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A Cell Cycle Phosphoproteome of the Yeast Centrosome

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Science  24 Jun 2011:
Vol. 332, Issue 6037, pp. 1557-1561
DOI: 10.1126/science.1205193

Abstract

Centrosomes organize the bipolar mitotic spindle, and centrosomal defects cause chromosome instability. Protein phosphorylation modulates centrosome function, and we provide a comprehensive map of phosphorylation on intact yeast centrosomes (18 proteins). Mass spectrometry was used to identify 297 phosphorylation sites on centrosomes from different cell cycle stages. We observed different modes of phosphoregulation via specific protein kinases, phosphorylation site clustering, and conserved phosphorylated residues. Mutating all eight cyclin-dependent kinase (Cdk)–directed sites within the core component, Spc42, resulted in lethality and reduced centrosomal assembly. Alternatively, mutation of one conserved Cdk site within γ-tubulin (Tub4-S360D) caused mitotic delay and aberrant anaphase spindle elongation. Our work establishes the extent and complexity of this prominent posttranslational modification in centrosome biology and provides specific examples of phosphorylation control in centrosome function.

Phosphorylation is a reversible posttranslational modification that regulates most cellular processes, including the duplication of centrosomes to form the mitotic spindle, which functions in chromosome segregation. Protein kinases, such as cyclin-dependent kinase Cdk1 (Cdc28), Mps1, and Polo kinase (Cdc5) (1, 2), phosphorylate the centrosome, known in yeast as the spindle pole body (SPB; Fig. 1A). The 18 centrosomal proteins (10 have human homologs; Figs. 1B and 2) can be organized into five functional subcomplexes (1): the γ-tubulin complex (Tub4, Spc98, and Spc97), which nucleates microtubules; the central core (Nud1, Spc42, Spc29, and Cnm67), which form the organelle’s structural foundation and precursor; the linker proteins connecting the core and γ-tubulin complexes; the membrane anchors; and the half-bridge components, where assembly begins. Previous studies examined phosphorylation of these components individually or within whole cell preparations (database S1, column 3). In contrast, we performed a comprehensive analysis of phosphorylation on enriched, intact centrosomes.

Fig. 1

Yeast centrosomes form the poles of the mitotic spindle and are composed of five subcomplexes. (A) Immunofluoresence (left) of a large-budded mitotic yeast cell showing centrosomes marked by Spc42–green fluorescent protein (GFP) (green), microtubules (red), and DNA (blue), and electron micrograph (right) showing trilaminar ultrastructure. Scale bar indicates 100 nm. [Credit: Eileen O’Toole, University of Colorado, Boulder] (B) Schematic of the five major functional centrosome subcomplexes. MT indicates microtubule.

Centrosomal complexes were isolated from yeast cells by using a modified affinity purification (3) (fig. S1A), and copurifying proteins were analyzed by solution digest and mass spectrometry (MS). Phosphopeptides were enriched with a metal affinity column, processed by liquid chromatography tandem mass spectrometry (LC MS/MS) on an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA), and identified with SEQUEST and DTASelect2 programs (4). DeBunker (5) and Ascore (6) programs were used to further validate phosphopeptides and phosphorylation assignments, respectively (database S2). Peptides from all 18 proteins were identified, with extensive peptide coverage for most proteins (Fig. 2). The centrosomal preparations (fig. S1B) were highly phosphorylated (fig. S1C), as observed by MS analysis (www.yeastrc.org/pdr/pages/front.jsp; search by protein name). In total, 297 phosphorylation events were mapped on 17 of the 18 yeast centrosomal proteins, of which 227 have not been previously reported. Among these are 49 potential Cdk1 sites [S/T-P, serine or threonine followed by proline, 5 are confirmed as Cdk sites (7)] and 22 tyrosines (Fig. 2). Combining data from this study (297 sites) and previous studies (29 sites), a total of 326 phosphorylation sites are now identified on the yeast centrosome (database S1).

Fig. 2

Phosphoproteomic analysis of enriched Sacchromyces cerevisiae centrosomes organized by centrosome complexes. Total sites indicates all phosphorylation sites found within all asynchronous, mitotic, and G1 preparations; numbers in parentheses are ambiguous assignments (databases S1 and S2); S/T(P) sites, potential Cdk1 sites; Y sites, tyrosine phosphorylation sites; Coverage, % of the total protein sequence recovered as peptides from mass spectrometry analyses of all centrosome preparations; and human homologs are indicated if applicable. Check marks indicate proteins that are known in vivo or in vitro substrates of Cdk1, Mps1, or Cdc5 kinases (references in fig. S4); dash entries, not observed; asterisks, homologous domains.

Because phosphorylation regulates cell cycle events (8), including centrosome duplication and mitotic spindle formation, we explored phosphorylation profile differences between centrosomes in cell cycle–arrested cells versus those growing asynchronously. Cells were arrested at an early step of centrosome duplication in late G1 phase with α-factor treatment or in mitosis after centrosome duplication and separation by depletion of the anaphase-promoting complex (APC) activator Cdc20 (fig. S2A). We detected 54 sites that were phosphorylated only in G1, 110 sites that were phosphorylated only in mitosis, and 68 sites phosphorylated in both phases (Fig. 3, fig. S2B, and database S1). The latter 68 sites are likely to be constitutively phosphorylated (because 61 were also found in asynchronous preparations). Of all the subcomplexes, the central core contained the largest number of sites (46% of the total, Fig. 2) and also the highest percentage of all shared sites (72%) (fig. S2C). The 29 mitotic sites on Nud1 may affect its subsequent role in recruitment of cell cycle regulatory proteins required for mitotic exit (9). In contrast to the central core, the γ-tubulin complex, linkers, and half-bridge have few constitutive sites and a large number of sites phosphorylated in mitosis.

Fig. 3

S. cerevisiae centrosome phosphorylation is dynamic during the cell cycle and is enriched in mitosis. Total Asynch, all sites found in asynchronous preparations or Unique, only in Asynch; Total G1, all sites found in G1 preparations or Unique in G1 (not in mitotic preparations); Total Mitotic, all sites found in mitotic preparations or Unique in mitotic (not in G1 preparations); Shared G1/M, sites found in mitotic and G1 preparations.

Analysis of phosphorylated residues that are likely within binding sites or targets of specific kinases showed distinct cell cycle patterns. For example, the majority of sites within Polo (Cdc5) binding motifs (fig. S3A) were observed in mitosis when its activity peaks (10). Cell cycle–specific phosphorylation was also observed in 21 of 22 tyrosine sites. In contrast, over half (27 of 49) of the potential Cdk consensus sites were phosphorylated throughout the cell cycle. The constitutive Cdk phosphorylation in Spc42 appeared to be essential, because mutating the Cdk motifs to nonphosphorylatable residues [8 sites out of 32 total phosphorylation sites in Spc42 (fig. S3B)] was lethal. The lethality may result, in part, from the decrease in Spc42 assembly into the centrosome (Fig. 4A). Furthermore, phosphorylation of these Cdk sites is critical for overall Spc42 phosphorylation, because phosphate incorporation decreased in the Spc42-8A mutant by 93% compared with the wild type (WT) (Fig. 4B).

Fig. 4

Effect of mutating all Cdk sites within the core protein, Spc42. (A) Incorporation of Spc42-GFP and Spc42-8A-GFP into the two centrosomes, analyzed by fluorescence microscopy (n = 200). Phase microscopy images show large-budded mitotic cells. Scale bar, 5 μm. (B) Relative 32P incorporation into GFP, Spc42-GFP (WT), and Spc42-8A-GFP (8A) proteins upon protein induction in yeast cells shown by anti-GFP Western blot (top) and autoradiograph (bottom). Quantified by phosphorimager (below) and normalized to α-GFP signal, n = 3.

Twelve centrosomal proteins are known substrates of Cdk1 or Mps1 (Fig. 2 and fig. S4A). We performed kinase reactions in vitro with either Cdk1 or Mps1 on our centrosome preparations and identified potential centrosomal substrates by in-gel digestion and MS analysis (fig. S4B). We observed kinase specificity on centrosomal substrates by distinct phosphorylation banding patterns, confirmed several substrates, and identified possible new substrates (Spc72 and Cnm67) for Mps1 and Cdk1, respectively.

Clustering of phosphorylation sites is a rare event that creates a charged region, which can affect protein interactions and contribute to structural integrity (11). A study of yeast Cdk phosphorylation showed that a cluster of sites, rather than individual residues, can be evolutionarily conserved (7). Clustering was prominent within our centrosomal phosphoproteome, with 174 of the 297 mapped sites clustered in seven proteins [fig. S5, ≥5 sites within 50 residues (4)]. Twenty-nine of the 49 Cdk consensus sites were included within these clustered regions. The importance of phosphorylation site clustering is exemplified by analysis of the N terminus of Spc110, which interacts with Spc97 to stabilize the γ-tubulin complex (12, 13). Mutating even 2 out of the 18 phosphorylation sites in this region (fig. S5) is lethal when combined with spc97 mutations (14).

Individual residues that are functionally and structurally important are also conserved through evolution (15, 16). We therefore examined fungal orthologs of centrosomal proteins to determine evolutionary constraint values [measured by positional conservation (17, 18)] for the 297 sites and also regional conservation throughout the proteins [fig. S6; see Fig. 5, A and B, for γ-tubulin (Tub4)]. This analysis identified 59 highly constrained sites in 13 proteins (>80% conserved) and 14 fully conserved residues in 8 proteins (fig. S7, A and B), of which three sites, Tub4-Y445, Spc29-T18, and Spc29-T240 (19), are essential for centrosome function (2022). Fourteen sites from this study are conserved in human centrosomal proteins (fig. S7C).

Fig. 5

Conservation and location of the γ-tubulin (Tub4) S360 residue and phenotype of the phosphomimetic, Tub4-S360D. (A) γ-Tubulin (Tub4) regional evolutionary constraint among fungi (20 amino acid sliding regions). Red dots represent phosphorylation sites identified in this study. Y axis, constraint; a value of 1 is fully conserved. X axis, amino acid position; purple lines, P-Fam domains (GTP binding and C-terminal domains). (B) Positional constraint histogram for amino acids 341 to 379 (19) within γ-tubulin (Tub4). Red columns are phosphorylated residues; star marks S360. Y axis, constraint; 1 is fully conserved. X axis, protein sequence. (C) Comparative model of yeast Tub4, generated by PyMOL (www.pymol.org), threaded onto the x-ray crystallographic structure of human γ-tubulin (4). Star marks position of Tub4-S360 in an exposed loop, expanded in inset and compared with human S364. (D) Immunofluorescence of mitotic spindles from WT and tub4-S360D cells at 25°C and 37°C. Centrosomes (Spc42-GFP, green), microtubules (anti–α-tubulin, red), and DNA [4′,6′-diamidino-2-phenylindole (DAPI), blue] are shown. % indicates the percentage of cells with shown spindle structure, n = 200 cells for each. Scale bar, 1.5 μm. (E) Live cell analysis of spindle length for WT and tub4-S360D cells at 25°C. Centrosomes labeled by Spc42-CFP (cyan fluorescent protein). Each time step is 20 s. Spindle length (μm) measured by mother and daughter pole displacement (4). Spindle lengths are shown in black (metaphase), green (fast anaphase), and red (slow anaphase) and are representative for WT (n = 17) and tub4-S360D (n = 18) strains. Green hashed line is mean transition length, and green shaded shows standard deviation.

The phosphorylated residue, S360 (19), within γ-tubulin (Tub4) (fig. S8A) is fully conserved in fungi (Fig. 5B and fig. S6) and in humans (fig. S8B). γ-Tubulin is part of an evolutionarily conserved complex (γ-tubulin small complex, γ-tuSC) that nucleates microtubules for chromosome segregation. Phosphorylation of γ-tubulin has been shown to promote centrosome duplication and microtubule assembly (20, 23). Tub4-S360 lies within a Cdk motif and is phosphorylated by Cdk1 in vitro (fig. S8, C and D). This site is located within a surface loop available for protein-protein interactions, as viewed in the γ-tubulin crystal structure (Fig. 5C, star). Furthermore, structural analysis using cryo-electron microscopy of the yeast γ-tubulin complex places this loop directly between Spc98 and Spc97 (13). Therefore, we mutated S360 to either a nonphosphorylatable alanine (A) or an aspartic/glutamic acid (D or E) to mimic constitutive phosphorylation. The tub4-S360A allele did not affect growth; however, tub4-S360D and tub4-S360E caused growth defects at 25°C and mitotic arrest resulting in cell death upon shift to a higher temperature (37°C) (fig. S9, A to C). Also, tub4-S360D was lethal in combination with mutations in SPC98 (spc98-2), by deletion of the spindle checkpoint gene MAD2 (mad2∆) that allows for correction of mitotic defects, and by deletion of the EB1 homolog BIM1 (bim1∆), which is involved in microtubule dynamics (fig. S9A).

These genetic interactions suggested that tub4-S360D cells had defects in mitotic spindle assembly, which we analyzed by immunofluorescence microscopy and live cell imaging. At 25°C the majority (66%) of spindles in tub4-S360D large-budded mitotic cells had not extended past metaphase length [~1.5 μm (24)], whereas WT cells (91%) had normal elongated anaphase spindles [6 to 10 μm (24)] (Fig. 5D, 25°C). This phenotype was exacerbated at 37°C, with 98% of tub4-S360D cells containing either adjacent or unresolvable spindle poles (54%) or metaphase-length spindles (44%) (Fig. 5D), compared with WT cells (89% normal anaphase spindles). Live cell analysis of microtubules in tub4-S360D cells grown at 25°C revealed that spindles persisted longer in the fast phase (25) of anaphase spindle elongation (Fig. 5E, green lines), resulting in a transition to the slow phase (25) with longer anaphase spindles [6.6 μm ± 1.5 (SEM)] than WT cells [4.1 μm ± 0.4 (SEM); P < 0.001] (Fig. 5E, dashed green lines). In addition, large spindle length fluctuations were observed in tub4-S360D cells before anaphase (Fig. 5E, black lines; quantified in fig. S10) and in the slow phase of anaphase (Fig. 5E, red lines). Thus, phosphorylation of a single Cdk site in γ-tubulin appears to contribute to proper dynamics of anaphase spindle microtubules.

Phosphoregulation of the centrosome is likely to be conserved, not only with respect to the protein kinases but also through specific residues in the respective human homologs. Illustrated by our analysis of γ-tubulin and Spc42, the conserved residues and phosphorylation patterns in yeast will be useful tools for studying the human centrosome, a much larger (>100 proteins) and more complicated microtubule organizing center.

Supporting Online Material

www.sciencemag.org/cgi/content/full/332/6037/1557/DC1

Materials and Methods

Figs. S1 to S10

Table S1

References

Databases S1 and S2

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
  3. Acknowledgments: We thank C. Pearson, A. Stemm-Wolf, and T. Su for comments on the manuscript; T. Davis and the Winey lab for helpful discussions; E. O'Toole for the electron micrograph; E. Nazarova for assisting with spindle dynamics analysis; M. Riffle for Yeast Resource Center assistance; and D. D'amours for the Cdc28 purification protocol. This work was supported by NIH U54 RR022220 and R01 GM062427 (M.P.R.), NIH R01 HG003039 (A.S.), NIH P41 RR011823 (J.R.Y. III, T.N. Davis, principal investigator), CIHR MOP-64404 (J.V.), and NIH GM51312 (M.W.). J.M.K. was supported by NIH F32 GM086038, and E.P.H. was supported by NIH T32 GM008759. Mass spectrometry data are provided at the Yeast Resource Center (www.yeastrc.org/pdr/pages/front.jsp).
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