Protein phosphatase 2A stabilizes human securin, whose phosphorylated forms are degraded via the SCF ubiquitin ligase

Sister chromatid segregation is triggered at the metaphase-to-anaphase transition by the activation of the protease separase. For most of the cell cycle, separase activity is kept in check by its association with the inhibitory chaperone securin. Activation of separase occurs at anaphase onset, when securin is targeted for destruction by the anaphase-promoting complex or cyclosome E3 ubiquitin protein ligase. This results in the release of the cohesins from chromosomes, which in turn allows the segregation of sister chromatids to opposite spindle poles. Here we show that human securin (hSecurin) forms a complex with enzymatically active protein phosphatase 2A (PP2A) and that it is a substrate of the phosphatase, both in vitro and in vivo. Treatment of cells with okadaic acid, a potent inhibitor of PP2A, results in various hyperphosphorylated forms of hSecurin which are extremely unstable, due to the action of the Skp1/Cull/F-box protein complex ubiquitin ligase. We propose that PP2A regulates hSecurin levels by counteracting its phosphorylation, which promotes its degradation. Misregulation of this process may lead to the formation of tumors, in which overproduction of hSecurin is often observed.Ministerio de Educación y Ciencia SAF 2002-04177-C0-

Heterologous viral expression systems in fosmid vectors increase the functional analysis potential of metagenomic libraries

The extraordinary potential of metagenomic functional analyses to identify activities of interest present in uncultured microorganisms has been limited by reduced gene expression in surrogate hosts. We have developed vectors and specialized E. coli strains as improved metagenomic DNA heterologous expression systems, taking advantage of viral components that prevent transcription termination at metagenomic terminators. One of the systems uses the phage T7 RNA-polymerase to drive metagenomic gene expression, while the other approach uses the lambda phage transcription anti-termination protein N to limit transcription termination. A metagenomic library was constructed and functionally screened to identify genes conferring carbenicillin resistance to E. coli. The use of these enhanced expression systems resulted in a 6-fold increase in the frequency of carbenicillin resistant clones. Subcloning and sequence analysis showed that, besides β-lactamases, efflux pumps are not only able contribute to carbenicillin resistance but may in fact be sufficient by themselves to convey carbenicillin resistance.España, Ministerio de Educación y Ciencia BIO2011-24003España, Ministerio de Educación y Ciencia CSD2007-000

UV-induced degradation of securin is mediated by SKP1-CUL1-βTrCP E3 ubiquitin ligase

Securin is a chaperone protein with bifunctional properties. It binds to separase to inhibit premature sister chromatid separation until the onset of anaphase, and it also takes part in cell-cycle arrest after UV irradiation. At metaphase-to-anaphase transition, securin is targeted for proteasomal destruction by the anaphase-promoting complex or cyclosome (APC/C), allowing activation of separase. However, although securin is reported to undergo proteasome-dependent degradation after UV irradiation, the ubiquitin ligase responsible for securing ubiquitylation has not been well characterized. In this study, we show that UV radiation induced a marked reduction of securin in both the nucleus and cytoplasm. Moreover, we show that GSK-3β inhibitors prevent securin degradation, and that CUL1 and βTrCP are involved in this depletion. We also confirmed that SKP1-CUL1-βTrCP (SCFβTrCP) ubiquitylates securin in vivo, and identified a conserved and unconventional βTrCP recognition motif (DDAYPE) in the securin primary amino acid sequence of humans, nonhuman primates and rodents. Furthermore, downregulation of βTrCP caused an accumulation of securin in non-irradiated cells. We conclude that SCFβTrCP is the E3 ubiquitin ligase responsible for securing degradation after UV irradiation, and that it is involved in securin turnover in nonstressed cells.Ministerio de Educación y Ciencia SAF 2005-07713-C03-0

Glycogen Synthase Kinase-3-ß (GSK3ß) negatively regulates PTTG1/human Securin protein stability, and GSK3ß inactivation correlates with securin accumulation in breast tumors.

PTTG1, also known as securin, is an inactivating partner of separase, the major effector for chromosome segregation during mitosis. At the metaphase-to-anaphase transition, securin is targeted for proteasomal destruction by the anaphase-promoting complex or cyclosome, allowing activation of separase. In addition, securin is overexpressed in metastatic or genomically instable tumors, suggesting a relevant role for securin in tumor progression. Stability of securin is regulated by phosphorylation; some phosphorylated forms are degraded out of mitosis, by the action of the SKP1-CUL1-F-box protein (SCF) complex. The kinases targeting securin for proteolysis have not been identified, and mechanistic insight into the cause of securin accumulation in human cancers is lacking. Here, we demonstrate that glycogen synthase kinase-3β (GSK3β) phosphorylates securin to promote its proteolysis via SCF(βTrCP) E3 ubiquitin ligase. Importantly, a strong correlation between securin accumulation and GSK3β inactivation was observed in breast cancer tissues, indicating that GSK3β inactivation may account for securin accumulation in breast cancers.Ministerio de Ciencia e Innovación de España SAF2008-03095 y SAF2008-05046Ministerio de Sanidad de España y Fondo Europeo de Desarrollo Regional (FEDER) ISCIII-RETIC-RD06/0020-FEDERDirección General de Investigación, Tecnología y Empresa, Junta de Andalucía P08-CVI-03603 y PI09-058

Regulación de la estabilidad de la oncoproteína PTTG1/hSecurina

El objetico de esta Tesis es el estudio de la relación de la estabilidad de la oncoproteína PTTG1/Sec. Para ello nos proponemos abordar dos objetivos concretos: 1. La securina humana, PTTG1/Sec, además de su función como inhibidor de la separación de las cromátidas hermanas antes de la anafase, interviene en la respuesta de la célula a los daños en el ADN. La radiación ultravioleta provoca un descenso drástico de la proteína. Este descenso es debido, en parte, a un aumento de la inestabilidad de PTTG1/Sec. No obstante, queda una proporción de PTTG1/Sec que no se destruye y que es necesaria para la parada del ciclo celular en respuesta a la radiación. Partiendo de estos antecedentes nos proponemos identificar la ligasa de ubiquitina implicada en la degradación de PTTG1/Sec en respuesta a la radiación ultravioleta. 2. La securina humana, PTTG1/Sec, es una fosfoproteína. El tratamiento con un inhibidor de fosfatasas, el ácido okadaico. OK, provoca la aparición de una serie de formas fosforiladas que aparentemente son más inestables. Por otra parte, PTTG1/Sec forma complejos y es un sustrato de la serina-treonina fosfatasa PP2A. partiendo de estos antecedentes nos hemos propuesto estudiar el papel de la fosforilación en la estabilidad de la proteína así como la función de la interacción PTTG1/Seg/PP2A

SCF(FBXW7)-mediated degradation of p53 promotes cell recovery after UV-induced DNA damage.

Eukaryotic cells have developed sophisticated mechanisms to ensure the integrity of the genome and prevent the transmission of altered genetic information to daughter cells. If this control system fails, accumulation of mutations would increase risk of diseases such as cancer. Ubiquitylation, an essential process for protein degradation and signal transduction, is critical for ensuring genome integrity as well as almost all cellular functions. Here, we investigated the role of the SKP1-Cullin-1-F-box protein (SCF)-[F-box and tryptophan-aspartic acid (WD) repeat domain containing 7 (FBXW7)] ubiquitin ligase in cell proliferation by searching for targets implicated in this process. We identified a hitherto-unknown FBXW7-interacting protein, p53, which is phosphorylated by glycogen synthase kinase 3 at serine 33 and then ubiquitylated by SCF(FBXW7) and degraded. This ubiquitylation is carried out in normally growing cells but primarily after DNA damage. Specifically, we found that SCF(FBXW7)-specific targeting of p53 is crucial for the recovery of cell proliferation after UV-induced DNA damage. Furthermore, we observed that amplification of FBXW7 in wild-type p53 tumors reduced the survival of patients with breast cancer. These results provide a rationale for using SCF(FBXW7) inhibitors in the treatment of this subset of tumors.-Galindo-Moreno, M., Giráldez, S., Limón-Mortés, M. C., Belmonte-Fernández, A., Reed, S. I., Sáez, C., Japón, M. Á., Tortolero, M., Romero, F. SCF(FBXW7)-mediated degradation of p53 promotes cell recovery after UV-induced DNA damage

Cisplatin-induced Cell Death Increases the degradation of the MRE11-RAD50-NBS1 Complex Through the Autophagy/lysosomal Pathway

Cisplatin and other platinum-based anticancer agents are among the most widely used chemotherapy drugs in the treatment of different types of cancer. However, it is common to find patients who respond well to treatment at first but later relapse due to the appearance of resistance to cisplatin. Among the mechanisms responsible for this phenomenon is the increase in DNA damage repair. Here, we elucidate the effect of cisplatin on the MRN (MRE11-RAD50-NBS1) DNA damage sensor complex. We found that the tumor suppressor FBXW7 is a key factor in controlling the turnover of the MRN complex by inducing its degradation through lysosomes. Inhibition of lysosomal enzymes allowed the detection of the association of FBXW7-dependent ubiquitylated MRN with LC3 and the autophagy adaptor p62/SQSTM1 and the localization of MRN in lysosomes. Furthermore, cisplatin-induced cell death increased MRN degradation, suggesting that this complex is one of the targets that favor cell death. These findings open the possibility of using the induction of the degradation of the MRN complex after genotoxic damage as a potential therapeutic strategy to eliminate tumor cells.Ministerio de Ciencia e Innovación SAF2017-87358-C2-1/2-R, PID2020-118774RB-C21, PID2020-118774RB-C22Junta de Andalucía 2021/BIO-211, 10102426

Regulación de los niveles de PLK1 por la E3 ligasa SKP1-CUL1-F-box

Trabajo presentado en el XXXVII Congreso de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celbrado en Granada del 9 al 12 de septiembre de 2014.PLK1 (Polo-Like Kinase1) es una quinasa de serina/treonina que interviene en muchos de los eventos que tienen lugar durante la mitosis, como la entrada en mitosis, la maduración de los centrosomas, el ensamblaje del huso mitótico, la separación de las cromátidas hermanas, la activación del complejo APC/C y la salida de mitosis y el inicio de la citocinesis. Además, PLK1 está implicada en la replicación del ADN y en el restablecimiento del ciclo celular tras el punto de control de G2 producido por los daños en el ADN. Durante el ciclo, PLK1 se degrada vía APC/C-proteasoma comenzando su degradación al final de la mitosis y continuando a lo largo de la fase G1. PLK1 también se degrada por el APC/C en G2 en respuesta al daño en el ADN. Por otra parte, recientemente hemos descrito que en la fase S los niveles de PLK1 están controlados por la ligasa de ubicuitina SCFFBXW7α. En este caso, PLK1 es ubicuitilada y degradada vía proteasoma en condiciones no estresantes e incrementándose este efecto tras radiación ultravioleta. Esta degradación contribuye a la parada del ciclo celular en la fase S al impedir la formación de los complejos de pre-replicación (pre- RC), lo que reduce la proliferación celular. Dado que se ha descrito que las ligasas de ubicuitina SCFFBXW7α y SCFβTrCP pueden regular la estabilidad de un mismo sustrato, tanto cooperando como interfiendo en la degradación del mismo, hemos estudiado el efecto de SCFβTrCP sobre PLK1 y sus posibles implicaciones en las funciones de la quinasa.N