Chk1 is a wee1 kinase in the G2 DNA damage checkpoint inhibiting cdc2 by Y15 phosphorylation

Abstract

The G2 DNA damage checkpoint ensures maintenance of cell viability by delaying progression into mitosis in cells which have suffered genomic damage. It is controlled by a number of proteins which are hypothesized to transduce signals through cell cycle regulators to delay activation of p34cdc2. Studies in mammalian cells have correlated induction of inhibitory tyrosine 15 (Y15) phosphorylation on p34cdc2 with the response to DNA damage. However, genetic studies in fission yeast have suggested that the major Y15 kinase, p107wee1, is not required for the cell cycle delay in response to DNA damage, although it is required for survival after irradiation. Thus, the target of the checkpoint, and hence the mechanism of cell cycle delay, remains unknown. We show here that Y15 phosphorylation is maintained in checkpoint‐arrested fission yeast cells. Further, wee1 is required for cell cycle arrest induced by up‐regulation of an essential component of this checkpoint, chk1. We observed that p107wee1 is hyperphosphorylated in cells delayed by chk1 overexpression or UV irradiation, and that p56chk1 can phosphorylate p107wee1 directly in vitro. These observations suggest that in response to DNA damage p107wee1 is phosphorylated by p56chk1 in vivo, and this results in maintenance of Y15 phosphorylation and hence G2 delay. In the absence of wee1, other Y15 kinases, such as p66mik1, may partially substitute for p107wee1 to induce cell cycle delay, but this wee1‐independent delay is insufficient to maintain full viability. This study establishes a link between a G2 DNA damage checkpoint function and a core cell cycle regulator.

Chk1 complements the G2/M checkpoint defect and radiosensitivity of ataxia-telangiectasia cells

“…In S. pombe several mutants have been described that are unable to arrest the cell cycle in response to DNA damage at the G2 checkpoint (Weinert and Hartwell, 1990;AlKhodairy et al, 1994;Ford et al, 1994). One of these genes, chk1, encodes a protein kinase that appears to function downstream of all other checkpoint genes to elicit a cell cycle arrest via cdc25 and wee1 to maintain tyrosine phosphorylation of p34 cdc2 (O'Connell et al, 1997;Rhind et al, 1997;Furnari et al, 1997;Sanchez et al, 1997). Irradiation of cells leads to phosphorylation of chk1 itself, and this response requires all of the upstream checkpoint components, including rad3 (Walworth and Bernards, 1996).…”

“…The exact mechanism(s) for G2 checkpoint activation in response to DNA damage remains unclear but recent data with S. pombe support a model where chk1 protein kinase maintains p34 cdc2 in its tyrosine phosphorylated, inactive state for the duration of the checkpoint arrest (O'Connell et al, 1997;Rhind et al, 1997). Overexpression of chk1 in wild type S. pombe led to cellular elongation and a failure to enter mitosis (Al-Khodairy et al, 1994;Ford et al, 1994).…”

“…This was the same phenotype as observed with a number of radmutants suggesting that these function upstream of chk1 in checkpoint arrest (Walworth and Bernards, 1996). More recently it has been shown the cell cycle arrest after chk1 overexpression is dependent on the wee1 tyrosine kinase, and chk1 may phosphorylate wee1 directly resulting in a maintenance of activity (O'Connell et al, 1997). However, this response is clearly more complex than this, as the cdc25 phosphatase has also been implicated as a chk1 target .…”

Instabilities in phosphorylation-dephosphorylation cascades and cell cycle checkpoints

“…When DNA is damaged, the cell cycle can be arrested at the G1 phase or the G2 phase of the cell cycle. Recently, details of the G2 DNA damage checkpoint (G2DDC) pathway that impinges on the cell cycle have been elucidated (Nurse, 1997;Weinert, 1997). It is now being suggested that the G2DDC pathway perturbs the cell cycle via Cdc25 (Furnari et al, 1996;Rhind et al, 1997).…”

“…When there is DNA damage, there is a sensing mechanism (called a checkpoint pathway) that detects this damage and transduces the information to the cell cycle machinery. The G1 DNA damage checkpoint pathway has been studied extensively (Carr, 1996) and, recently, more details about the G2 DNA damage checkpoint (G2DDC) pathway are being elucidated (Nurse, 1997;Weinert, 1997). A second objective of this paper is to study how the G2DDC pathway impinges on the cell cycle and to propose a resolution of the issue on the identity of the cell cycle protein targeted by the G2DDC pathway (Osmani and Ye, 1997).…”