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Binding of 14-3-3ß to the Carboxyl Terminus of Wee1 Increases Wee1 Stability, Kinase Activity, and G₂-M Cell Population

Departments of Molecular Biology [Y. W., C. J., H. D., Y. S.] and Cancer Research [K. E. H.], Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, Michigan 48105, and Onyx Pharmaceutical, Inc., Richmond, California 94806 [R. N. B.]

Abstract

Wee1 protein kinase plays an important regulatory role in cell cycle progression. It inhibits Cdc-2 activity by phosphorylating Tyr15 and arrests cells at G₂-M phase. In an attempt to understand Wee1 regulation during cell cycle, yeast two-hybrid screening was used to identify Wee1-binding protein(s). Five of the eight positive clones identified encode 14-3-3ß. In vivo binding assay in 293 cells showed that both full-length and NH₂-terminal truncated Wee1 bind with 14-3-3ß. The 14-3-3ß binding site was mapped to a COOH-terminal consensus motif, RSVSLT (codons 639 to 646). Binding with 14-3-3ß increases the protein level of full-length Wee1 but not of the truncated Wee1. Accompanying the protein level increases, the kinase activity of Wee1 also increases when coexpressed with 14-3-3ß. Increased Wee1 protein level/enzymatic activity is accountable, at least in part, to an increased Wee1 protein half-life when coexpressed with 14-3-3ß. The protein half-life of the NH₂-terminal truncated Wee1 is much longer than that of the full-length protein and is not affected by 14-3-3ß cotransfection. Biologically, 14-3-3ß/Wee1 coexpression increases the cell population at G₂-M phase. Thus, Wee1 binding with 14-3-3ß increases its biochemical activity as well as its biological function. The finding reveals a novel mechanism by which 14-3-3 regulates G₂-M arrest and suggests that the NH₂-terminal domain of Wee1 contains a negative regulatory sequence that determines Wee1 stability.

Introduction

Eukaryotic cell division is regulated by Cdks.² This mechanism of cell cycle control is conserved from yeast to mammalian cells (1) . One of the well-characterized Cdks is Cdc2, which was originally identified in Schizosaccharomyces pombe (2) . Both genetic and biochemical studies demonstrated that Cdc2 is essential for the G₂-M progression of the cell cycle, and activation of Cdc2 is sufficient to induce entry into M-phase (3 , 4) . The importance of Cdc2 in cell cycle regulation is evident by the observations that premature activation of Cdc2 leads to cell death (5, 6, 7, 8, 9, 10) . Furthermore, cells exposed to DNA-damaging agents show a delayed Cdc2 activation to repair damaged DNA (11, 12, 13, 14, 15) .

To ensure the proper timing of G₂-M transition, the Cdc2 kinase must be tightly regulated. That is achieved either by its association with cyclin B, which is required for Cdc2 kinase activity (3) , or mainly by phosphorylation/dephosphorylation of Cdc2 throughout the cell cycle. Phosphorylation of Thr161 in Cdc2 by Cdc-activating kinase (Cak) is essential for Cdc2 kinase activity. This phosphorylation stabilizes the kinase in the active conformation (16, 17, 18) . On the other hand, Cdc2 is subject to inhibitory phosphorylation, which is located in the ATP-binding domain of the kinase. The inhibitory phosphorylation is catalyzed by Wee1 and Mik1 kinases in yeast. In mammalian cells, Cdc2-inhibitory phosphorylations are catalyzed by Wee1 on Tyr15 and Myt 1 on both Thr14 and Tyr15 (19, 20, 21, 22) . At the G₂-M transition, inhibitory phosphorylation on Cdc2 is dephosphorylated by the Cdc25 phosphatase, which leads to an abrupt activation of Cdc2 (23, 24, 25) .

Wee1 protein kinase was originally isolated as a negative regulator of cell division in S. pombe (26) . Mutation in Wee1 resulted in smaller cell size, which indicated its role in cell division (26) . Subsequently, mammalian Wee1 kinase was isolated and shown to be able to complement the S. pombe Wee1 mutant, which indicated a functional conservation of Wee1 in cell cycle regulation (19 , 20 , 27) . Fission yeast Wee1 is a M_r 107,000 protein, consisting of a COOH-terminal catalytic domain, a NH₂-terminal regulatory domain, and a central region probably involved in substrate recognition (28) . The fission yeast Wee1 can be phosphorylated by Nim1 kinase at the COOH-terminal portion, and this phosphorylation inhibits Wee1 kinase activity (29 , 30) . Recently, it has been shown that DNA damage response kinases Cds1 and Chk1 can directly phosphorylate Wee1 (31 , 32) . Human Wee1 consists of 646 amino acids with a molecular mass of 94 kDa. It has a COOH-terminal catalytic domain and an inhibitory NH₂-terminal domain (27 , 33 , 34) . In mammalian cells, Wee1 kinase activity oscillates during the cell cycle. It increases during S and G₂ phases in parallel with the level of the protein. The kinase is inactivated at M phase because of hyperphosphorylation and protein degradation (33 , 34) .

In an attempt to gain a better understanding of how Wee1 activity is regulated in mammalian cells, we used the yeast two-hybrid screen approach to identify proteins that interact with Wee1. We report here that Wee1 specifically binds to 14-3-3ß both in vitro and in vivo and that association with 14-3-3ß increases Wee1 protein levels and kinase activity, at least in part by prolongation of Wee1 protein half-life. Significantly, this association increases Wee1 activity as a negative regulator of the cell cycle by increasing G₂-M cell population. Thus, our data show a novel regulation of mammalian Wee1 kinase by association with 14-3-3ß.

Results

Identification of 14-3-3ß as a Wee1-Binding Protein by the Yeast Two-Hybrid Screening.
The Wee1 kinase plays an important role in regulation of cell cycle progression through inhibitory phosphorylation on Tyr15 of Cdc2. To gain a better understanding of mammalian Wee1 regulation, we used the yeast two-hybrid screen to isolate proteins that may interact with Wee1. The COOH-terminal domain of Wee1, Wee11–288, which has a deletion of 288 amino acids at the NH₂ terminus, was used as the bait to screen a HeLa cell cDNA library. Among 2,000,00 transformants screened, we isolated eight clones that grew on His-deficient plate and showed blue color under selection with LacZ expression (not shown). These eight positive clones were sequenced, and five of them turned out to be the cDNA-encoding 14-3-3ß with stretches of bases of different lengths at the 5'-end untranslated region. To ensure the true binding, we performed an interaction assay in yeast by cotransformation of these five clones individually with the Wee1 bait and confirmed the finding (data not shown).

In Vivo Binding of Wee1 with 14-3-3ß.
To confirm the Wee1/14-3-3ß binding in vivo, we cotransfected myc-tagged 14-3-3ß with full-length Wee1 in 293 human embryonic kidney cells that had undetectable endogenous Wee1 (data not shown). We used IP-coupled Western analysis to immunoprecipitate 14-3-3ß, first with anti-myc-tag Ab, followed by Western blot analysis with Wee1 Ab. If Wee1 binds to 14-3-3ß, we should be able to detect it in the precipitant by myc-tag Ab. Indeed (as shown in Fig. 1, top ), Wee1 proteins were detected only in Wee1/14-3-3ß cotransfectant cells (Fig. 1 , top, Lane 3) , not in each of the individually transfected cell (Fig. 1 , top, Lanes 1 and 2). A similar result was seen when 14-3-3ß Ab, instead of the myc-tag Ab, was used for IP (data not shown). It is of note that two bands of Wee1 were detected. The exact nature of these two forms of Wee1 is not clear at the present time. The flag-tag Ab against NH₂-terminal tagged Wee1 mainly detects the upper band (with a molecular mass of 98 kDa; see Fig. 2 ).).

View larger version (54K): [in this window] [in a new window]   Fig. 1. Wee1 interacts with 14-3-3ß. Human 293 kidney cells were transfected with plasmids expressing various forms of Wee1 alone or in combination with 14-3-3ß. Two days after transfection, cells were harvested in lysis buffer, and 1 mg of cell lysate protein was immunoprecipitated with 1 µg of Myc-tagged Ab. The immunoprecipitated samples were resolved on a SDS-PAGE and blotted with anti-Wee1 Ab, as detailed in "Materials and Methods".

View larger version (25K): [in this window] [in a new window]   Fig. 3. 14-3-3ß coexpression increases Wee1 protein level and enzymatic activity. A, human 293 cells in 60-mm dish were transfected with plasmids (15 µg each) encoding Wee1 and/or 14-3-3ß. Twenty µg of indicated cell lysate protein were subjected to Western analysis with anti-Wee1 Ab. B, the 293 cells were transfected with the full-length Wee1 alone or in combination with 14-3-3ß as indicated. Transfected cells were harvested in lysis buffer and immunoprecipitated with anti-Wee1 Ab. The immunoprecipitated samples were then incubated with Cdc2/cyclin B in a kinase buffer for 15 min. The reaction was stopped with SDS-sample buffer, resolved on SDS-PAGE, and transferred to a polyvinylidine difluoride membrane. The membrane was then cut into two parts. The top portion was blotted with anti-Wee1 Ab, and the lower portion was blotted with anti-phospho-Tyr Ab. Densitometric quantitation was performed in a densitometer (Molecular Dynamics).

View larger version (33K): [in this window] [in a new window]   Fig. 2. The RSVSLT motif at the COOH terminus of Wee1 is required for 14-3-3ß binding. A, Wee1, but not its RSVSLT deletion mutant nor S642A mutant, binds to endogenous 14-3-3. Four flag-tagged Wee1 constructs, along with the vector control, were individually transfected into 293 cells. The cell lysates were immunoprecipitated with flag-tag Ab, followed by Western blot analysis with flag-tag Ab (top) and 14-3-3 Ab (bottom). B, binding of 14-3-3ß with Wee1, but not its RSVSLT deletion mutant nor S642A mutant. Four flag-tagged Wee1 constructs, along with the vector control, were cotransfected with Myc-tagged 14-3-3ß into 293 cells. The cell lysates were immunoprecipitated with Myc-tag Ab, followed by Western blot analysis with Flag-tag Ab (top) and 14-3-3 Ab (bottom).

Wee1 Binds to Endogenous 14-3-3: Requirement of RSXpSXT Motif at the COOH Terminus of Wee1.
Because endogenous Wee1 is not detectable, whereas 14-3-3 level was quite high in 293 cells, we next examined whether transfected Wee1 would interact with endogenous 14-3-3ß. The constructs expressing flag-tagged full-length Wee1 and NH₂-terminal truncated Wee1 were generated and transfected into 293 cells, along with the vector control. Cell lysates were first immunoprecipitated with flag-tag Ab, followed by Western blot with flag-tag Ab (for Wee1 expression level; Fig. 2A , top) or 14-3-3 Ab (for Wee1 bound endogenous 14-3-3; Fig. 2A , bottom). As shown in Fig. 2A , 14-3-3 protein was present in the immunoprecipitants pulled down by full-length Wee1 (Fig. 2A , bottom, Lane 1) and truncated Wee1 (Fig. 2A , bottom, Lane 4), but not the vector control (Fig. 2A , bottom, Lane 5), which indicated Wee1 interacts with endogenous 14-3-3.

Because 14-3-3 proteins bind to other proteins via phosphoserine residue and the consensus motifs for 14-3-3 binding are RSXpSXP or RXY/FXpSXP (35) , we, therefore, searched these consensus motifs in Wee1 protein but did not find a perfect match. However, if we allowed one mismatch in the RSXpSXP motif, we found a match with a substitution of consensus residue proline (P) to threonine (T) at codon 644 at the COOH terminus of the protein (RSVSLT, codons 639 to 644). To determine whether this motif is required for 14-3-3ß binding, the constructs expressing flag-tagged mutant Wee1S642A (with serine, a potential site for phosphorylation, substituted by alanine) and deletion mutant Wee1d638 (with this binding motif deleted) were transfected into 293 cells, followed by IP-Western analysis. Both of the Wee1 mutants were expressed at a comparable level as the wild type (Fig. 2A , Lanes 2 and 3, top), but none of them pulled down endogenous 14-3-3 (Fig. 2A , Lanes 2 and 3, bottom). In a reciprocal experiment, shown in Fig. 2B , all of the four Wee1 constructs were cotransfected with myc-tagged 14-3-3ß, followed by IP with myc-tag Ab and Western with flag-tag Ab (Fig. 2B , top) or 14-3-3 Ab (Fig. 2B , bottom). Although the expression level of 14-3-3ß is similar among the transfections (Fig. 2B , bottom), 14-3-3ß pulled down only wild-type full-length and NH₂-terminal truncated Wee1 (Fig. 2B , Lanes 1 and 4, top) but not the two Wee1 mutants (Fig. 2B , Lanes 2 and 3, top). A cross-react mouse IgG band was detected in every sample that overlapped with NH₂-terminal truncated Wee1 because both IP and Western blotting were performed with mouse antibodies (myc-tag and flag-tag). In a separate experiment, using myc-tag for IP and rabbit Ab against the COOH terminus of Wee1 (Santa Cruz) for Western blotting, the NH₂-terminal truncated Wee1 as well as full-length Wee1, but not the Wee1 mutants, were detected (data not shown). The result clearly indicated that the RSVSLT motif located in the COOH terminus of Wee1 is required for the 14-3-3ß binding. The result also provides a piece of indirect evidence that suggests that the phosphorylation of serine residue at codon 642 is required for the binding.

Binding with 14-3-3ß Increases Wee1 Protein Level As Well As Kinase Activity.
Having established in vivo interaction of the Wee1 with 14-3-3ß, we next examined the potential biochemical consequences of this interaction. We measured Wee1 protein levels by Western blot analysis in 293 cells transfected either with Wee1 alone or in combination with 14-3-3ß. As shown in Fig. 3A , in the absence of 14-3-3ß, expression of the full-length Wee1 (Fig. 3A , Lane 1), or kinase-inactive Wee 1 (Fig. 3A , Lane 4) was detectable. The NH₂-terminal truncated Wee1 (Wee11–214), however, expressed at a much higher level (Fig. 3A , Lane 3) and, as expected, no expression was detected in the antisense construct (Fig. 3A , Lane 2). When cotransfected with 14-3-3ß, both full-length and kinase-inactive Weel were expressed at a much higher level, as compared with Wee1 transfection alone (Fig. 3A , Lane 5 versus Lane 1 and Lane 8 versus Lane 4). Cotransfection with 14-3-3ß did not, however, change the expression of truncated Wee 1 (Fig. 3A , Lane 7 versus Lane 3). And again, no expression was detected in antisense construct (Fig. 3A , Lane 6). A slightly, but insignificantly, higher transfection efficiency was detected when cotransfected with 14-3-3ß in a ß-galactosidase luciferase assay but not in a CD20 fluorescence assay (data not shown). This result indicated that the binding of Wee1 with 14-3-3ß remarkably increased Wee1 protein levels.

We next examined whether this increased protein level was accompanied by an increase in Wee1 kinase activity, which is required for its biological function. Wee1 protein was immunoprecipitated with Wee1 Ab from transfected 293 cells. Kinase activity was determined with Cdc2/cyclin B as substrate (expressed and purified from baculovirus, see "Materials and Methods"). The IP-kinase reaction samples were resolved on a SDS-PAGE and transferred to polyvinylidine difluoride membranes. In Figure 3B , the top portion was blotted with Wee1 Ab to detect the amount of Wee1 protein precipitated and the bottom portion was blotted with antiphosphotyrosine Ab to detect the Cdc2 phosphorylation by Wee1. Cotransfection with 14-3-3ß induced a 2.4-fold increase of Wee1 protein level (Fig. 3B , Lane 4, compared with Lane 2, top). Wee1 protein was not detected in the vector control (Fig. 3B , Lane 1, top) or 14-3-3ß alone (Fig. 3B , Lane 3, top). As shown in Fig. 3B , bottom, transiently expressed Wee1 was able to phosphorylate Cdc2 in vitro (Lane 2) and the level of phosphorylation of Cdc2 was increased by 3.5-fold by cotransfection with 14-3-3ß (Lane 4, compared with Lane 2). Some background Cdc2 phosphorylation in 293 cells was also detected (Lanes 1 and 3). These reproducible results showed a close agreement between increased protein level and increased kinase activity, thus demonstrating that the binding of Wee1 with 14-3-3ß increases Wee1 protein level that is attributable to an increase in Wee1 kinase activity.

Binding with 14-3-3ß Stabilized Wee1 by Prolongation of Its Protein Half-Life.
We have shown that 14-3-3ß binding can increase Wee1 protein level. We then examined at which level this regulation occurred. We first examined potential transcriptional activation of Wee1 expression by 14-3-3ß. Northern analysis revealed that 14-3-3ß cotransfection did not change Wee1 messenger level (not shown), thus excluding the possibility of transcriptional regulation.

We next examined posttranslational regulation by a pulse-chase experiment. The 293 cells were first transfected with Wee1-expressing constructs either alone or in combination with 14-3-3ß, followed by [³⁵S]Met pulse-labeled for 30 min and chased up to 2 h. As shown in Fig. 4A , which is derived from three independent experiments, the Wee1 protein was rapidly degraded with a protein half-life of 25 min when transfected alone. In contrast, when cotransfected with 14-3-3ß, Wee1 protein seems to be stabilized with a half-life prolonged to about 50 min. One representative experiment was included in Fig. 3B to show Wee1 degradation in the absence or presence of 14-3-3ß. Expression of exogenous 14-3-3ß was confirmed in these 14-3-3ß cotransfected samples by Western blot using anti-14-3-3ß Ab (data not shown). These results suggest that 14-3-3ß increases Wee1 protein levels, at least in part, by stabilizing the Wee1 protein.

View larger version (20K): [in this window] [in a new window]   Fig. 4. 14-3-3ß coexpression stabilizes Wee1 protein. A and B, the 293 cells transfected with the full-length Wee1 alone or in combination with 14-3-3ß were [35S]Met-labeled for 30 min, followed by chasing up to 2 h, as indicated. Cell lysate was prepared and equal amounts of TCA precipitated counts were immunoprecipitated by anti-Wee 1 Ab and loaded on a SDS PAGE gel followed by autoradiography and densitometric quantitation in a densitometer (Molecular Dynamics). The densitometric value at 0 h postchase was arbitrarily set as 1. A, values are data from three independent experiments; bars, SE. B, one representative experiment. C, the same experiment was performed as indicated in A, except that the COOH-terminal truncated Wee1 (Wee11–214) instead of the full-length Wee1 was used for transfection, followed by a 30-min pulse and up to a 4-h chase.

Cotransfection of Wee1 with 14-3-3ß Increases Population of Cells Arrested at G₂-M Phase.
Previously published data have shown that overexpression of Wee1 can induce a cell cycle arrest at G₂-M phase (33 , 34 , 36) . To determine the biological significance of Wee1/14-3-3ß binding, we performed DNA flow cytometric analysis of 293 cells transfected with Wee1 alone or in combination with 14-3-3ß. The cDNA encoding CD20 cell surface marker was cotransfected and used to specifically monitor the transfected cells. The DNA content profiles shown in Fig. 5 were only for the CD20 positive cells except Fig. 5A , in which all of the cells are plotted. DNA flow cytometric analysis showed that the transfection of Wee1 caused an increase in the G₂-M population from 15.1 to 33.2% (compare Fig. 5A with Fig. 5B ), whereas transfection with 14-3-3ß alone had no effect (15.1 to 17.8%, compare Fig. 5A with Fig. 5C ). Significantly, cotransfection of Wee1 and 14-3-3ß increased the G₂-M fraction from 33.2 to 51.9% (compare Fig. 5B with Fig. 5D ). These results indicated that the elevated Wee1 protein level and kinase activity through 14-3-3ß binding did translate into an increased biological function of Wee1 as a G₂-M blocker.

View larger version (38K): [in this window] [in a new window]   Fig. 5. 14-3-3ß/Wee1 coexpression increases G2-M population. B-D, the 293 cells were cotransfected with pCMVCD20 and Wee1 (B), 14-3-3ß (C), or Wee1+14-3-3ß (D) in a 1:9 ratio. Cells in A were treated with calcium phosphate only. The cells were harvested 48 h posttransfection. DNA histograms (of propidium iodide fluorescence) are shown (only) for CD20-positive cells except those in A, in which all of the cells are plotted. Cell cycle distributions were estimated as described in "Materials and Methods."

Eukaroytic cell cycle regulation is controlled by the activity of Cdk. Wee1 as a key regulator of Cdc2 is likely to play a critical role in the G₂-M transition of the cell cycle. However, little is known about how Wee1 activity is regulated. Previous data have indicated that protein phosphorylation may play a role in the control of Wee1 activity in yeast (29, 30, 31, 32 , 37 , 38) . Most recently, the COOH-terminal portion of mouse Wee1 has been shown to bind with 14-3-3 (39) , but the functional significance of this protein-protein interaction was not addressed in the study (39) . Here we provide evidence that Wee1 activity in mammalian cells can be regulated by 14-3-3ß. Our data demonstrate that 14-3-3ß directly binds to Wee1 at the COOH-terminal RSVSLT motif, and phosphorylation of serine residue within this motif seems to be required for the binding. The binding with 14-3-3ß increases the Wee1 protein level by, at least in part, prolongation of Wee1 half-life and, thus, enhances Wee1 kinase activity. We also demonstrated that 14-3-3ß increases Wee1 function to cause a significant G₂-M cell cycle arrest. It is worth noting that in 293 cells, 14-3-3ß protein is quite abundant, whereas Wee1 level is undetectable.³ The lack of increased G₂-M population by 14-3-3ß transfection alone (compare Fig. 5C with Fig. 5A ) suggests a lack of activation of endogenous Wee1 by exogenous expression of 14-3-3ß. This is probably attributable to a saturation of limited endogenous Wee1, if any, by a high level of endogenous 14-3-3ß under normal growth conditions or probably attributable to the binding of 14-3-3ß to a variety of other proteins. Likewise, a significant increase of Wee1 activity, both biochemically and biologically when cotransfected with 14-3-3ß, suggests that endogenous 14-3-3 protein is also limiting and, again, probably attributable to its binding to many other cellular proteins. Furthermore, the binding of Wee1 with 14-3-3 may not be specific for the 14-3-3ß isoform, inasmuch as others have reported the binding of mouse Wee1 with 14-3-3 (39) . Moreover, we cannot exclude the possibility that 14-3-3ß could form heterodimer with other isoforms of 14-3-3 to mediate binding/interaction with Wee1.

14-3-3 proteins are a family of low-molecular-weight acidic proteins found in all eukaryotic cells. They are highly conserved evolutionarily and are present in high abundance in cells. This family of proteins has been identified in mammals, amphibians, insects, plants, and yeast (40) . There are seven distinct isoforms of 14-3-3 proteins in mammals (40) . The crystal structures of 14-3-3 proteins show that 14-3-3 protein forms dimers with conserved residues in the groove, suggesting that it plays an essential role as a molecular chaperone and adapter protein (41) . 14-3-3 has been shown to interact with many proteins involved in signal transduction. Many of them are kinases such as Raf-1, MEK, KSR, and Bcr-Abl (42, 43, 44) . Although the biological effects of these interactions need to be further elucidated, it is clear that 14-3-3 plays an important role in the signal transduction cascade.

The role of 14-3-3 family members in cell cycle control was recently demonstrated. In S. pombe, disruption of the 14-3-3 gene led to a loss of checkpoint control at the G₂ phase (45) . In mammalian cells, 14-3-3 was recently found to be subject to p53-regulation. It mediated DNA damage-induced p53 response by inhibiting G₂-M progression (46) . Accordingly, overexpression of 14-3-3 caused G₂ arrest (46) . How do the 14-3-3 family proteins regulate cell cycle? Several recent reports may shed light on this issue. It was found that 14-3-3 protein could interact with Cdc25C, a phosphatase that specifically dephosphorylates Thr14 and Tyr15 of Cdc2 and activates the Cdc2 kinase activity (47, 48, 49) . On DNA damage, Chk1 protein kinase is activated and phosphorylates Cdc25. The 14-3-3 protein, specifically bound to phosphorylated Cdc25C (Cdc25-Ser216P). The binding of Cdc25C by 14-3-3 has been shown to inhibit Cdc25C function, probably by sequestering Cdc25C from functionally interacting with Cdc2 in vivo (47 , 49 , 50) . In addition, in response to DNA damage, Rad24, a 14-3-3 protein in fission yeast, binds to phosphorylated Cdc25 (by Chk1) and exports the latter into the cytoplasm, in which it physically separates from nuclear Cdc2, thus preventing mitosis (51) . In addition, DNA-damage-induced p53 transactivates 14-3-3, which sequesters Cdc2-cyclin B1 complexes in the cytoplasm to prevent mitosis entry (52) . Our data, reported here, provide yet another mechanistic explanation for 14-3-3 to regulate cell cycle progression: it induces G₂-M arrest by increasing Wee1 protein level and kinase activity. Taken together, it seems that 14-3-3 proteins could induce G₂-M arrest via inhibition of Cdc2 activity by binding and activating the Wee1 kinase (mechanism 1; this report), or preventing Cdc2 activation by Cdc25 by (a) binding and inhibiting Cdc25 function (mechanism 2; Refs. 47, 48, 49 ); (b) nuclear export/exclusion of Cdc25 (mechanism 3; Ref. 51 ); or (c) both. In addition, 14-3-3 retains Cdc2/cyclin B in the cytoplasm to prevent its activation in the nucleus (mechanism 4; Fig. 6 ).

View larger version (18K): [in this window] [in a new window]   Fig. 6. Potential mechanisms by which 14-3-3 induces G2-M arrest: (a) 14-3-3 binds to Wee1 and increases Wee1 activity, which phosphorylates and inactivates Cdc2; (b) 14-3-3 binds to Cdc25-Ser216P to inhibit Cdc25 function (47 , 49 , 50) ; or (c) binding of 14-3-3 with Cdc25-Ser216P sequesters Cdc25P into the cytoplasm to separate it physically from nuclear Cdc2/cyclin B (51) ; and (d) 14-3-3 sequesters Cdc2/cyclin B in cytoplasm to prevent mitosis entry (52) .

In summary, we report here that 14-3-3ß physically binds to Wee1 at the consensus motif, RSVSLT, located in the COOH terminus of Wee1 protein, and this binding/interaction leads to an increased Wee1 protein level and kinase activity at least in part by protein stabilization. As a consequence of this binding, increased cell population is arrested in G₂-M phase. Future experiments are directed to determine: (a) whether the Wee1/14-3-3ß interaction is regulated in a cell-cycle-dependent manner; and (b) the physiological relevance of this interaction under stress conditions such as DNA-damage-induced growth arrest.

Materials and Methods

Materials and Plasmid Constructs.
Protease inhibitor cocktail was from Boehringer Mannheim Biochemicals. Antibodies against Wee1Hu (C-20) and 14-3-3ß (C-20) as well as ProteinG⁺ agarose were from Santa Cruz Biotechnology. Myc-tag Ab was from Invitrogen, and flag-tag Ab was from Sigma. Wild-type Cdc2/cyclin B was expressed in baculovirus and copurified as described previously (22) , and Cdc2 is in an active form with T161 phosphorylated. Anti-Myc Ab and plasmids, pcDNA3.1(-)/Myc-HisC and pcDNA3.1(-)/Myc-HisLacZ were from Invitrogen. Anti-CD20 fluorescein Leu-16 was from Becton Dickinson. Plasmids pCMV-CD20, pTA-Wee1^w (truncated Wee1, Wee11–214), pKS⁺HuWee1a/b (full-length Wee1), and pKS⁺HuWee1K328R were gifts from Dr. Tony Hunter at Salk Institute, La Jolla, CA. The pcDNA-Wee1 (NH₂-terminal truncated Wee1) was constructed from pTA-Wee1^w by subcloning a cDNA fragment encoding codons 215 to 646 of Wee1 protein into pcDNA3. pcDNA-Wee1 (full-length) and pcDNA-Wee1AS (full-length antisense) were constructed by subcloning the NotI/BamHI fragments from pKS⁺HuWee1a/b to pcDNA3. pcDNA-Wee1KR (kinase-inactive) was constructed from pKS⁺HuWee1K328Ra by subcloning the NotI/BamHI fragment to pcDNA3. Flag-tagged Wee1 constructs were generated by PCR using pcDNA-Wee1 as the template. The primers used are, for Wee1, full-length wild-type, Hwee-Flag-Bam01 (upstream) 5'-CGCGGATCCGCCACCATGGACTACAAGGACGACGATGACAAGAGCTTCCTGAGCCGA-CAG-3' and Hwee-XhoII (downstream) 5'-CCGCTCGAGTCAGTATATAGTAAGGCTGACAG; for NH₂-terminal truncated Wee1: Hwee-Flag-Bam03 (upstream) 5'-CGCGGATCCGCCACCATGGACTACAAGGACGACGATGACAAGGATACAGAAAAATCAGGAAAA-3' and Hwee-XhoII (down stream); for deletion mutant (Wee1d639), Hwee-Flag-Bam01 (upstream) and Hwee-Del-XhoII (downstream), 5'- CCGCTCGAGTCAGTTCATTTTCTTTCCAATAAGTCG-3'; and for Wee1 mutant (Wee1S642A), Hwee-Flag-Bam01 (upstream) and Hwee-MT-XhoII (downstream), 5'-CCGCTCGAGTCAGTATATAGTAAGGGCGACAGA-3'. The PCR products were digested with cloning restriction enzymes (BamHI and XhoI) and subcloned into pcDNA3. All of the clones were subjected to DNA sequence to confirm their identity and freedom of PCR-generated mutations. pcDNA-14-3-3ß-myc was generated by subcloning the 14-3-3ß PCR fragment to pcDNA3.1(-)/Myc-HisC. The 14-3-3ß PCR fragment flanks the entire coding region of 14-3-3ß with the introduction of the 5'-end EcoRI and the 3'-end HindIII restriction sites. The primers are as follows: P01, 5'-CAGGAATTCATGACAATGGATAAAAGTGA-3'; and P02, 5'-TAGAAAGCTTGGTTCTCTCCCTCCCCAGCGT-3'.

Yeast Two-Hybrid Screening.
The MATCHMAKER Two-Hybrid System from Clonetech was used to identify potential Wee1 interacting proteins. Two Wee1 constructs, the COOH-terminal containing the kinase domain (Wee11–288) as well as the COOH-terminal Wee1 with two potential phosphorylation sites mutated (Wee1 M1–288) were generated by PCR using the full-length Wee1 as the template. The primers used for Wee11–288 construction is Wee13 (5'-GGAATTCAGCAATATGAAGTCCCG-3') and Wee12 (5'-CGGGATCCTCAGTATATAGTAAGG-3'), and for Wee1 M1–288 construction, Wee13 MU (5'-GGAATTCGCCAATATGAAGGCCCGGTATACAGCA-3') and Wee12. The PCR fragments were gel-purified, cloned into the yeast two-hybrid vector, pGBT9, and used as the baits. pGBT9-Wee1 was cotransformed into the yeast HF-7c cells simultaneously with the human HeLa Matchmaker cDNA library, which was constructed in the plasmid pGAD GH (Clontech) and amplified according to the manufacturer’s recommendations. Approximately 2 x 10⁶ HeLa cDNA clones were screened. Transformed yeast cells were grown in agar plates containing the synthetic dropout media without leucine, tryptophan, or histidine. The colonies grown in the selection media were filter-lifted and assayed for ß-galactosidase activity in the presence of X-gal. The plasmid DNA from the positive clones was extracted and cotransformed with the bait to confirm the true binding in the yeast cells.

Cell Culture and Transfection.
Human kidney cell line 293 was cultured in Eagle’s MEM with 10% FBS. A calcium phosphate method was used to transfect plasmid DNA to 293 cells at a ratio of 30 µg of DNA per 60-mm dish (54) . For the Wee1-alone transfection, 15 µg of Wee1 encoding plasmid and 15 µg of the vector plasmid were used; and, for the Wee1/14-3-3ß cotransfection, 15 µg of each encoding plasmid was used. Transfection was terminated 7 h later. Forty h posttransfection, cells were harvested in lysis buffer [50 mM Tris (pH7.5), 150 mM NaCl, 0.5% NP40, 50 mM NaF, 1 mM NaVO₃, 1 mM DTT, and protease inhibitors, BMB] for biochemical analysis or fixed in 85% ethanol for FACS analysis.

Immunoblot.
Cell lysates prepared above were subjected to SDS-PAGE (4–20% gradient gel), followed by transferring to nitrocellulose membranes with a Bio-Rad semidry transfer unit. The membranes were incubated with TBST [50 mM Tris (pH 7.5), 150 mM NaCl, 0.1% Tween 20] containing 5% dry milk or 3% BSA for 1 h at room temperature to block nonspecific binding. The membranes were then incubated with 1 µg of Ab in 10 ml of TBST+5% dry milk or 3% BSA for 1 h at room temperature with shaking. After washing twice with TBST, each time for 15 min, the membranes were incubated with 10 ml of corresponding horseradish peroxidase-conjugated secondary Ab (1:3000 dilution in TBST+5% milk or 3% BSA). After washing twice with TBST, each time for 15 min, protein bands were detected with Amersham enhanced chemiluminescence kit. To quantitate Wee1 protein levels, a densitometer (Molecular Dynamics) was used.

IP-Western.
Cells posttransfection were lysed in a lysis buffer. Cell extracts (1 mg of protein) in 500 µl of cell lysis buffer were first incubated with 5 µl of Protein G⁺ agarose at 4°C for 30 min, with rotation to block the nonspecific binding. The sample was then subjected to centrifugation at 2000 rpm for 3 min at 4°C to remove the agarose beads. One µg of Myc-tag Ab was incubated with the sample at 4°C for 3–4 h with rotation. During the last hour, 5 µl of protein G⁺ agarose were added. For IP with Flag-tag Ab, 20 µl of agarose conjugated with flag-tag Ab were used. The immunoprecipitated samples were washed four times with cell lysis buffer without proteinase inhibitor and spun at 2000 rpm for 3 min to collect the proteinG-Ab-Ag complex. The proteinG-Ab-Ag complex was then resuspended in 20 µl of 2x SDS sample buffer and boiled at 100°C for 5 min. The sample was then resolved on SDS-PAGE. The immunoblot was performed as described above.

IP-Kinase.
IP was performed as described above except anti-Wee1 Ab and protein A agarose were used. The precipitated protein A agarose-Ab-Ag complex was first washed with cell lysis buffer, then with kinase buffer (50 mM Tris, 10 mM MgCl₂, and 1 mM DTT). The sample was incubated with kinase buffer in the presence of 50 µM ATP and 100 nM purified Cdc2/cyclinB for 15 min at room temperature. The reaction was terminated with 2x SDS sampling buffer. The samples were resolved on SDS-PAGE gel and transferred to nitrocellulose membrane. The membrane was cut into two portions. The top portion was blotted with anti-Wee1 Ab, and the lower portion was blotted with antiphosphotyrosine Ab.

Pulse-Chase Experiments.
Cells were plated on 60-mm dishes with EMEM+10% FBS, 1 x 10⁶ cells/dish. The next day, the cells were fed with fresh medium, and transfection was performed as described above. Forty h posttransfection, the cells were washed once with PBS and once with a pulse medium (Cys-free, Met-free DMEM containing 10% dialyzed FBS). The cells were subsequently preincubated with the pulse medium for 15 min at 37°C, and then pulsed with 1 ml of pulse medium containing 100 µCi [³⁵S]Met for 30 min at 37°C. The cells were washed once with a chase medium (4 mM Met in DMEM containing 10% FBS), and then chased with 5 ml of the chase medium for various time periods. Cells were harvested at 0, 0.5, 1, 1.25, 2, or 4 h after chase and lysed in 0.5 ml of lysis buffer at 4°C for 20 min with shaking. An aliquot (5 µl) of the cell lysate was precipitated with 10% TCA pelleted by centrifugation at 14,000 rpm for 10 min and analyzed for ³⁵S incorporation on a liquid scintillation counter. Equal amounts of TCA-precipitated proteins were used for IP analysis.

FACS Analysis.
Cells were cotransfected with Wee1 and/or 14-3-3ß and pCMV-CD20 in a ratio of 9:1. The cells were harvested by incubating with 5 mM EDTA in PBS at 37°C for 5 min. Cells were pelleted by centrifugation at 1000 x g for 5 min and then were washed with PBS. The pellet was resuspended in 20 µl of anti-CD20 fluoroscein and incubated at 4°C for 30 min in darkness. The cells were then washed with PBS containing 1% FBS, resuspended in 0.5 ml of PBS and fixed by dropwise addition of 10 ml of 85% ice-cold ethanol. The cells were left at 4°C for at least 15 min and then were centrifuged at 1000 x g for 5 min. After washing with PBS containing 1% FBS, the cells were suspended in PBS containing 1% FBS, 1 µg/ml propidium iodide and 0.25 mg/ml RNaseA. After incubation at 37°C for 30 min, the cells were filtered through 35 µm nylon mesh (Falcon 2235 tubes), and were analyzed for green (CD20-FITC) or red (propidium iodide) fluorescence on a Becton Dickinson FACScan. The distribution of cell cycle phase was estimated by the broadened trapezoid model of Bagwell (55) , as implemented by the program Modfit LT (Verity Software House).

Acknowledgments

We thank Dr. Tony Hunter (Salk Institute) for providing the full-length Wee 1 cDNA, Drs. Wilbur Leopold and Steve Hunt (Parke-Davis) for helpful discussions, and Jobi Wong for graphic assistance. We would also like to thank Dr. Shi-Hua Li (Emery University) and Dr. Junhui Bian (Abilene Christian University) for helpful discussion with the yeast two-hybrid screening.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom requests for reprints should be addressed, at Department of Molecular Biology, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, 2800 Plymouth Road, Ann Arbor, MI 48105. Phone: (734) 622-1959; Fax: (734) 622-7158; E-mail: yi.sun{at}wl.com

² The abbreviations used are: Cdk, cyclin-dependent kinase; IP, immunoprecipitation; FBS, fetal bovine serum; Ab, antibody; TCA, trichloroacetic acid; FACS, fluorescence-activated cell sorting.

³ Y. Wang and Y. Sun, unpublished observations.

Received for publication 8/ 5/99. Revision received 2/ 3/00. Accepted for publication 3/13/00.

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