Sequential activation of protein kinases within the mitogen-activated protein kinase (MAPK) cascades is a common mechanism of signal transduction in many cellular processes. Four such cascades have been elucidated thus far, and named according to their MAPK tier component as the ERK1/2, JNK, p38MAPK, and ERK5 cascades. These cascades cooperate in transmitting various extracellular signals, and thus control cellular processes such as proliferation, differentiation, development, stress response, and apoptosis. Here we describe the classic ERK1/2 cascade, and concentrate mainly on the properties of MEK1/2 and ERK1/2, including their mode of regulation and their role in various cellular processes and in oncogenesis. This cascade may serve as a prototype of the other MAPK cascades, and the study of this cascade is likely to contribute to the understanding of mitogenic and other processes in many cell lines and tissues.
Mitogen-activated protein kinase kinase (MEK) is a dual-specificity protein kinase that is located primarily in the cellular cytosol, both prior to and upon mitogenic stimulation. The existence of a nuclear export signal in the N-terminal domain of MEK [Fukuda, M., Gotoh, I., Gotoh, Y. & Nishida, E. (1996) J. Biol. Chem. 271, [20024][20025][20026][20027][20028] suggests that there are circumstances under which MEK enters the nucleus and must be exported. Using mutants of MEK, we show that the deletion of the nuclear export signal sequence from constitutively active MEK caused constitutive localization of MEK in the nucleus of COS7 and HEK-293T cells. However, when the same region was deleted from a catalytically inactive MEK, cytoplasmic localization was observed in resting cells, which turned nuclear upon stimulation. Confocal microscopy of COS7 cells expressing the above mutants showed localization of the active MEK in the nuclear envelope and also in the cell periphery. The differences in cellular localization between the wild-type and mutant MEKs are not due to severe changes in specificity because the recombinant, constitutively active MEK that lacked its Nterminal region exhibited the same substrate specificity as the wild-type MEK, both in vitro and in intact cells. Taken together, our results indicate that upon mitogenic stimulation, MEK, like extracellular signal responsive kinase and p90 RSK , is massively translocated to the nucleus. Rapid export from the nucleus, which is mediated by the nuclear export signal, is probably the cause for the cytoplasmic distribution observed with wild-type MEK.The transmission of extracellular signals from the cell surface into the nucleus involves several groups of protein kinases, which are collectively known as the mitogen-activated protein kinase (MAPK) signaling cascades. One of these cascades, the extracellular signal responsive kinase (ERK) signaling cascade, involves a sequential phosphorylation and activation of Raf1, MAPK kinase (MAPKK, also known as MEK), ERK, p90 RSK , and under some conditions also GSK3 (reviewed in ref. 1). Other MAPK signaling cascades are the JNK (stress-activated protein kinases) cascade, the p38RK (HOG, reviewed in ref.2), and other, less-characterized cascades. A key step in the signaling mechanism of the ERK cascade is the translocation of both ERK1 and 2 and p90 RSK into the nucleus (3, 4). This translocation, which occurs in response to mitogenic stimulation, is rapid (occurs within 5-30 min), and might be a prerequisite for activation of nuclear processes such as transcription (5, 6). The mechanism by which protein kinases are translocated to the nucleus upon stimulation is not yet known. The sequences of the ERKs and p90 RSK do not contain a nuclear localization signal (NLS), and although the size of ERK may permit a simple diffusion via nuclear pores, such diffusion would probably be much slower than the rapid movement observed upon activation. However, because kinase-deficient mutants of ERK can translocate to the nucl...
A key step in the signaling mechanism of the mitogenactivated protein kinase/extracellular signal-responsive kinase (ERK) cascade is its translocation into the nucleus where it regulates transcription and other nuclear processes. In an attempt to characterize the subcellular localization of ERK2, we fused it to the 3-end of the gene expressing green fluorescent protein (GFP), resulting in a GFP-ERK2 protein. The expression of this construct in CHO cells resulted in a nuclear localization of the GFP-ERK2 protein. However, coexpression of the GFP-ERK2 with its upstream activator, MEK1, resulted in a cytosolic retention of the GFP-ERK2, which was the result of its association with MEK1, and was reversed upon stimulation. We then examined the role of the C-terminal region of ERK2 in its subcellular localization. Substitution of residues 312-319 of GFP-ERK2 to alanine residues prevented the cytosolic retention of ERK2 as well as its association with MEK1, without affecting its activity. Most important for the cytosolic retention are three acidic amino acids at positions 316, 319, and 320 of ERK2. Substitution of residues 321-327 to alanines impaired the nuclear translocation of ERK2 upon mitogenic stimulation. Thus, we conclude that residues 312-320 of ERK2 are responsible for its cytosolic retention, and residues 321-327 play a role in the mechanism of ERK2 nuclear translocation. Mitogen-activated protein kinase (MAPK)1 signaling cascades are main routes of communication between the plasma membrane and regulatory intracellular targets and thus initiate a large array of cellular responses (1-4). The first MAPK cascade elucidated is the one that signals through the extracellular signal-responsive kinases 1 and 2 (ERK1/2), which are activated via a sequential phosphorylation and activation of the protein kinases Raf1 and MAPK/ERK kinase (MEK). Upon activation, ERK phosphorylates and activates several regulatory targets, which eventually culminate in regulation of proliferation, differentiation, and other cellular processes.Key steps in the signaling mechanism of the ERK cascade are the changes in localization of its components upon stimulation. In resting cells, all components of the cascade seem to be localized primarily in the cell cytosol. However, this localization is rapidly changed upon extracellular stimulation, which causes Raf1 recruitment to the plasma membrane (5) and translocation of MEK (6), ERK (7), and RSK (7) into the nucleus. After translocation, MEK seems to be rapidly exported from the nucleus by its nuclear export signal (NES; Ref. 8), although the timing and role of its translocation are still controversial (9, 10). ERK and RSK on the other hand are retained in the nucleus for longer times after stimulation, and this longer time is correlated with the effects of ERK on mitogenesis and neurite outgrowth in PC12 cells (11,12).The mechanism of nuclear translocation of the different kinases is not fully understood. Recently, it was shown that in resting cells ERK is retained in the cytosol by its assoc...
The mitogen-activated protein kinase, ERK is activated by a dual phosphorylation on threonine and tyrosine residues. Using a synthetic diphospho peptide, we have generated a monoclonal antibody directed to the active ERK. The antibody specifically identified the active doubly phosphorylated, but not the inactive mono-or non-phosphorylated forms of ERKs. A direct correlation was observed between ERK activity and the intensity in Western blot of mitogen-activated protein kinases from several species. The antibody was proven suitable for immunofluorescence staining, revealing a transient reactivity with ERKs that were translocated to the nucleus upon stimulation. In conclusion, the antibody can serve as a useful tool in the study of ERK signaling in a wide variety of organisms.
Rapamycin and its analogues have significant antiproliferative action against a variety of tumors. However, sensitivity to rapamycin is reduced by Akt activation that results from the ablative effects of rapamycin on a p70 S6K-induced negative feedback loop that blunts phosphoinositide 3-kinase (PI3K)-mediated support for Akt activity. Thus, sensitivity to rapamycin might be increased by imposing an upstream blockade to the PI3K/Akt pathway. Here, we investigated this model using the somatostatin analogue octreotide as a tool to decrease levels of activated
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