Budding Yeast Centrosome Duplication Requires Stabilization of Spc29 via
Mps1-mediated
Phosphorylation |
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Authors: | Eric P Holinger William M Old Thomas H Giddings Jr Catherine Wong John R Yates III and Mark Winey |
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Institution: | Departments of ‡Molecular, Cellular and Developmental Biology and §Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0347 and the ¶Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037 |
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Abstract: | Protein phosphorylation plays an important role in the regulation of
centrosome duplication. In budding yeast, numerous lines of evidence suggest a
requirement for multiple phosphorylation events on individual components of
the centrosome to ensure their proper assembly and function. Here, we report
the first example of a single phosphorylation event on a component of the
yeast centrosome, or spindle pole body (SPB), that is required for SPB
duplication and cell viability. This phosphorylation event is on the essential
SPB component Spc29 at a conserved Thr residue, Thr240. Mutation of
Thr240 to Ala is lethal at normal gene dosage, but an increased
copy number of this mutant allele results in a conditional phenotype.
Phosphorylation of Thr240 was found to promote the stability of the
protein in vivo and is catalyzed in vitro by the Mps1
kinase. Furthermore, the stability of newly synthesized Spc29 is reduced in a
mutant strain with reduced Mps1 kinase activity. These results demonstrate the
first evidence for a single phosphorylation event on an SPB component that is
absolutely required for SPB duplication and suggest that the Mps1 kinase is
responsible for this protein-stabilizing phosphorylation.Centrosomes are critical for organizing microtubules that make up the
mitotic and meiotic spindles that segregate chromosomes during cell division.
The duplication of these organelles must be tightly regulated to occur once
and only once during each cell cycle to prevent the formation of monopolar or
multipolar mitotic spindles that can cause chromosomal instability. The yeast
centrosome is called the spindle pole body
(SPB)3 and is one of
the best characterized microtubule-organizing centers. Although the SPB and
the centrosome are morphologically distinct, they share the common function of
spindle organization. Many SPB components and regulators of SPB assembly and
function are conserved throughout evolution
(1). This has made the yeast
SPB an excellent model in which to study the regulation of centrosome
duplication.The regulation of centrosome function and duplication by phosphorylation is
well documented
(2–10).
Although several yeast SPB components are phosphoproteins in vivo
(11–16),
little is known about the specific sites of phosphorylation or the roles these
modifications play in the regulation of SPB duplication and function. The
yeast cyclin-dependent kinase Cdc28 and the multifunctional Mps1 kinase have
both been implicated in the regulation of SPB components by phosphorylation
(17–20).
Two essential SPB components, Spc42 and Spc110, are phosphorylated by both of
these kinases. Prevention of modification by either kinase alone is not
detrimental, but the two kinases work in concert with each other to produce a
fully functional protein. These examples demonstrate that some SPB components
are coordinately regulated by the actions of more than one protein kinase and
that an accumulation of hyperphosphorylation, rather than specific individual
phosphorylation events, is the predominant mechanism of phosphoregulation of
SPB components.In this study, we demonstrate that a single phosphothreonine,
phospho-Thr240, near the C terminus of the SPB component Spc29 is
absolutely required for SPB duplication and mitotic progression. The
modification promotes the stability of the Spc29 protein and appears to be
catalyzed by the Mps1 kinase. These results reveal the first single
phosphorylation event known to be essential for SPB duplication and elucidate
a mechanism by which cells can achieve tight regulation of centrosome
duplication through a cascade of phosphorylation-mediated protein
stabilization wherein the yeast cyclin-dependent kinase stabilizes the Mps1
kinase by phosphorylation
(19), and the Mps1 kinase in
turn stabilizes the Spc29 protein by phosphorylation, ensuring adequate levels
of this critical SPB component for the assembly of new spindle poles. |
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