From the Department of Biochemistry, Purdue
University, West Lafayette, Indiana 47907, the ¶ Division of
Biology, California Institute of Technology, Pasadena, California
91125, and the
Department of Biomolecular Chemistry, University
of Wisconsin, Madison, Wisconsin, 53706
Received for publication, December 26, 2000, and in revised form, March 26, 2001
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ABSTRACT |
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In the budding yeast Saccharomyces
cerevisiae, the multifunctional protein Net1 is implicated in
regulating the cell cycle function of the Cdc14 protein phosphatase.
Genetic and cell biological data suggest that during interphase and
early mitosis Net1 holds Cdc14 within the nucleolus where its activity
is suppressed. Upon its transient release from Net1 at late anaphase,
active Cdc14 promotes exit from mitosis by dephosphorylating targets in
the nucleus and cytoplasm. In this paper we present evidence supporting the proposed role of Net1 in regulating Cdc14 and exit from mitosis. We
show that the NH2-terminal fragment Net1(1-600) directly
binds Cdc14 in vitro and is a highly specific competitive
inhibitor of its activity (Ki = 3 nM)
with five different substrates including the physiologic targets Swi5
and Sic1. An analysis of truncation mutants indicates that the Cdc14
binding site is located within a segment of Net1 containing residues
1-341. We propose that Net1 inhibits by occluding the active site of
Cdc14 because it acts as a competitive inhibitor, binds to a site
located within the catalytic domain (residues 1-374), binds with
reduced affinity to a Cdc14 C283S mutant in which an active site Cys is
replaced, and is displaced by tungstate, a transition state analog
known to bind in the catalytic site of protein-tyrosine phosphatases.
The Cdc14 phosphatases are a conserved subset of dual specificity
enzymes (1-3) of the protein-tyrosine phosphatase
(PTP)1 family (4-7). The
essential Cdc14 phosphatase from budding yeast (1, 8-10),
Saccharomyces cerevisiae, is involved in driving cells from
late anaphase into G1 of the subsequent cell cycle, a
series of events known as exit from mitosis (for review, see Refs.
11-13). The onset of mitosis occurs when cyclin-dependent kinases are activated following their association with mitotic cyclins.
Exit from mitosis requires the inactivation of
cyclin-dependent kinases, a process that involves the
ubiquitination and subsequent destruction of cyclins and other
regulatory proteins. The anaphase-promoting complex/cyclosome (APC/C)
is a tightly regulated multisubunit ubiquitin ligase that first
initiates anaphase, then exit from mitosis, by targeting proteins for
degradation in an ordered and tightly coordinated fashion (for review,
see Ref. 14). In budding yeast, inactivation of the mitotic
cyclin-dependent kinase (Cdc28) occurs by two processes,
the APC/C-dependent ubiquitination and subsequent
destruction of B-type cyclins (Clb1-6) and the synthesis of the
Clb/Cdc28 inhibitor Sic1 (12, 13). Cdc14 drives both of these processes
by dephosphorylating at least three targets: Hct1, Swi5, and Sic1 (8,
10). Cdc14 dephosphorylates inhibitory sites and thereby activates the
APC/C regulator Cdh1/Hct1 so that a subset of mitotic cyclins, Clb2 and
Clb3, is targeted for ubiquitination by the APC/C (8, 10). Swi5 is a
zinc finger transcription factor that is required for expression of the
SIC1 gene. The dephosphorylation of Swi5 permits its
translocation from the cytoplasm to the nucleus where it can activate
Sic1 transcription (8). The Sic1 protein itself may be protected from
premature degradation when dephosphorylated by Cdc14 (8).
Net1 (also known as Cfi1) is a core component of the nucleolar
RENT complex that regulates Cdc14 during the cell cycle (for review, see Refs. 15-17). From G1 to anaphase, Net1
sequesters Cdc14 in the nucleolus, where its access to substrate is
limited, and its phosphatase activity is suppressed (18-20). At late
anaphase, Cdc14 is transiently released from Net1 permitting the active phosphatase to reach targets in the nucleus and cytoplasm (18, 19). The
RENT complex appears to have multiple functions besides Cdc14
regulation including roles in maintaining the integrity of the
nucleolus (20, 21) and sequestering Sir2 to tandem rDNA repeats (21).
The NAD-dependent histone deacetylase activity of Sir2
silences rDNA chromatin and represses recombination among tandem rDNA
repeats, a deleterious process that leads to senescence of cells
(22-24).
The mechanism by which Cdc14 is released from Net1 is not yet clear,
but it is dependent in part on activation of a signaling pathway known
as the mitotic exit network (9, 11, 12). When the dividing nucleus
spans the bud neck during late mitosis, the mitotic exit network
pathway is activated, and a signal is propagated which promotes release
of Cdc14 (25, 26). The APC/C-mediated destruction of the anaphase
inhibitor Pds1 is not only necessary for sister-chromatid separation
but also for the subsequent release of Cdc14 (27-29). However, Pds1
degradation and Cdc14 release are not sufficient for exit from mitosis
unless the Clb5 cyclin has also been destroyed by the APC/C at the
metaphase to anaphase transition (28). Thus, multiple controls ensure
that exit from mitosis occurs only after chromosome segregation and
correct partitioning of the dividing nucleus to the mother and daughter
cells (25, 26, 28-30).
Previous studies have not determined whether Net1 on its own is
sufficient to inhibit Cdc14 activity. In this paper we have characterized the direct effect of Net1 on Cdc14 activity in
vitro. We have shown that a 600-residue NH2-terminal
fragment of Net1 alone binds the catalytic domain of Cdc14 and acts as
a potent and specific competitive inhibitor of its activity toward
physiologic substrates. We have defined a segment of Net1 spanning
residues 1-341 which contains the Cdc14 binding site and fully
inhibits phosphatase activity. We also provide evidence that Net1 acts by occluding the active site of Cdc14.
Construction of Plasmids--
Plasmids were constructed using
standard cloning and polymerase chain reaction techniques as briefly
outlined below. The authenticity of all plasmids was verified by DNA
sequencing. For two-hybrid analyses, the coding sequence of the
CDC14 gene was cloned into the pGBDU-C1 vector (31) to
create pGBDU-CDC14, which encodes a fusion protein with the
GAL4 DNA binding domain. Using polymerase chain reaction,
the NET1 coding sequence was inserted into pGAD-C1 (31) to
produce pGAD-NET1, which expresses a fusion protein with the
GAL4 activation domain. Restriction fragments derived from
pGAD-NET1 were used to construct a series of six pGAD plasmids encoding
fusion proteins comprised of Net1 truncation mutants containing
residues 1-601, 1-341, 1-207, 92-341, 342-1189, and 92-1189. A
pGAD vector encoding a fusion protein with Net1(1-146) was constructed
using a fragment amplified by polymerase chain reaction. The complete
SWI5 coding sequence and codons 1-600 of NET1
were amplified by polymerase chain reaction and inserted into pET21a to
generate the pET-SWI5-His6 and
pET-NET1(1-600)-His6 expression plasmids. A restriction
fragment from pGAD-Net1(1-341) was inserted into pET21a to give
pET-NET1(1-341)-His6 expression plasmid. All of these pET
expression plasmids encode proteins bearing NH2-terminal T7 tags.
Yeast Two-hybrid Screens and Assays--
A two-hybrid screen was
performed by transforming the yeast strain PJ69-4A (31) with the
pGBDU-CDC14 plasmid expressing the Cdc14 bait protein. This strain was
then transformed with each of three S. cerevisiae Y2HL
genomic libraries (31), and about 2 × 106 colonies
with each library were screened for activation of the HIS3
and ADE2 reporter genes (see below). After establishing that reporter activation was dependent on the presence of both bait and
target proteins, plasmids from positive clones were isolated and
sequenced. This screen identified several positive clones containing
5'-coding sequences from the yeast YJL076W (NET1) gene.
Two-hybrid assays were used to assess the ability of Net1 truncation
mutants to interact with Cdc14. After transformation of the yeast
strain PJ69-4A (31) with the pGBDU-CDC14 plasmid and a pGAD vector
encoding one of the Net1 truncation mutants (see above) cells were
plated on medium permitting detection of reporter gene activation.
Ade+ cells were identified by growth on SC medium lacking
adenine, leucine, and uracil, whereas His+ cells grew on SC
medium lacking histidine, leucine, and uracil but containing 1 mM 3-aminotriazole. Activation of the lacZ
reporter gene was assessed by performing liquid Protein Expression and Purification--
Yeast GST-Cdc14 and
GST-Cdc14(1-374) were overexpressed in Escherichia coli
BL21 (DE3) cells as described (1) except that lysates were treated with
polyethyleneimine (PEI) prior to affinity purification. Using a stock
solution of 5% (w/v) PEI at pH 8, lysates were adjusted to a final PEI
concentration of 0.15% (w/v) and centrifuged at 10,000 × g for 10 min at 4 °C. The pellet was resuspended in 1 volume of lysis buffer containing 500 mM NaCl, mixed and
clarified by centrifugation (10,000 × g for 10 min) prior to purification using glutathione-Sepharose (1). The GST-HCdc14A
(1) and GST-HCdc14B (1) fusion proteins were expressed in
E. coli BL21 (DE3) cells from pET-GST vectors (1) and
purified from PEI-treated extracts as described above. The TC45 splice variant of the human T cell phosphatase was expressed and purified using the method of Hao et al. (32). Purified recombinant
VHR and PPT1 protein phosphatases were provided by Drs. Zhong-Yin Zhang
and S. Rossie, respectively.
E. coli BL21 (DE3) cells transformed with expression
plasmids encoding Net1(1-600)-His6,
Net1(1-341)-His6, and Swi5-His6 were grown at
37 °C in LB medium containing 100 µg/ml ampicillin until the
A600 was about 0.7. Following the addition of 50 µM isopropyl-1-thio-
For further purification, Net1(1-600)-His6 (1-2 mg) was
dialyzed with buffer A (50 mM Tris, pH 8.0, 2 mM EDTA, 0.1% (v/v) 2-mercaptoethanol), loaded on a Mono Q
HR 5/5 column, and eluted with a 25-ml linear gradient from 0 to 500 mM NaCl in buffer A using a fast protein liquid
chromatography system (Amersham Pharmacia Biotech). Peak fractions were
combined, dialyzed with 50 mM imidazole, pH 6.6, 1 mM EDTA, 0.1% (v/v) 2-mercapthoethanol for phosphatase assays or with 25 mM Tris, pH 7.4, 2.6 mM KCl,
137 mM NaCl, 0.1% (v/v) 2-mercaptoethanol for in
vitro binding assays and concentrated with Centricon-10 (Amicon)
membranes. Net1(1-341)-His6 was purified further on a
Sephacryl S300 HR 1 × 80-cm column (Amersham Pharmacia Biotech)
equilibrated in 50 mM imidazole, pH 6.6, 350 mM
NaCl, 1 mM EDTA, 0.1% (v/v) 2-mercapthoethanol and eluted
at a flow rate of 0.5 ml/min using the fast protein liquid
chromatography system. Peak fractions were combined and concentrated by
either ultrafiltration with a Centriprep-10 (Amicon) membrane or
dehydration in a dialysis bag covered with dry Sephadex G-25.
Sic1-Myc-His6 was expressed as a fusion protein with
maltose-binding protein and purified as described (33).
The Cdc28 cyclin-dependent kinase was immunopurified from
yeast as a complex with either the Clb2 or Cln2 cyclin carrying epitope
tags derived from human influenza virus hemagglutinin (HA) as described
(34). Yeast with copies of either GAL1-HA-CLB2 or
GAL1-HA-CLN2 genes were grown in medium containing 2%
galactose to induce expression and disrupted with glass beads. The
HA-Clb2·Cdc28 and HA-Cln2·Cdc28 kinase complexes were
immunopurified from extracts using 12CA5 monoclonal antibodies
cross-linked to protein A-Sepharose beads (34).
In Vitro Binding Assays--
GST, GST-Cdc14, and GST-Cdc14 C283S
affinity matrices were prepared by adding 0.5 nmol of each protein to
200 µl of a 50% slurry of glutathione-Sepharose in binding buffer B
(25 mM Tris, pH 7.4, 137 mM NaCl, 2.6 mM KCl, 0.1% (v/v) 2-mercaptoethanol). Each mixture was
incubated for 30 min at 4 °C in a final volume of 1 ml buffer B. The
supernatant was removed, and each of the three affinity matrices was
suspended in buffer B and split into two aliquots of equal volume. 1 nmol of Net1 in buffer B was added to one aliquot of each affinity
matrix (final volume of 1 ml of buffer B), whereas the second sample of
affinity resin received an equal volume of buffer B without Net1. After
mixing by inversion for 30 min at 4 °C, the resin was centrifuged
and washed four times with 1 ml of buffer B containing 0.01% (v/v)
Triton X-100. Washed beads were suspended in 50 µl of 2 × SDS-PAGE loading buffer, boiled for 5 min, and an aliquot (20 µl) was
separated on a 12% SDS-polyacrylamide gel. Proteins were visualized by
staining the gel with Coomassie Blue. Densitometry was used to estimate
the relative amounts of Net1 fragment and GST-Cdc14 proteins in the SDS
gels assuming that the two proteins have equal staining with Coomassie
dye. Images of gels dried between translucent membranes were obtained
from a scanner (Astra 2200, Umax) used in the transparency mode and
analyzed using ImageQuant software (Molecular Dynamics).
Preparation of 32P-labeled Substrates--
Swi5 and
histone H1 were phosphorylated with [ Phosphatase Assays--
The phosphatase activity of yeast Cdc14,
HCdc14A, HCdc14B, VHR, and TC45 toward pNPP and protein substrates was
measured as described (1, 32) in reactions carried out at 30 °C for
5-40 min in buffer containing 50 mM imidazole, pH 6.6, 1 mM EDTA, 1 mM dithiothreitol, and 0.5 mg/ml
bovine serum albumin. Assays with radiolabeled Sic1, Swi5, histone H1,
and Tyr(P)-MBP substrates were performed in a total volume of 30 µl;
50-µl reactions were employed for pNPP and casein. The final assay
buffer for yeast PPT1 contained 56 mM Tris, 36 mM imidazole, pH 7.2, 1.8 mM EDTA, 1.2 mM EGTA, 0.1% 2-mercaptoethanol, 0.5 mg/ml bovine serum
albumin, and 6 µM phosphocasein.
Limited Proteolysis of
Net1(1-600)-His6--
Digestion with Staphylococcus
aureus V8 protease (0.15 µg) was performed for 60 min at
room temperature with 75.5 µg Net1(1-600) in 0.5 ml of buffer
containing 40 mM ammonium bicarbonate, pH 7.8, 4 mM Tris, 22 mM NaCl, 0.4 mM KCl,
3% (v/v) glycerol, and 0.02% (v/v) 2-mercaptoethanol. Cleavage with
1.5 µg of endoproteinase Arg-C (stored at Determination of the Mode of Inhibition and
Ki--
Because of its high affinity for Cdc14, the
Henderson method (37) for tight binding inhibitors was used for the
analysis of Net1 inhibition. For these experiments, the activity of
GST-Cdc14 (5 nM) was measured at six different substrate
concentrations ranging from 0.27 to 17 µM Tyr(P)-MBP. For
each concentration of substrate, six assays were performed with
Net1(1-600) concentrations between 0 and 100 nM. For each
substrate concentration, the value It/(1 Net1 (1)-His6 Alone Binds Cdc14 Directly--
In
addition to a genetic screen described by Shou et al. (18),
we and others (19) also identified Net1 in a yeast two-hybrid screen
for regulators or substrates of Cdc14 (Table
I). Along with the immunolocalization and
genetic studies (18-20), these two-hybrid results support the notion
that Net1 and Cdc14 interact in vivo, but they fail to
establish whether this interaction is direct or mediated by other
proteins. To determine whether there is a direct interaction between
Net1 and Cdc14 in vitro, we used binding assays employing
recombinant GST-Cdc14 (1) and Net1(1-600)-His6. This
NH2-terminal fragment of Net1 was used to preclude problems associated with the bacterial expression of the 1,189-residue full-length protein. Sequence analysis of Net1 clones obtained in our
two-hybrid screens indicated that the NH2-terminal half of
Net1 (residues 1-641) interacted with Cdc14. A two-hybrid assay confirmed that a fragment of Net1 containing residues 1-601 could interact with Cdc14 (Table I, third row).
Purified GST-Cdc14 or GST alone was immobilized on
glutathione-Sepharose beads and incubated with a 4-fold molar excess of Net1(1-600). SDS-PAGE analysis (Fig. 1)
revealed that Net1(1-600) was retained by the GST-Cdc14 affinity
support but not by immobilized GST alone. The molar ratio of GST-Cdc14
to Net1(1-600) on the affinity matrix (Fig. 1, lane 4) was
estimated to be 1:1 using densitometric analysis of the SDS-gel. This
experiment corroborates results from two-hybrid analyses and
demonstrates that in vitro Net1(1-600) binds GST-Cdc14 with
high affinity and in a manner that is not dependent on other
proteins.
Net1 Is a Potent Inhibitor of Cdc14 Activity--
Evidence that
Net1 negatively regulates Cdc14 activity includes the ability of a
recessive net1 mutant to bypass the requirement for Tem1 and
Cdc15 in promoting exit from mitosis (18), an elevation of Cdc14
activity in net1 mutants (18), the ability of Net1 overproduction to suppress cell cycle defects induced by Cdc14 overexpression (19, 20), and the inactivation of GST-Cdc14 by
immunopurified Net1 (18). To corroborate these findings and to
determine whether Net1 alone directly inhibits Cdc14 activity, we
measured the effect of purified Net1(1-600) on GST-Cdc14 activity in vitro.
The transcription factor Swi5 and the Cdc28 inhibitor Sic1, which are
physiologic Cdc14 substrates (8), were employed to evaluate the
efficacy of Net1 as an inhibitor. Both substrates are phosphorylated
in vivo by the Cdc28 protein kinase in a complex with either
mitotic or G1 cyclins, respectively (33, 38). For use in
inhibition assays, recombinant Sic1 and Swi5 were phosphorylated in vitro using immunopurified complexes of Cln2·Cdc28 and
Clb2·Cdc28 kinases, respectively. Net1(1-600) potently inhibited the
dephosphorylation of both Sic1 and Swi5 by GST-Cdc14 with
IC50 values of 2 and 1 nM, respectively (Fig.
2A). Net1(1-600) also
inhibited activity toward three artificial substrates, pNPP, histone H1
phosphorylated on Ser/Thr residues (data not shown) and as shown below,
Tyr(P)-MBP. We observed a dependence of IC50 values on the
total GST-Cdc14 concentration over a range from 1 to 34 nM,
indicating that Net1 binds in a stoichiometric manner, which is
consistent with a dissociation constant of 10 nM or
less.
The Noncatalytic Domain of Cdc14 Is Not Required for Net1 Binding
and Inhibition--
The Asn/Ser-rich COOH-terminal domain (residues
375-551) of yeast Cdc14 is not required for its activity or its role
in promoting cell cycle progression (1). To assess the role of this
noncatalytic domain in binding Net1, in vitro binding and
inhibition studies were performed with GST-Cdc14(1-374), a fully
active mutant of Cdc14 containing the catalytic domain. The amount of
Net1(1-600) bound by immobilized GST-Cdc14(1-374) was comparable to
that observed with equivalent quantities of the full-length GST-Cdc14
affinity matrix (data not shown). Using Tyr(P)-MBP as an artificial
substrate (1), Net1(1-600) inhibited both GST-Cdc14 and
GST-Cdc14(1-374). The IC50 value measured with the
catalytic domain, Cdc14(1-374), was about 1.5-fold greater than that
for the full-length enzyme (data not shown). Similar results were
obtained using pNPP as substrate. These data show that Net1 binds to a
site residing within the catalytic domain and indicate that the
Asn/Ser-rich COOH-terminal domain has little influence on the affinity
of Cdc14 for Net1.
Effect of Ionic Strength on Net1 Inhibition--
Because of the
substantial reduction in GST-Cdc14 activity in the presence of more
than 100 mM monovalent salt (1), the inhibition studies
described above were performed in low salt buffers having
nonphysiologic ionic strength. Therefore, the sensitivity of GST-Cdc14
to Net1(1-600) was also measured in buffer having an ionic strength
comparable to that in the cell. In the presence of 120 mM
KCl, the IC50 value for Net1(1-600) increased from 5.6 to
74 nM (data not shown). The 10-fold reduction in affinity
of Net1 for Cdc14 was not accompanied by changes in the shape of the
inhibition curve.
Net1 Is a Specific Inhibitor of Yeast Cdc14 Activity--
To
assess its specificity for Cdc14, we examined the ability of
Net1(1-600) to inhibit other enzymes of the PTP family. The activity
of the dual-specific phosphatase VHR (39) and the TC45 variant of the
tyrosine-specific T cell phosphatase (32) were not affected by the
addition of Net1(1-600) at concentrations 300-fold greater than those
giving half-maximal inhibition of GST-Cdc14 (data not shown). Likewise,
up to 1 µM Net1(1-600) had no effect on the activity of
yeast PPT1, a PP5-like enzyme of the Ser/Thr phosphatase family
(40).
The two human homologs of yeast Cdc14 were also not inhibited by Net1
(data not shown). At concentrations up to 0.3 µM,
Net1(1-600) had no effect on GST-HCdc14A (1) and GST-HCdc14B
(1)2, fusion proteins
containing the catalytic domains from each of the human Cdc14
phosphatases (2). At 3 µM Net1(1-600), HCdc14A and
HCdc14B were stimulated 1.5- and 2.5-fold, respectively. Under conditions identical to those employed with yeast Cdc14, there was no
detectable binding of Net1(1-600) to affinity matrices containing the
HCdc14A and B catalytic domains (data not shown). Thus, Net1 not only
fails to inhibit, but is also incapable of binding the human enzymes.
These data indicate that Net1 exhibits a high degree of specificity for
yeast Cdc14.
Net1 Is a Competitive Inhibitor of Cdc14--
To determine the
type of inhibition and Ki value, the Henderson
method (37) for analyzing tight binding inhibitors was used with
Net1(1-600). A representative experiment using Tyr(P)-MBP as substrate
is shown in Fig. 2, B and C. The linear increase in slope with increasing substrate concentration shown in the slope
replot of Fig. 2C indicates that Net1(1-600) is a
competitive inhibitor of Cdc14. The intercept of the slope replot
yields a Ki value of 3 nM. A similar
Ki was obtained using pNPP as substrate (data not shown).
To evaluate the possibility that Net1 binds at the active site, we
examined the effect of sodium tungstate on the interaction with Cdc14.
Tungstate, a competitive inhibitor of PTPs including yeast Cdc14 (1),
binds within the active site of these enzymes. Pretreatment with sodium
tungstate reduced in a concentration-dependent manner the
amount of Net1(1-600) that was bound to a GST-Cdc14 affinity matrix
(Fig. 3, A and B).
These results show that Net1 and tungstate binding are mutually
exclusive and suggest that access to the phosphatase active site is
necessary for the interaction with Net1. The reduced affinity of
Net1(1-600) for the Cdc14 C283S active site mutant is consistent with
this conclusion. Under identical conditions, the amount of Net1(1-600)
bound to an affinity matrix comprised of the GST-Cdc14 C283S mutant was
54% of that bound to a matrix formed with the wild type enzyme
(compare lanes 4 and 6 of Fig. 1).
Identification of an NH2-terminal Net1
Fragment That Binds and Inhibits Cdc14--
To delineate further the
Cdc14 binding region of Net1 a series of truncation mutants lacking
both NH2- and COOH-terminal sequences was analyzed using
yeast two-hybrid assays. The Net1(1-341) truncation mutant interacted
with Cdc14 as shown by activation of all three reporter genes in the
two-hybrid system (Table I, fourth row). To assess its functional
properties and confirm the two-hybrid results,
Net1(1-341)-His6 was expressed in E. coli. An
in vitro binding experiment performed in a manner comparable
to that used for Net1(1-600) showed that purified Net1(1-341) was
bound to the GST-Cdc14 affinity matrix (Fig.
4A, lane 2). A relatively small but detectable amount of Net1(1-341) was retained by GST alone
(Fig. 4A, lane 4). The addition of 300 mM NaCl
eliminated the association of Net1(1-341) with GST without a
significant reduction in binding to GST-Cdc14 (data not shown). This
effect may be explained by the ability of salt to suppress the
aggregation of Net1(1-341) and thereby prevent the retention of small
amounts of insoluble protein on the affinity matrices. Using Tyr(P)-MBP as substrate, the IC50 for Net1(1-341) was 9.7 nM, about 3-fold higher than that measured for Net1(1-600)
under identical conditions (Fig. 4B). These results
demonstrate that Net1(1-341) retains the ability to bind and fully
inhibit Cdc14 albeit with a modest reduction in affinity compared with
Net1(1-600).
Among 11 truncation mutants examined, no
NH2-terminal fragments shorter than Net1(1-341) were shown
conclusively to bind Cdc14. In two-hybrid assays, Net1(1-146) was
unable to associate with Cdc14, and the results with a segment
comprised of residues 1-207 were interpreted as being negative because
only one of the three reporter genes was activated (Table I, sixth and
seventh rows). Consistent with these results, a 27-kDa product obtained
from limited cleavage by the Arg-C protease failed to bind a GST-Cdc14 affinity matrix despite containing as many as the first 242 residues from the NH2 terminus of Net1 (see below). In addition, a
preparation of 35S-labeled Net1(1-242) which was produced
using the reticulocyte in vitro translation system also
failed to bind a GST-Cdc14 affinity matrix. The failure to express
His-tagged Net1(1-276) and Net1(1-207) in E. coli
precluded the analysis of these fragments in binding and inhibition assays.
Net1 truncation mutants with 112 or more residues deleted from the
NH2 terminus failed to interact with Cdc14 in two-hybrid assays (data not shown). The Net1(92-1189) mutant interacted with Cdc14 (Table I, eighth row), whereas deletion of the same 91-residue segment from Net1(1-341) abrogated the interaction (Table I, ninth
row). These results indicate that the first 91 residues at the
NH2 terminus of Net1 may not be necessary for Cdc14
binding, but they may influence the stability or folding of Net1,
particularly in the context of the smaller Net1(1-341) fragment.
Limited Proteolytic Cleavage of Net1(1-600)--
To examine
its domain and structural organization, recombinant Net1(1-600) was
subjected to limited proteolysis using trypsin, chymotrypsin,
subtilisin, endoproteinase Arg-C, and S. aureus V8
proteases. Utilizing low protease to substrate ratios (1:500-1:2,000) typically employed in limited proteolysis studies, Net1(1-600) was
degraded rapidly by trypsin, chymotrypsin, and subtilisin, providing no
evidence for canonical protease-resistant domains (data not shown).
However, digestion with Arg-C and V8 proteases released in modest yield
fragments exhibiting partial resistance to proteolysis (Fig.
5, A and B). These
products were tested for their ability to bind GST-Cdc14 using in
vitro binding assays as described above. A major 42-kDa fragment
and multiple minor products from the V8 protease digest were bound by
the GST-Cdc14 affinity matrix (Fig. 5A) but not by GST alone
(data not shown). The amino acid sequence of the 42-kDa fragment
matched that of the NH2 terminus of recombinant
Net1(1-600) (Fig. 5C). The COOH-terminal residue of this
fragment is unknown, but its size (estimated from SDS-PAGE mobility)
suggests that it could extend up to Glu-369.
Cleavage with endoproteinase Arg-C yielded three major products, a
27-kDa fragment and two polypeptides migrating as a doublet around
57-kDa (Fig. 5B). The NH2 termini of the 27-kDa
fragment and the major component of the doublet were determined by
sequence analysis, but the location of their COOH termini was based on size estimates derived from SDS-gel mobilities. The 27-kDa fragment is
derived from the NH2 terminus and could extend from residue 1 to either Arg-216 or Arg-241 (Fig. 5C). The 57-kDa product
was generated by cleavage after Arg-184, and its large size suggests that it contains residues 185-600 of Net1 (Fig. 5C).
Because neither product binds GST-Cdc14 (Fig. 5B), it
appears that Arg-C cleavage disrupts a contiguous sequence surrounding
Arg-184 which is necessary for an interaction with Cdc14 (Fig.
5C). These studies are fully consistent with results from
truncation mutagenesis showing that residues 1-341 encompass a Cdc14
binding region.
Current models (15-18) posit that Net1 retains inactive
Cdc14 in the nucleolus during interphase and early mitosis until the transient release of the active phosphatase at late anaphase triggers exit from mitosis. The intrinsic biochemical properties of Net1(1-600) which we have observed in vitro fully support this model.
Nanomolar concentrations of Net1(1-600) alone fully inhibited the
activity of Cdc14 through a direct interaction that was not dependent
on other proteins. The effect of Net1(1-600) was specific to Cdc14 and
observed with all substrates examined including the physiologic targets, Swi5 and Sic1. Importantly, Net1(1-600) retained its ability
to act as a high affinity inhibitor (IC50 = 70 nM) when buffers approximating physiologic ionic strength
and pH were employed. These findings demonstrate that Net1 alone has
the capacity to regulate all cellular functions requiring Cdc14 by
sequestering the phosphatase from the nucleoplasm to the nucleolus and
fully inhibiting its activity. Our results also suggest that
post-translational modifications of the type commonly observed in
eukaryotes are unlikely to be required for inhibition because
Net1(1-600) was produced in bacteria. However, post-translational
modification or the binding of other protein ligands could influence
the affinity of Net1 for Cdc14 and may mediate the release of Cdc14 at
late anaphase as suggested previously (18).
Results from in vitro binding, inhibition assays, and
limited proteolytic cleavage demonstrate that residues 1-341 of Net1 are sufficient for both binding and inhibiting Cdc14 (Fig.
5C). Two-hybrid assays suggest that the first 91 residues of
Net1 are not essential for binding, but the possibility that this
segment is needed for inhibition has not been excluded. Net1 also
sequesters Sir2 within the nucleolus where it is involved in rDNA
silencing. Cuperus et al. (41) have shown that residues
566-801 of Net1 interact with an NH2-terminal fragment of
Sir2 (Fig. 5C). Their results together with our data
establish that Sir2 and Cdc14 bind to distinct, non-overlapping sites
within the linear sequence of Net1 and show that neither protein is
required for binding of the other (Fig. 5C). Additional
structure-function analyses of Net1 will be required to define the
minimal sequence(s) required for binding and inhibiting Cdc14.
Several findings suggest that Net1 inhibits by occluding the
active site of Cdc14. Net1 acts as a competitive inhibitor of Cdc14,
indicating that inhibitor and substrate binding are mutually exclusive.
The Net1 binding site has been mapped to a position within the
conserved domain of Cdc14 (residues 1-374) which encompasses the
entire active site region (residues 279-291). The structures (4,
42-45) of several different PTPs reveal that tungstate, a general PTP
inhibitor that mimics the transition state, is bound within the active
site in a manner similar to the phosphate of substrates. Sodium
tungstate prevents Net1 binding in a
concentration-dependent manner (Fig. 3), suggesting that
contacts within the Cdc14 active site are required for binding. The
significant reduction in Net1 affinity which occurs upon replacement of
the Cdc14 active site cysteine also supports the idea that binding
involves the catalytic site. Several studies indicate that replacement
of the active site Cys can induce local changes in the conformation and
properties of PTP catalytic sites (46, 47). For example, a
Yersinia PTP mutant, in which the analogous Cys is replaced
by Ser, exhibits a 10-fold reduction in affinity for tungstate (47).
Although we propose an interaction within the catalytic cleft of Cdc14, it is likely that contacts with flanking surfaces are also involved. Interactions with adjacent surfaces could account for the selectivity of Net1 for the yeast enzyme over human Cdc14 and other PTPs despite the high degree of sequence similarity in their catalytic sites. These
data favor a model in which Net1 binds at the active site, but we
cannot rule out the possibility that it binds to a distinct inhibitory
site and competes indirectly through long range interactions that
result in conformational changes at the substrate binding site.
Additional studies will be required to delineate further the precise
location of the Net1 binding site.
Residues 1-220 of Net1 exhibit 30% sequence identity to residues
1-233 of another yeast protein Tof2 (Fig. 5C). Other
than its ability to interact with DNA topoisomerase I (48), little is
known about the function of Tof2. Although the Tof2-like
segment is contained within the Cdc14 binding region defined by these studies (Fig. 5C), it is not yet clear whether these
sequences are involved directly in binding and/or inhibition. The lack
of binding observed with the 27-kDa cleavage product of Net1, which spans most if not all of these conserved sequences, suggests the Tof2-like region may not have a direct role in regulating Cdc14.
The Caenorhabditis elegans, Drosophila, and human
genomes and the sequence data bases contain no open reading frames
exhibiting significant sequence similarity to Net1. The lack of an
extant Net1 homolog in metazoans is surprising because Cdc14
phosphatase genes are found in the C. elegans,
Drosophila, zebrafish, chicken, mouse, and human genomes.
This observation coupled with the insensitivity of human Cdc14 to Net1
suggests that the regulation of metazoan and budding yeast Cdc14
differ. In contrast to fungi, the nuclear membrane and nucleolus of
metazoans break down during mitosis. As a result of its disassembly,
higher eukaryotes may not employ the nucleolus as a site to regulate
exit from mitosis. It will be important to determine how Cdc14 activity
and its potential function in mitotic exit are controlled in metazoans.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase
assays as described (31).
-D-galactopyranoside, cells expressing Net1(1-600)-His6 and
Swi5-His6 were grown for 14-16 h at room temperature,
whereas cells producing Net1(1-341)-His6 were induced with
400 µM
isopropyl-1-thio-
-D-galactopyranoside and grown for
4 h at 30 °C. Swi5-His6 and
Net1(1-600)-His6 were purified directly from extracts
using His-Bind resin (Novagen) according to protocols from the
manufacturer, whereas lysates containing Net1(1-341)-His6
were treated with PEI as described above before purification on
His-Bind resin.
-32P]ATP using
the partially purified HA-Clb2·Cdc28 complex, whereas Sic1 was phosphorylated with the HA-Cln2·Cdc28 kinase (see above). Myelin basic protein (MBP) (Life Technologies, Inc.) was phosphorylated on
tyrosine using GST-lyn kinase as described (35).
Radiolabeled casein phosphorylated on Ser residues was provided by Dr.
S. Rossie. Protein substrate concentrations given herein represent the
total concentration of phosphorylated residues used in each assay.
20 °C in water) was
carried out at 30 °C as described above for staphylococcal V8
protease except that the digestion buffer contained 80 mM
ammonium bicarbonate at pH 8. Aliquots (50 µl) of each digest were
precipitated with trichloroacetic acid as described previously (32) and
analyzed on 12% SDS-polyacrylamide gels. The remainder of each
reaction (equivalent to 1 nmol of the Net1(1-600) fragment) was
stopped by adding soybean trypsin inhibitor to a final concentration of
1 mg/ml and used for in vitro binding assays with affinity
matrices containing 0.25 nmol of GST-Cdc14 or GST (see above). For
amino acid sequencing, proteolytic fragments were resolved by SDS-PAGE
and transferred to polyvinylidene difluoride membranes as described
(36). Protein bands on the membrane were stained with Coomassie Blue,
excised, and analyzed by automated gas phase sequencing.
vi/v0) was plotted on the
y axis versus
v0/vi on the x axis, where It is the total Net1(1-600) concentration, and
vi and v0 are the
velocities with and without inhibitor, respectively. This plot
generates a series of lines for each concentration of substrate which
were fit by unweighted linear regression using a fixed intercept value
of 5 nM, which was the total enzyme concentration (37). The
Ki and mechanism of inhibition were determined from
a replot of the slopes of these lines versus substrate concentration.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Two-hybrid interaction of Net1 or Net1 mutants with Cdc14
-galactosidase assays.
View larger version (39K):
[in a new window]
Fig. 1.
Net1(1-600) binds Cdc14 in
vitro. GST, GST-Cdc14, and GST-Cdc14 C283S affinity
matrices were prepared by binding 0.25 nmol of each protein to
glutathione-Sepharose beads. Purified Net1(1-600)-His6 (1 nmol) was incubated for 30 min at 4 °C with each affinity matrix in
1 ml of buffer B. After washing with buffer B containing 0.01%
(v/v) Triton X-100, an aliquot (20%) of each
Net1(1-600)-His6-treated matrix (lanes 2,
4, and 6) and the untreated controls (lanes
1, 3, and 5) were mixed with sample buffer
and separated on 12% SDS-polyacrylamide gels. An aliquot (7 µg) of
purified Net1(1-600)-His6 used in these studies was also
subjected to SDS-PAGE (lane 7). Protein was visualized by
staining with Coomassie Blue. The positions of molecular mass
markers are shown on the left, and the locations of
GST-Cdc14 and Net1(1-600)-His6 are indicated on the
right. These results are representative of those obtained in
multiple experiments.
View larger version (29K):
[in a new window]
Fig. 2.
Net1(1-600) is a potent competitive
inhibitor of Cdc14 activity. Panel A, the phosphatase
activity of 4 nM and 1 nM GST-Cdc14 with 3 µM phospho-Sic1 ( ) and 2 µM phospho-Swi5
(
), respectively, was measured in the presence of the indicated
concentrations of recombinant Net1(1-600)-His6.
Phosphatase activity is plotted as the percent of the control value
measured in the absence of Net1(1-600)-His6. Each point is
the mean of three to five determinations. Inhibition curves were
obtained using nonlinear regression analysis to fit the data to an
equation for a rectangular hyperbola. Protein was estimated by the
method of Bradford (49) using bovine serum albumin as standard.
Panel B, the mode of inhibition for
Net1(1-600)-His6 was determined using the methods for
tight binding inhibitors described by Henderson (37). GST-Cdc14
activity was determined in the presence of varying
Net1(1-600)-His6 concentrations at the following
Tyr(P)-MBP substrate concentrations: 0.27 µM (
), 0.54 µM (
), 2.2 µM (
), 4.3 µM (
), 8.7 µM (
), and 17 µM (
). For each substrate concentration,
It/(1
vi/v0) versus
v0/vi was plotted where
It is the total Net1(1-600)-His6
concentration, and vi and
v0 are the velocities measured with and without
inhibitor, respectively. Each data point is the mean of three
determinations. Panel C, the Ki was
derived from a replot of the slopes (
) (± S.E.) obtained for each
line in panel B versus Tyr(P)-MBP concentration. The
y intercept of this plot yields the
Ki value.
View larger version (41K):
[in a new window]
Fig. 3.
Sodium tungstate blocks the binding of
Net1(1-600) to Cdc14. Panel A, an in vitro
binding assay was performed as described for Fig. 1 except that the
GST-Cdc14 affinity matrices were incubated with 0.1, 1.0, and 10 mM sodium tungstate for 20 min prior to the addition of 1 nmol of Net1(1-600)-His6 (lanes 3-5). The
incubation with Net1(1-600)-His6 and subsequent washing
steps were done in the presence of the indicated concentration of
sodium tungstate. After washing, the affinity matrices were collected
and separated on a 12% SDS-polyacrylamide gel. Lane 1 shows
the GST-Cdc14 affinity matrix alone, and lane 2 shows the
amount of Net1(1-600)-His6 bound in the absence of sodium
tungstate. Protein was visualized by staining with Coomassie Blue. The
positions of molecular mass markers are shown on the left,
and the locations of GST-Cdc14 and Net1(1-600)-His6 are
indicated on the right. Panel B, the relative amount of
Net1(1-600)-His6 bound to the GST-Cdc14 affinity matrix in
the presence of the indicated concentrations of sodium tungstate. The
relative amount of bound Net1(1-600)-His6 was estimated by
densitometry of the SDS-gel in panel A as outlined under
"Experimental Procedures."
View larger version (27K):
[in a new window]
Fig. 4.
Net1(1-341) binds and inhibits Cdc14
in vitro. Panel A, purified
Net1(1-341)-His6 (1 nmol) was incubated for 30 min at
4 °C with GST and GST-Cdc14 affinity matrices containing 0.25 nmol
of each protein. Affinity matrices and Net1(1-341) were mixed in 1 ml
of buffer B. After washing with buffer B containing 0.01% (v/v) Triton
X-100, the affinity matrices, both those mixed with
Net1(1-341)-His6 (lanes 2 and 4) and
untreated controls (lanes 1 and 3), were treated
with sample buffer and resolved on 12% SDS-polyacrylamide gels that
were stained with Coomassie Blue. The positions of molecular mass
markers are shown on the left, and the positions of
GST-Cdc14 and Net1(1-341)-His6 are indicated on the
right. Panel B, the relative phosphatase activity of 2.5 nM GST-Cdc14 with 3.2 µM Tyr(P)-MBP as
substrate was measured in the presence of the indicated concentrations
of recombinant Net1(1-600)-His6 ( ) and
Net1(1-341)-His6 (
). Phosphatase activity is given as
the percent of the control value measured in the absence of Net1
fragment, and each data point is the mean of at least three
determinations.
View larger version (30K):
[in a new window]
Fig. 5.
Analysis of proteolytic fragments of
Net1(1-600). Panel A, Net1(1-600)-His6
was digested with staphylococcal V8 protease for 60 min at room
temperature using a protease:Net1weight ratio of 1:500. Aliquots of an
untreated control (lane 1) and the digested sample
(lane 2) were precipitated with trichloroacetic acid and
separated on a 12% SDS-polyacrylamide gel. The remaining sample was
treated with soybean trypsin inhibitor and analyzed for its ability to
bind to a GST-Cdc14 affinity matrix (see Fig. 1). Lane 3,
washed GST-Cdc14 matrix without added V8 digest; lane 4,
washed GST-Cdc14 matrix mixed with the V8 digest; lane 5,
digested material not bound by the affinity matrix. Panel B,
Net1(1-600)-His6 was digested with Arg-C protease for 60 min at 30 °C using a protease:Net1 weight ratio of 1:50. Samples of
untreated (lane 1) and digested (lane 2)
Net1(1-600)-His6 were analyzed as above for panel
A. An in vitro Cdc14 binding assay was performed on an
aliquot of the Arg-C digest as in panel A. Lane
3, washed GST-Cdc14 matrix without added Arg-C digest; lane
4, washed GST-Cdc14 matrix mixed with the Arg-C digest; lane
5, digested material not bound by the affinity matrix. Protein was
visualized by staining with Coomassie Blue. In panels A and
B, the positions of GST-Cdc14,
Net1(1-600)-His6, and proteolytic fragments are indicated
on the right. Panel C, schematic diagram illustrating the
structural organization of full-length Net1 (upper bar) and
the location of the major fragments (black lines) derived
from limited proteolysis. The NH2 termini of fragments
were mapped by amino acid sequence analysis, whereas the dashed
lines indicate that the locations of COOH termini were deduced
from size as estimated by SDS-PAGE and were not determined directly.
The shaded box within the upper bar delineates
the location of sequences containing the Cdc14 binding site, and the
black box denotes the position of the Sir2 binding region as
determined by Cuperus et al. (41). The cross-hatched
bar below indicates the location of Net1 sequences displaying a
high degree of sequence similarity to Tof2.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. S. Rossie and Z.-Y. Zhang for generously providing samples of the PPT1 and VHR protein phosphatases, respectively; the Purdue Laboratory for Macromolecular Structure for performing amino acid sequence analyses; and Dr. S. Rossie for critical comments on the manuscript.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant CA59935 (to H. C.), a Howard Hughes Medical Institute predoctoral fellowship (to W. S.), a Beckman Young Investigator award (to R. J. D), and a fellowship (to Y. L.) from the Indiana Elks Charities. This is Journal Paper 16424 from the Purdue University Agriculture Experiment Station.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
** Work in the laboratory of Dr. Elizabeth A. Craig was supported by National Institutes of Health Grant GM31107.
To whom correspondence should be addressed: 1153 Dept. of
Biochemistry, Purdue University, West Lafayette, IN 47907-1153. Tel.: 765-494-4754; Fax: 765-494-7897; E-mail: charb@purdue.edu.
Published, JBC Papers in Press, March 27, 2001, DOI 10.1074/jbc.M011689200
2 L. Has, T. Despande, Y. Liu, G. S. Taylor, C. Baskerville, and H. Charbonneau, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are: PTP, protein-tyrosine phosphatase; APC/C, anaphase-promoting complex/cyclosome; SC, synthetic complete; GST, glutathione S-transferase; PEI, polyethyleneimine; HCdc14, human homologs of yeast Cdc14; HA, hemagglutinin; PAGE, polyacrylamide gel electrophoresis; MBP, myelin basic protein; pNPP, p-nitrophenyl phosphate; Tyr(P)-MBP, myelin basic protein phosphorylated on tyrosine residues.
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