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J. Biol. Chem., Vol. 276, Issue 21, 17958-17967, May 25, 2001
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From the Departments of a Biochemistry and
h Molecular Biotechnology, University of Washington, Seattle,
Washington 98195 and the d Department of Molecular, Cellular and
Developmental Biology, b Howard Hughes Medical Institute, and
k Department of Chemistry and Biochemistry, University of
Colorado, Boulder, Colorado 80309
The yeast spindle pole body (SPB)
component Spc110p (Nuf1p) undergoes specific serine/threonine
phosphorylation as the mitotic spindle apparatus forms, and this
phosphorylation persists until cells enter anaphase. We demonstrate
that the dual-specificity kinase Mps1p is essential for the
mitosis-specific phosphorylation of Spc110p in vivo and
that Mps1p phosphorylates Spc110p in vitro. Phosphopeptides
generated by proteolytic cleavage were identified and sequenced by mass
spectrometry. Ser60, Thr64, and
Thr68 are the major sites in Spc110p phosphorylated by
Mps1p in vitro, and alanine substitution at these sites
abolishes the mitosis-specific isoform in vivo. This is the
first time that phosphorylation sites of an SPB component have been
determined, and these are the first sites of Mps1p phosphorylation
identified. Alanine substitution for any one of these phosphorylated
residues, in conjunction with an alanine substitution at residue
Ser36, is lethal in combination with alleles of
SPC97, which encodes a component of the Tub4p complex.
Consistent with a specific dysfunction for the alanine substitution
mutations, simultaneous mutation of all four serine/threonine residues
to aspartate does not confer any defect. Sites of Mps1p phosphorylation
and Ser36 are located within the N-terminal globular domain
of Spc110p, which resides at the inner plaque of the SPB and binds the
Tub4p complex.
Yeast Mps1p Phosphorylates the Spindle Pole Component Spc110p in
the N-terminal Domain*
*
This work was supported in part by National Institutes of
Health (NIH) Grant GM40506 (to T. N. D.), NIH Grant GM-51312 (to M. W.), and NIH Grant AR39730 (to K. A. R.); by the Howard Hughes Medical Institute (to N. G. A.); and by NCRR, NIH Grant P41RR11823 (to J. Y.). Mass spectra presented were generated at the University of
Colorado, Boulder.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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