MAPK-activated protein kinase 5 (MK5) was recently identified as a physiological substrate of the atypical MAPK ERK3.Complex formation between ERK3 and MK5 results in phosphorylation and activation of MK5, concomitant stabilization of ERK3, and the nuclear exclusion of both proteins. However, ablation of ERK3 in HeLa cells using small interfering RNA or in fibroblasts derived from ERK3 null mice reduces the activity of endogenous MK5 by only 50%, suggesting additional mechanisms of MK5 regulation. Here we identify the ERK3-related kinase ERK4 as a bona fide interaction partner of MK5. Binding of ERK4 to MK5 is accompanied by phosphorylation and activation of MK5. Furthermore, complex formation also results in the relocalization of MK5 from nucleus to cytoplasm. However unlike ERK3, ERK4 is a stable protein, and its half-life is not modified by the presence or absence of MK5. Finally, although knock-down of ERK4 protein in HeLa cells reduces endogenous MK5 activity by ϳ50%, a combination of small interfering RNAs targeting both ERK4 and ERK3 causes a further reduction in the MK5 activity by more than 80%. We conclude that MK5 activation is dependent on both ERK3 and ERK4 in these cells and that these atypical MAPKs are both physiological regulators of MK5 activity.

Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase family of serine/threonine kinases. Little is known on the regulation of ERK3 function. Here, we report that ERK3 is constitutively localized in the cytoplasmic and nuclear compartments. In contrast to other mitogenactivated protein kinases, the cellular distribution of ERK3 remains unchanged in response to common mitogenic or stress stimuli and is independent of the enzymatic activity or phosphorylation of the kinase. The cytoplasmic localization of ERK3 is directed by a CRM1-dependent nuclear export mechanism. Treatment of cells with leptomycin B causes the nuclear accumulation of ERK3 in a high percentage of cells. Moreover, ectopic expression of CRM1 promotes the cytoplasmic relocalization of ERK3, whereas overexpression of snurportin 1, which binds CRM1 with high affinity, inhibits the nuclear export of ERK3. We also show that CRM1 binds to ERK3 in vitro. Importantly, we show that enforced localization of ERK3 in the nucleus or cytoplasm markedly attenuates the ability of the kinase to induce cell cycle arrest in fibroblasts. Our results suggest that nucleocytoplasmic shuttling of ERK3 is required for its negative regulatory effect on cell cycle progression.Recent studies have highlighted the importance of the nucleocytoplasmic transport of signaling proteins in the regulation of eukaryotic cellular responses such as cell cycle progression and cell proliferation, differentiation, stress responses, and circadian clocks (1). Small regulatory proteins can, in principle, enter and leave the nucleus by simple diffusion through the nuclear pore complexes. However, molecules larger than ϳ 60 kDa are actively translocated from one side of the nuclear envelope to the other by a family of soluble transport receptors, classified as importins or exportins, which belong to the importin-␤ family of RanGTP-binding proteins. Importins bind their cargo in the cytoplasm and release it upon binding to RanGTP in the nucleus; on the opposite, exportins form a trimeric complex with cargo and RanGTP in the nucleus and release their transport substrate into the cytoplasm upon GTP hydrolysis (for review, see Refs. 2-4). At steady state, the subcellular localization of proteins is determined by the balance between import and export rates. Cargo proteins interact directly with their cognate transport receptor through specific nuclear localization signal (NLS) 1 or nuclear export signal (NES) sequences or indirectly via adaptor molecules (2, 4).MAP kinases are a family of evolutionary conserved protein kinases that play a critical role in transducing extracellular chemical and physical signals into intracellular responses (for review, see Refs. 5-7). Upon activation, these enzymes phosphorylate a variety of substrates that are present in all compartments of the cell. Consequently, the spatial distribution of MAP kinases, as well as the temporal regulation of their activity, is an important determinant of the ultimate biological...

The extracellular signal-regulated kinase 1 and 2 (ERK1/2) mitogen-activated protein (MAP) kinase signaling pathway plays an important role in the proliferative response of mammalian cells to mitogens. However, the individual contribution of the isoforms ERK1 and ERK2 to cell proliferation control is unclear. The two ERK isoforms have similar biochemical properties and recognize the same primary sequence determinants on substrates. On the other hand, analysis of mice lacking individual ERK genes suggests that ERK1 and ERK2 may have evolved unique functions. In this study, we used a robust genetic approach to analyze the individual functions of ERK1 and ERK2 in cell proliferation using genetically matched primary embryonic fibroblasts. We show that individual loss of either ERK1 or ERK2 slows down the proliferation rate of fibroblasts to an extent reflecting the expression level of the kinase. Moreover, RNA interference-mediated silencing of ERK1 or ERK2 expression in cells genetically disrupted for the other isoform similarly reduces cell proliferation. We generated fibroblasts genetically deficient in both Erk1 and Erk2. Combined loss of ERK1 and ERK2 resulted in a complete arrest of cell proliferation associated with G 1 arrest and premature replicative senescence. Together, our findings provide compelling genetic evidence for a redundant role of ERK1 and ERK2 in promoting cell proliferation.