Yeast Mps1p phosphorylates the spindle pole component Spc110p in the N-terminal domain.
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. Ser(60), Thr(64), and Thr(68) 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 Ser(36), 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 Ser(36) are located within the N-terminal globular domain of Spc110p, which resides at the inner plaque of the SPB and binds the Tub4p complex.
Friedman
D B
DB
Departments of Biochemistry and Molecular Biotechnology, University of Washington, Seattle, Washington 98195, USA.
Kern
J W
JW
Huneycutt
B J
BJ
Vinh
D B
DB
Crawford
D K
DK
Steiner
E
E
Scheiltz
D
D
Yates
J
J
Resing
K A
KA
Ahn
N G
NG
Winey
M
M
Davis
T N
TN
D002148:Calmodulin-Binding Proteins; D003598:Cytoskeletal Proteins; D005656:Fungal Proteins / Q000378:metabolism*; D008938:Mitosis; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
D002148:Calmodulin-Binding Proteins; D003598:Cytoskeletal Proteins; D005656:Fungal Proteins; D009687:Nuclear Proteins; C073277:SPC110 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; C093416:MPS1 protein, S cerevisiae