Global analysis of Cdk1 substrate phosphorylation sites provides insights into evolution

Abstract

To explore the mechanisms and evolution of cell-cycle control, we analyzed the position and conservation of large numbers of phosphorylation sites for the cyclin-dependent kinase Cdk1 in the budding yeast Saccharomyces cerevisiae. We combined specific chemical inhibition of Cdk1 with quantitative mass spectrometry to identify the positions of 547 phosphorylation sites on 308 Cdk1 substrates in vivo. Comparisons of these substrates with orthologs throughout the ascomycete lineage revealed that the position of most phosphorylation sites is not conserved in evolution; instead, clusters of sites shift position in rapidly evolving disordered regions. We propose that the regulation of protein function by phosphorylation often depends on simple nonspecific mechanisms that disrupt or enhance protein-protein interactions. The gain or loss of phosphorylation sites in rapidly evolving regions could facilitate the evolution of kinase-signaling circuits.

Fig. 1

Large-scale identification of Cdk1 substrates in vivo. (A) Distributions of log₂ heavy/light (H/L) ratios for unphosphorylated peptides (gray), all phosphopeptides (black) and phosphopeptides containing a minimal (orange) or full (red) Cdk1 consensus phosphorylation motif. (B) Log₁₀ p-values (binomial distribution) for the enrichment (red) or depletion (blue) of each amino acid (columns) at each position flanking the phosphorylated serine or threonine (rows) in phosphopeptides that changed greatly in abundance (log₂ H/L < −3) relative to residues flanking serines and threonines proteome-wide. (C) Venn diagram representing the number of proteins and unique phosphorylation sites identified in the three experiments (2). The blue square indicates the total number of proteins and phosphorylation sites for which H/L ratios could be determined rigorously and for which the precise position of the phosphate could be assigned with 95% confidence. The orange square indicates proteins and phosphopeptides containing a minimal consensus motif, and the red square indicates proteins and phosphopeptides that decreased in abundance over 50% after Cdk1 inhibition (log₂ H/L < −1). Cdk1 substrates (listed in Table S1) were defined by the overlap between the orange and red squares. Squares are scaled to the number of proteins.

Fig. 2

Selected Cdk1 substrates grouped by cellular process. A subset of proteins phosphorylated in a Cdk1-dependent manner are organized into functional groups. The color of the box surrounding each protein corresponds to the fold-change of the most dynamically regulated phosphorylation site of each protein.

Fig. 3

Structural analysis of Cdk1-dependent phosphorylation sites. (A) The predicted structural environment of residues in all proteins in the S. cerevisiae genome (black) and the residues that are phosphorylated in a Cdk1-dependent manner (red). Secondary structure (PsiPred) and disorder (PONDR) prediction algorithms (2) were used to predict the structural environment, and pre-computed domain predictions were downloaded from SGD (ftp://ftp.yeastgenome.org/yeast/). All differences are significant at p < 10⁻⁶⁹ (binomial distribution). See Table S4 for details. (B) The distribution of Cdk1-dependent phosphorylation sites per protein (red) is compared to the distribution of sites per protein from simulations in which the same number of phosphorylation sites is randomly scattered across a set of mitotic proteins with probability proportional to protein length (gray). To conservatively estimate the number of proteins present in mitosis, we used the set of 3838 proteins that are detectable by western blotting (21). 1000 simulations were performed and each simulated distribution was compared to the true distribution by calculating the Mann-Whitney p-value. The “Cdk1 Expected” distribution is the average of the 1000 simulations. (C) The distribution of average distances between phosphates within a given protein. The average distances between Cdk1 sites were calculated for all proteins with two or more detected phosphorylation sites (red) and compared to the expected distribution generated by averaging the results of 1000 simulations in which the same number of phosphates was randomly assigned positions within each protein (gray). Because the expected distance between phosphates in a protein depends on the length of the protein, the average distances between phosphates shown here are normalized to protein length. The median Mann-Whitney p-value from comparison of each of the 1000 simulated distributions to the true distribution is shown.

Fig. 4

Evolution of Cdk1-dependent phosphorylation sites. (A) Representative multiple sequence alignment of 27 orthologs of S. cerevisiae Shp1. The ascomycete phylogeny (Fig. S3) is shown to the left of the alignment. Amino acid conservation is indicated by blue boxes, and minimal Cdk1 consensus motifs are highlighted in yellow. Blue arrows indicate predicted domains and the green arrow indicates a predicted disordered region (PONDR). (B) Hierarchical clusters summarize the evolution of all 547 Cdk1 phosphorylation sites: each row is a different species and each column is a different phosphorylation site. The phylogeny (32 species; see Fig. S3) is represented by a tree at the left. In the top clustergram, yellow indicates that a consensus site (S/T-P) aligns with the phosphorylation site detected in S. cerevisiae (top row). Gray indicates that no single ortholog was detected in that species. In the bottom clustergram, yellow indicates that there is an enrichment of Cdk1 consensus sites in the ortholog of the S. cerevisiae protein we identified as a Cdk1 substrate. Enrichment in each ortholog was assessed by assuming that the expected frequency of a consensus motif is equal to the global frequency across all ORFs in the species, and then using the Poisson distribution to calculate the probability of observing greater than or equal to the actual number of consensus sites. Enrichment was defined by a p-value of less than 0.01 (for example, a typical 400-residue protein is expected to contain 2.8 sites, but must contain 8 or more sites to achieve p<0.01; see Table S5 for details). Two groups are highlighted within the clustergrams: one with conservation of precise site position (red box) and one with conservation of enrichment of consensus sites (blue box). Beneath each clustergram is a metric termed “age”, which summarizes each column as a single conservation score (Fig. S4). More intense yellow indicates greater conservation.