OP11-Wee1A is active in Δ90-cyclin B-treated extracts.
Since activation of Cdk1 causes Wee1A to become hyperphosphorylated and inactivated and Cdks phosphorylate serine and threonine residues that are followed by prolines, we addressed whether any of the 11 SP and TP motifs present in Wee1A (Ser 38, Thr 53, Ser 62, Ser 68, Ser 74, Thr 86, Thr 101, Thr 104, Ser 115, Thr 150, and Thr 450) are required for mitotic hyperphosphorylation and inactivation of Wee1A. To this end, we generated OP11-Wee1A, which has all 11 potential SP/TP phosphorylation sites changed to alanines (Fig. ). N-terminally His6/Flag-tagged wild-type Wee1A and OP11-Wee1A were overexpressed in Sf9 cells and affinity purified with anti-Flag beads. Bead-bound Wee1A was incubated for 40 min in interphase Xenopus egg extracts or extracts pretreated (for 20 to 30 min) with Δ90-cyclin B (200 nM). Control experiments established that this concentration of Δ90-cyclin B was sufficient to cause Cdk1 activation and M-phase entry (as assessed by H1 kinase assay and the morphology of sperm chromatin) even in the presence of the immobilized wild-type Wee1A or OP11-Wee1A. The extract-treated beads were washed and mixed with a complex of kinase-minus T161A-Cdk1, nondegradable Δ65-cyclin B1, and MgATP. The resulting tyrosine phosphorylation of Cdk1 was assessed by anti-phosphotyrosine immunoblotting (Fig. ).
In interphase extracts, both wild-type Wee1A (Fig. , lanes 2 to 5) and OP11-Wee1A (Fig. , lanes 6 to 9) phosphorylated Cdk1 on tyrosine in a dose-dependent manner, with the activities of the two Wee1A proteins being similar. Incubation of wild-type Wee1A with Δ90-cyclin B-treated extracts (M-phase extracts) resulted in a marked inhibition of Wee1A kinase activity (lanes 11 to 14) without significantly decreasing the amount of Wee1A present (data not shown). In contrast, OP11-Wee1A was resistant to mitotic inactivation; its activity after incubation with M-phase extracts (Fig. , lanes 15 to 18) was similar to its activity after incubation with interphase extracts (Fig. , lanes 6 to 9). These data demonstrate that the 11 SP/TP motifs include sites that are required for mitotic inactivation of Wee1A.
Previous work has shown that Xenopus
Wee1A can autophosphorylate on Tyr 90, Tyr 103, and Tyr 110 and that the level of Wee1A autophosphorylation is regulated during the first mitotic cell cycle and development (23
). As mentioned above, these studies also suggested that the extent of Wee1A autophosphorylation might be a more faithful indicator of Wee1A's biological activity than is its in vitro kinase activity (23
). We therefore assessed the autophosphorylation of wild-type Wee1A and OP11-Wee1A in interphase extracts and M-phase extracts. As shown in Fig. , addition of wild-type baculovirus-expressed Wee1A to interphase extracts caused a small increase in the apparent molecular weight of Wee1A (top blot, lanes 1 to 2) and caused Wee1A to become tyrosine phosphorylated (bottom blot, lanes 1 to 2). This small shift was also seen in OP11-Wee1A (Fig. ) but not in kinase-minus K239I-Wee1A (KM-Wee1A) (Fig. , lane 7, and data not shown), suggesting that it might be directly due to autophosphorylation (which occurs in interphase wild-type Wee1A and OP11-Wee1A but not in KM-Wee1A; Fig. , lanes 7 to 9). Note that if the tyrosine phosphorylation of Wee1A were actually due to some other kinase or occurred by intermolecular trans-autophosphorylation, KM-Wee1A would have been expected to exhibit strong tyrosine phosphorylation. Thus, the low levels of KM-Wee1A tyrosine phosphorylation seen in Fig. , lanes 7 and 10, imply that, under these conditions (20 nM recombinant Wee1A added to an extract containing approximately 16 nM endogenous Wee1A (22
), Wee1A tyrosine phosphorylation occurs primarily by intramolecular autophosphorylation.
Incubation with M-phase extracts caused Wee1A to shift to a still higher apparent molecular weight and caused a marked decrease in Wee1A tyrosine phosphorylation (Fig. , lane 3). In contrast, OP11-Wee1A showed similar high levels of tyrosine phosphorylation and similar electrophoretic mobilities when treated with either interphase extracts or M-phase extracts (Fig. , lanes 5 to 6). The difference in autophosphorylation between mitotic wild-type Wee1A and OP11-Wee1A was fivefold on average, which was similar to the difference in autophosphorylation between interphase- and M-phase-treated wild-type Wee1A (Fig. , lanes 3, 6, 11, and 12; also see Fig. ). These findings support the conclusion that OP11-Wee1 has a normal, high protein kinase activity toward itself and toward Cdk1-cyclin B during interphase but is resistant to inactivation during M phase.
FIG. 3. Mutating putative phosphorylation sites. (A and B) Effects of single alanine mutations on interphase (A) and M-phase (B) electrophoretic mobility and autophosphorylation of Wee1A. Wild-type (WT) Wee1A, OP11-Wee1A, and 11 individual alanine mutants were (more ...) MALDI-TOF analysis of interphase and M-phase Wee1A.
Next we attempted to map the mitotic phosphorylation sites in wild-type Wee1A by MALDI-TOF MS. Flag bead-bound recombinant Wee1A was treated with interphase extracts, M-phase extracts, or no agent; separated on an SDS-polyacrylamide gel; visualized by Coomassie staining (Fig. ); excised from the gel; digested with trypsin; and subjected to MS. The peptides identified covered 44 to 60% of the total Wee1A sequence and included 5 of the 11 SP/TP residues (Fig. ). We found evidence for three phosphorylations in mitotic Wee1A (Fig. ). The first was on a peptide that comprised amino acids 45 to 56 and included one TP site, Thr 53 (TNNCPFPIT53PQR). The interphase Wee1A sample yielded a peak corresponding to the mass of nonphosphorylated TNNCPFPITPQR (1,458.7 Da), whereas in the M-phase sample the 1,458.7-Da peak disappeared and new peaks corresponding to phosphorylated TNNCPFPITPQR (1,538.7 Da) and phosphorylated TNNCPFPITPQRNER (1,937.9 Da) appeared (Fig. ). The second phosphorylation was on a peptide comprising amino acids 130 to 155 and again including one TP site, Thr 150 (FVAGTGAELDDPSLVNINPFT150PESYR). A peak corresponding to the nonphosphorylated peptide (2,809.4 Da) was detected in interphase Wee1A and was replaced by a peak corresponding to the phosphorylated peptide (2,889.4 Da) in M-phase Wee1A (Fig. ). The third possible phosphorylation was on a peptide that included amino acids 37 to 44, with one SP site, Ser 38 (GS38PVSSWR). A peak corresponding to nonphosphorylated GSPVSSWR (875.4 Da) was detected in interphase Wee1A and was not detected in M-phase Wee1A. No peptide corresponding to phosphorylated GSPVSSWR (955.4 Da) was detected in either interphase Wee1A or M-phase Wee1A; however, as described below, mutational analysis supported the conclusion that this peptide was in fact phosphorylated at Ser 38 in M phase.
FIG. 2. Identification of mitotic phosphorylation sites in Wee1A. (A) Coomassie-stained gels of recombinant Wee1A incubated with buffer or extracts. (B) Summary of the peptides identified by MALDI-TOF MS. Coverage ranged from 44 to 60%. Peptides containing 5 (more ...) Mutational analysis of the SP/TP motifs.
Each of the three phosphorylated tryptic peptides identified in M-phase Wee1A contained one SP or TP motif, but each contained other potential phosphorylation sites as well. To determine whether the SP/TP motifs in these three peptides were actual phosphorylation sites, and to look for additional SP/TP phosphorylation sites in the regions of Wee1A from which tryptic peptides were not recovered, we constructed a series of Wee1A mutants in which each of the 11 SP/TP sites was individually replaced with alanine (S38A, T53A, S62A, S68A, S74A, T86A, T101A, T104A, S115A, T150A, and T450A). Each of these proteins was expressed and purified, incubated with interphase or M-phase extract, and subjected to anti-Flag immunoblotting to determine protein levels and electrophoretic mobilities and anti-phosphotyrosine immunoblotting to determine autophosphorylation.
In interphase, the electrophoretic mobilities and autophosphorylation of all of the Wee1A alanine mutants were similar to those of wild-type Wee1A and OP11-Wee1A (Fig. ). However, in M phase there were clear differences among the various mutants. First, the S38A-, T53A-, and S62A-Wee1A bands were only partially shifted in M phase (Fig. ). This finding established Ser 38 and Thr 53 as the phosphorylation sites in GS38PVSSWR and TNNCPFPIT53PQR, peptides whose phosphorylation had been established by the MALDI-TOF data in Fig. , and added Ser 62 as an additional mitotic phosphorylation site. Although their mitotic gel shifts were incomplete, all of these mutants exhibited normal low levels of autophosphorylation in M phase (Fig. ). Note that the high T53A-Wee1A tyrosine phosphorylation signal in the top part of Fig. reflects uneven protein loading; when normalized to the Flag-Wee1A signal and averaged, the T53A-Wee1A tyrosine phosphorylation was no higher than that of wild-type Wee1A (Fig. ).
Two additional mutants, T104A-Wee1A and T150A-Wee1A, were found to be normal with respect to the mitotic gel shift (Fig. ) but resistant to mitotic inactivation (Fig. ). The T104A and T150A mutants were approximately three- to sixfold higher in mitotic autophosphorylation than was wild-type Wee1A (Fig. ). In addition, their mitotic autophosphorylation was similar to the interphase autophosphorylation of all of the Wee1A proteins (Fig. and ). This evidence established Thr 150 as the phosphorylation site in FVAGTGAELDDPSLVNINPFT150PESYR, consistent with the MALDI-TOF data (Fig. ), and added a fifth SP/TP site (Thr 104) to the list of mitotic phosphorylation sites.
Taken together, the MALDI-TOF MS data and mutagenesis data established five SP/TP sites as being phosphorylated in M-phase Wee1A. Three of the sites, Ser 38, Thr 53, and Ser 62, were required for the mitotic gel shift of Wee1A but were largely dispensable for mitotic inactivation of Wee1A, whereas two other sites, Thr 104 and Thr 150, were required for mitotic inactivation of Wee1A but were dispensable for the gel shift.
We next examined how combinations of phosphorylation site mutations affected the mitotic inactivation and mitotic gel shift of Wee1A. In the first group, mutations at Ser 38, Thr 53, and Ser 62 were combined (S38A/T53A, T53A/S62, S38A/S62A, and S38A/T53A/S62A). All of these mutations decreased the mitotic gel shift of Wee1A, with the decrease being most pronounced in the S38A/T53A/S62A triple mutant. None of these mutations compromised the mitotic inactivation of Wee1A (data not shown).
Next we combined mutations of the two sites implicated in mitotic inactivation of Wee1A, Thr 104 and Thr 150. As shown in Fig. , mutation of Thr 150 fully blocked mitotic inactivation of Wee1A whereas mutation of Thr 104 partially blocked it. The T104A/T150A double mutant was no more resistant to inactivation than the T150A single mutant (Fig. ). We also examined combinations of T101A with T104A and T150A. T101A/T104A-Wee1A had no greater activity than the T104A single mutant, and T101A/T104A/T150A-Wee1A had no greater activity than T104A/T150A (Fig. ). None of these mutations compromised the mitotic gel shift of Wee1A (Fig. ).
Finally, mutations at Ser 38, Thr 53, and Ser 62 were combined with a mutation at Thr 104 or Thr 150 (data not shown). In all cases, the status of Ser 38/Thr 53/Ser 62 determined the mitotic gel shift and the status of Thr 104/Thr 150 determined whether the protein could be inactivated in M phase.
Taken together, these findings indicate that the activity of Wee1A and the electrophoretic mobility of Wee1A are independently regulated by distinct sets of phosphorylations. The phosphorylations of three N-terminal sites, Ser 38, Thr 53, and Ser 62, combined to produce the gel shift seen in mitotic Wee1A. Moreover, they combined in a graded, additive fashion: OP11-Wee1A and S38A/T53A/S62A-Wee1A showed no mitotic gel shift, whereas the Wee1A mutants with one or two of the N-terminal sites showed partial shifts. Two other phosphorylation sites, Thr 104 and Thr 150, are required for mitotic inactivation of Wee1A. Mutating Thr 150 tended to have a stronger effect than mutation of Thr 104, but both Thr 104 and Thr 150 were required for full mitotic inactivation of Wee1A.
Previous work has demonstrated that Wee1A is relatively unstable in postreplication interphase extracts (21
). Ser 38 phosphorylation has been implicated in the control of Wee1A degradation (2
). We therefore asked whether any of our alanine mutations resulted in a change in Wee1A stability. Wild-type and mutant 35
S-labeled Wee1A proteins were translated in reticulocyte lysates and added to chromatin-supplemented interphase Xenopus
egg extracts. The amount of 35
S-labeled Wee1A remaining was then assessed as a function of time. As shown in Fig. , wild-type Wee1A was degraded with a half-life of 260 ± 20 min. The S38A mutant had a significantly longer half-life, 364 ± 49 min, in qualitative (but not quantitative) agreement with the trend seen by Ayad et al. (2
). The T53A mutant had a similarly long half-life, 353 ± 48 min, suggesting that Thr 53 phosphorylation is also required for Wee1A degradation. The half-lives of the S38A/T53A/S62A triple mutant and OP11-Wee1A were similar (373 ± 27 min and 380 ± 49 min, respectively) to those of the S38A and T53A single mutants (Fig. ). These data suggest that the phosphorylation of multiple poorly conserved phosphorylation sites in the N terminus of Wee1A contributes to the regulation of Wee1A degradation.
FIG. 4. Degradation of Wee1A mutants in interphase Xenopus egg extracts. Wild-type (wt) Wee1A and four phosphorylation site mutants (S38A-Wee1A, T53A-Wee1, S38A/T53A/S62A-Wee1A, and OP11-Wee1A) were translated in vitro in reticulocyte lysates in the presence (more ...) Generation of a phospho-Thr 150 antibody and phosphorylation of Thr 150 in vivo.
To see whether and when the Thr 150 residue is phosphorylated in vivo, we generated an affinity-purified antibody to phospho-Thr 150 (pT150). The specificity of the antibody was verified by incubating wild-type Wee1A, OP11-Wee1A, and each of the 11 single alanine mutations with buffer or M-phase extract and then subjecting the Wee1A proteins to immunoblotting with Flag antibody and pT150 antibody. As shown in Fig. , the pT150 antibody recognized M-phase wild-type Wee1A but not buffer-treated Wee1A. Moreover, the pT150 antibody recognized the M-phase forms of all of the Wee1A mutants except OP11-Wee1A and T150A-Wee1A (Fig. ), the two mutants that lacked the Thr 150 phosphorylation site. These findings established the specificity of the antibody for Thr 150-phosphorylated forms of Wee1A and furthermore demonstrated that Thr 150 phosphorylation does not depend upon prior phosphorylation of Thr 104 (the other site whose phosphorylation was required for mitotic inactivation of Wee1A) or any of the other SP/TP sites. However, one Wee1 multisite alanine mutant—OP9-Wee1A, which has all of the SP/TP sites mutated except Thr 150 and Thr 450—was found not to undergo Thr 150 phosphorylation (data not shown). This could mean that Thr 150 phosphorylation depends upon the prior phosphorylation of at least one of the N-terminal SP/TP sites, even though no single individual site is required. It is also possible that the regulatory domain of OP9-Wee1A is misfolded, rendering Thr 150 unphosphorylatable.
FIG. 5. Phosphorylation of Thr 150 in vitro and in cycling extracts. (A) Recognition of T150-phosphorylated forms of recombinant Wee1A by an affinity-purified pT150 antibody (Ab). (B) Endogenous Wee1A is phosphorylated at Thr 150 in CSF-arrested extracts but (more ...)
Next we immunoblotted endogenous Wee1A from interphase extracts or CSF-arrested M-phase extracts. As shown in Fig. , Wee1A antibodies detected Wee1A bands in both interphase and M phase. In contrast, the pT150 antibody detected only the hyperphosphorylated M-phase Wee1A protein.
Finally, we subjected cycling Xenopus
egg extracts to immunoblot analysis with a Wee1A antibody and the pT150 antibody. As shown in Fig. , Thr 150 was transiently phosphorylated just prior to nuclear envelope breakdown. The mitotic gel shift of Wee1A also preceded nuclear envelope breakdown, consistent with previous findings (43
). However, the gel shift was half maximal by 30 min, at which time Thr 150 phosphorylation was barely detectable (Fig. ). Assuming that the shift was due to phosphorylation of Ser 38, Thr 53, and Ser 62, this finding implies that those phosphorylations generally precede the phosphorylation of Thr 150, even though they are not strictly required for Thr 150 phosphorylation (Fig. ). These results also imply that the “mitotic” phosphorylation of Wee1A may actually occur during late interphase.
Functional significance of the mitotic inactivation of Wee1A.
Having identified two sites (Thr 104 and Thr 150) required for mitotic inactivation of Wee1A, we can assess whether the inactivation of Wee1A is important for the mitotic activation of Cdk1 or, alternatively, is a backup mechanism that is normally unimportant because of the various additional ways of inactivating Wee1A (decreased transcription, decreased translation, and proteolysis). To this end, we compared the efficacies of wild-type Wee1A, T104A-Wee1A, T150A-Wee1, and OP11-Wee1A at blocking cyclin-driven Cdk1 activation. As shown in Fig. , both T104A-Wee1A and T150A-Wee1A inhibited mitotic entry more strongly than did wild-type Wee1A. This could be seen as a subtle shift to the right in the cyclin-versus-Cdk1 activity dose-response curves (Fig. ) and as a more dramatic decrease in the mitotic phosphorylation of p42 MAPK (Fig. ) (see reference 36
for other examples of relatively small changes in Cdk1 activity resulting in more pronounced changes in p42 MAPK phosphorylation). These findings suggest that the phosphorylation-mediated inactivation of Wee1A is important for mitotic entry.
FIG. 6. Effectiveness of wild-type (WT) Wee1A, T104A-Wee1A, T150A-Wee1A, and OP11-Wee1A at inhibiting mitotic entry. Interphase extracts were supplemented with 40 nM Wee1A and various concentrations of nondegradable Δ90-cyclin B. Extracts were incubated (more ...)
OP11-Wee1A was somewhat more effective at inhibiting mitotic entry than either the T104A- or T150A-Wee1A mutant, as shown by both H1 kinase assays (Fig. ) and phospho-MAPK blots (Fig. ). This finding was somewhat surprising given that M-phase OP11-Wee1A was not detectably higher in its autophosphorylation than M-phase T150A-Wee1A (Fig. ). This suggests that the phosphorylation of Ser 38, Thr 53, and/or Ser 62, or possibly some additional unidentified sites, has some sort of negative effect on Wee1A function.