SUPPLEMENTARY INFORMATION

Supplemental Methods

Expression and Purification of Recombinant Cdc14 phosphatases — The budding yeast

CDC14 and fission yeast CLP1 open reading frames were subcloned into a pET15 vector

(Novagen) to generate constructs expressing N-terminal His6-tagged proteins. The A285G

mutation was introduced into the pET15b-Cdc14 expression construct by site-directed

mutagenesis using the QuikChange mutagenesis kit (Stratagene) to produce a pET15b-Cdc14

A285G construct.

E. coli BL21 (DE3) cells containing pET15b constructs expressing budding yeast His6-

Cdc14, His6-Cdc14 A285G mutant, or fission yeast His6-Clp1, were grown up to A600=0.6 in

one liter LB with ampicillin at 37 °C with shaking at 250 rpm. Protein expression was induced for

6 h at room temperature by addition of IPTG to a final concentration of 0.1 mM. Cells were

collected by centrifugation, resuspended in 20 ml of 20 mM Tris-HCl (pH 7.5 at 25 °C), 5 mM

imidazole, 500 mM NaCl (Ni2+ Buffer A), and lysed on ice by sonication using a Branson

sonifier. Lysates were clarified by centrifugation at 25,000 x g for 20 min at 4 °C.

The soluble extract was loaded onto a 5 ml Ni2+- charged column at 2 ml/min and

washed with 50 ml Ni2+ Buffer A using an FPLC system (Pharmacia). The column was further

washed with 20 ml of 10% Ni2+ Buffer B [20 mM Tris-HCl (pH 7.5 at 25 °C), 300 mM imidazole,

500 mM NaCl] in Ni2+ Buffer A and 20 ml of 20% Ni2+ Buffer B. Protein was eluted with 100%

Ni2+ Buffer B and passed through a G-25 fast flow desalting column to exchange the buffer to

50 mM Tris (pH 7.5), 300 mM NaCl, 2 mM EDTA, and 0.1% β- mercaptoethanol.

GST fusion proteins of the catalytic domains of hCdc14A (residues 1-379) and hCdc14B

(residues 1-418) were expressed in E. coli under the control of the T7 polymerase promoter

using pGST-HCA379 and pGST-HCB418 vectors, respectively, constructed using a pET-GST

plasmid (1). E. coli BL21(DE3) cells transformed with pGST-HCA379 or pGST-HCB418 were

induced with IPTG, lysed by sonication, and proteins affinity purified as described previously for

yeast GST-Cdc14 (1). A portion of each affinity-purified GST fusion protein was stored at - 80°C

in aliquots containing 20% glycerol, 20 mM Tris (pH 7.5), 170 mM NaCl, 2 mM KCl, 2 mM

EDTA, 2 mM DTT, 8 mM glutathione, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 µM pepstatin

and 1 µg/ml each aprotinin and leupeptin. The remainder of the purified enzymes was

exchanged into a high salt buffer by gel filtration and stored in aliquots at – 80°C in a storage

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buffer identical to that described above except that there was no glutathione and the final NaCl

concentration was 270 mM.

SDS-PAGE indicated that GST-hCdc14A(1-379) constituted at least 90% of the total

protein in the purified sample. The GST-hCdc14B(1-418) sample contained the full-length fusion

protein (about 12% of the total protein) and fragments ranging in size from 36 to 38 kDa

assumed to be a mixture of truncated GST fusion proteins and polypeptides containing the free

hCdc14B catalytic domain. The activity of this sample towards pNPP suggested some

fragments were active since the estimated amount of full-length fusion protein alone could not

account for the total activity measured. This is consistent with the work of Gray et al. (2), who

crystallized an active, 40-kDa fragment encompassing the catalytic domain of hCdc14B. Protein

concentration was determined by the Bradford assay using BSA as a standard.

Purification of Recombinant Cdk Substrates — Acm1, Cdc6, and Fin1 were expressed in E.

coli BL21 (DE3) as GST fusion proteins from pGEX6P-1 (GE Healthcare) by induction with 0.5

mM IPTG at room temperature for 8 hours in 2 liters of LB/ampicillin. Cell pellets were lysed in

50 mM Tris-HCl (pH 7.8), 250 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EDTA, 1 mM

dithiothreitol, 1 mM PMSF, 1 µM pepstatin, and 10 µM leupeptin by incubation with 1 mg/ml

lysozyme for 30 min on ice followed by sonication. Extracts were clarified by centrifugation at

35,000 x g for 30 min. Clarified extracts were passed through 1 ml GSTrap FF glutathione

sepharose columns (GE Healthcare) using an Akta FPLC system and washed to baseline A280

with 50 mM Tris-HCl (pH 7.8), 250 mM NaCl, 10% glycerol, and 1 mM dithiothreitol (for GST-

Acm1 and GST-Cdc6 the wash buffer also contained 0.1% Triton X-100). GST fusion proteins

were eluted with the same buffer containing 10 mM glutathione, collected in 500 µl fractions,

and peak fractions dialyzed into 20 mM HEPES (pH 7.5), 100 mM NaCl, 10 mM MgCl2, 0.5 mM

dithiothreitol, and 40% glycerol.

Phosphorylation of Recombinant Protein Substrates — Purified GST fusion proteins were

prepared as substrates for Cdc14 by phosphorylation in vitro with budding yeast Cdk1 (Cdc28)

bound to the mitotic cyclin Clb2. To purify the Clb2-Cdc28 complex, Clb2 bearing a protein A

tag was overexpressed in a sic1Δ yeast strain and purified using IgG-coupled agarose resin. To

phosphorylate recombinant GST fusion proteins, 125 µl of each substrate was diluted 4-fold in

kinase buffer, supplemented with 1 mM ATP, and ~10 µl purified Cdk1 beads were added. The

mixture was incubated at 30 °C for 30 min with gentle mixing, the Cdk beads were pelleted by

centrifugation, and phosphorylated proteins in the supernatant recovered. Each sample of

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phosphorylated recombinant protein was analyzed by MS as described below to evaluate the

stiochiometry of phosphorylation, and used for phosphatase assays.

LC-MS Analysis and Label-Free Quantification — Peptide samples were resuspended in or

adjusted to 3% acetonitrile and 0.1% TFA and were first injected onto a Zorbax C18 trap column

(Agilent) using an Agilent 1100 nano HPLC system. Peptides were eluted with a gradient of 3-

40% acetonitrile in 0.1 % formic acid over 60 min at 300 nl/min through a 75 µm i.d. fused silica

capillary (Biotaq) electrospray ionization tip packed with Magic C18 resin (Microm Bioresources,

Inc.) and into an LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific). The Orbitrap was

operated in two modes. For phosphorylation site mapping, and stoichiometry and phosphatase

analysis of singly modified peptides, the Orbitrap was operated in data-dependent MS2 mode,

collecting a single MS scan followed by MS2 scans of the four most abundant MS peaks with a

1 min dynamic exclusion window. The product ion spectra were used to ensure phosphate

groups were located at the expected Cdk sequences and to identify appropriate fragment ions

for selected reaction monitoring (SRM) analysis. Phosphorylation of non-proline directed serine

and threonine residues was negligible, as expected. For monitoring two Cdk phosphorylation

sites on individual peptides, pseudo-SRM analysis was performed by operating the Orbitrap in

targeted MS2 mode. A list of peptide m/z values was programmed along with narrow product

ion scan ranges bracketing the fragment ions selected to distinguish the two phosphorylation

sites from each other. The same set of standard peptides used for the conventional analysis

was monitored as well for normalization purposes.

Extracted ion chromatograms for individual phosphopeptides and non-phosphorylated

standard peptides used for normalization were generated from the XCalibur software (Thermo

Fisher) and exported into Graphpad Prism for peak integration and data processing. Similarly,

for Cdc6 analysis by SRM, chromatograms for each transition were generated and integrated in

Xcalibur. The stoichiometry of phosphorylation at individual sites on recombinant proteins was

determined using label-free quantitative approaches described previously for conventional MS

data (3) and SRM data (4). To generate graphs of relative phosphorylation over time, the raw

signals for phosphopeptides at each timepoint were first normalized using the average percent

deviation of at least three standard peptides (not containing Cdk phosphorylation sequences)

generated from the same protein relative to the zero timepoint. Subsequently, the normalized

phosphopeptide signals were converted to percentages of the zero timepoint signal. To

distinguish Fin1 Thr68 from Ser74 phosphorylation, the initial Lys-C digest was additionally

digested with trypsin to generate distinct peptides containing Thr68 and Ser74.

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Sequence Comparisons and Analysis of the hCdc14B Structure — Multiple alignments of

the Cdc14 and KAP phosphatase sequences were generated using the MAFFT program with

the Jalview (version 2.6.1) multiple alignment editor. Table S5 lists the accession number,

source organism (common name and genus species), and residue numbers defining the

sequences depicted in the alignment shown in Fig. S2. The DaliLite server (5) was used to

assess the structural similarity between the active site regions of hCdc14B (residues 292-345)

and KAP phosphatases (residues115-172). MacPyMOL (The PyMOL Molecular Graphics

System, Schrodinger, LLC) was used to visualize the hCdc14B structure (PDB ID:1OHE) (2),

mutate the bound phosphopeptide substrate by utilizing the site mutagenesis function, and to

measure distances between atoms.

Supplementary References

1. Taylor, G. S., Liu, Y., Baskerville, C. and Charbonneau, H. (1997) J. Biol. Chem. 272, 24054-

24063

2. Gray, C. H., Good, V. M., Tonks, N. K. and Barford, D. (2003) EMBO J. 22, 3524-3535

3. Steen, H., Jebanathirajah, J. A., Springer, M. and Kirschner, M. W. (2005) Proc. Natl. Acad.

Sci. U S A 102, 3948-3953

4. Balasubramaniam, D., Eissler, C. L., Stauffacher, C. V. and Hall, M. C. (2010) Proteomics 10,

4301-4305

5. Holm, L. and Rosenstrom, P. (2010) Nucleic Acids Res. 38, W545-9

6. Ubersax, J. A., Woodbury, E. L., Quang, P. N., Paraz, M., Blethrow, J. D., Shah, K., Shokat,

K. M. and Morgan, D. O. (2003) Nature 425, 859-864

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TABLE S1Steady-state kinetic parameters for budding yeast and human Cdc14 phosphatases towards phosphopeptide substratesKinetic parameters for ScCdc14 (His6-tagged Cdc14 from Saccharomyces cerevisiae) and hCdc14A(1-379) (a GST fusion protein of the catalytic domain from human Cdc14A) were determined by measuring initial velocity as a function of peptide substrate concentration at pH 8.0 and 30 °C as described. Data were fit with the Michaelis-Menten equation using nonlinear regression. The mean ± standard error from at least three independent trials are shown.

Peptide Sequencea kcat Km kcat/Km(s-1) (μM) (M-1 s-1)

ScCdc14Acm1pS31b VKGNELRSPSKRRSQI 5.9 ± 1.3 220 ± 60 27,000Acm1pS3b MISPSKKRTIL 2.0 ± 0.3 100 ± 20 20,000Acm1pS48 TDYALRRSPIKTIQIS 3.6 ± 0.8 500 ± 200 7,200Cdh1pS42b SSASLLSSPSRRSRPS 2.2 ± 0.2 330 ± 50 6,700Cdh1pS239 DSKQLLLSPGKQFRQI 1.06 ± 0.05 250 ± 40 4,200Fin1pS148 ARFKNGLMSPERIQQQ 0.71 ± 0.05 210 ± 50 3,400Cdc6pS43 TDVTPESSPEKLQFGS 2.2 ± 0.1 1,400 ± 200 1,600Cdh1pS16 NNTPSSSPLKGSESK 0.83 ± 0.06 700 ± 100 1,200Fin1pS54 FLKPPMRISPNKTDGMK 2.7 ± 0.5 6,000 ± 1,300 450Cdh1pS169 AAGLEEFSPHSTPVTP NDc ND NDFin1pS74 SIQVTPRRIMSPECLK ND ND ND

Acm1pS202 KKVVRKLSFHVYEDE ND ND NDCdh1pS125 DTVGSESSIDRIKNTR ND ND NDScc1pS175 GAAPWDTSLEVGRRFS ND ND ND

Cdc6pT7 MSAIPITPTKRIRRN 0.04 ± 0.01 8,000 ± 2200 5Acm1pT161 ISLPSFITPPRNSKIS ND ND NDCdh1pT157 YELERVSTPPPEAAGL ND ND NDCdh1pT173 EEFSPHSTPVTPRRLF ND ND NDCdh1pT176 SPHSTPVTPRRLFTSQ ND ND NDCdc6pT23 LFDDAPATPPRPLKRK ND ND NDFin1pT68 SIQVTPRRIMSPECLK ND ND ND

hCdc14A(1-379)Acm1pS31 VKGNELRSPSKRRSQI 0.055 ± 0.001 11 ± 2 5,000 Cdh1pS239 DSKQLLLSPGKQFRQI 0.090 ± 0.002 31 ± 4 2,900Cdh1pS42 SSASLLSSPSRRSRPS 0.049 ± 0.005 600 ± 100 82Cdc6pT7 MSAIPITPTKRIRRN 0.077 ± 0.014 10,000 ± 2,000 8Cdh1pT176 SPHSTPVTPRRLFTSQ ND ND ND

a Peptide sequences are given in single-letter code with phosphorylated residues underlined in bold. b A modified form of the Michaelis-Menten equation that included a term for substrate inhibition was fit to the

data giving Ki values of 95 ± 30 µM, 400 ± 100 µM, and 400 ± 50 µM for Acm1pS31, Acm1pS3, and Cdh1pS42, respectively.

cND, in reactions containing from 200-1,000 nM Cdc14 and 1-5 mM substrate, activity was at or below the limit of detection of the inorganic phosphate assay and steady state kinetic parameters were not determined.

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TABLE S2Steady-state kinetic parameters for phosphopeptide substrates and their variantsKinetic parameters (kcat and Km) for budding yeast Cdc14 were determined by measuring initial velocity as a function of peptide substrate concentration and fitting the data with the Michaelis-Menten equation using nonlinear regression. The mean ± standard error from at least three independent trials are shown.

Peptide Sequencea kcat Km kcat/Km(s-1) (μM) (M-1 s-1)

Acm1pS31 VKGNELRSPSKRRSQI 5.9 ± 0.8 220 ± 60 27,000Acm1pT31 VKGNELRTPSKRRSQI 0.01 ± 0.003 1,200 ± 500 8Acm1pY31 VKGNELRYPSKRRSQI 0.12 ± 0.01 1,200 ± 300 100Acm1pS31A32 VKGNELRSASKRRSQI 0.16 ± 0.01 3,400 ± 500 47Acm1pY31A32 VKGNELRYASKRRSQI 0.12 ± 0.01 1,800 ± 200 67

Cdc6pT7 MSAIPITPTKRIRRN 0.04 ± 0.01 8,000 ± 2000 5Cdc6pS7b MSAIPISPTKRIRRN 0.47 ± 0.06 80 ± 20 5,900

Cdh1pS169 AAGLEEFSPHSTPVTP NDd ND NDCdh1pS169(KTP) AAGLEEFSPHKTPVTP 1.2 ± 0.2 2,100 ± 400 570Cdh1pS169(KRP) AAGLEEFSPHKRPVTP 0.65 ± 0.03 570 ± 50 1,100Cdh1pS169(KRR) AAGLEEFSPHKRRVTP 1.16 ± 0.08 370 ± 60 3,100

Acm1pS31b,c VKGNELRSPSKRRSQI 1.08 ± 0.06 60 ± 10 18,000Acm1pS31(KRA)c VKGNELRSPSKRASQI 1.5 ± 0.2 140 ± 40 11,000Acm1pS31(KAA)c VKGNELRSPSKAASQI 0.60 ± 0.06 510 ± 90 1,200Acm1pS31(AAA)c VKGNELRSPSAAASQI ND ND ND

aPeptide sequences are given in single-letter code with phosphorylated residues underlined in bold. Positions differing from the wild-type sequence are shown in red.

bPeptides Cdc6pS7 and Acm1pS31 exhibited substrate inhibition, and using nonlinear regression, data were fit with a modified form of the Michaelis-Menton equation including a term for substrate inhibition to give Ki values of 230 ± 20 µM and 1,400 ± 500 µM, respectively.

cData for Acm1pS31 and its three variants were collected with an independent preparation of both the Acm1pS31 peptide and Cdc14 phosphatase.

dND, in reactions containing from 200-1,000 nM Cdc14 and 1-5 mM substrate, activity was at or below the limit of detection of the inorganic phosphate assay and steady state kinetic parameters were not determined.

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TABLE S3Estimation of phosphorylation stoichiometry of recombinant protein substrates

Protein Phosphorylation Site(s) Peptide Sequencea Stoichiometryb

(%)

Acm1 Ser 3 SDLEVLFQGPLGSMISPSKc 97Ser 48 RRSQIDTDYALRRSPIK 25Thr 161 KISLPSFITPPRNSK 19

Fin1 Ser 36 DNVFVRLSMSPLRTTSQK 92Ser 54 EFLKPPMRISPNK 80

Thr 68 Ser 74 HSIQVTPRRIMSPECLKd 80Ser 117 IQNSDHITNIIFPTSPTK 44

Cdc6 Thr 23 NLFDDAPATPPRPLK 63Thr 39 LQFTDVTPESSPEK 3Ser 43 LQFTDVTPESSPEK 7Thr 135 FQSLPLSLSTPR 22Thr 368 GSLNSAQVPLTPTTSPVKe 58Ser 372 GSLNSAQVPLTPTTSPVKe 72

aPeptide sequences are given in single-letter code with phosphorylated residues shown in red and underlined in bold.

bAverage stoichiometries for 8 pSer and 6 pThr sites were 62% and 41%, respectively.cThis peptide contains sequence from GST fused to the Acm1 N-terminus.dThe doubly phosphorylated species was by far the most abundant and was therefore used to estimate

stoichiometry for both Thr68 and Ser74.eStoichiometry was measured from SRM data because the two phosphorylated peptides were not

chromatographically separable. See supplemental methods for the estimation procedure.

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TABLE S4Analysis of Cdk consensus sequences in substrates targeted by Cdc14 in early anaphase

Protein Name SP : TP χ2 analysisa SPx(K/R):SP χ2 analysisb

Acm1 4:1 4:0Ase1 8:4 5:3

Cdc15 8:2 5:3Fin1 5:1 4:1Pds1 3:2 2:1Sld2 8:3 4:4Sli15 15:4 5:10

Average 3.0:1 1.3:1

Yeast Proteomec 1.3:1 p<0.01 0.12:1 p<0.001Cdk Substratesd 1.5:1 p<0.05 0.43:1 p<0.001

aFor χ2 analysis, frequencies of SP and TP in early anaphase Cdc14 substrates were used as observed data and frequencies in the yeast proteome or Cdk substrates as the expected data (null hypothesis, degrees of freedom = 1).

bFor χ2 analysis, frequencies of SPx(K/R) and SP in early anaphase Cdc14 substrates were used as observed data and frequencies in the yeast proteome or Cdk substrates as the expected data (null hypothesis, degrees of freedom = 1). x = any amino acid.

cThe PatMatch algorithm was used to determine frequencies in the yeast proteome (www.yeastgenome.org).

dCdk substrates taken from Ubersax et al. (6). The top 50 substrate sequences were used to determine average frequency of SP, TP, and SPx(K/R) sequences (x = any amino acid).

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TABLE S5Source of sequences used in multiple alignment of Figure S2

Residues AlignedOrganismCommon Name

Organism(Genus species)

AccessionNumber

ActiveSite

AcidicGroove

Cdc14Budding Yeast Saccharomyces cerevisiae NP_116684.1 278-294 165-181Fission Yeast Schizosaccharomyces pombe NP_594716.1 281-297 170-186Human A Homo sapiens NP_201569.1 273-289 167-183Human B Homo sapiens NP_201588.1 309-325 203-219Fruit Fly Drosophila melanogaster NP_001162904.1 278-294 171-187Sea Urchin Strongylocentrotus purpuratus XP_781590.2 271-287 165-181Hydra Hydra magnipapillata XP_002162225.1 272-288 166-182Sea Squirt Ciona intestinalis XP_002127684.1 338-354 232-248

KapHuman Homo sapiens NP_005183.2 135-151Mouse Mus musculus NP_082498.1 135-150Salmon Salmo salar NP_001134613.1 131-147Lizard Anolis carolinensis XP_003227865.1 128-144Sea Urchin Strongylocentrotus purpuratus XP_785719.2 150-166

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A

C

B

D

E

Velo

city

(pm

ol m

in-1

)

1

0

2

3

4

5

STP KTP KRP KRRVe

loci

ty (p

mol

min

-1)

0

2

1

3

4

5

AAA KAA KRA KRR

Figure S1. Preference for basic residues is conserved in hCdc14A and hCdc14B. Sequences of the wild type Cdh1pS169 (A) and Acm1pS31 (B) peptides are shown on the first line with the amino acid sequence of the +3 to +5 region in each variant shown below in red. (C) Rate of dephosphorylation of 300 µM Cdh1pS169 and variants by GST-hCdc14A(1-379). Rate of dephosphorylation of 300 µM Acm1pS31 and variants by (D) GST-hCdc14A(1-379) and (E) GST-hCdc14B(1-418). The amino acid sequence of the +3 to +5 region in each peptide is shown below the x axis. Rates are expressed per pmol Cdc14. Data represent the mean of three independent experiments with standard errors.

+3 +4 +5

Cdh1pS169- A A G L E E F pS P H S T P V T P K T P K R P K R R

Acm1pS31- V K G N E L R pS P S K R R S Q I K R A K A A A A A

Velo

city

(pm

ol m

in-1

)

0

0.02

0.04

0.06

0.08

0.10

AAA KRA KRR

+3 +4 +5

10

285 168 171 177

active site acidic groove

Budding yeast Cdc14 K I A V H C K A G L G R T G C L I E S Y E K Y E H V E F G D F NFission yeast Cdc14 V I A V H C K A G L G R T G C L I H D Y E T Y E R V E N G D F NHuman A Cdc14 A I A V H C K A G L G R T G T L I D E Y E H Y E R V E N G D F NHuman B Cdc14 A I A V H C K A G L G R T G T L I D E Y E H Y E K A E N G D L NFruit fly Cdc14 A I A V H C K A G L G R T G S L I E E Y E Y F E R V E N G D F NSea Urchin Cdc14 S L A V H C K A G L G R T G T L I E E Y E H Y E R V E N G D F NHydra Cdc14 A V A V H C K A G L G R T G T L I M E Y E H Y E R V E N G D F NSea Squirt Cdc14 A I A V H C K A G L G R T G T L I D E Y E H Y E R V E N G D F N

Human KAP K T L I H C Y G G L G R S C L V AMouse KAP K T L I H C Y G G L G R S C L - AAtlantic Salmon KAP K T V I H C Y G G L G R S G L I ALizard KAP K T L I H C Y G G L G R S C L I ASea Urchin KAP G T L V H C F G G I G R S S L I I

•

Figure S2. Alignment of the active site sequences of Cdc14 and KAP phosphatases and the acidic groove of Cdc14 phosphatases from diverse eukaryotic species. The essential active site Cys, selectivity determinant, and three negatively charged acidic groove residues are highlighted with gray, red, and yellow shading, respectively. Numbers refer to the budding yeast sequence. See supplemental methods and Table S5 for details.

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