Regulation of the anaphase-promoting complex/cyclosome by bimAAPC3 and proteolysis of NIMA.

Surprisingly, although highly temperature-sensitive, the bimA1(APC3) anaphase-promoting complex/cyclosome (APC/C) mutation does not cause arrest of mitotic exit. Instead, rapid inactivation of bimA1(APC3) is shown to promote repeating oscillations of chromosome condensation and decondensation, activation and inactivation of NIMA and p34(cdc2) kinases, and accumulation and degradation of NIMA, which all coordinately cycle multiple times without causing nuclear division. These bimA1(APC3)-induced cell cycle oscillations require active NIMA, because a nimA5 + bimA1(APC3) double mutant arrests in a mitotic state with very high p34(cdc2) H1 kinase activity. NIMA protein instability during S phase and G2 was also found to be controlled by the APC/C. The bimA1(APC3) mutation therefore first inactivates the APC/C but then allows its activation in a cyclic manner; these cycles depend on NIMA. We hypothesize that bimAAPC3 could be part of a cell cycle clock mechanism that is reset after inactivation of bimA1(APC3). The bimA1(APC3) mutation may also make the APC/C resistant to activation by mitotic substrates of the APC/C, such as cyclin B, Polo, and NIMA, causing mitotic delay. Once these regulators accumulate, they activate the APC/C, and cells exit from mitosis, which then allows this cycle to repeat. The data indicate that bimAAPC3 regulates the APC/C in a NIMA-dependent manner.

The G2/M DNA damage checkpoint inhibits mitosis through Tyr15 phosphorylation of p34cdc2 in Aspergillus nidulans.

It is possible to cause G2 arrest in Aspergillus nidulans by inactivating either p34cdc2 or NIMA. We therefore investigated the negative control of these two mitosis-promoting kinases after DNA damage. DNA damage caused rapid Tyr15 phosphorylation of p34cdc2 and transient cell cycle arrest but had little effect on the activity of NIMA. Dividing cells deficient in Tyr15 phosphorylation of p34cdc2 were sensitive to both MMS and UV irradiation and entered lethal premature mitosis with damaged DNA. However, non-dividing quiescent conidiospores of the Tyr15 mutant strain were not sensitive to DNA damage. The UV and MMS sensitivity of cells unable to tyrosine phosphorylate p34cdc2 is therefore caused by defects in DNA damage checkpoint regulation over mitosis. Both the nimA5 and nimT23 temperature-sensitive mutations cause an arrest in G2 at 42 degrees C. Addition of MMS to nimT23 G2-arrested cells caused a marked delay in their entry into mitosis upon downshift to 32 degrees C and this delay was correlated with a long delay in the dephosphorylation and activation of p34cdc2. Addition of MMS to nimA5 G2-arrested cells caused inactivation of the H1 kinase activity of p34cdc2 due to an increase in its Tyr15 phosphorylation level and delayed entry into mitosis upon return to 32 degrees C. However, if Tyr15 phosphorylation of p34cdc2 was prevented then its H1 kinase activity was not inactivated upon MMS addition to nimA5 G2-arrested cells and they rapidly progressed into a lethal mitosis upon release to 32 degrees C. Thus, Tyr15 phosphorylation of p34cdc2 in G2 arrests initiation of mitosis after DNA damage in A. nidulans.

Proteolysis and tyrosine phosphorylation of p34cdc2/cyclin B. The role of MCM2 and initiation of DNA replication to allow tyrosine phosphorylation of p34cdc2.

Previously, it has been shown that Aspergillus cells lacking the function of nimQ and the anaphase-promoting complex (APC) component bimEAPC1 enter mitosis without replicating DNA. Here nimQ is shown to encode an MCM2 homologue. Although mutation of nimQMCM2 inhibits initiation of DNA replication, a few cells do enter mitosis. Cells arrested at G1/S by lack of nimQMCM2 contain p34(cdc2)/cyclin B, but p34(cdc2) remains tyrosine dephosphorylated, even after DNA damage. However, arrest of DNA replication using hydroxyurea followed by inactivation of nimQMCM2 and bimEAPC1 does not abrogate the S phase arrest checkpoint over mitosis. nimQMCM2, likely via initiation of DNA replication, is therefore required to trigger tyrosine phosphorylation of p34(cdc2) during the G1 to S transition, which may occur by inactivation of nimTcdc25. Cells lacking both nimQMCM2 and bimEAPC1 are deficient in the S phase arrest checkpoint over mitosis because they lack both tyrosine phosphorylation of p34(cdc2) and the function of bimEAPC1. Initiation of DNA replication, which requires nimQMCM2, is apparently critical to switch mitotic regulation from the APC to include tyrosine phosphorylation of p34(cdc2) at G1/S. We also show that cells arrested at G1/S due to lack of nimQMCM2 continue to replicate spindle pole bodies in the absence of DNA replication and can undergo anaphase in the absence of APC function.

Two S-phase checkpoint systems, one involving the function of both BIME and Tyr15 phosphorylation of p34cdc2, inhibit NIMA and prevent premature mitosis.

We demonstrate that there are at least two S-phase checkpoint mechanisms controlling mitosis in Aspergillus. The first responds to the rate of DNA replication and inhibits mitosis via tyrosine phosphorylation of p34cdc2. Cells unable to tyrosine phosphorylate p34cdc2 are therefore viable but are unable to tolerate low levels of hydroxyurea and prematurely enter lethal mitosis when S-phase is slowed. However, if the NIMA mitosis-promoting kinase is inactivated then non-tyrosine-phosphorylated p34cdc2 cannot promote cells prematurely into mitosis. Lack of tyrosine-phosphorylated p34cdc2 also cannot promote mitosis, or lethality, if DNA replication is arrested, demonstrating the presence of a second S-phase checkpoint mechanism over mitotic initiation which we show involves the function of BIME. In order to overcome the S-phase arrest checkpoint over mitosis it is necessary both to prevent tyrosine phosphorylation of p34cdc2 and also to inactivate BIME. Lack of tyrosine phosphorylation of p34cdc2 allows precocious expression of NIMA during S-phase arrest, and lack of BIME then allows activation of this prematurely expressed NIMA by phosphorylation. The mitosis-promoting NIMA kinase is thus a target for S-phase checkpoint controls.

The NIMA protein kinase is hyperphosphorylated and activated downstream of p34cdc2/cyclin B: coordination of two mitosis promoting kinases.

Initiation of mitosis in Aspergillus nidulans requires activation of two protein kinases, p34cdc2/cyclin B and NIMA. Forced expression of NIMA, even when p34cdc2 was inactivated, promoted chromatin condensation. NIMA may therefore directly cause mitotic chromosome condensation. However, the mitosis-promoting function of NIMA is normally under control of p34cdc2/cyclin B as the active G2 form of NIMA is hyperphosphorylated and further activated by p34cdc2/cyclin B when cells initiate mitosis. To see the p34cdc2/cyclin B dependent activation of NIMA, okadaic acid had to be added to isolation buffers to prevent dephosphorylation of NIMA during isolation. Hyperphosphorylated NIMA contained the MPM-2 epitope and, in vitro, phosphorylation of NIMA by p34cdc2/cyclin B generated the MPM-2 epitope, suggesting that NIMA is phosphorylated directly by p34cdc2/cyclin B during mitotic initiation. These two kinases, which are both essential for mitotic initiation, are therefore independently activated as protein kinases during G2. Then, to initiate mitosis, we suggest that each activates the other's mitosis-promoting functions. This ensures that cells coordinately activate p34cdc2/cyclin B and NIMA to initiate mitosis only upon completion of all interphase events. Finally, we show that NIMA is regulated through the cell cycle like cyclin B, as it accumulates during G2 and is degraded only when cells traverse mitosis.