Modulation of p27(Kip1) levels by the cyclin encoded by Kaposi's sarcoma-associated herpesvirus.

DNA tumour viruses have evolved a number of mechanisms by which they deregulate normal cellular growth control. We have recently described the properties of a cyclin encoded by human herpesvirus 8 (also known as Kaposi's sarcoma-associated herpesvirus) which is able to resist the actions of p16(Ink4a), p21(Cip1) and p27(Kip1) cdk inhibitors. Here we investigate the mechanism involved in the subversion of a G1 blockade imposed by overexpression of p27(Kip1). We demonstrate that binding of K cyclin to cdk6 expands the substrate repertoire of this cdk to include a number of substrates phosphorylated by cyclin-cdk2 complexes but not cyclin D1-cdk6. Included amongst these substrates is p27(Kip1) which is phosphorylated on Thr187. Expression of K cyclin in mammalian cells leads to p27(Kip1) downregulation, this being consistent with previous studies indicating that phosphorylation of p27(Kip1) on Thr187 triggers its downregulation. K cyclin expression is not able to prevent a G1 arrest imposed by p27(Kip1) in which Thr187 is mutated to non-phosphorylatable Ala. These results imply that K cyclin is able to bypass a p27(Kip1)-imposed G1 arrest by facilitating phosphorylation and downregulation of p27(Kip1) to enable activation of endogenous cyclin-cdk2 complexes. The extension of the substrate repertoire of cdk6 by K cyclin is likely to contribute to the deregulation of cellular growth by this herpesvirus-encoded cyclin.     

Cell cycle arrest mediated by WEE1 is involved in the unfolded protein response in plants

Activation of the unfolded protein response (UPR) in mammalian cells leads to cell cycle arrest at the G1 phase (Thomas et al., J Biol Chem 288:7606–7617, 2013). However, how UPR signaling affects cell cycle arrest remains largely unknown in plants. Here, we examined UPR and endoreduplication in Col-0, wee1, and ER stress sensing-deficient ire1a&b plants during DNA replication and ER stress conditions. We found that WEE1, an essential negative regulator of the cell cycle, is involved in the maintenance of ER homeostasis during genotoxic stress and the ER stress hypersensitivity of ire1a&b is alleviated by loss-of-function mutation in WEE1. WEE1-mediated cell cycle arrest was required for IRE1–bZIP60 pathway activation during ER stress. In contrast, loss-of-function mutation in WEE1 caused increased expression of UPR-related genes during DNA replication stress. WEE1 and IRE1 were required for endoreduplication during DNA replication stress and ER stress, respectively. Taken together, these findings suggest that cell cycle regulation is associated with UPR activation in different manners during ER stress and DNA replication stress in Arabidopsis.     

High-intensity Raf signal causes cell cycle arrest mediated by p21Cip1.

Activated Raf has been linked to such opposing cellular responses as the induction of DNA synthesis and the inhibition of proliferation. However, it remains unclear how such a switch in signal specificity is regulated. We have addressed this question with a regulatable Raf-androgen receptor fusion protein in murine fibroblasts. We show that Raf can cause a G1-specific cell cycle arrest through induction of p21Cip1. This in turn leads to inhibition of cyclin D- and cyclin E-dependent kinases and an accumulation of hypophosphorylated Rb. Importantly, this behavior can be observed only in response to a strong Raf signal. In contrast, moderate Raf activity induces DNA synthesis and is sufficient to induce cyclin D expression. Therefore, Raf signal specificity can be determined by modulation of signal strength presumably through the induction of distinct protein expression patterns. Similar to induction of Raf, a strong induction of activated Ras via a tetracycline-dependent promoter also causes inhibition of proliferation and p21Cip1 induction at high expression levels. Thus, p21Cip1 plays a key role in determining cellular responses to Ras and Raf signalling. As predicted by this finding we show that Ras and loss of p21 cooperate to confer a proliferative advantage to mouse embryo fibroblasts.     

Raf-induced proliferation or cell cycle arrest is determined by the level of Raf activity with arrest mediated by p21Cip1.

The Raf family of protein kinases display differences in their abilities to promote the entry of quiescent NIH 3T3 cells into the S phase of the cell cycle. Although conditional activation of deltaA-Raf:ER promoted cell cycle progression, activation of deltaRaf-1:ER and deltaB-Raf:ER elicited a G1 arrest that was not overcome by exogenously added growth factors. Activation of all three deltaRaf:ER kinases led to elevated expression of cyclin D1 and cyclin E and reduced expression of p27Kip1. However, activation of deltaB-Raf:ER and deltaRaf-1:ER induced the expression of p21Cip1, whereas activation of deltaA-Raf:ER did not. A catalytically potentiated form of deltaA-Raf:ER, generated by point mutation, strongly induced p21Cip1 expression and elicited cell cycle arrest similarly to deltaB-Raf:ER and deltaRaf-1:ER. These data suggested that the strength and duration of signaling by Raf kinases might influence the biological outcome of activation of this pathway. By titration of deltaB-Raf:ER activity we demonstrated that low levels of Raf activity led to activation of cyclin D1-cdk4 and cyclin E-cdk2 complexes and to cell cycle progression whereas higher Raf activity elicited cell cycle arrest correlating with p21Cip1 induction and inhibition of cyclin-cdk activity. Using green fluorescent protein-tagged forms of deltaRaf-1:ER in primary mouse embryo fibroblasts (MEFs) we demonstrated that p21Cip1 was induced by Raf in a p53-independent manner, leading to cell cycle arrest. By contrast, activation of Raf in p21Cip1(-/-) MEFs led to a robust mitogenic response that was similar to that observed in response to platelet-derived growth factor. These data indicate that, depending on the level of kinase activity, Raf can elicit either cell cycle progression or cell cycle arrest in mouse fibroblasts. The ability of Raf to elicit cell cycle arrest is strongly associated with its ability to induce the expression of the cyclin-dependent kinase inhibitor p21Cip1 in a manner that bears analogy to alpha-factor arrest in Saccharomyces cerevisiae. These data are consistent with a role for Raf kinases in both proliferation and differentiation of mammalian cells.