We show that the major cell cycle kinase Cdc28 and its Clb6 cyclin subunit specifically phosphorylate Swi6 at serine 160 and direct export of Swi6 from the nucleus. We also show that dephosphorylation of Swi6 at serine 160 and nuclear import are controlled by Cdc14, a phosphatase active in the final stages of M phase.
The pleiotropic and essential activity of Cdc28 and the redundancy of cyclin function have historically made it difficult to identify the substrate specificities of this kinase. Nevertheless, we conclude that Clb6/Cdc28 activity is responsible for phosphorylation at serine 160 of Swi6 because of the thermosensitive phosphorylation of Swi6 and histone H1 by Cdc28-13, because of the cyclin-specific stimulation of this activity, and because phosphorylation occurred at a putative Cdc28 consensus site. Phosphorylation at serine 160 had earlier been identified in vivo as the only site in Swi6 subject to cell cycle-dependent phosphorylation (46
). The same phosphorylation at serine 160 in vivo and in vitro indicates that the availability of this kinase site is independent of interaction of Swi6 with Mbp1 or Swi4. Note that this specificity of in vitro phosphorylation occurred despite the presence of other putative Cdc28 kinase sites and other sites subject to non-cell-cycle-dependent phosphorylation in vivo (45
In addition, by purifying all nine cyclins of Cdc28, we show that the specific phosphorylation of serine 160 is directed largely by Clb6-Cdc28 and not by other G1 or mitotic cyclin-Cdc28 kinase complexes. The specificity of this approach is emphasized by identical assays with a different substrate, where Ndd1 was phosphorylated by Clb2 rather than Clb6 (6). Clb6 specificity was confirmed with Cdc28 purified from cells depleted for different cyclins. This approach also suggested a minor contribution of Clb5 to Swi6 phosphorylation. However, as we were unable to detect this in other assays and as there is a near overlap of error bars of kinase activity in wild-type and Δclb5 extracts, we do not consider Clb5 to be a major source of Swi6 phosphorylation.
A specific interaction was also detected between Clb6 and Swi6 in vitro. The interaction occurred with the central section of Swi6 encompassing the ankyrin repeat domain and an associated transcriptional activation region (13
). However, an RXL motif in this region was not required even though this motif has been equated with cyclin-substrate binding in other systems (8
). Nevertheless, ankyrin repeats have a well-established role in protein-protein interactions (reviewed in reference 43
), and Clb2 is known to interact with the structurally related ankyrin repeats of Swi4 (47
Prior to this work, extensive characterization of S-phase cyclin activities focused mainly on their effects at replication origins (19
), but a direct demonstration of the substrate specificity of the Clb6-Cdc28 kinase has remained elusive. Thus, for the first time, we provide direct evidence for Clb6/Cdc28 substrate specificity.
To relate the in vitro phosphorylation of serine 160 by Clb6/Cdc28 to in vivo events, we demonstrated that Clb6 and presumably its associated kinase, Cdc28, are required for nuclear export of Swi6. Nevertheless, a small amount of cytoplasmic Swi6 was seen in Δclb6 mutants (Fig. ), and Cdc28 purified from Δclb6 cells also had a residual ability to phosphorylate Swi6 (Fig. ). Thus, in accord with the apparent in vivo redundancy of Clb activity, it is possible that other cyclins may have a minor activity which in vivo is sufficient to stimulate nuclear export.
We also examined how Swi6 dephosphorylation and nuclear import of Swi6 might be governed by Cdc14. Cdc14 is a phosphatase that acts as an antagonist of Clb/Cdc28 activity in the final phases of mitosis (32
). Our finding that arrested cdc14-1
cells had predominantly cytoplasmic Swi6 is consistent with earlier observations that phosphorylated Swi6 accumulated during a cdc15
). Moreover, we show that Cdc14 can also stimulate nuclear import of Swi6 in metaphase-arrested cells and, at least in vitro, is capable of dephosphorylating the serine 160 residue of Swi6. Together these results implicate Cdc14 in nuclear import of Swi6. Although a direct effect of Cdc14 on Swi6 dephosphorylation is likely, we cannot exclude the intervention of another phosphatase that is directly or indirectly controlled by Cdc14.
The idea of nuclear trafficking as a mechanism of Swi6 regulation was first proposed by Taba et al. (51
). In this context, Clb6 can be seen as a negative regulator that curtails G1
by promoting nuclear export of Swi6 in addition to other mechanisms of antagonizing G1
Cdc28 activity (4
). This conclusion is supported by the inhibitory effects of Clb6 overexpression and the stimulatory effects of CLB6
deletion on SBF- and MBF-dependent gene expression (4
). Likewise, the effects of CLB6
overexpression and swi6
deletion are similar in that both deregulate CDC6
). We propose that Swi6 phosphorylation by Clb6/Cdc28 generates a negative regulatory feedback mechanism. After passing Start, Clb6 is synthesized by Swi6 acting in MBF. The subsequent phosphorylation of Swi6 by Clb6 then helps to reduce further G1
-specific gene expression by localizing Swi6 to the cytoplasm (Fig. ).
FIG. 7. Scheme for cell cycle regulation of nuclear export and import of Swi6. Swi6 is represented by grey shading in the lower cartoon. The broken line indicates reduction in MBF and SBF activity by loss of Swi6 from the nucleus. See the Discussion for further (more ...)
Localization of Swi6 to the cytoplasm was initially envisaged to result from a stimulation of nuclear export (46
). However, this idea is not consistent with a more recent proposal that nuclear trafficking of Swi6 is an essential step in licensing Swi6 for optimal SBF activity after reentry to the nucleus (36
). In particular, the increased SBF activity in clb6
mutants would not be expected as reduced export of Swi6 would reduce licensing and hence attenuate G1
-specific gene expression. These conflicting ideas can be reconciled, if as suggested (36
), there is continuous nucleocytoplasmic shuttling of Swi6. In this model, phosphorylation of serine 160 would inhibit reentry into the nucleus rather than stimulating nuclear export. This would result in an apparent nuclear localization of Swi6 in a Δclb6
mutant throughout the cell cycle, but having been licensed by nuclear shuttling, the Swi6 or Swi6S160A would be transcriptionally active. Clearly, the question of whether phosphorylation triggers nuclear export or prevents import requires further examination, but in either case Clb6 would play a determining role. It is interesting that the S160A mutation that leads to nuclear localization of Swi6 does not reveal any major changes in SBF- or MBF-driven transcription (46
). As Δclb6
mutants show increased G1
-specific transcription (4
), Clb6 may have other roles in controlling Swi6-dependent transcription in addition to cellular localization of Swi6.
Our conclusion that Cdc28 has cell cycle-dependent activity towards Swi6 appears to contradict two earlier experiments which were used as evidence against Swi6 phosphorylation by Cdc28 (46
). In one, Swi6 was not phosphorylated when protein synthesis was inhibited after release from a G1
arrest. It was argued that Cdc28 was unlikely to phosphorylate Swi6 at serine 160, as Cdc28 is active whether or not protein synthesis is permitted. However, if Clb6 specifies Cdc28 activity against Swi6, this cyclin would first have to be synthesized in G1
to allow Swi6 to be phosphorylated. Thus, this earlier result is completely consistent with our findings that Clb6/Cdc28 phosphorylates Swi6.
In a second experiment, cdc28-4
mutants were released from a hydroxyurea block into the restrictive temperature, where they arrested at M phase. Under these conditions, it was concluded that Cdc28 did not phosphorylate Swi6 because phosphorylation, albeit reduced, still occurred, even though the thermosensitive Cdc28-4 kinase was unable to perform an essential step in mitosis (46
). It is important to stress that this result does not actually exclude Cdc28 from being the kinase that phosphorylates Swi6 at serine 160. Although the conditions used do impair an essential step at G2
), they do not exclude the possibility of there being sufficient residual Cdc28 activity to phosphorylate a substrate implicated in a nonessential step of the cell cycle. Note that similar arguments have to be made to explain why thermosensitive CDC28
mutants normally arrest in G1
even though Cdc28 has multiple essential targets throughout the cell cycle (38
Unfortunately, assays of possible residual activities of thermosensitive Cdc28 kinase at the restrictive temperature are problematic. Cdc28-13 assayed at 37°C has no activity against histone H1 or Sic1 even though cdc28-13
cells are able to pass the Cdc28-dependent step in mitosis and arrest only in G1
). Moreover, no activity towards histone H1 can be detected with Cdc28-4 even at the permissive temperature (37
). Given these reservations, our observations of altered Swi6 cellular localization in Δclb6
mutants strongly indicate that Cdc28 coupled with Clb6 is required for driving and/or maintaining Swi6 in the cytoplasm. Importantly, after submission of this report, the specific inhibition of Cdc28-as1 in vivo was shown to block phosphorylation of Swi6 (54
). Although neither the phosphorylated residue nor the specific cyclin were identified, this observation is in complete agreement with the conclusions made here that Swi6 is a substrate for the Cdc28 kinase.
In summary, we show how phosphorylation and nuclear export of Swi6 are integrated with changes in cyclin-dependent kinase in the transition from Cln-dependent G1 to the subsequent Clb-dependent phase of the cell cycle. Similarly, with the elimination of Clb kinase activity at the end of M phase, Cdc14 also triggers nuclear import of Swi6 in preparation for SBF- and MBF-driven transcription in the ensuing G1 phase (Fig. ).