Weihong Wen

MST1 promotes apoptosis through phosphorylation of histone H2AX

MST1 (mammalian STE20-like kinase 1) is a serine/threonine kinase that is cleaved and activated by caspases during apoptosis. Overexpression of MST1 induces apoptotic morphological changes such as chromatin condensation, but the mechanism is not clear. Here we show that MST1 induces apoptotic chromatin condensation through its phosphorylation of histone H2AX at Ser-139. During etoposide-induced apoptosis in Jurkat cells, the cleavage of MST1 directly corresponded with strong H2AX phosphorylation. In vitro kinase assay results showed that MST1 strongly phosphory-lates histone H2AX. Western blot and kinase assay results with a mutant S139A H2AX confirmed that MST1 phos-phorylates H2AX at Ser-139. Direct binding of MST1 and H2AX can be detected when co-expressed in HEK293 cells and was also confirmed by an endogenous immunoprecipitation study. When overexpressed in HeLa cells, both the MST1 full-length protein and the MST1 kinase domain (MST1-NT), but not the kinase-negative mutant (MST1-NT-KN), could induce obvious endogenous histone H2AX phosphorylation. The caspase-3 inhibitor benzyloxycarbonyl-DEVD-fluoromethyl ketone (Z-DEVD-fmk) attenuates phosphorylation of H2AX by MST1 but cannot inhibit MST1-NT-induced histone H2AX phosphorylation, indicating that cleaved MST1 is responsible for H2AX phosphory-lation during apoptosis. Histone H2AX phosphorylation and DNA fragmentation were suppressed in MST1 knockdown Jurkat cells after etoposide treatment. Taken together, our data indicated that H2AX is a substrate of MST1, which functions to induce apoptotic chromatin condensation and DNA fragmentation.

Phosphorylation of Caspase-7 by p21-activated Protein Kinase (PAK) 2 Inhibits Chemotherapeutic Drug-induced Apoptosis of Breast Cancer Cell Lines

p21-activated kinase (PAK) 2, a member of the PAK family of serine/threonine protein kinases, plays an important role in physiological processes such as motility, survival, mitosis, and apoptosis. However, the role of PAK2 in resistance to chemotherapy is unclear. Here we report that PAK2 is highly expressed in human breast cancer cell lines and human breast invasive carcinoma tissue compared with a human non-tumorigenic mammary epithelial cell line and adjacent normal breast tissue, respectively. Interestingly, we found that PAK2 can bind with caspase-7 and phosphorylate caspase-7 at the Ser-30, Thr-173, and Ser-239 sites. Functionally, the phosphorylation of caspase-7 decreases its activity, thereby inhibiting cellular apoptosis. Our data indicate that highly expressed PAK2 mediates chemotherapeutic resistance in human breast invasive ductal carcinoma by negatively regulating caspase-7 activity.

Phosphorylation of Caspase-8 (Thr-263) by Ribosomal S6 Kinase 2 (RSK2) Mediates Caspase-8 Ubiquitination and Stability

The ribosomal S6 kinase 2 (RSK2) is a member of the p90 ribosomal S6 kinase (p90RSK) family of proteins and plays a critical role in proliferation, cell cycle, and cell transformation. Here, we report that RSK2 phosphorylates caspase-8, and Thr-263 was identified as a novel caspase-8 phosphorylation site. In addition, we showed that EGF induces caspase-8 ubiquitination and degradation through the proteasome pathway, and phosphorylation of Thr-263 is associated with caspase-8 stability. Finally, RSK2 blocks Fas-induced apoptosis through its phosphorylation of caspase-8. These data provide a direct link between RSK2 and caspase-8 and identify a novel molecular mechanism for caspase-8 modulation by RSK2.

RSK2 mediates NF-κB activity through the phosphorylation of IκBα in the TNF-R1 pathway

The ribosomal S6 kinase 2 (RSK2) is a well‐known serine/threonine kinase and a member of the p90 ribosomal S6 kinase (p90RSK) family of proteins. It is activated downstream of the MEK/ERKs cascade by mitogenic stimuli such as EGF or TPA. Here, we show that RSK2 is activated by treatment with tumor necrosis factor‐α (TNF‐α) and directly phosphorylates IκBα at Ser‐32, leading to lκBα degradation. The phosphorylation of IκBα promotes the activation and translocation of the nuclear factor‐κB (NF‐κB) subunits p65 and p50 to the nucleus. The net result is an increased NF‐κB activity, which serves as a mechanism for RSK2 blockade of TNF‐α‐induced apoptosis and enhanced cell survival.—Peng, C., Cho, Y.‐Y., Zhu, F., Xu, Y.‐M., Wen, W., Ma, W.‐Y., Bode, A. M., Dong, Z. RSK2 mediates NF‐κB activity through the phosphorylation of IκBα in the TNF‐R1 pathway. FASEB J. 24, 3490–3499 (2010). www.fasebj.org

P21-activated protein kinase (PAK2)-mediated c-Jun phosphorylation at 5 threonine sites promotes cell transformation

The oncoprotein c-Jun is one of the components of the activator protein-1 (AP-1) transcription factor complex. AP-1 regulates the expression of many genes and is involved in a variety of biological functions such as cell transformation, proliferation, differentiation and apoptosis. AP-1 activates a variety of tumor-related genes and therefore promotes tumorigenesis and malignant transformation. Here, we found that epidermal growth factor (EGF) induces phosphorylation of c-Jun by P21-activated kinase (PAK) 2. Our data showed that PAK2 binds and phosphorylates c-Jun at five threonine sites (Thr2, Thr8, Thr89, Thr93 and Thr286) in vitro and ex vivo. Knockdown of PAK2 in JB6 Cl41 (P+) cells had no effect on c-Jun phosphorylation at Ser63 or Ser73 but resulted in decreases in EGF-induced anchorage-independent cell transformation, proliferation and AP-1 activity. Mutation at all five c-Jun threonine sites phosphorylated by PAK2 decreased the transforming ability of JB6 cells. Knockdown of PAK2 in SK-MEL-5 melanoma cells also decreased colony formation, proliferation and AP-1 activity. These results indicated that PAK2/c-Jun signaling plays an important role in EGF-induced cell proliferation and transformation.

Knockdown of RNF2 induces apoptosis by regulating MDM2 and p53 stability

RNF2, also known as Ring1B/Ring2, is a component of the polycomb repression complex 1. RNF2 is highly expressed in many tumors, suggesting that it might have an oncogenic function, but the mechanism is unknown. Here, we show that knockdown of RNF2 significantly inhibits both cell proliferation and colony formation in soft agar, and induces apoptosis in cancer cells. Knockdown of RNF2 in HCT116 p53+/+ cells resulted in significantly more apoptosis than was observed in RNF2 knockdown HCT116 p53−/− cells, indicating that RNF2 knockdown-induced apoptosis is partially dependent on p53. Various p53-targeted genes were increased in RNF2 knockdown cells. Further studies revealed that in RNF2 knockdown cells, the p53 protein level was increased, the half-life of p53 was prolonged and p53 ubiquitination was decreased. In contrast, cells overexpressing RNF2 showed a decreased p53 protein level, a shorter p53 half-life and increased p53 ubiquitination. Importantly, we found that RNF2 directly binds with both p53 and MDM2 and promotes MDM2-mediated p53 ubiquitination. RNF2 overexpression could also increase the half-life of MDM2 and inhibit its ubiquitination. The regulation on p53 and MDM2 stability by RNF2 was also observed during the etoposide-induced DNA damage response. These results provide a possible mechanism explaining the oncogenic function of RNF2, and because RNF2 is important for cancer cell survival and proliferation, it might be an ideal target for cancer therapy or prevention.

Tumor Necrosis Factor Receptor-associated Factor Family Protein 2 Is a Key Mediator of the Epidermal Growth Factor-induced Ribosomal S6 Kinase 2/cAMP-responsive Element-binding Protein/Fos Protein Signaling Pathway

Background: TRAF2 function in the EGF pathway and colon cancer development is unclear. Results: Knockdown TRAF2 blocked EGF- induced cell transformation and EGF signaling pathway through RSK2 ubiquitination. Conclusion: TRAF2 has an important function in the EGF pathway and is overexpressed and required in colon cancer development. Significance: TRAF2 regulates the EGF pathway through RSK2/CREB/c-Fos and is important for colon cancer development. TRAF2 has an important function in mediating the TNF-R signaling pathway toward activation of NF-κB and JNKs. Here we reveal a novel function of TRAF2 in the epidermal growth factor (EGF) signaling pathway. Knockdown of TRAF2 blocked EGF-induced AP-1 activity and anchorage- independent cell transformation. Notably, we showed that EGF induces ribosomal S6 kinase 2 (RSK2) ubiquitination, and knocking down TRAF2 suppresses ubiquitination of RSK2 induced by EGF. We also found that TRAF2 affects RSK2 activity through RSK2 ubiquitination. RSK2 plays a critical role in AP-1 activity mediated through CREB and c-Fos, which regulates anchorage-independent cell transformation. In addition, TRAF2 is overexpressed in colon cancer and required for colon cancer development, suggesting that TRAF2 might be a potential molecular target for cancer prevention and treatment.

Abstract 4277: TRAF2 is a key mediator of the EGF-induced signaling pathway.

TRAF2 plays a critical role in the mediation of the TNF-R signaling pathway toward activation of NF-κB and JNKs. Here, we reveal a novel function of TRAF2 in the epidermal growth factor (EGF) signaling pathway. Knockdown of TRAF2 blocked EGF-induced AP-1 activity and anchorage-independent cell transformation. We also showed that TRAF2 is required for RSK2 activity through RSK2 ubiquitination and that RSK2 plays a critical role in AP-1 activity mediated through CREB/c-Fos, which regulate anchorage-independent cell transformation. Citation Format: Cong Peng, Feng Zhu, Weihong Wen, Ke Yao, Weiya Ma, Ann Bode, Zigang Dong. TRAF2 is a key mediator of the EGF-induced signaling pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4277. doi:10.1158/1538-7445.AM2013-4277

Abstract 5153: PAK2-mediated c-Jun phosphorylation at 5 threonine sites promotes cell transformation.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC PAK2 plays an important role in cell proliferation, but the direct downstream proteins and precise regulatory mechanism involved in mediating PAK2’s role in proliferation are not yet clear. Here, we found that epidermal growth factor (EGF) induces phosphorylation of c-Jun by P21-activated kinase (PAK2). Knockdown of PAK2 in JB6 Cl41 (P+) cells had no effect on c-Jun phosphorylation at Ser63 or Ser73 but resulted in decreases in EGF-induced anchorage-independent cell transformation, proliferation and AP-1 activity. The oncoprotein c-Jun is one of the components of the activator protein-1 (AP-1) transcription factor complex. AP-1 activates a variety of tumor-related genes and promotes tumorigenesis and malignant transformation. Knockdown of PAK2 in SK-MEL-5 melanoma cells also decreased colony formation, proliferation and AP-1 activity. We found that PAK2 binds and phosphorylates c-Jun at five threonine sides (Thr2, Thr8, Thr89, Thr93 and Thr286) in vitro and in vivo. Mutation at all five sites decreased the transforming ability of JB6 cells. These results indicated that PAK2/c-Jun signaling plays an important role in tumorigenesis and provides an increased understanding of the signaling transduction mechanism of skin tumor cells and suggests that PAK2 might be a new chemopreventive or chemotherapeutic target. Citation Format: Tingting Li, Jishuai Zhang, Feng Zhu, Weihong Wen, Tatyana Zykova, Xiang Li, Kangdong Liu, Cong Peng, Wei-Ya Ma, Zigang Dong, Ann M. Bode. PAK2-mediated c-Jun phosphorylation at 5 threonine sites promotes cell transformation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5153. doi:10.1158/1538-7445.AM2013-5153

Abstract 1034: A natural inhibitor of c-Jun N-terminal kinase 1

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The c-Jun N-terminal kinases (JNKs) pathway plays a important role in many physiological processes including cell proliferation, survival, death, DNA repair and metabolism induced by a variety of stress signals and pro-inflammatory stimuli. The JNK proteins are encoded by three genes, jnk1, jnk2, jnk3. The protein products of jnk1 and jnk2 are found in all cells and tissues, whereas the JNK3 protein is found primarily in the brain, but is also found in the heart and the testes. The unequal expression of the different isoforms of JNK is possibly determined by some unique characteristics. Each JNK appears to have a distinct function in cancer, asthma, diabetes, and Parkinsons disease. Recent studies demonstrate that JNK1 is a key contributor for the serine phosphorylation of the IRS-1. Several other studies also demonstrated the importance of JNK1, but not JNK2, in lung fibrosis and gastric cancer. We have identified a novel flavonoid isolated from natural sources, herein referred to Compound-A, that suppressed JNK1 activity, with no effect on JNK2 in vitro. At 10 μM, Compound-A specifically inhibited JNK1 by about 50%. We also found that compound-A inhibits UVB-induced c-Jun phosphorylation in a cell culture system. In contrast to SP600125, a non-selective JNKs inhibitor that binds to the ATP active site, compound-A appears to target the protein-protein interaction between JNK1 and its binding proteins, including the JNK-interacting protein-1(JIP1) rather than the ATP binding site. In a computer simulation model, compound-A complexed with JNK1, but not JNK2, and formed strong hydrogen bonding at Glu329. Glu329 is a key residue for the JNK complex with JIP1. This interaction induces a hinge motion between the JNK N- and C-terminal domains and decreases the affinity of the kinase and ATP. These results demonstrate that compound-A is a selective JNK1 inhibitor. In humans, JNK1 play a key role in obesity-induced type 2 diabetes mellitus and the absence of JNK1 results in decreased adiposity, significantly improved insulin sensitivity and an enhanced insulin receptor signaling capacity in mouse obesity models. Therefore, we suggest that because of JNK1s critical role in diabetes, compound-A might have therapeutic potential against this devastating disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1034. doi:10.1158/1538-7445.AM2011-1034