Changes in Regulatory Phosphorylation of Cdc25C Ser287 and Wee1 Ser549 during Normal Cell Cycle Progression and Checkpoint Arrests

Abstract

Entry into mitosis is catalyzed by cdc2 kinase. Previous work identified the cdc2-activating phosphatase cdc25C and the cdc2-inhibitory kinase wee1 as targets of the incomplete replication-induced kinase Chk1. Further work led to the model that checkpoint kinases block mitotic entry by inhibiting cdc25C through phosphorylation on Ser287 and activating wee1 through phosphorylation on Ser549. However, almost all conclusions underlying this idea were drawn from work using recombinant proteins. Here, we report that in the early Xenopus egg cell cycles, phosphorylation of endogenous cdc25C Ser287 is normally high during interphase and shows no obvious increase after checkpoint activation. By contrast, endogenous wee1 Ser549 phosphorylation is low during interphase and increases after activation of either the DNA damage or replication checkpoints; this is accompanied by a slight increase in wee1 kinase activity. Blocking mitotic entry by adding the catalytic subunit of PKA also results in increased wee1 Ser549 phosphorylation and maintenance of cdc25C Ser287 phosphorylation. These results argue that in response to checkpoint activation, endogenous wee1 is indeed a critical responder that functions by repressing the cdc2-cdc25C positive feedback loop. Surprisingly, endogenous wee1 Ser549 phosphorylation is highest during mitosis just after the peak of cdc2 activity. Treatments that block inactivation of cdc2 result in further increases in wee1 Ser549 phosphorylation, suggesting a previously unsuspected role for wee1 in mitosis. INTRODUCTIONEntry into mitosis is initiated by activation of cyclin B/cdc2. Preformed complexes of cyclin B/cdc2 accumulate during interphase, but their activity is repressed by inhibitory phosphorylations on cdc2 at Tyr15 (catalyzed by wee1 and myt1) and Thr14 (catalyzed by myt1). These phosphorylations are removed by the phosphatase cdc25C (reviewed in Berry and Gould, 1996;Lew and Kornbluth, 1996). Early work led to the conclusion that cdc2 and cdc25C activities both increase rapidly during the G 2 /M transition as the result of positive feedback loops between cyclin B/cdc2 and cdc25C, ultimately leading to the full activation of both cdc25C and cyclin B/cdc2 (Izumi et al., 1992;Hoffmann et al., 1993;Izumi and Maller, 1993;Strausfeld et al., 1994). The mechanisms that trigger this abrupt activation are still not well understood (Margolis and Kornbluth, 2004;Perdiguero and Nebreda, 2004).Over the past decade, there has been a large increase in the molecular understanding of how checkpoint pathways become activated, and how they affect the basic cell cycle regulatory machinery. Genetic and biochemical approaches have identified sensors that detect incompletely replicated or damaged DNA and that stimulate signal transduction pathways that lead to activation of the kinases Chk1 and Chk2/Cds1 (reviewed in Sancar et al., 2004). Both Chk1 and Chk2 can phosphorylate cdc25C on Ser287 (human Ser216), which creates a binding site for 14-3-3 proteins; this is thought to inhibit cdc25C activation and thus...

Aurora A, mitotic entry, and spindle bipolarity

“…At the molecular level ( Fig. 1D), mitotic entry was reflected by (i) removal of the inhibitory phosphorylation on Ser-287 on cdc25C (29,30), the phosphatase that removes the inhibitory Tyr-15 phosphorylation from cdc2, (ii) dephosphorylation of cdc2 Tyr-15, and (iii) a rise in cdc2's histone H1 kinase activity. The mock-depleted extract continued through mitosis, as judged both morphologically and by the mid-mitotic activation of MAPK that is required to maintain chromosomes condensed after destruction of cyclin B and inactivation of cdc2 (31, 32).…”

Antagonism of Chk1 Signaling in the G2 DNA Damage Checkpoint by Dominant Alleles of Cdr1

“…Chk1 Antagonism by Cdr Kinases2002; Stumpff et al 2004;Stanford and Ruderman 2005). Although Cdr1 overexpression has been shown to cause a ''wee'' phenotype (Russell and Nurse 1987;Wu et al 1996), the only function for Cdr1 determined previously from the cdr1D cells had been an inability to arrest in G 1 upon nitrogen starvation (Young and Fantes 1987;Feilotter et al 1991).…”

“…In S. pombe, phosphorylation of Wee1 by Chk1 leads to a transient stabilization of this short-lived protein and hence to increased cellular levels of Wee1 kinase (O'Connell et al 1997;Raleigh and O'Connell 2000). Similarly, Chk1 has been implicated in the regulation of Wee1 homologs in Xenopus (Lee et al 2001;Stanford and Ruderman 2005), Drosophila (Price et al 2000;Stumpff et al 2004), 1 and humans (Li et al 2002). For Cdc25, its phosphorylation by Chk1 in S. pombe leads to its nuclear exclusion and catalytic inactivation LopezGirona et al 1999LopezGirona et al , 2001).…”

Ubiquitin and SUMO systems in the regulation of mitotic checkpoints

“…Finally, it has been reported that Chk1-catalyzed phosphorylation of Xenopus Wee1 at Ser549 (Ser642 in human Wee1A) helps to activate Wee1 after checkpoint induction [23,24]. It is likely that this phosphorylation event also contributes to the stabilization of Wee1.…”