Cyclin B Proteolysis and the Cyclin-dependent Kinase Inhibitor rum1p Are Required for Pheromone-induced G₁Arrest in Fission Yeast

Abstract

The blocking of G 1 progression by fission yeast pheromones requires inhibition of the cyclin-dependent kinase cdc2p associated with the B-cyclins cdc13p and cig2p. We show that cyclosome-mediated degradation of cdc13p and cig2p is necessary for down-regulation of B-cyclin-associated cdc2p kinase activity and for phermone-induced G 1 arrest. The cyclin-dependent kinase inhibitor rum1p is also required to maintain this G 1 arrest; it binds both cdc13p and cig2p and is specifically required for cdc13p proteolysis. We propose that rum1p acts as an adaptor targeting cdc13p for degradation by the cyclosome. In contrast, the cig2p-cdc2p kinase can be down-regulated, and the cyclin cig2p can be proteolyzed independently of rum1p. We suggest that pheromone signaling inhibits the cig2p-cdc2p kinase, bringing about a transient G 1 arrest. As a consequence, rum1p levels increase, thus inhibiting and inducing proteolysis of the cdc13p-cdc2p kinase; this is necessary to maintain G 1 arrest. We have also shown that pheromoneinduced transcription occurs only in G 1 and is independent of rum1p. INTRODUCTIONEntry into S-phase and mitosis in the eukaryotic cell cycle is controlled by the activation of cyclin-dependent kinases (CDKs). In the yeasts, both processes are initiated by a single CDK core enzyme encoded by cdc2 in fission yeast and CDC28 in budding yeast. Cdc2p and Cdc28p associate with mitotic B-type cyclins to initiate mitosis, cdc13p in fission yeast (Booher and Beach, 1988;Hagan et al., 1988;Booher et al., 1989;Moreno et al., 1989), and Clb1-4p in budding yeast (Ghiara et al., 1991;Surana et al., 1991;Fitch et al., 1992;Richardson et al., 1992) and with S-phase B-cyclins to trigger S-phase, usually cig2p in fission yeast (Fisher and Nurse, 1996;Martin-Castellanos et al., 1996;Mondesert et al., 1996) and Clb5-6p in budding yeast (Epstein and Cross, 1992;Kü hne and Linder, 1993;Schwob and Nasmyth, 1993;Schwob et al., 1994). There is considerable overlap between mitotic and S-phase B-cyclins (Schwob et al., 1994;Fisher and Nurse, 1996;Mondesert et al., 1996), and in fission yeast a single cyclin cdc13p can bring about both S-phase and mitosis (Fisher and Nurse, 1996;Mondesert et al., 1996). In budding yeast, activation of S-phase Clbp-Cdc28p protein kinase depends on the prior activation of Cdc28p associated with another class of G 1 cyclins, Cln1-3p.The mechanisms ensuring the timely inactivation and activation of cyclin B-CDK in G 1 have been studied mainly in budding yeast. S-phase Clbp-Cdc28p protein kinase is up-regulated by three independent mechanisms, all of which involve Clnp-Cdc28p kinase activity. Clnp-Cdc28p protein kinase 1) activates transcription of CLB genes (Epstein and Cross, 1992;Schwob and Nasmyth, 1993) and 2) inactivates Clbp proteolysis (Amon et al., 1994). The latter involves ubiquitin-mediated degradation of B-type cyclins, which requires the cyclosome (Sudakin et al., 1995) or anaphase-promoting complex consisting of eight subunits, including Apc1p/bimEp/cut4p (Peters et al., 1996;Yamashita et ...

Cdk phosphorylation of the Ste11 transcription factor constrains differentiation-specific transcription to G1

“…Consistent with this, the Ste11 target genes mat1-Mm and fus1 can be induced by pheromone in G 1 -arrested cells but not in cells arrested at S phase or in G 2 (Stern and Nurse 1998). Similarly, the meiotic activator gene mei3 is induced by starvation in diploid G 1 -arrested but not G 2 -arrested cells (Watanabe et al 2001).…”

Fission yeast Tor2 promotes cell growth and represses cell differentiation

“…3C). This delay was not unexpected because pheromone-induced genes regulated by Ste11 are not required for G1 arrest and are expressed only once the cells have entered the G1 phase of the cell cycle (Stern and Nurse, 1998). We conclude that inactivation of Tor2 induces differentiation by upregulating the expression of the same set of genes as those expressed under nitrogen starvation.…”

The dynamics of cell cycle regulation

“…In fission yeast cells, there is evidence that the pheromone signal works through increasing the amount of Rum1. (35) Notice that cells may over-ride pheromone-induced G 1 arrest simply by growing large enough to get around the big nose of the G 1 steady-state curve. This is called adaptation; in fission yeast cells it occurs after about 6 hours.…”

“…In the absence of Rum1, cells cycle just like wild type, with short G 1 phase, but they cannot stop in G 1 . (22,43) For example, pheromone cannot induce G 1 arrest in rum1D cells, (35) and hence they are sterile. When both Wee1 and Rum1 are missing (in wee1 ts rum1D double mutants), both G 1 and G 2 control modules are compromised, (44) and the oscillatory character of the mitotic module is revealed (Fig.…”