Cdc14 phosphatases preferentially dephosphorylate a subset of cyclin-dependent kinase (Cdk) sites containing phosphoserine

Abstract

Mitotic cell division is controlled by cyclin-dependent kinases (Cdks), which phosphorylate hundreds of protein substrates responsible for executing the division program. Cdk inactivation and reversal of Cdk-catalyzed phosphorylation are universal requirements for completing and exiting mitosis and resetting the cell cycle machinery. Mechanisms that define the timing and order of Cdk substrate dephosphorylation remain poorly understood. Cdc14 phosphatases have been implicated in Cdk inactivation and are thought to be generally specific for Cdk-type phosphorylation sites. We show that budding yeast Cdc14 possesses a strong and unusual preference for phosphoserine over phosphothreonine at Pro-directed sites in vitro. Using serine to threonine substitutions in the Cdk consensus sites of the Cdc14 substrate Acm1, we demonstrate that phosphoserine specificity exists in vivo. Furthermore, it appears to be a conserved property of all Cdc14 family phosphatases. An invariant active site residue was identified that sterically restricts phosphothreonine binding and is largely responsible for phosphoserine selectivity. Optimal Cdc14 substrates also possessed a basic residue at the +3 position relative to the phosphoserine, whereas substrates lacking this basic residue were not effectively hydrolyzed. The intrinsic selectivity of Cdc14 may help establish the order of Cdk substrate dephosphorylation during mitotic exit and contribute to roles in other cellular processes.

FIGURE 1.

Budding yeast Cdc14 is highly selective for Ser(P) at Cdk phosphorylation sites in phosphopeptide substrates. A, time-dependent dephosphorylation of phosphopeptide substrates Acm1pS3 (●), Acm1pT161 (○), and Cdh1pT157 (X) by budding yeast Cdc14. The concentration of all substrates was 300 μm. B, same as A showing Ser(P)-containing peptide substrates Acm1pS3 (●), Acm1pS31 (■), Acm1pS48 (▴), Cdh1pS42 (⧫), Cdh1pS169 (▾), and Cdh1pS239 (○). All substrates were 100 μm. Peptide sequences are shown in Table 1 and supplemental Table S1. C and D, the rate of dephosphorylation of peptides Acm1pS31 (■) and Acm1pT31 (●) (C) and of peptides Cdc6pS7 (■) and Cdc6pT7 (●) (D) was measured as a function of peptide concentration. Data are the average of three independent experiments, and using non-linear regression, data were fit with a form of the Michaelis-Menten equation containing a substrate inhibition term. The amino acid sequence and kinetic parameters for peptides in C and D are given in Table 1 or supplemental Table S2.

FIGURE 2.

Budding yeast Cdc14 preferentially dephosphorylates Ser(P)-containing Cdk phosphorylation sites in physiologic protein substrates. A–C, dephosphorylation of the indicated residues from full-length recombinant GST-Acm1 (A), GST-Fin1 (B), and GST-Cdc6 (C) by budding yeast Cdc14 was measured over time (black bars, 0 min; white bars, 5 min; gray bars, 30 min) using a quantitative mass spectral assay. Single-letter codes were used to indicate amino acid residues in this panel. Data are means of three trials with standard errors. Remaining phosphorylation at each site is plotted relative to 0 min, which was set at 100%.

FIGURE 3.

Activity of Cdc14 phosphatases is dependent on basic residues C-terminal to Ser(P) at Cdk sites. A and C, the complete sequences of the wild-type Acm1pS31 (A) and Cdh1pS169 (C) peptides are shown in black on the first line with the residues of the +3 to +5 region (relative to Ser(P) (pS)) in each variant shown below in red. B and D, rates of dephosphorylation of Acm1pS31 and its variants (B) and of Cdh1pS169 and its variants (D) by budding yeast Cdc14 were compared at a single substrate concentration of 300 μm. The amino acid sequence of the +3 to +5 region in each peptide is shown below the x axis. Data represent the mean of three independent experiments with standard errors.

FIGURE 4.

Cdc14 Ser(P) selectivity exists in vivo. A, schematic of Acm1 protein showing the location and sequence of the four Ser(P)-containing Cdk consensus sites and the corresponding amino acid substitutions made to create the Acm1-S4T mutant. B, extracts from asynchronous cultures of cells expressing Acm1 or Acm1-S4T from the natural ACM1 promoter before and after galactose-induced overexpression of 3HA-Cdc14 were analyzed by SDS-PAGE and immunoblotting. G6PD is a loading control. pAcm1 represents a slow mobility Cdk-phosphorylated form of Acm1. C, anti-HA antibody resin was used to isolate 3HA-Cdc14-C283S and interacting proteins from soluble extracts of asynchronous cultures expressing either wild-type Acm1 or Acm1-S4T from the natural ACM1 promoter. Immunoblotting was used to detect the indicated proteins in the initial extracts and after anti-HA IP. G6PD is a loading control.

FIGURE 5.

Human Cdc14A, human Cdc14B, and fission yeast Clp1 phosphatases exhibit selectivity for Ser(P). A, the rate of dephosphorylation of the indicated phosphopeptides by the hCdc14A(1–379) catalytic domain was compared at single substrate concentrations (250 μm for Ser(P) peptides and for Acm1pT31 and Cdc6pT7; 1 mm for all other Thr(P)-containing peptides). pS, Ser(P); pT, Thr(P). B, the rate of dephosphorylation of the indicated phosphopeptides by the hCdc14B(1–418) catalytic domain was compared at single substrate concentrations (500 μm for Acm1pS3 and Acm1pS31, 1 mm for all others). Data in A and B are averages of four trials with standard errors. C, dephosphorylation of Acm1pS31 (●) and Acm1pT31 (■) by fission yeast Clp1 was measured as a function of peptide concentration. Data were fit with the Michaelis-Menten equation containing a substrate inhibition term. Rates are expressed per pmol of Cdc14.

FIGURE 6.

Cdc14 selectivity for Ser(P) arises from the structure of Cdc14 active site. A, the active site of human Cdc14B (Protein Data Bank (PDB) ID: 1OHE) (17) with the bound peptide substrate modified in silico to contain a Thr(P) side chain. A surface representation of the protein and peptide is depicted with purple indicating the Thr(P) side chain methyl group and the mesh delineating the surface of Ala-316. The distance between the carbon atoms of the methyl groups on Ala 316 and the Thr(P) side chain is 2.3 Å, a value that is substantially less than the sum of the Van der Waals radii of the two atoms, indicating severe steric clash. The surface after a Gly (orange) substitution at 316 is also shown. The side chain of Lys-315 was hidden for optimal visualization of the active site pocket. MacPyMOL (30) was used to visualize the hCdc14B structure, mutate the bound phosphopeptide substrate by utilizing the site mutagenesis function, and measure distances between atoms. B, relative rates of Cdc6pT7 dephosphorylation by wild-type budding yeast Cdc14 (●) and the Cdc14 A285G mutant (■) were measured as a function of peptide concentration. Activities were normalized to activity on Cdc6pS7 to directly compare differences in selectivity. Data are averages of three trials and were fit with the Michaelis-Menten equation. C, catalytic efficiencies (k_cat/K_m) determined from B and similar experiments on Cdc6pS7. Selectivity (Ser(P)/Thr(P) (pS/pT)) is k_cat/K_m for Cdc6pS7 divided by k_cat/K_m for Cdc6pT7.