ERK promotes tumorigenesis by inhibiting FOXO3a via MDM2-mediated degradation

The RAS-ERK pathway is known to play a pivotal role in differentiation, proliferation and tumour progression. Here, we show that Erk downregulates Forkhead box O 3a (FOXO3a) by directly interacting with and phosphorylating FOXO3a at Ser 294, Ser 344 and Ser 425, which consequently promotes cell proliferation and tumorigenesis. The ERK-phosphorylated FOXO3a degrades via an MDM2-mediated ubiquitin-proteasome pathway. However, the non-phosphorylated FOXO3a mutant is resistant to the interaction and degradation by murine double minute 2 (MDM2), thereby resulting in a strong inhibition of cell proliferation and tumorigenicity. Taken together, our study elucidates a novel pathway in cell growth and tumorigenesis through negative regulation of FOXO3a by RAS-ERK and MDM2.     

Disrupted interaction between CFTR and AF-6/afadin aggravates malignant phenotypes of colon cancer

How mutations or dysfunction of CFTR may increase the risk of malignancies in various tissues remains an open question. Here we report the interaction between CFTR and an adherens junction molecule, AF-6/afadin, and its involvement in the development of colon cancer. We have found that CFTR and AF-6/afadin are co-localized at the cell–cell contacts and physically interact with each other in colon cancer cell lines. Knockdown of CFTR results in reduced epithelial tightness and enhanced malignancies, with increased degradation and reduced stability of AF-6/afadin protein. The enhanced invasive phenotype of CFTR-knockdown cells can be completely reversed by either AF-6/afadin over-expression or ERK inhibitor, indicating the involvement of AF-6/MAPK pathway. More interestingly, the expression levels of CFTR and AF-6/afadin are significantly downregulated in human colon cancer tissues and lower expression of CFTR and/or AF-6/afadin is correlated with poor prognosis of colon cancer patients. The present study has revealed a previously unrecognized interaction between CFTR and AF-6/afadin that is involved in the pathogenesis of colon cancer and indicated the potential of the two as novel markers of metastasis and prognostic predictors for human colon cancer.     

Sumoylation of the GTPase Ran by the RanBP2 SUMO E3 Ligase Complex

The SUMO E3 ligase complex RanBP2/RanGAP1*SUMO1/Ubc9 localizes at cytoplasmic nuclear pore complex (NPC) filaments and is a docking site in nucleocytoplasmic transport. RanBP2 has four Ran binding domains (RBDs), two of which flank RanBP2''s E3 ligase region. We thus wondered whether the small GTPase Ran is a target for RanBP2-dependent sumoylation. Indeed, Ran is sumoylated both by a reconstituted and the endogenous RanBP2 complex in semi-permeabilized cells. Generic inhibition of SUMO isopeptidases or depletion of the SUMO isopeptidase SENP1 enhances sumoylation of Ran in semi-permeabilized cells. As Ran is typically associated with transport receptors, we tested the influence of Crm1, Imp β, Transportin, and NTF2 on Ran sumoylation. Surprisingly, all inhibited Ran sumoylation. Mapping Ran sumoylation sites revealed that transport receptors may simply block access of the E2-conjugating enzyme Ubc9, however the acceptor lysines are perfectly accessible in Ran/NTF2 complexes. Isothermal titration calorimetry revealed that NTF2 prevents sumoylation by reducing RanGDP''s affinity to RanBP2''s RBDs to undetectable levels. Taken together, our findings indicate that RanGDP and not RanGTP is the physiological target for the RanBP2 SUMO E3 ligase complex. Recognition requires interaction of Ran with RanBP2''s RBDs, which is prevented by the transport factor NTF2.     

Autophagy promotes resistance to photodynamic therapy-induced apoptosis selectively in colorectal cancer stem-like cells

Recent studies have indicated that cancer stem-like cells (CSCs) exhibit a high resistance to current therapeutic strategies, including photodynamic therapy (PDT), leading to the recurrence and progression of colorectal cancer (CRC). In cancer, autophagy acts as both a tumor suppressor and a tumor promoter. However, the role of autophagy in the resistance of CSCs to PDT has not been reported. In this study, CSCs were isolated from colorectal cancer cells using PROM1/CD133 (prominin 1) expression, which is a surface marker commonly found on stem cells of various tissues. We demonstrated that PpIX-mediated PDT induced the formation of autophagosomes in PROM1/CD133⁺ cells, accompanied by the upregulation of autophagy-related proteins ATG3, ATG5, ATG7, and ATG12. The inhibition of PDT-induced autophagy by pharmacological inhibitors and silencing of the ATG5 gene substantially triggered apoptosis of PROM1/CD133⁺ cells and decreased the ability of colonosphere formation in vitro and tumorigenicity in vivo. In conclusion, our results revealed a protective role played by autophagy against PDT in CSCs and indicated that targeting autophagy could be used to elevate the PDT sensitivity of CSCs. These findings would aid in the development of novel therapeutic approaches for CSC treatment.     

Toc GTPases

Chloroplasts import more than 90% of their protein constituents from the cytosol. The import is mediated by translocon complexes located in the chloroplast envelope and the stroma. This review focuses on the two GTPases in the Toc (translocon at the outer envelope membrane of chloroplasts) complex. Hypotheses are presented about gating across the outer membrane and the possible functional states of the GTPases. This article is for the Special Issue of IMB 20th anniversary.     

Cell cycle propagation is driven by light–dark stimulation in a cultured symbiotic dinoflagellate isolated from corals

Endosymbiosis is an intriguing plant–animal interaction in the dinoflagellate–Cnidaria association. Throughout the life span of the majority of corals, the dinoflagellate Symbiodinium sp. is a common symbiont residing inside host gastrodermal cells. The mechanism of regulating the cell proliferation of host cells and their intracellular symbionts is critical for a stable endosymbiotic association. In the present study, the cell cycle of a cultured Symbiodinium sp. (clade B) isolated from the hermatypic coral Euphyllia glabrescens was investigated using flow cytometry. The results showed that the external light–dark (L:D) stimulation played a pivotal role in regulating the cell cycle process. The sequential light (40–100 μmol m⁻² s⁻¹ ~ 12 h) followed by dark (0 μmol m⁻² s⁻¹ ~ 12 h) treatment entrained a single cell cycle from the G₁ to the S phase, and then to the G₂/M phase, within 24 h. Blue light (~450 nm) alone mimicked regular white light, while lights of wavelengths in the red and infrared area of the spectrum had little or no effect in entraining the cell cycle. This diel pattern of the cell cycle was consistent with changes in cell motility, morphology, and photosynthetic efficiency (F _v /F _m ). Light treatment drove cells to enter the growing/DNA synthesis stage (i.e., G₁ to S to G₂/M), accompanied by increasing motility and photosynthetic efficiency. Inhibition of photosynthesis by 3-(3, 4-dichlorophenyl)-1, 1-dimethyl-urea (DCMU) treatment blocked the cell proliferation process. Dark treatment was required for the mitotic division stage, where cells return from G₂/M to G₁. Two different pools of adenylyl cyclase (AC) activities were shown to be involved in the growing/DNA synthesis and mitotic division states, respectively. Communicated by Biology Editor Dr Michael Lesser     

Evaluation on the effect of different in-gel peptide isoelectric focusing parameters in global proteomic profiling

Peptide isoelectric focusing (IEF) is a common technique used in two-dimensional liquid chromatography tandem mass spectrometry (2D–LC–MS/MS) proteomic workflow, in which the tryptic peptide is first pre-fractionated based on pI values before being subjected to reverse phase LC–MS analysis. Although this method has been widely used by many research groups, a systemic study on the optimal conditions and fundamental parameters influencing the experimental outcomes has been lacking, including the effect of peptide extraction methods, the extent of pre-fractionation, and the choice of pH range. In this study, we compared the effect of different parameters on the numbers of peptides and proteins identified using two complex mouse proteomes. The results indicated that extraction of peptides from immobilized pH gradient (IPG) strips by sequential elution of increasingly organic solvents provided the highest number of peptide identification. In addition, we showed that approximately 45 more unique proteins were identified for every additional fraction collected during peptide IEF. Although narrow pH ranges provided higher resolution in peptide separation as expected, different pH ranges yielded similar numbers of peptide and protein identification. Overall, we demonstrated that the extraction solvent influenced the numbers of peptide and protein identification and quantitatively demonstrated the advantage of extensive fractionation and the performance of different pH ranges in practice.