Abstract
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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 17958-17967 |
| Number of pages | 10 |
| Journal | Journal of Biological Chemistry |
| Volume | 276 |
| Issue number | 21 |
| DOIs | |
| State | Published - Jan 25 2001 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
- Cell Biology
Cite this
Yeast Mps1p Phosphorylates the Spindle Pole Component Spc110p in the N-terminal Domain. / Friedman, David B.; Kern, Joshua W.; Huneycutt, Brenda J.; Vinh, Dani B.N.; Crawford, Douglas K.; Steiner, Estelle; Scheiltz, David; Yates, John; Resing, Katheryn A.; Ahn, Natalie G.; Winey, Mark; Davis, Trisha N.
In: Journal of Biological Chemistry, Vol. 276, No. 21, 25.01.2001, p. 17958-17967.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Yeast Mps1p Phosphorylates the Spindle Pole Component Spc110p in the N-terminal Domain
AU - Friedman, David B.
AU - Kern, Joshua W.
AU - Huneycutt, Brenda J.
AU - Vinh, Dani B.N.
AU - Crawford, Douglas K.
AU - Steiner, Estelle
AU - Scheiltz, David
AU - Yates, John
AU - Resing, Katheryn A.
AU - Ahn, Natalie G.
AU - Winey, Mark
AU - Davis, Trisha N.
PY - 2001/1/25
Y1 - 2001/1/25
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.M010461200
DO - 10.1074/jbc.M010461200
M3 - Article
VL - 276
SP - 17958
EP - 17967
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 21
ER -