Institute of Molecular Biology, Pathology, CNR (National Research Council), c\\\/o Sapienza University of Rome, via degli Apuli 4, 00185 Rome, Italy
Mitotic spindle regulation
In mitosis the nuclear envelope breaks down and the nucleus and cytoplasmic compartments merge. At this stage, RAN takes on a second role as a global regulator of the mitotic apparatus. A large number of studies describe mitotic phenotypes associated with an imbalance in the RAN cycle, indicating that multiple steps of the mitotic division are under RAN control. Here it would not be possible to summarise all relevant studies, but many reviews address possible mechanisms of RAN in mitotic control and converge in identifying RAN as a master regulator of mitosis (Clarke and Zhang, 2008; Kalab and Heald, 2008; Ciciarello et al., 2007; Arnaoutov and Dasso, 2005; Di Fiore et al., 2004; Weis, 2003; Hetzer et al., 2002; Moore, 2001). Relevant aspects are highlighted below.
- Centrosome function. A fraction of RAN localizes at centrosomes through the anchoring protein AKAP450, a large coil-coiled scaffolding protein that tethers several factors to the centrosomes; RAN displacement from centrosomes, induced by expressing a dominant negative mutant of the anchoring protein AKAP450, prevents the formation of centrosomal microtubule asters (Keryer et al., 2003). RAN is also indirectly involved in control of centrosome duplication because it cooperates with CRM1 in regulating the centrosomal recruitment of the centrosome duplication licensing factor nucleophosmin (NPM) (Wang et al., 2005). Interestingly, some viral proteins that cause abnormal centrosome organisation and/or duplication, e.g. HLTV-1 Tax (Peloponese et al., 2005) and HPV E7 (De Luca et al., 2003), interact with the centrosomal RAN fraction and disrupt the centrosomal regulatory functions of RAN (Lavia et al., 2003).
- Mitotic spindle assembly and function. RAN is indispensable for the organisation and dynamic functions of microtubules in the mitotic spindle (Ohba et al., 1999; Carazo-Salas et al., 1999; Wilde and Zheng, 1999; Kalab et al., 1999; Zhang et al., 2009). RAN regulates the organisation of mitotic microtubules, both from centrosomes, in what is regarded as the most classical microtubule nucleation pathway, and from kinetochores (Tulu et al., 2006; Torosantucci et al., 2008; OConnell et al., 2009); the latter is regarded as an alternative pathway that is activated when the centrosomal activity is impaired, for example by mutation in centrosomal factors, or when microtubule nucleation is reactivated after treatment with microtubule-targeting drugs (OConnell and Khodjakov, 2007). A variety of mitotic factors are targets of RAN control and mediate the spindle-organising role of RAN (Clarke and Zhang, 2008; Kalab and Heald, 2008; Ciciarello et al., 2007). Mutations, or silencing, of regulators of the nucleotide-bound state of RAN imbalance the RAN cycle and result in a variety of mitotic abnormalities; recurrent elements include: a) the failure to establish a bipolar spindle, with the formation of multipolar spindles that drive unequal chromosome segregation to more than one pole; b) chromosome misalignment at the cell equator; c) altered microtubule dynamics, eventually resulting in chromosome segregation errors. All of these conditions ultimately contribute to the loss of fidelity in chromosome segregation.
- Microtubule/kinetochore interactions and spindle checkpoint function (Arnaoutov and Dasso, 2005). Imbalance in the RAN cycle is associated with abnormal attachments between microtubules and the kinetochores of chromosomes, with an ensuing failure of chromosome biorientation; cells carrying such imbalance often progress to segregate chromosomes in the presence of incomplete or incorrect microtubule attachments to kinetochores, suggesting that the microtubule defects go undetected by the mitotic spindle checkpoint. RAN mechanisms in the process are not fully understood, but it has been observed that the nucleoporin RANBP2/NUP358, a RAN-binding protein that is endowed with E3 SUMO ligase activity, as well as RANGAP1, the hydrolysis factor for RAN, which is a substrate of SUMOylation by RANBP2/NUP358, must both be recruited at kinetochores in a RANGTP-dependent manner in order to control the spindle checkpoint schedule in response to microtubule attachments to kinetochores (Joseph et al., 2002; Joseph et al., 2004; Salina et al., 2003; Arnaoutov et al., 2005; Zuccolo et al., 2007).
Nuclear envelope organisation and nuclear pore assembly
When mitosis terminates, RAN has roles in the organisation of the nuclear envelope and nuclear pores in the reforming interphase nucleus. The requirement for RAN in the process is indicated by experiments with in vitro cell-free reconstitution systems, as well as in genetic experiments in yeast and in C.elegans in vivo (reviewed by Clarke and Zhang, 2001; Hetzer et al., 2002). Some of these experiments, in particular with yeast mutants, also indicates a requirement for RAN activity in initiation of nuclear pore formation, without which the nuclear envelope precursors would fuse in a sealed envelope incapable of supporting nucleo-cytoplasmic transport. In human cells, some RAN enrichment is seen at the nuclear envelope with a punctuate pattern coinciding with nuclear pores; this reflects the accumulation of a fraction of RAN therein, possibly through interactions with the nucleoporin RANBP2/NUP358, which contains four Ran-binding domains.
A role of RAN in regulating the apoptotic response to a variety of stimuli is increasingly being recognized (Woo et al., 2008; Tietze et al., 2008; Wong et al., 2009). RAN function in apoptosis is just emerging and is therefore incompletely understood as yet. Available studies implicate RAN in at least two major apoptotic pathways.
- The first pathway, triggered by DNA damage, involves the delocalisation of RAN network members from the nucleus and entails a key role of RCC1 as a sensor of apoptotic modification in chromatin; one downstream factor that the RAN system targts in this response is NF-kB (Wong et al., 2009).
- The second pathway is triggered by microtubule-emanating signals. RAN network members have roles in microtubule dynamics and interplay with many factors that can increase, or decrease, mitotic microtubule stability, hence inducing abnormal mitotic delay. RAN and its regulators can trigger the apoptotic response via microtubules and can modulate apoptosis induction by microtubule-targeting chemotherapeutic agents. The underlying molecular pathway seems to be independent on p53 but dependent on caspases (Woo et al., 2008; Rensen et al., 2009).
- the subcellular localisation of nuclear and cytoplasmic macromolecules in interphase,
- the organisation and function of the mitotic apparatus after nuclear envelope breakdown, and
- the reorganisation of the nuclear envelope after mitosis,
- the apoptotic response to a variety of conditions.
RAN roles in these basic processes underlie the requirement for RAN function for cell viability and duplication and account for the abnormal proliferation and genetic instability observed in cells with RAN deregulated activity.
To be noted
In addition to classical RAN-dependent functions described above, a recent work highlights a novel interactions of RAN with RASSF1, a tumor suppressor protein sharing similarities with RAS effector proteins (Dallol et al., 2009). RASSF1 regulates apoptosis and loss of RASSF1 is common in a variety of human cancers.
Wilhelmina Maria Rensen ; Patrizia Lavia
RAN (RAN, member RAS oncogene family)
Atlas Genet Cytogenet Oncol Haematol. 2009-11-01
Online version: http://atlasgeneticsoncology.org/gene/42039/ran-(ran-member-ras-oncogene-family)