Cells entering G1 after mitosis inherit an active APCCdh1
complex from the previous cycle, which targets a number of proteins, including Clb1, Clb2, Cin8, Kip1 and Cdc5, for proteolytic destruction (ref. 23
). For successful entry into mitosis in the subsequent division cycle, cells need to re-accumulate these proteins. Therefore, cells must ensure that Cdh1 is inactivated as they progress towards M phase. It has been shown previously that the Cdc28–Clb3, –Clb4 and –Clb5 are collectively responsible for accumulation of the mitotic Clb1 and Clb2 by inactivating Cdh1 (ref. 24
). However, cdc28-as1
cells treated with 1NM-PP1 show a substantial amount of Clb2, but continue to degrade Cin8 and Kip1 in a Cdh1-dependent manner, and are unable to separate the SPBs. This suggests that Cdh1 is not fully inactivated in these cells. On the basis of our results, we propose a regulatory scheme for Cdh1 inactivation leading to SPB separation during a normal cell cycle (): inactivation of Cdh1 (fully active during G1) is initiated in early S phase by Cdc28–Clb3, –Clb4 and –Clb5, which leads to accumulation of Clb1, Clb2 and Cdc5 polo kinase. Collective phosphorylation of Cdh1 by Cdc28–Clb kinase complexes then creates PBB sites which allow binding of Cdc5 to Cdh1. Cdc5 in turn phosphorylates Cdh1 at Ser 125 and Ser 258, rendering it completely inactive and allowing accumulation of microtubule-associated proteins, such as Cin8, and subsequently, separation of SPBs. Thus Cdc28/Cdk1 acts as a priming kinase for the recruitment of Cdc5 polo kinase to Cdh1.
This is consistent with the findings of a previous study15
, in which pSer/pThr was originally identified as a PBB site using a proteomic approach, and suggested a mechanism by which polo kinase is recruited to specific sites in response to Cdk1 phosphorylation. Such a synergistic scheme involving Cdk and polo kinase has a number of parallels in other systems25
. It should be noted that, although our results strongly suggest a role for Cdc5 polo kinase in Cdh1 inactivation, resulting in SPB separation, the possibility that Cdc5 also contributes to SPB separation by phosphorylating specific SPB-associated protein(s) necessary for the breaking of inter-SPB bridge cannot be ruled out. Such a model would be consistent with the observation that Cdc5 is localized to the SPBs during late S phase and early mitosis.
Inactivation of polo kinase is known to result in monopolar spindles in other systems, suggesting its involvement in centrosome separation26
, but its exact role has not been elucidated. Cdc5 polo kinase has never been implicated in SPB separation in S. cerevisiae
prior to this study because cdc5
mutants or cdc5Δ
cells do assemble a spindle, albeit inefficiently, and go on to arrest in telophase22
. Our results suggest that Cdc5 is non-essential for SPB separation in wild-type cells because Acm1 () partially inhibits Cdh1 and promotes sluggish accumulation of Cin8. The failure of cdc5Δ acm1Δ
double mutants to separate SPBs is consistent with this notion. Hence Cdc28–Clb, Cdc5 polo kinase and Acm1–Bmh1–Bmh2 cooperatively inactivate Cdh1 to allow accumulation of Cin8 and Kip1, resulting in timely separation of SPBs. It is unclear why cells would use three concerted ways to inactivate Cdh1; perhaps this elaborate mechanism has some relevance to substrate selection or the timing of their proteolytic destruction. Nevertheless, our findings provide a new model for the inactivation of important mitotic regulators by the synergistic action of Cdk1 and polo kinase. It remains to be seen whether the role of polo kinase in centrosome separation in mammalian cells also involves modulation of the proteolytic machinery.