c-Cbl is not required for ERK1/2-dependent degradation of BimEL

Bim_EL the most abundant Bim splice variant, is subject to ERK1/2-catalysed phosphorylation, which targets it for ubiquitination and proteasome-dependent destruction. In contrast, inactivation of ERK1/2, following withdrawal of survival factors, promotes stabilization of Bim_EL. It has been proposed that the RING finger protein Cbl binds to Bim_EL and serves as its E3 ubiquitin ligase. However, this is controversial since most Cbl substrates are tyrosine phosphoproteins and yet Bim_EL is targeted for destruction by ERK1/2-catalysed serine phosphorylation. Consequently, a role for Cbl could suggest a second pathway for Bim_EL turnover, regulated by direct tyrosine phosphorylation, or could suggest that Bim_EL is a coincidence detector, requiring phosphorylation by ERK1/2 and a tyrosine kinase. Here we show that degradation of Bim_EL does not involve its tyrosine phosphorylation; indeed, Bim_EL is not a tyrosine phosphoprotein. Furthermore, Bim_EL fails to interact with Cbl and growth factor-stimulated, ERK1/2-dependent Bim_EL turnover proceeds normally in Cbl-containing or Cbl−/− fibroblasts. These results indicate that Cbl is not required for ERK1/2-dependent Bim_EL turnover in fibroblasts and epithelial cells and any role it has in other cell types is likely to be indirect.     

RACK1 associates with CLEC-2 and promotes its ubiquitin-proteasome degradation

CLEC-2 is a C-type lectin-like receptor and plays an important role in platelet activation. Snake venom toxin rhodocytin and the endogenous sialoglycoprotein podoplanin are identified as ligands for CLEC-2 and function as stimulators in platelet activation. We also previously indentified two splice variants of murine CLEC-2 as well as a soluble fragment cleaved from the full-length form. However, little is known about the interacting partners with the cytoplasmic region of CLEC-2. In this study, we reported that RACK1, the receptor for activated C-kinase 1, associated with the cytoplasmic tail of CLEC-2. Moreover, overexpression of RACK1 decreased the stability of CLEC-2 through promoting its ubiquitin-proteasome degradation, without impairing surface expression and downstream signaling of CLEC-2. Taken together, these results suggest RACK1 as a novel modulator of CLEC-2 expression.     

RACK1 promotes the proliferation of THP1 acute myeloid leukemia cells

The receptor for activated C kinase 1 (RACK1), an adaptor protein implicated in the regulation of multiple signaling pathways, has been reported to contribute to the survival of leukemic progenitor cells by enhancing the activity of glycogen synthase kinase 3β (GSK3β). However, it remains unknown whether RACK1 also contributes to the oncogenic growth of acute myeloid leukemia (AML) cells. Here, we report that transient or stable silencing of endogenous RACK1 expression by RACK1 short hairpin RNAs (shRNAs) led to impaired proliferation of THP1 AML cells without inducing terminal differentiation. Further exploration revealed that RACK1 loss-of-function resulted in reduced GSK3β activity. GSK3β shRNA treatment showed similar effects to RACK1 loss-of-function. Our data collectively suggest that RACK1 contributes to THP1 cell proliferation through, at least partially, enhancing GSK3β activity. Thus, targeting RACK1 may have some important therapeutic implications in the treatment of AML.     

RACK1 inhibits the serum- and anchorage-independent growth of v-Src transformed cells

Cancer cells are capable of serum- and anchorage-independent growth, and focus formation on monolayers of normal cells. Previously, we showed that RACK1 inhibits c-Src kinase activity and NIH3T3 cell growth. Here, we show that RACK1 partially inhibits v-Src kinase activity, and the serum- and anchorage-independent growth of v-Src transformed cells, but has no effect on focus formation. RACK1-overexpressing v-Src cells show disassembly of podosomes, which are actin-rich structures that are distinctive to fully transformed cells. Together, our results demonstrate that RACK1 overexpression in v-Src cells partially reverses the transformed phenotype of the cells. Our results identify an endogenous inhibitor of the oncogenic Src tyrosine kinase and of cell transformation.     

XBP-1u suppresses autophagy by promoting the degradation of FoxO1 in cancer cells

Autophagy is activated to maintain cellular energy homeostasis in response to nutrient starvation. However, autophagy is not persistently activated, which is poorly understood at a mechanistic level. Here, we report that turnover of FoxO1 is involved in the dynamic autophagic process caused by glutamine starvation. X-box-binding protein-1u (XBP-1u) has a critical role in FoxO1 degradation by recruiting FoxO1 to the 20S proteasome. In addition, the phosphorylation of XBP-1u by extracellular regulated protein kinases1/2 (ERK1/2) on Ser61 and Ser176 was found to be critical for the increased interaction between XBP-1u and FoxO1 upon glutamine starvation. Furthermore, knockdown of XBP-1u caused the sustained level of FoxO1 and the persistent activation of autophagy, leading to a significant decrease in cell viability. Finally, the inverse correlation between XBP-1u and FoxO1 expression agrees well with the expression profiles observed in many human cancer tissues. Thus, our findings link the dynamic process of autophagy to XBP-1u-induced FoxO1 degradation.     

MCM7与RACK1在肺癌中的表达及其临床意义

目的探讨MCM7与RACK1在各类型肺癌中的表达及其相关性。方法收集肺腺癌150例、鳞癌150例、大细胞癌20例和小细胞癌50例及其癌旁正常肺组织石蜡标本,采用免疫组织化学S-P法检测各类型肺癌组织和癌旁正常肺组织中MCM7与RACK1的表达,并分析二者与肺癌临床病理因素的关系。结果 MCM7与RACK1在癌组织中的表达明显高于癌旁正常肺组织;χ~2检验结果显示肺癌中MCM7与RACK1的表达均与肺癌的病理分级、淋巴结转移、组织学分类和临床TNM分期相关;Pearson相关性分析结果显示,肺癌中MCM7与RACK1的表达呈正相关;Kaplan-Meier法分析显示MCM7与RACK1高表达患者生存时间缩短。结论肺癌中MCM7与RACK1的表达呈正相关,二者表达在肺癌中起促进作用,可作为检测肺癌细胞的增殖状态、新的判断预后的重要因子,以其为靶向进行临床治疗的重要指标。     

ROS mediate selenite-induced apoptosis in colon cancer cells

Selenite-induced oxidative stress and its relationship to mitochondrial apoptosis was studied in human adenocarcinoma HT-29 cells. It is shown that selenite induces caspase-dependent apoptosis, which is mediated by mitochondria via released cytochrome c, apoptosis-inducing factor (AIF) and Smac/Diablo. Selenite activates stress kinases p38 and JNK while suppressing reduced glutathione (GSH) and thioredoxin reductase (TrxR) levels, transiently inducing heme oxygenase (HO-1) system as well as reducing Akt expression. Pre-treatment of cells with selected antioxidants and stress kinase inhibitors significantly prevented selenite-induced cell death, thereby implicating oxidative stress as a direct (Bax) as well as indirect (via kinases) cause of HT-29 cells demise. These results thus demonstrate for the first time active proapoptotic and anti-survival effects of selenite in colon cancer cells.     

Phosphorylation of RACK1 in plants

Receptor for Activated C Kinase 1 (RACK1) is a versatile scaffold protein that interacts with a large, diverse group of proteins to regulate various signaling cascades. RACK1 has been shown to regulate hormonal signaling, stress responses and multiple processes of growth and development in plants. However, little is known about the molecular mechanism underlying these regulations. Recently, it has been demonstrated that Arabidopsis RACK1 is phosphorylated by an atypical serine/threonine protein kinase, WITH NO LYSINE 8 (WNK8). Furthermore, RACK1 phosphorylation by WNK8 negatively regulates RACK1 function by influencing its protein stability. These findings promote a new regulatory system in which the action of RACK1 is controlled by phosphorylation and subsequent protein degradation.     

Tumor-infiltrating programmed death receptor-1+ dendritic cells mediate immune suppression in ovarian cancer

Within the ovarian cancer microenvironment, there are several mechanisms that suppress the actions of antitumor immune effectors. Delineating the complex immune microenvironment is an important goal toward developing effective immune-based therapies. A dominant pathway of immune suppression in ovarian cancer involves tumor-associated and dendritic cell (DC)-associated B7-H1. The interaction of B7-H1 with PD-1 on tumor-infiltrating T cells is a widely cited theory of immune suppression involving B7-H1 in ovarian cancer. Recent studies suggest that the B7-H1 ligand, programmed death receptor-1 (PD-1), is also expressed on myeloid cells, complicating interpretations of how B7-H1 regulates DC function in the tumor. In this study, we found that ovarian cancer-infiltrating DCs progressively expressed increased levels of PD-1 over time in addition to B7-H1. These dual-positive PD-1(+) B7-H1(+) DCs have a classical DC phenotype (i.e., CD11c(+)CD11b(+)CD8(-)), but are immature, suppressive, and respond poorly to danger signals. Accumulation of PD-1(+)B7-H1(+) DCs in the tumor was associated with suppression of T cell activity and decreased infiltrating T cells in advancing tumors. T cell suppressor function of these DCs appeared to be mediated by T cell-associated PD-1. In contrast, ligation of PD-1 expressed on the tumor-associated DCs suppressed NF-κB activation, release of immune regulatory cytokines, and upregulation of costimulatory molecules. PD-1 blockade in mice bearing ovarian cancer substantially reduced tumor burden and increased effector Ag-specific T cell responses. Our results reveal a novel role of tumor infiltrating PD-1(+)B7-H1(+) DCs in mediating immune suppression in ovarian cancer.     

植物RACK1蛋白研究进展

RACK1（蛋白激酶C受体）是一种色氨酸-天门冬氨酸域（WD40结构）重复蛋白。它是一种多功能支架蛋白, 结合来自不同转导通路的信号分子并在多种哺乳动物发育过程中起关键作用。在植物中也存在RACK1同源基因, 如拟南芥基因组有3个编码RACK1蛋白质的基因, 这3个蛋白质与哺乳动物RACK1在氨基酸水平的相似性都超过75%。此外, 植物RACK1蛋白质包含的WD40数量、位置和蛋白激酶C结合位点的结构域在很大程度上是保守的。该文对植物RACK1蛋白的发现、结构及其在信号转导方面的功能进行综述。     

SGT1 regulates Akt signaling by promoting beta-TrCP-dependent PHLPP1 degradation in gastric cancer cells

SGT1 (suppressor of G2 allele of Skp1) plays a role in various cellular processes including kinetochore assembly and protein ubiquitination by interacting with Skp1, a component of SCF E3 ligase complex. However, the function of SGT1 in cancer is largely unknown. Here, we showed that SGT1 was over-expressed in gastric cancer tissues and silencing of SGT1 by siRNAs significantly inhibited the growth and colony formation of gastric cancer cells. We further showed that SGT1 could regulate Akt signaling pathway by modulating Akt ser473 phosphorylation status. Moreover, we found that SGT1 was able to regulate the stability of PHLPP1, which is the direct phosphatase for Akt ser473 phosphorylation. Immunoprecipitation assay revealed that SGT1 could enhance the binding between PHLPP1 and beta-TrCP which has been documented to be able to target PHLPP1 for destruction. Decreased PHLPP1 in SGT1 over-expressed gastric cancer cells failed to dephosphorylate Akt and resulted in increased Akt ser473 phosphorylation and amplified downstream Akt signaling. Thus, our data revealed a previously uncovered role of SGT1 in gastric cancer development, and suggested that SGT1 could be a promising anti-cancer target to against gastric cancer.     

Evidence that Ser87 of BimEL is phosphorylated by Akt and regulates BimEL apoptotic function

Bim, the Bcl-2 interacting mediator of cell death, is a member of the BH3-only family of pro-apoptotic proteins. Recent studies have demonstrated that the apoptotic activity of Bim can be regulated through a post-translational mechanism whereby ERK phosphorylation serves as a signal for Bim ubiquitination and proteasomal degradation. In this report, we investigated the signaling pathways leading to Bim phosphorylation in Ba/F3 cells, an interleukin-3 (IL-3)-dependent B-cell line. IL-3 stimulation induced phosphorylation of Bim(EL), one of the predominant isoforms of Bim expressed in cells, at multiple sites, as evidenced by the formation of at least three to four bands by Western blotting that were sensitive to phosphatase digestion. The appearance of multiple, phosphorylated species of Bim(EL) correlated with Akt, and not ERK, activation. The PI3K inhibitor, LY294002, blocked IL-3-stimulated Akt activity and partially blocked Bim(EL) phosphorylation. In vitro kinase assays showed that recombinant Akt could directly phosphorylate a GST-Bim(EL) fusion protein and identified the Akt phosphorylation site in the Bim(EL) domain as Ser(87). Further, we demonstrated that cytokine stimulation promotes Bim(EL) binding to 14-3-3 proteins. Finally, we show that mutation of Ser(87) dramatically increases the apoptotic potency of Bim(EL). We propose that Ser(87) of Bim(EL) is an important regulatory site that is targeted by Akt to attenuate the pro-apoptotic function of Bim(EL), thereby promoting cell survival.     

Metalloproteinases mediate extracellular matrix degradation by cells from mouse blastocyst outgrowths.

The maintenance and developmental remodeling of extracellular matrix is crucial to such processes as uterine implantation and the cell migratory events of morphogenesis. When mouse blastocysts are placed in culture they adhere to extracellular matrix, and trophoblast giant cells migrate out onto the matrix and degrade it. The secretion of functional proteinases by developing mouse embryos increases dramatically at the time of implantation. By zymography we identified the major secreted gelatin-degrading proteinase, also known as type IV collagenase, as one migrating at 92 x 10(3) Mr. Several casein-degrading proteinases were also secreted. The tissue inhibitor of metalloproteinases (TIMP) inhibited all of the embryo-derived proteinases detected by gelatin gel zymography, indicating that they are metalloproteinases, whereas TIMP did not inhibit all of the caseinases. Urokinase was also secreted. Addition of TIMP at 5-500 nM effectively inhibited the degradation of matrix by the trophoblast outgrowths. Blocking antibodies directed against 92 x 10(3) Mr gelatinase abolished matrix degradation by the trophoblast cells. These observations suggest that several metalloproteinases are regulated in early development and that 92 x 10(3) Mr gelatinase, in particular, has a rate-limiting function in degradation of the maternal extracellular matrix by trophoblast cells.     

An enhanced association of RACK1 with Abl in cells transfected with oncogenic ras

The cellular RACK1 was shown in association with Abl in BALB/3T3 cells transfected with S-ras(Q(61)K) by immunoprecipitation. An identical finding was demonstrated with cells transfected with the embryonic E-ras, but not in cells without transformation. The Abl-RACK1 of transformed cells as resolvable with Triton X-114 was found with little affinity for FAK, PY(397)-FAK and integrin. Of interests, PY(397)-FAK in the membrane skeleton of transformed cells was shown in significant quantities on the Western blot. However the PY(397)-FAK of transformed cells was not functionally able to react with RACK1 and recruit cytokeratin-1, a substrate of Src, indicating that PY(397)-FAK is not operative to transmit integrin signals. In other words, the Abl-RACK1 of transformed cells cannot replace the Src-RACK1 of cells without transformation to bridge PY(397)-FAK and cytokeratin-1 for integrin signals, and the formation of Abl-RACK1 in transformed cells may block the association of PY(397)-FAK-RACK1. We characterized Abl and RACK1 from transformed cells by chromatography on a HiTrap-PEP(Taxol) affinity column, constructed from a beta-tubulin peptide specific for Taxol binding (PEP(Taxol)). However, the Triton X-100 cannot achieve the same resolution of Abl-RACK1 from plasma membrane as is shown with Triton X-114. A significant fraction of Abl was deposited at the membrane skeleton and was therefore not accessible with Triton X-100. Half of Abl resolved with Triton X-100 was demonstrated to have catalytic activity as shown with positive phosphotyrosine staining on the Western blot and competitive elution with a specific phosphate, such as sodium beta-glycerophosphate, from HiTrap-PEP(Taxol), but this was not associated with RACK1. No significant difference of RACK1 was found in Triton X-100 resolvable membrane preparations from cells with and without transformations. Future studies are planned to differentiate the mechanism operative for RACK1 associated and RACK1 freed Abl in cells transformed with oncogenic ras.