Region-Specific Targets of p42/p44MAPK Signaling in Rat Brain

Abstract: In vitro studies indicate that p42/p44^MAPK phosphorylate both nuclear and cytoplasmic proteins. However, the functional targets of p42/p44^MAPK activation in vivo remain unclear. To address this question, we localized activated p42/p44^MAPK in hippocampus and cortex and determined their signaling effects after electroconvulsive shock treatment (ECT) in rats. Phosphorylated p42/p44^MAPK content increased in the cytoplasm of hippocampal neurons in response to ECT. Consistent with this cytoplasmic localization, inhibition of ECT-induced p42/p44^MAPK activation by the extracellular signal-regulated kinase kinase inhibitor PD098059 blocked phosphorylation of the cytoplasmic protein microtubule-associated protein 2c (MAP2c), but failed to inhibit the induction of the nuclear protein c-Fos in response to ECT. In contrast to hippocampal neurons, cortical neurons exhibited an increase in amount of phosphorylated p42/p44^MAPK in both the nucleus and cytoplasm after ECT. Accordingly, PD098059 blocked the induction of Fos-like immunoreactivity in the nuclei of cortical neurons as well as MAP2c phosphorylation in the cytoplasm. Our data indicate that both nuclear and cytoplasmic substrates can be activated by p42/p44^MAPK in vivo. However, the functional targets of p42/p44^MAPK signaling depend on the precise location of p42/p44^MAPK within different subcellular compartments of brain regions. These results indicate unique functional pathways of p42/p44^MAPK-mediated signal transduction within different brain regions in vivo.     

Fidelity and spatio-temporal control in MAP kinase (ERKs) signalling.

The mitogen activated protein (MAP) kinase module: (Raf -->MEK-->ERKs) is central to the control of cell growth, cell differentiation and cell survival. The fidelity of signalling and the spatio-temporal activation are key determinants in generating precise biological responses. The fidelity is ensured by scaffold proteins - protein kinase 'insulators' - and by specific docking sites. The duration and the intensity of the response are in part controlled by the compartmentalization of the signalling molecules. Growth factors promote rapid nuclear translocation and persistent activation of p42/p44 MAP kinases, respectively and ERK2/ERK1, during the entire G1 period with an extinction during the S-phase. These features are exquisitely controlled by the temporal induction of the MAP kinase phosphatases, MKP1-3. MKP1 and 2 induction is strictly controlled by the activation of the MAP kinase module providing evidence for an auto-regulatory mechanism. This negative regulatory loop is further enhanced by the capacity of p42/p44 MAPK to phosphorylate MKP1 and 2. This action reduces the degradation rate of MKPs through the ubiquitin-proteasomal system. Whereas the two upstream kinases of the module (Raf and MEK) remain cytoplasmic, ERKs (anchored to MEK in the cytoplasm of resting cells) rapidly translocate to the nucleus upon mitogenic stimulation. This latter process is rapid, reversible and controlled by the strict activation of the MAPK cascade. Following long-term MAPK stimulation, p42/p44 MAPKs progressively accumulate in the nucleus in an inactive form. Therefore we propose that the nucleus represents a site for ERK action, sequestration and signal termination. With the generation of knockdown mice for each of the ERK isoforms, we will illustrate that besides controlling cell proliferation the ERK cascade also controls cell differentiation and cell behaviour.     

Growth factors induce nuclear translocation of MAP kinases (p42mapk and p44mapk) but not of their activator MAP kinase kinase (p45mapkk) in fibroblasts

Mitogen-activated protein kinases (p42mapk and p44mapk) are serine/threonine kinases that are activated rapidly in cells stimulated with various extracellular signals. This activation is mediated via MAP kinase kinase (p45mapkk), a dual specificity kinase which phosphorylates two key regulatory threonine and tyrosine residues of MAP kinases. We reported previously that the persistent phase of MAP kinase activation is essential for mitogenically stimulated cells to pass the "restriction point" of the cell cycle. Here, using specific polyclonal antibodies and transfection of epitope-tagged recombinant MAP kinases we demonstrate that these signaling protein kinases undergo distinct spatio-temporal localization in growth factor-stimulated cells. In G0-arrested hamster fibroblasts the activator p45mapkk and MAP kinases (p42mapk, p44mapk) are mainly cytoplasmic. Subsequent to mitogenic stimulation by serum or alpha-thrombin both MAP kinase isoforms translocate into the nucleus. This translocation is rapid (seen in 15 min), persistent (at least during the entire G1 period up to 6 h), reversible (by removal of the mitogenic stimulus) and apparently 'coupled' to the mitogenic potential; it does not occur in response to nonmitogenic agents such as alpha-thrombin-receptor synthetic peptides and phorbol esters that fail to activate MAP kinases persistently. When p42mapk and p44mapk are expressed stably at high levels, they are found in the nucleus of resting cells; this nuclear localization is also apparent with kinase-deficient mutants (p44mapk T192A or Y194F). In marked contrast the p45mapkk activator remains cytoplasmic even during prolonged growth factor stimulation and even after high expression levels achieved by transfection. We propose that the rapid and persistent nuclear transfer of p42mapk and p44mapk during the entire G0-G1 period is crucial for the function of these kinases in mediating the growth response.     

MAP kinase subcellular localization controls both pattern and proliferation in the developing Drosophila wing

Mitogen-activated protein kinases (MAPKs) phosphorylate target proteins in both the cytoplasm and nucleus, and a strong correlation exists between the subcellular localization of MAPK and resulting cellular responses. It was thought that MAPK phosphorylation was always followed by rapid nuclear translocation. However, we and others have found that MAPK phosphorylation is not always sufficient for nuclear translocation in vivo. In the developing Drosophila wing, MAPK-mediated signaling is required both for patterning and for cell proliferation, although the mechanism of this differential control is not fully understood. Here, we show that phosphorylated MAPK (pMAPK) is held in the cytoplasm in differentiating larval and pupal wing vein cells, and we show that this cytoplasmic hold is required for vein cell fate. At the same time, we show that MAPK does move into the nucleus of other wing cells where it promotes cell proliferation. We propose a novel Ras pathway bifurcation in Drosophila and our results suggest a mechanism by which MAPK phosphorylation can signal two different cellular outcomes (differentiation versus proliferation) based on the subcellular localization of MAPK.     

Cytoplasmic localization of Wis1 MAPKK by nuclear export signal is important for nuclear targeting of Spc1/Sty1 MAPK in fission yeast

Mitogen-activated protein kinase (MAPK) cascade is a ubiquitous signaling module that transmits extracellular stimuli through the cytoplasm to the nucleus; in response to activating stimuli, MAPKs translocate into the nucleus. Mammalian MEK MAPK kinases (MAPKKs) have in their N termini an MAPK-docking site and a nuclear export signal (NES) sequence, which are known to play critical roles in maintaining ERK MAPKs in the cytoplasm of unstimulated cells. Herein, we show that the Wis1 MAPKK of the stress-activated Spc1 MAPK cascade in fission yeast also has a MAPK-docking site and an NES sequence in its N-terminal domain. Unexpectedly, an inactivating mutation to the NES of chromosomal wis1(+) does not affect the subcellular localization of Spc1 MAPK, whereas this NES mutation disturbs the cytoplasmic localization of Wis1. However, when Wis1 is targeted to the nucleus by fusing to a nuclear localization signal sequence, stress-induced nuclear translocation of Spc1 is abrogated, indicating that cytoplasmic Wis1 is required for nuclear transport of Spc1 upon stress. Moreover, we have observed that a fraction of Wis1 translocates into the nucleus in response to stress. These results suggest that cytoplasmic localization of Wis1 MAPKK by its NES is important for stress signaling to the nucleus.     

Nuclear activation and translocation of mitogen-activated protein kinases modulated by ethanol in embryonic liver cells

Activation and nuclear translocation of mitogen activated protein (MAP) kinases in ethanol-treated embryonic liver cells (BNLCL2) was investigated. The relative amount of MAPK proteins, MAP kinase activity and MAPK/LDH (lactate dehydrogenase) ratios were determined in nuclear and cytosolic fractions before and after serum stimulation. In ethanol-treated cells, serum-stimulated MAPK activation was potentiated in both cytosolic and nuclear fractions. Levels of both the p42 and p44 MAPK proteins increased in nuclear fractions from cells treated with ethanol alone for 24 h. Serum-stimulated nuclear translocation of both p42 and p44 MAPK was potentiated in ethanol-treated cells. Nuclear fractions from ethanol-treated cells had a modest increase in MAP kinase activity concurrent with the increased MAPK protein levels. The ratio of MAPK/LDH increased in nuclear fractions with increasing concentrations of ethanol and after serum stimulation. This further confirmed the nuclear translocation of MAPK and also demonstrated that it is not a non-specific effect of ethanol. These results demonstrate, for the first time, that in BNLCL2 liver cells ethanol treatment has dual effects. First, ethanol triggered nuclear translocation of MAPK without causing its activation. Second, it potentiated serum-stimulated activation and translocation of MAPK in the nucleus. These findings provide a novel mechanism through which ethanol may affect cellular and nuclear processes in liver cells.     

p42 mitogen-activated protein kinase and p90 ribosomal S6 kinase are selectively phosphorylated and activated during thrombin-induced platelet activation and aggregation.

Human platelets provide an excellent model system for the study of phosphorylation events during signal transduction and cell adhesion. Platelets are terminally differentiated cells that exhibit rapid phosphorylation of many proteins upon agonist-induced activation and aggregation. We have sought to identify the kinases as well as the phosphorylated substrates that participate in thrombin-induced signal transduction and platelet aggregation. In this study, we have identified two forms of mitogen-activated protein kinase (MAPK), p42mapk and p44mapk, in platelets. The data demonstrate that p42mapk but not p44mapk becomes phosphorylated on serine, threonine, and tyrosine during platelet activation. Immune complex kinase assays, gel renaturation assays, and a direct assay for MAPK activity in platelet extracts all support the conclusion that p42mapk but not p44mapk shows increased kinase activity during platelet activation. The activation of p42mapk, independently of p44mapk, in platelets is unique since in other systems, both kinases are coactivated by a variety of stimuli. We also show that platelets express p90rsk, a ribosomal S6 kinase that has previously been characterized as a substrate for MAPK. p90rsk is phosphorylated on serine in resting platelets, and this phosphorylation is enhanced upon thrombin-induced platelet activation. Immune complex kinase assays demonstrate that the activity of p90rsk is markedly increased during platelet activation. Another ribosomal S6 protein kinase, p70S6K, is expressed by platelets but shows no change in kinase activity upon platelet activation with thrombin. Finally, we show that the increased phosphorylation and activity of both p42mapk and p90rsk does not require integrin-mediated platelet aggregation. Since platelets are nonproliferative cells, the signal transduction pathways that include p42mapk and p90rsk cannot lead to a mitogenic signal and instead may regulate cytoskeletal or secretory changes during platelet activation.     

Interaction of MAP kinase with MAP kinase kinase: its possible role in the control of nucleocytoplasmic transport of MAP kinase.

The mitogen-activated protein kinase (MAPK) cascade consisting of MAPK and its direct activator, MAPK kinase (MAPKK), is essential for signaling of various extracellular stimuli to the nucleus. Upon stimulation, MAPK is translocated to the nucleus, whereas MAPKK stays in the cytoplasm. It has been shown recently that the cytoplasmic localization of MAPKK is determined by its nuclear export signal (NES) in the near N-terminal region (residues 33-44). However, the mechanism determining the subcellular distribution of MAPK has been poorly understood. Here, we show that introduction of v-Ras, active STE11 or constitutively active MAPKK can induce nuclear translocation of MAPK in mammalian cultured cells. Furthermore, we show evidence suggesting that MAPK is localized to the cytoplasm through its specific association with MAPKK and that nuclear accumulation of MAPK is accompanied by dissociation of a complex between MAPK and MAPKK following activation of the MAPK pathway. We have identified the MAPK-binding site of MAPKK as its N-terminal residues 1-32. Moreover, a peptide encompassing the MAPK-binding site and the NES sequence of MAPKK has been shown to be sufficient to retain MAPK to the cytoplasm. These findings reveal the molecular basis regulating subcellular distribution of MAPK, and identify a novel function of MAPKK as a cytoplasmic anchoring protein for MAPK.     

巨噬细胞免疫调变信号：Raf—1,MAPKp44,MAPKp42和p38MAPK的研究

为了了解巨噬细胞免疫调变机理,我们应用ＬＰＳ和ＰＭＡ处理小鼠抑制性巨噬细胞,观察到Ｒａｓ下游信号分子ＡＦ－１,分裂原激活蛋白激酶ＭＡＰＫｐ４４,ＭＡＰＫｐ４２和ｐ３８ＭＡＰＫ均被活化,发现ｆｏｒｓｋｏｌｉｎ能增强ｐ３８ＭＡＰＫ的活性,进一步提示ＰＫＣ和ＰＡＫ途径增强了ｐ３８ＭＡＰＫ的磷酸化效应,为我们了解ＬＰＳ如何激活ｐ３８ＭＡＰＫ信号通路提供了一个新的机会／