Ran in Mitosis - Ras Superfamily Small G Proteins: Biology and Mechanisms 2 - page 127

The structure of RCC1 resembles a seven-bladed propeller (Renault et al. 1998 ),
whose N-terminus seems to mediate chromatin docking by promoting interaction
with the DNA (Chen et al. 2007 ), while other sequence elements bind to the Histone
2A/B dimer (Nemergut et al. 2001 ). A recent structural analysis of two RCC1
molecules on a nucleosome shows insights into the mode of interaction with
nucleosomes: Two Arginines in an unstructured region in RCC1 named the
“switchback loop” mediate binding to an acidic patch in the H2A/B dimer. A
motif next to the switchback loop further contributes to RCC1 DNA binding
directly and promotes contacts with the negatively charged DNA phospho-
backbone. Although not visible in the crystal structure, the model also supports
an important function in DNA binding of the unstructured N-terminal RCC1 tail
(Makde et al. 2010 ). Consistently, mitotic phosphorylation of RCC1 in the
N-terminal tail at Serine 11 by Cdk1 modulates chromatin binding (Hutchins
et al. 2004 ; Li and Zheng 2004 ; Hitakomate et al. 2010 ), a mechanism that targets
in particular one ( γ ) out of three human RCC1 isoforms ( ʱ , ʲ , γ ) to mitotic

chromatin. The -isoform shows high mitotic phosphorylation levels, interacts

tightly with mitotic chromatin and, in the form of a phosphomimetic mutant,
efficiently rescues the temperature-dependent tsBN2 phenotype of premature chro-
mosome condensation (Hood and Clarke 2007 ). Furthermore, N-terminal
tri-methylation after trimming the starting methionine of RCC1 promotes direct
DNA binding, which is essential for proper mitosis in intact human cells (Chen
et al. 2007 ). Strong chromatin binding of RCC1 in mitosis is also coupled to Ran's
nucleotide exchange. The binary RCC1-RanGDP complex stably binds to chroma-
tin, while RCC1-RanGTP quickly dissociates. This mechanism ensures local,
effective RanGTP production on mitotic chromatin (Li et al. 2003 ) and is consistent
with the structural data of RCC1 on nucleosomes (Hondele and Ladurner 2010 ;
Makde et al. 2010 ). Although recent data question the role of N-terminal phos-
phorylations of RCC1 in chromatin binding, they confirm, using fluorescence
correlation spectroscopy, that the interaction of RCC1 with chromatin is positively
regulated in mitosis. Tight binding also increases the enzymatic activity in nucle-
otide exchange (Bierbaum and Bastiaens 2013 ), which validates the idea that the
generation of RanGTP not only continues in mitosis but that RCC1 on mitotic
chromosomes produces more RanGTP than during interphase (Fig. 7.1 ).
To ensure efficient mitotic RanGTP production, Ran also undergoes modifica-
tions in mitosis, in particular at Serine 135 (S135), which is targeted by p21
activated kinase 4 (PAK4). PAKs best understood function lies in the regulation
of exit from G1 and G2 phases of the cell cycle, but recent data indicate that it also
regulates proper chromosome alignment and spindle function in mitosis (Bompard
et al. 2013 ). Phosphorylation of Ran at S135 makes the GTP conformation more
resistant to GTP hydrolysis, thus increasing the effective RanGTP concentration in
a PAK4-dependent manner. Interestingly, colocalisation studies in human cells
indicate that active PAK4 and Ran colocalize not only on mitotic chromosomes
but also at centrosomes and the spindle midzone (Bompard et al. 2010 ). This further
suggests a complex, non-uniform pattern of RanGTP activity that regulates key
activities in spindle formation locally even at a distance from chromatin.

γ