rapidly decreases following several different apoptotic stimuli and that knockdown of Sh3rf2 activates the pro-apoptotic JNK pathway in neuronal cells. These findings support a model in which Sh3rf2 promotes proteasomal degradation of pro-apoptotic POSH in healthy cells and in which apoptotic stimuli lead to rapid loss of Sh3rf2 expression, and consequently to stabilization of POSH and JNK activation and cell death. On the basis of these observations, we propose the alternative name POSHER (POSH-eliminating RING protein) for the Sh3rf2 protein.     

The beta1 integrin activates JNK independent of CagA, and JNK activation is required for Helicobacter pylori CagA+-induced motility of gastric cancer cells

The Helicobacter pylori CagA protein is translocated into gastric epithelial cells through a type IV secretion system (TFSS), and published studies suggest CagA is critical for H. pylori-associated carcinogenesis. CagA is thought to be necessary and sufficient to induce the motogenic response observed in response to CagA+ strains, as CagA interacts with proteins involved in adhesion and motility. We report that H. pylori strain 60190 stimulated AGS cell motility through a CagA- and TFSS-dependent mechanism, because strains 60190DeltacagA or 60190DeltacagE (TFSS-defective) did not increase motility. The JNK pathway is critical for H. pylori-dependent cell motility, as inhibition using SP600125 (JNK1/2/3 inhibitor) or a JNK2/3-specific inhibitor blocked motility. JNK mediates H. pylori-induced cell motility by activating paxillin, because JNK inhibition blocked paxillinTyr-118 phosphorylation, and paxillin expression knockdown completely abrogated bacteria-induced motility. Furthermore, JNK and paxillinTyr-118 were activated by 60190DeltacagA but not 60190DeltacagE, demonstrating CagA-independent signaling critical for cell motility. A beta1 integrin-blocking antibody significantly inhibited JNK and paxillinTyr-118 phosphorylation and cell scattering, demonstrating that CagA-independent signaling required for cell motility occurs through beta1. The requirement of both Src and focal adhesion kinase for signaling and motility further suggests the importance of integrin signaling in H. pylori-induced cell motility. Finally, we show that JNK activation occurs independent of known upstream kinases and signaling molecules, including Nod1, Cdc42, Rac1, MKK4, and MKK7, which demonstrates novel signaling leading to JNK activation. We report for the first time that H. pylori mediates CagA-independent signaling that promotes cell motility through the beta1 integrin pathway.     

RNF13, a RING Finger Protein,Mediates Endoplasmic Reticulum Stress-induced Apoptosis through the Inositol-requiring Enzyme (IRE1α)/c-Jun NH2-terminal Kinase Pathway

Disturbance of homeostasis at endoplasmic reticulum (ER) causes stress to cells that in turn triggers an adaptive signaling pathway termed unfolded protein response for the purpose of restoring normal cellular physiology or initiating signaling events leading to apoptosis. Identification of those genes that are involved in the unfolded protein response-mediated apoptotic signaling pathway would be valuable toward elucidating the molecular mechanism underlying the relationship between ER stress and apoptosis. We initiated a genetic screen by using the retroviral insertion mutation system to search for genes whose inactivation confers resistance to apoptosis induction by staurosporine. Using this approach, RING finger protein 13 (RNF13) was identified. Interestingly, RNF13 is highly enriched in ER. RNF13 knockdown cells are resistant to apoptosis and JNK activation triggered by ER stress. Conversely, overexpression of RNF13 induces JNK activation and caspase-dependent apoptosis. The RING and transmembrane domains of RNF13 are both required for its effects on JNK activation and apoptosis. Moreover, systematic analysis of the involvement of individual signaling components in the ER stress pathway using knockdown approach reveals that RNF13 acts upstream of the IRE1α-TRAF2 signaling axis for JNK activation and apoptosis. Finally, RNF13 co-immunoprecipitates with IRE1α, and the intact RING domain is also required for mediating its interaction. Together, our data support a model that RNF13 is a critical mediator for facilitating ER stress-induced apoptosis through the activation of the IRE1α-TRAF2-JNK signaling pathway.     

Regulation of myostatin signaling by c-Jun N-terminal kinase in C2C12 cells

Myostatin, a member of the transforming growth factor beta (TGF-beta) superfamily, is a negative regulator of skeletal muscle growth. We found that myostatin could activate c-Jun N-terminal kinase (JNK) signaling pathway in both proliferating and differentiating C2C12 cells. Using small interfering RNA (siRNA) mediated activin receptor type IIB (ActRIIB) knockdown, the myostatin-induced JNK activation was significantly reduced, indicating that ActRIIB was required for JNK activation by myostatin. Transfection of C2C12 cells with TAK1-specific siRNA reduced myostatin-induced JNK activation. In addition, JNK could not be activated by myostatin when the expression of MKK4 was suppressed with MKK4-specific siRNA, suggesting that TAK1-MKK4 cascade was involved in myostatin-induced JNK activation. We also found that blocking JNK signaling pathway by pretreatment with JNK specific inhibitor SP600125, attenuated myostatin-induced upregulation of p21 and downregulation of the differentiation marker gene expression. Furthermore, it was also observed that the presence of SP600125 almost annulled the growth inhibitory role of myostatin. Our findings provide the first evidence to reveal the involvement of JNK signaling pathway in myostatin's function as a negative regulator of muscle growth.     

Roles of phosphatidylinositol 3-kinase and Rac in the nuclear signaling by tumor necrosis factor-alpha in rat-2 fibroblasts.

We investigated the extent to which phosphatidylinositol 3-kinase (PI 3-kinase) and Rac, a member of the Rho family of small GTPases, are involved in the signaling cascade triggered by tumor necrosis factor (TNF)-alpha leading to activation of c-fos serum response element (SRE) and c-Jun amino-terminal kinase (JNK) in Rat-2 fibroblasts. Inhibition of PI 3-kinase by LY294002 or wortmannin, two specific PI 3-kinase antagonists, or co-transfection with a dominant negative mutant of PI 3-kinase dose-dependently blocked stimulation of c-fos SRE by TNF-alpha. Similarly, LY294002 significantly diminished TNF-alpha-induced activation of JNK, suggesting that nuclear signaling triggered by TNF-alpha is dependent on PI 3-kinase-mediated activation of both c-fos SRE and JNK. We also found nuclear signaling by TNF-alpha to be Rac-dependent, as demonstrated by the inhibitory effect of transient co-transfection with a dominant negative Rac mutant, RacN17. Our findings suggest that Rac is situated downstream of PI 3-kinase in the TNF-alpha signaling pathway to the nucleus, and we conclude that PI 3-kinase and Rac each plays a pivotal role in the nuclear signaling cascade triggered by TNF-alpha.     

Lipopolysaccharide-induced c-Jun NH2-terminal kinase activation in human neutrophils: role of phosphatidylinositol 3-Kinase and Syk-mediated pathways

Polymorphonuclear leukocytes (neutrophils) respond to lipopolysaccharide (LPS) through the up-regulation of several pro-inflammatory mediators. We have recently shown that LPS-stimulated neutrophils express monocyte chemoattractant protein 1 (MCP-1), an AP-1-dependent gene, suggesting that LPS activates the c-Jun N-terminal kinase (JNK) pathway in neutrophils. Previously, we have shown the activation of p38 MAPK, but not JNK, in suspended neutrophils stimulated with LPS but have recently shown activation of JNK by TNF-alpha in an adherent neutrophil system. We show here that exposure to LPS activates JNK in non-suspended neutrophils and that LPS-induced MCP-1 expression, but not tumor necrosis factor-alpha (TNF-alpha) or interleukin-8 (IL-8), is dependent on JNK activation. In addition, LPS stimulation of non-suspended neutrophils activates Syk and phosphatidylinositol 3-kinase (PI3K). Inhibition of Syk with piceatannol or PI3K with wortmannin inhibited LPS-induced JNK activation and decreased MCP-1 expression after exposure to LPS, suggesting that both Syk and PI3K reside in a signaling pathway leading to LPS-induced JNK activation in neutrophils. This Syk- and PI3K-dependent pathway leading to JNK activation after LPS exposure in non-suspended neutrophils is specific for JNK, because inhibition of neither Syk nor PI3K decreased p38 activation after LPS stimulation. Furthermore we show that PI3K inhibition decreased LPS-induced Syk activation suggesting that PI3K resides upstream of Syk in this pathway. Finally, we show that Syk associates with Toll-like receptor 4 (TLR4) upon LPS stimulation further implicating Syk in the LPS-induced signaling pathway in neutrophils. Overall our data suggests that LPS induces JNK activation only in non-suspended neutrophils, which proceeds through Syk- and PI3K-dependent pathways, and that JNK activation is important for LPS-induced MCP-1 expression but not for TNF-alpha or IL-8 expression.     

HPK1, a hematopoietic protein kinase activating the SAPK/JNK pathway.

In mammalian cells, a specific stress-activated protein kinase (SAPK/JNK) pathway is activated in response to inflammatory cytokines, injury from heat, chemotherapeutic drugs and UV or ionizing radiation. The mechanisms that link these stimuli to activation of the SAPK/JNK pathway in different tissues remain to be identified. We have developed and applied a PCR-based subtraction strategy to identify novel genes that are differentially expressed at specific developmental points in hematopoiesis. We show that one such gene, hematopoietic progenitor kinase 1 (hpk1), encodes a serine/threonine kinase sharing similarity with the kinase domain of Ste20. HPK1 specifically activates the SAPK/JNK pathway after transfection into COS1 cells, but does not stimulate the p38/RK or mitogen-activated ERK signaling pathways. Activation of SAPK requires a functional HPK1 kinase domain and HPK1 signals via the SH3-containing mixed lineage kinase MLK-3 and the known SAPK activator SEK1. HPK1 therefore provides an example of a cell type-specific input into the SAPK/JNK pathway. The developmental specificity of its expression suggests a potential role in hematopoietic lineage decisions and growth regulation.     

Epidermal growth factor receptor regulates osteoclast differentiation and survival through cross-talking with RANK signaling

The epidermal growth factor receptor (EGFR) functions in various cellular physiological processes such as proliferation, differentiation, and motility. Although recent studies have reported that EGFR signaling is involved in osteoclast recruitment and formation, the molecular mechanism of EGFR signaling for the regulation of osteoclastogenesis remains unclear. We investigated the role of the EGFR in osteoclast differentiation and survival and show that the expression of the EGFR was highly up-regulated by receptor activator of nuclear factor-kappaB ligand (RANKL) during osteoclast differentiation. EGFR-specific tyrosine kinase inhibitors and EGFR knockdown blocked RANKL-dependent osteoclast formation, suggesting that EGFR signaling plays an important role in osteoclastogenesis. EGFR inhibition impaired the RANKL-mediated activation of osteoclastogenic signaling pathways, including c-Jun N-terminal kinase (JNK), NF-kappaB, and Akt/protein kinase B (PKB). In addition, EGFR inhibition in differentiated osteoclasts caused apoptosis through caspase activation. Inhibition of the phosphoinositide-3 kinase (PI3K)-Akt/PKB pathway and subsequent activation of BAD and caspases-9 and -3 may be responsible for the EGFR inhibition-induced apoptosis. The EGFR physically associated with receptor activator of nuclear factor-kappaB (RANK) and Grb2-associated binder 2 (Gab2). Moreover, RANKL transactivated EGFR. These data indicate that EGFR regulates RANKL-activated signaling pathways by cross-talking with RANK, suggesting that the EGFR may play a crucial role as a RANK downstream signal and/or a novel type of RANK co-receptor in osteoclast differentiation and survival.     

SIZ1/SIZ2 control of chromosome transmission fidelity is mediated by the sumoylation of topoisomerase II

The Smt3 (SUMO) protein is conjugated to substrate proteins through a cascade of E1, E2, and E3 enzymes. In budding yeast, the E3 step in sumoylation is largely controlled by Siz1p and Siz2p. Analysis of Siz- cells shows that SUMO E3 is required for minichromosome segregation and thus has a positive role in maintaining the fidelity of mitotic transmission of genetic information. Sumoylation of the carboxy-terminus of Top2p, a known SUMO target, is mediated by Siz1p and Siz2p both in vivo and in vitro. Sumoylation in vitro reveals that Top2p is an extremely potent substrate for Smt3p conjugation and that chromatin-bound Top2p can still be sumoylated, unlike many other SUMO substrates. By combining mutations in the TOP2 sumoylation sites and the SIZ1 and SIZ2 genes we demonstrate that the minichromosome segregation defect and dicentric minichromosome stabilization, both characteristic for Smt3p-E3-deficient cells, are mediated by the lack of Top2p sumoylation in these cells. A role for Smt3p-modification as a signal for Top2p targeting to pericentromeric regions was suggested by an analysis of Top2p-Smt3p fusion. We propose a model for the positive control of the centromeric pool of Top2p, required for high segregation fidelity, by Smt3p modification.     

Hipk promotes photoreceptor differentiation through the repression of Twin of eyeless and Eyeless expression

Organogenesis is a complex developmental process, which requires tight regulation of selector gene expression to specify individual organ types. The Pax6 homolog Eyeless (Ey) is an example of such a factor and its expression pattern reveals it is dynamically controlled during development. Ey?s paralog Twin of eyeless (Toy) induces its expression during embryogenesis, and the two genes are expressed in nearly identical patterns during the larval stages of development. While Ey must be expressed to initiate retinal specification, it must subsequently be repressed behind the morphogenetic furrow to allow for neuronal differentiation. Thus far, a few factors have been implicated in this repression including the signaling pathways Hedgehog (Hh) and Decapentaplegic (Dpp), and more recently downstream components of the retinal determination gene network (RDGN) Sine oculis (So), Eyes absent (Eya), and Dachshund (Dac). Homeodomain-interacting protein kinase (Hipk), a conserved serine–threonine kinase, regulates numerous factors during tissue patterning and development, including the Hh pathway. Using genetic analyses we identify Hipk as a repressor of both Toy and Ey and show that it may do so, in part, through Hh signaling. We also provide evidence that Ey repression is a critical step in ectopic eye development and that Hipk plays an important role in this process. Because Ey repression within the retinal field is a critical step in eye development, we propose that Hipk is a key link between eye specification and patterning.     

The yeast homologue of the microtubule-associated protein Lis1 interacts with the sumoylation machinery and a SUMO-targeted ubiquitin ligase

Microtubules and microtubule-associated proteins are fundamental for multiple cellular processes, including mitosis and intracellular motility, but the factors that control microtubule-associated proteins (MAPs) are poorly understood. Here we show that two MAPs—the CLIP-170 homologue Bik1p and the Lis1 homologue Pac1p—interact with several proteins in the sumoylation pathway. Bik1p and Pac1p interact with Smt3p, the yeast SUMO; Ubc9p, an E2; and Nfi1p, an E3. Bik1p interacts directly with SUMO in vitro, and overexpression of Smt3p and Bik1p results in its in vivo sumoylation. Modified Pac1p is observed when the SUMO protease Ulp1p is inactivated. Both ubiquitin and Smt3p copurify with Pac1p. In contrast to ubiquitination, sumoylation does not directly tag the substrate for degradation. However, SUMO-targeted ubiquitin ligases (STUbLs) can recognize a sumoylated substrate and promote its degradation via ubiquitination and the proteasome. Both Pac1p and Bik1p interact with the STUbL Nis1p-Ris1p and the protease Wss1p. Strains deleted for RIS1 or WSS1 accumulate Pac1p conjugates. This suggests a novel model in which the abundance of these MAPs may be regulated via STUbLs. Pac1p modification is also altered by Kar9p and the dynein regulator She1p. This work has implications for the regulation of dynein''s interaction with various cargoes, including its off-loading to the cortex.