Signalling and Cell Cycle Group by steepslope9876

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									             Signalling and Cell Cycle Group
             Summary:
             We are interested in understanding basic mechanisms of cell proliferation and
             differentiation, in particular how external signals are interpreted by cells to elaborate
             the appropriate responses. Our studies focus on two main areas:

             1. Mechanisms of signal transduction by the stress-activated MAP kinases p38α
                and p38β and their role in carcinogenesis

             2. Regulation and role of the RINGO/Speedy proteins, a new family of CDK
                activators

             Strategic goals:
             ■■   Understand signal integration by p38 MAP kinases and their role in carcinogenesis
             ■■   Characterise the regulation and function of the CDK activators RINGO/Speedy
             ■■   Investigate how signalling pathways control the cell cycle machinery
             ■■   Generate mouse models to study in vivo roles of p38 MAP kinases and RINGO proteins




                                                                                                     Ángel R. Nebreda
                                                                                                              Group Leader

                  Ángel R. Nebreda was born in 1961 in Benavente, Spain. He studied Biology at the Universidad de Salamanca
                  (Spain) and received his PhD in 1986 from the same university working on the cloning and characterisation
                  of fl-glucanase genes in Saccharomyces cerevisiae.
                  In 1987, he moved to the National Institutes of Health, Bethesda, USA, as a Post-doctoral Fellow in E.
                  Santos’ laboratory, where he studied signal transduction mechanisms by Ras and other oncogenes using
                  biochemical and cellular assays, mainly Xenopus oocytes and cultured mammalian cells. He then returned
                  to Europe in 1992 to join T. Hunt’s group at the Cancer Research UK Clare Hall Laboratories, South Mimms,
                                UK. During this period, his work focused on the regulation of the cell cycle machinery by
                                 signalling pathways, especially at the G2/M transition. He also cloned a new vertebrate MAP
                                  kinase, which is known today as p38 MAP kinase.
                                  In 1995, he started his own group at the European Molecular Biology Laboratory (EMBL),
                                  Heidelberg, Germany, where he and his team worked on signal transduction by MAP
                                 kinase pathways and the mechanisms that control CDK activation during oocyte meiotic
                                 maturation. His main contributions during this period relate to the mechanisms of activation
                                 and biological functions of p38 MAP kinases, using both cultured cells and genetically
                                  modified mice, and the signalling pathways that orchestrate the meiotic cell cycle, using
                                     the Xenopus system. His group also cloned and characterised a new family of proteins
                                      named RINGO/Speedy that can directly activate Cdk1 and Cdk2.
                                       Ángel R. Nebreda joined the CNIO as a Group Leader of the Signalling and Cell Cycle
                                       Group in 2004. Recent work by his group has elucidated new mechanisms by which
                                        p38 MAP kinase suppresses tumour formation as well as in vivo roles of this signaling
                                         pathway in tissue homeostasis and mouse cancer models. His laboratory has also
                                          demonstrated that RINGO proteins can change the substrate specificity of CDKs.
                                          He was elected EMBO member in 2003 and is an Editor of the journal FEBS Letters
                                           since 2004.




22 | Molecular Oncology Programme
                                                                                                                                  Scientific
                                                                                                                                    Report
                                                                                                                                         2008




Staff Scientists:            Post-doctoral             Graduate Students:                                     Technicians:
Ana Cuadrado                 Fellows:                  Nadia Corrado                Vanesa Lafarga            Soraya Ardila
Iván Del Barco               Matthieu Lemaire          (until October)              Carmen L. Pereira         (since February)
Patricia González            Silvana A. Mouron         Ignacio Dolado               E. Josué Ruiz             Irene Chicote
                                                       (until December)                                       Laura E. Doglio
Marcial Vilar                                                                       Aneta Mª Swat
(since July)                                           Jalaj Gupta                                            (since September)
                                                                                                              Esther Seco
                                                                                                              Stephan Tenbaum
                                                                                                              (until April)


                                    Signal integration by p38 MAP                      tumourigenesis. In non-transformed cells,
                                    kinases                                            confluent cell-cell contacts normally impose
                                                                                       a G0/G1 cell cycle arrest via poorly identified
                                    Contact inhibition is a fundamental process        signalling cues. We have previously reported
                                    in tissue homeostasis and one of the earliest      that cells deficient in p38α MAP kinase
                                    regulatory mechanisms bypassed during              display impaired contact inhibition,



                                                        Sparse




                                                                WT cells
                                                                                                                  Higher
                                       Confluent                                                                  cellurar density

                      Figure 1                                             oncogene
       p38α plays an important
   role in the contact inhibition
        response: the cell cycle
     arrest induced by cell-cell
  contacts. As a consequence,
              the inactivation or
       downregulation of p38α                                                                                     Enhanced
    results in higher saturation    Transformed                                                                   multi-layer growth
   density of confluent cultures
            as well as enhanced
    susceptibility to oncogenic
                 transformation.




                                                                                                        Signalling and Cell Cycle Group |   23
                                         making them more sensitive to oncogenic           tumourigenesis and inflammatory responses
                                         transformation.                                   as well as in the interplay between both
                                                                                           processes. Our results indicate that p38α
                                         We have now found that the cell cycle inhibitor   may play quite different roles depending on
                                         p27Kip1 plays a key role in this process          the tissues and types of cancer.
                                         downstream of p38α. Interestingly, p38α
                                         indirectly stabilises p27Kip1 in confluent        Cell cycle regulation by RINGO
                                         cells by inhibiting mitogenic signals from the    proteins
                                         epidermal growth factor receptor (EGFR). This
                                         p38α function is mediated by the ubiquitin        Cell cycle progression is regulated by Cyclin-
                                         ligase Siah-2 that induces degradation of         Dependent Kinases (CDKs), whose activation
                                         Sprouty 2, an inhibitor of the EGFR ubiquitin     is usually associated with the binding of cyclins.
                                         ligase cCbl. Thus, Sprouty 2 downregulation       CDK1 and CDK2 can also be activated by
                                         results in enhanced activity of cCbl, which       non-cyclin proteins named RINGO or Speedy,
                                         in turn induces EGFR ubiquitination and           which were originally identified as potent
                                         degradation with the consequent termination       inducers of the G2/M transition in Xenopus
                                         of mitogenic signalling in confluent cells.       oocytes. However, it is unclear how XRINGO
                                         Accordingly, confluent p38α-deficient cells       triggers M-phase entry in oocytes.
                                         show impaired accumulation of p27Kip1 that
                                         correlates with higher levels of both Sprouty     We have found that XRINGO-activated CDKs
                                         2 and EGFR signalling.                            can target a specific set of phosphorylation
                                                                                           sites in the regulatory domain of Myt1, a
                                         Collectively, our results indicate that           Wee1-family protein kinase that plays a key
                                         downregulation of p27Kip1 by the p38α-            role in the G2 arrest of oocytes by inhibiting
                                         Sprouty2-EGFR network plays a key role in         CDK1/cyclin B (pre-MPF). We have identified
                                         the cell cycle arrest induced by high cellular    three serines that are the major phospho-
                                         density and also constitutes a barrier against    acceptor sites for CDK/XRINGO but are
                                         cell transformation (Figure 1).                   poorly phosphorylated by CDK/cyclin.
                                                                                           Phosphorylation of these Ser inhibits Myt1
                                         We have generated genetically modified            activity in vitro. Importantly, these Myt1
                                         mice to investigate the functions of p38α in      residues are phosphorylated during XRINGO-
                                         vivo. Using these animal models we have           induced oocyte maturation, and mutation of
                                         previously reported an important role for p38α    the three Ser to Ala makes Myt1 resistant to
                                         in lung homeostasis. We have now found that       the inhibition by CDK/XRINGO.
                                         p38α also plays important and specific roles
                                         in hematopoietic organs, which impinge on         Our results demonstrate that XRINGO-
                                         the regulation of the immune response.            activated CDKs have different substrate
                                                                                           specificity than the equivalent CDK/cyclin
                                         We are using mouse models to address              complexes. We have also identified a
                                         the role of p38α in different types of            mechanism of Myt1 regulation based on



                            Figure 2
            XRINGO-activated CDK1                                                                                 Cyclin B
             phosphorylates Myt1 on
              three residues: Ser410,                             Myt1
                  Ser414 and Ser444.                                                                                 CDK1
             These phosphorylations
           downregulate Myt1 kinase                                                         CDK1
                                                                                                                      p        p
          activity on Thr14 and Tyr15
             of CDK1, resulting in the
                                                                 Active                 phosphorylation
                                                                                                               T14
            activation of CDK1/cyclin
             B, which in turn triggers                                                                               Inactive
                       M-phase entry.



                                                                      S410 S414
             XRINGO                                                     p p                                       Cyclin B
                                                                               S444
                                                                                    p
               CDK1                                                Myt1                                              CDK1
                                          Myt1
                                     phosphorylation
                                                                 Inactive                                             Active

24 | Molecular Oncology Programme
                                                                                                                                      Scientific
                                                                                                                                        Report
                                                                                                                                             2008
site-specific phosphorylation, which is likely     the RINGO E-activated CDKs have different
to mediate the induction of G2/M transition        substrate specificity than the CDKs activated
in oocytes by XRINGO (Figure 2).                   by other RINGO proteins, which may account
                                                   for their different effects on the cell cycle. These
Five different mammalian RINGO/Speedy              results indicate that although all RINGO/Speedy
proteins have been identified, all sharing a       family members can activate CDKs, they may
conserved central ‘core’ of about 75 residues.     differently regulate cell cycle progression.
We have biochemically and functionally
characterised RINGO E, a distant member            We are characterising RINGO A, the
of this protein family, which is apparently only   mammalian RINGO protein that is most
expressed in human cells.                          similar to Xenopus XRINGO. We have found
                                                   that RINGO A is a highly unstable protein
In contrast with other RINGO proteins,             whose expression and phosphorylation are
we found that RINGO E negatively affects           periodically regulated during the cell cycle.
cell cycle progression. Thus, RINGO E
overexpression inhibits the meiotic progression    RINGO A is normally degraded by the
in Xenopus oocytes as well as the proliferation    proteasome and the process involves the
of mammalian cells in culture.                     ubiquitin ligase Skp2. We have evidence
                                                   suggesting that tight regulation of RINGO
RINGO E can bind to endogenous CDK1                A is important for the somatic cell cycle of
and CDK2 in both cellular systems. However,        mammalian cells. ■




Publications
Dolado, I., Nebreda, A.R. (2008). AKT         Jagemann, L.R., Pérez-Rivas, L.G., Ruiz,        Hernández-Torres, F., Martínez-Fernández,
and oxidative stress team up to kill          E.J., Ranea, J.A., Sánchez-Jiménez,             S., Zuluaga, S., Nebreda, A., Porras, A.,
cancer cells. Cancer Cell 14, 427-429.        F., Nebreda, A.R., Alba, E., Lozano, J.         Aránega, A.E., Navarro, F. (2008). A role
                                              (2008). The functional interaction of 14-       for p38alpha mitogen-activated protein
Ruiz, E.J., Hunt, T., Nebreda, A.R. (2008).                                                   kinase in embryonic cardiac differentiation.
                                              3-3 proteins with the ERK1/2 scaffold
Meiotic inactivation of Xenopus Myt1 by                                                       FEBS Lett 582, 1025-1031.
                                              KSR1 occurs in an isoform-specific
CDK/XRINGO, but not CDK/cyclin, via
                                              manner. J Biol Chem 283, 17450-
site-specific phosphorylation. Mol Cell                                                       Awards and recognition
                                              17462.
32, 210-220.
                                              Dinarina, A., Ruiz, E.J., O’Loghlen, A.,        •	    “Best Biomedical Research Work
Heinrichsdorff, J., Luedde, T., Perdiguero,                                                         in 2007” Award from the Fundación
                                              Mouron, S., Perez, L., Nebreda, A.R.
E., Nebreda, A.R., Pasparakis, M. (2008).                                                           de Investigación Médica Mutua
                                              (2008). Negative regulation of cell-
p38 alpha MAPK inhibits JNK activation                                                              Madrileña
                                              cycle progression by RINGO/Speedy E.
and collaborates with IkappaB kinase
                                              Biochem J 410, 535-542.
2 to prevent endotoxin-induced liver
failure. EMBO Rep 9, 1048-1054.




                                                                                                          Signalling and Cell Cycle Group |    25

								
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