Yeast Mps1p Phosphorylates the Spindle Pole Component Spc110p in the N-terminal Domain

David B. Friedman,Natalie G. Ahn,Katheryn A. Resing,Estelle Steiner,Dani B.N. Vinh,John Yates,David Scheiltz,Joshua W. Kern,Mark Winey,Brenda J. Huneycutt,Trisha N. Davis,Douglas K. Crawford

doi:10.1074/jbc.m010461200

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. 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.

Highlights

We have shown previously that the mitosis-specific serine/ threonine phosphorylation of Spc110p occurs after SPB duplication is completed as the mitotic spindle first forms, and persists up to the metaphase/anaphase transition (26)
We show here that the dual-specificity kinase Mps1p phosphorylates Spc110p and that this phosphorylation is necessary for the production of the mitosis-specific Spc110p isoform in vivo
Mitosis-specific Spc110p phosphorylation can occur in the absence of proper SPB duplication but cannot occur in the absence of Mps1p activity, and Mps1p production during G1 arrest can drive formation of the mitosis-specific Spc110p isoform

Summary

EXPERIMENTAL PROCEDURES

Strains, and Genetic Manipulations—SD complete, SD-uracil (34), SD-uracil low adenine (17), YPD and YPD low adenine, and LB (35) were described previously. Plasmid pDV29 encoding GST-Spc110p-(1–183) (SPC110 GenBank௢ accession number Z11582) was constructed by cloning the NcoI-EcoRI fragment from pDV17 (25) into the SmaI and EcoRI sites of pGEX-2T (Amersham Pharmacia Biotech, Piscataway, NJ). PDF48 expresses 12xHIS-Spc[110] P-(1–225) (truncated at residue 225 of Spc110p) and was constructed by cloning the SphI-NsiI fragment from pDF30 into the SphI and PstI sites of pQE32. Wild-type and mutant forms of recombinant GST-Spc110p-(1–183) were co-expressed with plasmid pCL5 in strain GM1. Kinase assays used to generate phosphorylated GST-Spc110p-(1–183) for analysis of phosphorylation sites by mass spectrometry used 200 ␮l of GST-Mps1p bound to GSH-Sepharose in a 50% slurry, which was washed once in kinase assay buffer without ATP (KAB-ATP: 50 mM Tris, pH 7.5, 10 mM MgCl2, 2 mM DTT) and resuspended in 50 ␮l of KAB-ATP and 10 ␮l of GSTSpc110p-(1–183) at a concentration of 1 mg/ml. Matrixassisted laser desorption ionization, time of flight (MALDI-TOF) mass spectrometry was carried out on 0.5 ␮l of analyte mixed with 0.5 ␮l of ␣-cyano-4-hydroxycinnamic acid matrix (Agilent Technologies, Hewlett-Packard), using a Voyager DE-STR mass spectrometer (PerSeptive Biosystems, Foster City, CA)

RESULTS

DISCUSSION

S36A pJK8