Yeast Mps1p phosphorylates the spindle pole component Spc110p in the N-terminal domain

The yeast spindle pole body (SPB) component Spc110p (Nuf1p) undergoes speci fic serine/threonine phosphorylation as the mitotic spindle apparatus forms , and this phosphorylation persists until cells enter anaphase. We demonstr ate that the dual-specificity kinase Mps1p is essential for the mitosis-spe cific phosphorylation of Spc110p in vivo and that Mps1p phosphorylates Spc1 10p in vitro. Phosphopeptides generated by proteolytic cleavage were identi fied 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 su bstitution at these sites abolishes the mitosis-specific isoform in vivo, T his is the first time that phosphorylation sites of an SPB component have b een determined, and these are the first sites of Mps1p phosphorylation iden tified. Alanine substitution for any one of these phosphorylated residues, in conjunction with an alanine substitution at residue Ser(36), is lethal i n combination with alleles of SPC97, which encodes a component of the Tub4p complex. Consistent with a specific dysfunction for the alanine substituti on mutations, simultaneous mutation of all four serine/threonine residues t o aspartate does not confer any defect. Sites of Mps1p phosphorylation and Ser36 are located within the N-terminal globular domain of Spc110p, which r esides at the inner plaque of the SPB and binds the Tub4p complex.