Extracellular Signal-regulated Kinase 1/2 Activity Is Not Required in Mammalian Cells during Late G₂for Timely Entry into or Exit from Mitosis

Abstract

Extracellular signal-regulated kinase (ERK)1/2 activity is reported to be required in mammalian cells for timely entry into and exit from mitosis (i.e., the G 2 -mitosis [G 2 /M] and metaphase-anaphase [M/A] transitions). However, it is unclearwhether this involvement reflects a direct requirement for ERK1/2 activity during these transitions or for activating gene transcription programs at earlier stages of the cell cycle. To examine these possibilities, we followed live cells in which ERK1/2 activity was inhibited through late G 2 and mitosis. We find that acute inhibition of ERK1/2 during late G 2 and through mitosis does not affect the timing of the G 2 /M or M/A transitions in normal or transformed human cells, nor does it impede spindle assembly, inactivate the p38 stress-activated checkpoint during late G 2 or the spindle assembly checkpoint during mitosis. Using CENP-F as a marker for progress through G 2 , we also show that sustained inhibition of ERK1/2 transiently delays the cell cycle in early/mid-G 2 via a p53-dependent mechanism. Together, our data reveal that ERK1/2 activity is required in early G 2 for a timely entry into mitosis but that it does not directly regulate cell cycle progression from late G 2 through mitosis in normal or transformed mammalian cells. INTRODUCTIONThe extracellular signal-regulated (ERK)1/2 pathway consists of Raf1, mitogen-activated protein kinase kinase (MEK)1/2, and ERK1/2 kinases, which upon pathway activation are sequentially and specifically phosphorylated . Once activated, ERK1/2 phosphorylates numerous cytoplasmic and nuclear substrates that lead to diverse cellular responses, including proliferation and differentiation, via both transcription-dependent and independent mechanisms (Pearson et al., 2001;Yoon and Seger, 2006). Basically, ERK1/2 activity is essential for cell growth; it mediates the G 1 /S transition by facilitating nucleotide and protein syntheses as well as the transcription of cell cycle regulators, including cyclins D and E (Widmann et al., 1999;Roovers and Assoian, 2000). Not unexpectedly, constitutive activation of ERK1/2 via mutations in Raf or its upstream Ras G protein leads to uncontrolled cell growth, whereas the inability to activate ERK1/2 is lethal in utero (Wojnowski et al., 1998;Giroux et al., 1999) and inhibits the growth of cultured cells (Pages et al., 1993).In addition to its essential role in promoting the G 1 /S transition, enhanced ERK1/2 activity is also required in mammalian cells for a timely G 2 -mitosis (G 2 /M) transition. Long-term (hours to days) suppression of ERK1/2 by using pharmacological inhibitors of MEK1/2, dominant-negative MEK1, or RNA interference (RNAi) produces a delay in "G 2 /M" (Wright et al., 1999;Hayne et al., 2000;Roberts et al., 2002;Liu et al., 2004;Knauf et al., 2006). Gene expression profiling of nontransformed mammary epithelial cells also reveals that constitutive activation of ERK1/2 via MEK1 leads to increased levels of mRNAs that encode mitotic proteins such as Cyclin B, CDK1, CENP-E, Bub1, Mad...

The ERK1/2 mitogen-activated protein kinase pathway as a master regulator of the G1- to S-phase transition

“…However, more recent studies have shown that phospho-specific antibodies to MEK1/MEK2 strongly cross-react with phosphorylated nucleophosmin, a multifunctional nuclear phosphoprotein involved in centrosome duplication (Cha et al, 2004;Hayne et al, 2004). Also, the labeling of centromeric regions, centrosomes and midbodies observed with the phospho-ERK1/2 antibody could not be blocked by pretreatment with the MEK1/2 inhibitor U0126, although the drug completely suppressed the weak phospho-ERK1/2 signal observed by immunoblot analysis in mitotic cells (Shinohara et al, 2006). Thus, there is no conclusive evidence that ERK1/ERK2 MAP kinases are active in mitosis.…”

“…However, the interpretation of these studies is complicated by the fact that MEK1/MEK2 inhibitors were added for long periods of time and it is therefore difficult to distinguish whether the delay in mitosis entry results from a direct effect of ERK1/ERK2 in late G2 or from an indirect consequence of earlier cell cycle defects. The role of ERK1/2 signaling in the G2/M transition and mitotic progression was revisited in a recent study using time-lapse video microscopy in live cells to precisely monitor the timing of these transitions (Shinohara et al, 2006). The inhibitors of MEK1/ MEK2 were added to cell cultures 15 min before recording the observations, which is enough to suppress ERK1/ERK2 activity completely.…”

Mitosis in vertebrates: the G2/M and M/A transitions and their associated checkpoints

“…Thus, although inhibiting the ERK1/2 kinase delays the G2/M transition, this is not, as originally concluded, because its activity is required during this transition for timely entry into mitosis. Rather, it is because ERK1/2 activity is required during very early G2 for timely progression through G2 (Shinohara et al 2006).…”

Knockdown of B-Raf impairs spindle formation and the mitotic checkpoint in human somatic cells

“…10,[14][15][16] However, this may occur in an ERK1/2-dependent transcriptional manner as rapid inhibition of ERK activity by pharmacological MEK inhibitors does not appear to delay the G 2 /M transition in tissue culture cells. 17 Members of the Raf family of cytosolic serine/threonine kinases are key activators of the MAPK cascade. In mammals there are three Raf members: A-Raf, B-Raf and C-Raf (also called Raf-1) and each can directly phosphorylate and activate MEKs 1 and 2, which in turn, directly phosphorylate and activate ERKs 1 and 2 (reviewed in ref.…”