David Glover - Overview
Mitotic kinases
The cyclin dependent kinase, cdk1, is required to establish the mitotic
state, but a number of other conserved protein kinases mediate specific
aspects of mitotic progression. Polo kinase is required for centrosome
maturation at mitotic entry through the recruitment of g-tubulin and the
activation of the Abnormal Spindle protein (Asp). polo mutants show metaphase
arrest and fail to activate the anaphase promoting complex (APC/C). Finally,
Polo like kinase activity is also required for cytokinesis in organisms
as diverse as fission yeast and fruit flies. In concert with Polo kinase,
the A-type Aurora kinase is required to recruit other proteins to the
centrosome including D-TACC and its partner the Minispindles protein,
a homologue of the microtubule associated protein ch-TOG, upregulated
in several tumour types. Lack of Aurora A kinase results in spindle pole
defects including a failure of centrosomes to separate to establish bipolar
spindles. The B-type Aurora kinase, on the other hand, is required for
the phosphorylation of histone H3 and recruitment of condensins upon chromosome
condensation. It is a passenger protein that is transferred from the chromosomes
to the central spindle at the metaphase-anaphase transition where it is
subsequently required for cytokinesis. We are searching for proteins that
interact with these mitotic kinases to regulate their activity, and use
proteomics to identify substrates.
The centrosome
Asp is required in addition to the g-tubulin ring complex (gTuRC) for
the integrity of the mitotic centrosome. We study the molecular interactions
made by Asp in a 19S complex and the effects of protein phosphorylation
on microtubule nucleation by centrosomes in vitro. We have shown that
dd4 encodes the 91kDa component of the gTuRC and now examine the mitotic
and meiotic phenotypes of dd4 alleles in combination with different alleles
of g-tubulin and asp. We also examine the interactions between microtubules
nucleated by the centrosome and MAPs: Orbit, Minispindles (CH-tog), and
Dmbim.
Late mitotic pathways
Inactivation of cdk1 at the metaphase-anaphase transition occurs in part
through degradation of cyclin B by the APC/C. In addition to studies of
the APC/C, we have also found E2 ubiquitin conjugating enzymes confer
specificity for cyclin B degradation. As anaphase proceeds, the establishment
of the central spindle is then required for the correct execution of cytokinesis.
This requires the activity of the Pavarotti kinesin-like protein or its
MKLP1 counterparts in other organisms. Pav-KLP appears also to be required
to recruit cortical molecules to the contractile ring. We would like to
identify the equivalent pathway used by animal cells corresponding to
the late mitotic regulatory network identified in both fission and budding
yeasts. As part of this work we have identified genes for four Drosophila
isoforms of one component of these pathways, MOB1. We are generating mutations
in these genes in order to study their late mitotic roles.
