A novel mechanism for mitogen-activated protein kinase localization

Mitogen-activated protein kinases/extracellular signal regulated kinases (MAPKs/ERKs) are typically thought to be soluble cytoplasmic enzymes that translocate to the nucleus subsequent to their phosphorylation by their activating kinases or mitogen-activated protein/extracellular signal regulated kinase kinase. We report here the first example of nuclear translocation of a MAPK that occurs via temporally regulated exit from a membranous organelle. Confocal microscopy examining the subcellular localization of ERK3 in several cell lines indicated that this enzyme was targeted to the Golgi/endoplasmic reticulum Golgi intermediate compartment. Deletion analysis of green fluorescent protein (GFP)-ERK3 uncovered a nuclear form that was carboxy-terminally truncated and established a Golgi targeting motif at the carboxy terminus. Immunoblot analysis of cells treated with the proteasome inhibitor MG132 further revealed two cleavage products, suggesting that in vivo, carboxy-terminal cleavage of the full-length protein controls its subcellular localization. In support of this hypothesis, we found that deletion of a small region rich in acidic residues within the carboxy terminus eliminated both the cleavage and nuclear translocation of GFP-ERK3. Finally, cell cycle synchronization studies revealed that the subcellular localization of ERK3 is temporally regulated. These data suggest a novel mechanism for the localization of an MAPK family member, ERK3, in which cell cycle-regulated, site-specific proteolysis generates the nuclear form of the protein.     

Posttranslational regulation of tristetraprolin subcellular localization and protein stability by p38 mitogen-activated protein kinase and extracellular signal-regulated kinase pathways

The p38 mitogen-activated protein kinase (MAPK) signaling pathway, acting through the downstream kinase MK2, regulates the stability of many proinflammatory mRNAs that contain adenosine/uridine-rich elements (AREs). It is thought to do this by modulating the expression or activity of ARE-binding proteins that regulate mRNA turnover. MK2 phosphorylates the ARE-binding and mRNA-destabilizing protein tristetraprolin (TTP) at serines 52 and 178. Here we show that the p38 MAPK pathway regulates the subcellular localization and stability of TTP protein. A p38 MAPK inhibitor causes rapid dephosphorylation of TTP, relocalization from the cytoplasm to the nucleus, and degradation by the 20S/26S proteasome. Hence, continuous activity of the p38 MAPK pathway is required to maintain the phosphorylation status, cytoplasmic localization, and stability of TTP protein. The regulation of both subcellular localization and protein stability is dependent on MK2 and on the integrity of serines 52 and 178. Furthermore, the extracellular signal-regulated kinase (ERK) pathway synergizes with the p38 MAPK pathway to regulate both stability and localization of TTP. This effect is independent of kinases that are known to be synergistically activated by ERK and p38 MAPK. We present a model for the actions of TTP and the p38 MAPK pathway during distinct phases of the inflammatory response.     

Time Courses of Changes in Phospho- and Total- MAP Kinases in the Cochlea after Intense Noise Exposure

Mitogen-activated protein kinases (MAP kinases) are intracellular signaling kinases activated by phosphorylation in response to a variety of extracellular stimuli. Mammalian MAP kinase pathways are composed of three major pathways: MEK1 (mitogen-activated protein kinase kinase 1)/ERK 1/2 (extracellular signal-regulated kinases 1/2)/p90 RSK (p90 ribosomal S6 kinase), JNK (c-Jun amino (N)-terminal kinase)/c-Jun, and p38 MAPK pathways. These pathways coordinately mediate physiological processes such as cell survival, protein synthesis, cell proliferation, growth, migration, and apoptosis. The involvement of MAP kinase in noise-induced hearing loss (NIHL) has been implicated in the cochlea; however, it is unknown how expression levels of MAP kinase change after the onset of NIHL and whether they are regulated by transient phosphorylation or protein synthesis. CBA/J mice were exposed to 120-dB octave band noise for 2 h. Auditory brainstem response confirmed a component of temporary threshold shift within 0–24 h and significant permanent threshold shift at 14 days after noise exposure. Levels and localizations of phospho- and total- MEK1/ERK1/2/p90 RSK, JNK/c-Jun, and p38 MAPK were comprehensively analyzed by the Bio-Plex® Suspension Array System and immunohistochemistry at 0, 3, 6, 12, 24 and 48 h after noise exposure. The phospho-MEK1/ERK1/2/p90 RSK signaling pathway was activated in the spiral ligament and the sensory and supporting cells of the organ of Corti, with peaks at 3–6 h and independently of regulations of total-MEK1/ERK1/2/p90 RSK. The expression of phospho-JNK and p38 MAPK showed late upregulation in spiral neurons at 48 h, in addition to early upregulations with peaks at 3 h after noise trauma. Phospho-p38 MAPK activation was dependent on upregulation of total-p38 MAPK. At present, comprehensive data on MAP kinase expression provide significant insight into understanding the molecular mechanism of NIHL, and for developing therapeutic models for acute sensorineural hearing loss.     

Functional characterisation of the regulatory subunit of cyclic AMP-dependent protein kinase A homologue of Giardia lamblia: Differential expression of the regulatory and catalytic subunits during encystation

To understand the functional roles of protein kinase A (PKA) during vegetative and differentiating states of Giardia parasites, we studied the structural and functional characteristics of the regulatory subunit of PKA (gPKAr) and its involvement in the giardial encystment process. Molecular cloning and characterisation showed that gPKAr contains two tandem 3'5'-cyclic adenosine monphosphate (cyclic AMP) binding domains at the C-terminal end and the interaction domain for the catalytic subunit. A number of consensus residues including in vivo phosphorylation site for PKAc and dimerisation/docking domain are present in gPKAr. The regulatory subunit physically interacts with the catalytic subunit and inhibits its kinase activity in the absence of cyclic AMP, which could be partially restored upon addition of cyclic AMP. Western blot analysis showed a marked reduction in the endogenous gPKAr concentration during differentiation of Giardia into cysts. An increased activity of gPKAc was also detected during encystation without any significant change in the protein concentration. Distinct localisations of gPKAc to the anterior flagella, basal bodies and caudal flagella as noted in trophozoites were absent in encysting cells at later stages. Instead, PKAc staining was punctate and located mostly to the cell periphery. Our study indicates possible enrichment of the active gPKAc during late stages of encystation, which may have implications in completion of the encystment process or priming of cysts for efficient excystation.     

Control of MAP kinase signaling to the nucleus

MAP kinase (MAPK) signaling is among central signaling pathways that regulate cell proliferation, cell differentiation and apoptosis. As MAPK should transmit extracellular signals to proper regions or compartments in cells, controlling subcellular localization of MAPK is important for regulating fidelity and specificity of MAPK signaling. The ERK1/2-type of MAPK is the best characterized member of the MAPK family. In response to extracellular stimulus, ERK1/2 translocates from the cytoplasm to the nucleus by passing through the nuclear pore by several independent mechanisms. Sef (similar expression to fgf genes), a transmembrane protein, has been shown to be a regulator of subcellular distribution of ERK1/2. Sef binds to activated MEK1/2, the specific activator of ERK1/2, and tethers the activated MEK1/2/activated ERK1/2 complex to the Golgi apparatus and the plasma membrane. Thus, Sef blocks ERK1/2 signaling to the nucleus and allows signaling to the cytoplasm. Here we review recent findings on spatial regulation of MAPK, especially on nucleocytoplasmic trafficking of ERK1/2.     

Regulation of Cdc25C by ERK-MAP kinases during the G2/M transition

Induction of G(2)/M phase transition in mitotic and meiotic cell cycles requires activation by phosphorylation of the protein phosphatase Cdc25. Although Cdc2/cyclin B and polo-like kinase (PLK) can phosphorylate and activate Cdc25 in vitro, phosphorylation by these two kinases is insufficient to account for Cdc25 activation during M phase induction. Here we demonstrate that p42 MAP kinase (MAPK), the Xenopus ortholog of ERK2, is a major Cdc25 phosphorylating kinase in extracts of M phase-arrested Xenopus eggs. In Xenopus oocytes, p42 MAPK interacts with hypophosphorylated Cdc25 before meiotic induction. During meiotic induction, p42 MAPK phosphorylates Cdc25 at T48, T138, and S205, increasing Cdc25's phosphatase activity. In a mammalian cell line, ERK1/2 interacts with Cdc25C in interphase and phosphorylates Cdc25C at T48 in mitosis. Inhibition of ERK activation partially inhibits T48 phosphorylation, Cdc25C activation, and mitotic induction. These findings demonstrate that ERK-MAP kinases are directly involved in activating Cdc25 during the G(2)/M transition.     

Identification of Giardia lamblia DHHC Proteins and the Role of Protein S-palmitoylation in the Encystation Process

Protein S-palmitoylation, a hydrophobic post-translational modification, is performed by protein acyltransferases that have a common DHHC Cys-rich domain (DHHC proteins), and provides a regulatory switch for protein membrane association. In this work, we analyzed the presence of DHHC proteins in the protozoa parasite Giardia lamblia and the function of the reversible S-palmitoylation of proteins during parasite differentiation into cyst. Two specific events were observed: encysting cells displayed a larger amount of palmitoylated proteins, and parasites treated with palmitoylation inhibitors produced a reduced number of mature cysts. With bioinformatics tools, we found nine DHHC proteins, potential protein acyltransferases, in the Giardia proteome. These proteins displayed a conserved structure when compared to different organisms and are distributed in different monophyletic clades. Although all Giardia DHHC proteins were found to be present in trophozoites and encysting cells, these proteins showed a different intracellular localization in trophozoites and seemed to be differently involved in the encystation process when they were overexpressed. dhhc transgenic parasites showed a different pattern of cyst wall protein expression and yielded different amounts of mature cysts when they were induced to encyst. Our findings disclosed some important issues regarding the role of DHHC proteins and palmitoylation during Giardia encystation.     

The stimulus-dependent co-localization of serum- and glucocorticoid-regulated protein kinase (Sgk) and Erk/MAPK in mammary tumor cells involves the mutual interaction with the importin-alpha nuclear import protein

In Con8 rat mammary epithelial tumor cells, indirect immunofluorescence revealed that Sgk (serum- and glucocorticoid-regulated kinase) and Erk/MAPK (extracellular signal-regulated protein kinase/mitogen activated protein kinase) co-localized to the nucleus in serum-treated cells and to the cytoplasmic compartment in cells treated with the synthetic glucocorticoid dexamethasone. Moreover, the subcellular distribution of the importin-alpha nuclear transport protein was similarly regulated in a signal-dependent manner. In vitro GST-pull down assays revealed the direct interaction of importin-alpha with either Sgk or Erk/MAPK, while RNA interference knockdown of importin-alpha expression disrupted the localization of both Sgk and Erk into the nucleus of serum-treated cells. Wild type or kinase dead forms of Sgk co-immunoprecipitated with Erk/MAPK from either serum- or dexamethasone-treated mammary tumor cells, suggesting the existence of a protein complex containing both kinases. In serum-treated cells, nucleus residing Sgk and Erk/MAPK were both hyperphosphorylated, indicative of their active states, whereas, in dexamethasone-treated cells Erk/MAPK, but not Sgk, was in its inactive hypophosphorylated state. Treatment with a MEK inhibitor, which inactivates Erk/MAPK, caused the relocalization of both Sgk and ERK to the cytoplasm. We therefore propose that the signal-dependent co-localization of Sgk and Erk/MAPK mediated by importin-alpha represents a new pathway of signal integration between steroid and serum/growth factor-regulated pathways.     

Giardia lamblia: regulation of secretory vesicle formation and loss of ability to reattach during encystation in vitro

Encystation of Giardia lamblia is required for survival outside the host, as well as for initiation of new infections. Previously, we induced cultured G. lamblia trophozoites to encyst in vitro for the first time. During encystation, we observed the appearance of a new class of large secretory vesicle (encystation-specific vesicle; ESV) within which cyst antigens are concentrated and transported to the nascent wall. The present kinetic and physiologic studies now show that ESV are the earliest morphologic change observed in encystation. Expression of ESV, as well as subsequent encystation, are regulated by exposure to bile at the slightly alkaline pH which is typical of the human intestinal tract. ESV formation appears to be less stringently regulated than formation of water-resistant cysts because omission of either encystation stimuli or alkaline pH preferentially inhibits encystation. Since cysts do not attach, we asked when in encystation this physiologic transition occurs. We found that most encysting trophozoites remain attached until they begin to round up (greater than 24 hr). However, if they are made to detach, as early as 12 hr in encystation, well before they round up, they are defective in the ability to reattach. If trophozoites also become less able to reattach to the intestinal epithelium early in encystation in vivo, this would increase their exposure to lumenal encystation stimuli and promote encystation. These studies have provided new insights into the complex sequence of morphologic and physiologic alterations which occur during encystation of G. lamblia in vitro and their regulation by host intestinal factors.     

MEK kinases are regulated by EGF and selectively interact with Rac/Cdc42.

MEK kinases (MEKKs) 1, 2, 3 and 4 are members of sequential kinase pathways that regulate MAP kinases including c-Jun NH2-terminal kinases (JNKs) and extracellular regulated kinases (ERKs). Confocal immunofluorescence microscopy of COS cells demonstrated differential MEKK subcellular localization: MEKK1 was nuclear and in post-Golgi vesicular-like structures; MEKK2 and 4 were localized to distinct Golgi-associated vesicles that were dispersed by brefeldin A. MEKK1 and 2 were activated by EGF, and kinase-inactive mutants of each MEKK partially inhibited EGF-stimulated JNK activity. Kinase-inactive MEKK1, but not MEKK2, 3 or 4, strongly inhibited EGF-stimulated ERK activity. In contrast to MEKK2 and 3, MEKK1 and 4 specifically associated with Rac and Cdc42 and kinase-inactive mutants blocked Rac/Cdc42 stimulation of JNK activity. Inhibitory mutants of MEKK1-4 did not affect p21-activated kinase (PAK) activation of JNK, indicating that the PAK-regulated JNK pathway is independent of MEKKs. Thus, in different cellular locations, specific MEKKs are required for the regulation of MAPK family members, and MEKK1 and 4 are involved in the regulation of JNK activation by Rac/Cdc42 independent of PAK. Differential MEKK subcellular distribution and interaction with small GTP-binding proteins provides a mechanism to regulate MAP kinase responses in localized regions of the cell and to different upstream stimuli.     

Fine structure of the biogenesis of Giardia lamblia encystation secretory vesicles

Synthesis, transport, and assembly of the extracellular cyst wall is the hallmark of Giardia lamblia encystation. Much is known of the biochemical pathways and their regulation. However, from a cell biology point of view, the biogenesis of the encystation specific vesicles (ESVs) that transport cyst wall proteins to the periphery of the cell is poorly understood. Therefore, we exploited a number of complementary ultrastructural approaches to test the hypothesis that the formation of ESVs utilizes a novel regulated secretory pathway. We analyzed parasites at different stages of encystation in vitro by electron microscopy of thin sections, freeze fracture replicas, and three-dimensional reconstruction from serial sections of cells fixed for cytochemical localization of the endoplasmic reticulum (ER) marker, glucose 6-phosphatase. We also used a stereological approach to determine the area occupied by the ER, clefts, ESVs, and cyst wall. Taken together, our kinetic data suggest that some ER cisternae first dilate to form clefts, which enlarge into the ESVs. Living non-encysting and early-encysting trophozoites were labeled around the periphery of both nuclei with C(6)-NBD-ceramide. At 18-21 h, outward migration of some ESVs frequently caused protrusions at the periphery of encysting trophozoites. The presence of lysosome-like peripheral vesicles between the ESV and plasma membrane of the cell was confirmed using acridine orange, an acidic compartment marker. Our data suggest that G. lamblia has a novel secretory pathway in which certain functions of the ER and Golgi co-localize spatially and temporally. These studies will increase understanding of the evolutionary appearance of regulated secretory pathways for assembly of a primitive extracellular matrix in an early diverging eukaryote.     

Mitogen-activated protein kinases in cell-cycle control

The mitogen-activated protein kinase (MAPK) family of kinases connects extracellular stimuli with diverse cellular responses ranging from activation or suppression of gene expression to the regulation of cell mortality, growth, and differentiation. The MAPK family has been studied extensively; however, the role of these kinases in cell growth and cell-cycle control has become increasingly complex. Patterns have begun to emerge from these studies that show the functions of MAPK subfamilies at different stages of the cell cycle. Their patterns of subcellular localization and movement during the cell cycle are subfamily-specific and have raised many questions about possible cell-cycle functions that have yet to be demonstrated. This article will compare and contrast our current understanding of the functions and localization patterns of the MAPK subfamilies (ERK, BMK, p38, and JNK) in cell-cycle control.     

Novel Reporter for Faithful Monitoring of ERK2 Dynamics in Living Cells and Model Organisms

Uncoupling of ERK1/2 phosphorylation from subcellular localization is essential towards the understanding of molecular mechanisms that control ERK1/2-mediated cell-fate decision. ERK1/2 non-catalytic functions and discoveries of new specific anchors responsible of the subcellular compartmentalization of ERK1/2 signaling pathway have been proposed as regulation mechanisms for which dynamic monitoring of ERK1/2 localization is necessary. However, studying the spatiotemporal features of ERK2, for instance, in different cellular processes in living cells and tissues requires a tool that can faithfully report on its subcellular distribution. We developed a novel molecular tool, ERK2-LOC, based on the T2A-mediated coexpression of strictly equimolar levels of eGFP-ERK2 and MEK1, to faithfully visualize ERK2 localization patterns. MEK1 and eGFP-ERK2 were expressed reliably and functionally both in vitro and in single living cells. We then assessed the subcellular distribution and mobility of ERK2-LOC using fluorescence microscopy in non-stimulated conditions and after activation/inhibition of the MAPK/ERK1/2 signaling pathway. Finally, we used our coexpression system in Xenopus laevis embryos during the early stages of development. This is the first report on MEK1/ERK2 T2A-mediated coexpression in living embryos, and we show that there is a strong correlation between the spatiotemporal subcellular distribution of ERK2-LOC and the phosphorylation patterns of ERK1/2. Our approach can be used to study the spatiotemporal localization of ERK2 and its dynamics in a variety of processes in living cells and embryonic tissues.     

Molecular cloning and characterization of a novel dual specificity phosphatase, MKP-5.

A group of dual specificity protein phosphatases negatively regulates members of the mitogen-activated protein kinase (MAPK) superfamily, which consists of three major subfamilies, MAPK/extracellular signal-regulated kinase (ERK), stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), and p38. Nine members of this group of dual specificity phosphatases have previously been cloned. They show distinct substrate specificities for MAPKs, different tissue distribution and subcellular localization, and different modes of inducibility of their expression by extracellular stimuli. Here we have cloned and characterized a novel dual specificity phosphatase, which we have designated MKP-5. MKP-5 is a protein of 482 amino acids with a calculated molecular mass of 52.6 kDa and consists of 150 N-terminal amino acids of unknown function, two Cdc25 homology 2 regions in the middle, and a C-terminal catalytic domain. MKP-5 binds to p38 and SAPK/JNK, but not to MAPK/ERK, and inactivates p38 and SAPK/JNK, but not MAPK/ERK. p38 is a preferred substrate. The subcellular localization of MKP-5 is unique; it is present evenly in both the cytoplasm and the nucleus. MKP-5 mRNA is widely expressed in various tissues and organs, and its expression in cultured cells is elevated by stress stimuli. These results suggest that MKP-5 is a novel type of dual specificity phosphatase specific for p38 and SAPK/JNK.     

PB1 domains of MEKK2 and MEKK3 interact with the MEK5 PB1 domain for activation of the ERK5 pathway

MEKK2 and MEKK3 are MAPK kinase kinases that activate the ERK5 pathway by phosphorylating and activating the MAPK kinase, MEK5. Activated MEK5 then phosphorylates and activates ERK5. PB1 domains were first defined in the p67phox and Bem1p proteins and have been shown to mediate protein-protein heterodimerization. A PB1 domain is encoded within the N-terminal portion of MEKK2, MEKK3, and MEK5. Herein, we analyze the functional role of MEKK2, MEKK3, and MEK5 PB1 domains in the ERK5 activation pathway. The PB1 domains of MEKK2 and MEKK3 bind the PB1 domain of MEK5 but do not significantly homo- or heterodimerize with one another in vitro. Furthermore, co-immunoprecipitation of MEKK2 and MEK5 from cell lysates shows that they form a complex in vivo. Deletion or mutation of the MEKK2 PB1 domain abolishes MEKK2-MEK5 complexes, demonstrating that the PB1 domain interaction is required for MEKK2-MEK5 interactions. Expression in cells of the MEKK2 or MEKK3 PB1 domain inhibits ERK5 activation, whereas expression of a mutant MEKK2 unable to bind the MEK5 PB1 domain or expression of the p67phox PB1 domain has no effect on ERK5 activation. These findings demonstrate that the PB1 domain mediates the association of MEKK2 and MEKK3 with MEK5 and that the respective PB1 domains of these kinases are critical for regulation of the ERK5 pathway. The free PB1 domain of MEKK2 or MEKK3 functions effectively to inhibit the ERK5 pathway but not the p38 or JNK pathways, demonstrating the specific and unique requirement of the MEKK2 and MEKK3 PB1 domain in regulating ERK5 activation.     

WNK4 inhibits NCC protein expression through MAPK ERK1/2 signaling pathway

WNK [with no lysine (K)] kinase is a subfamily of serine/threonine kinases. Mutations in two members of this family (WNK1 and WNK4) cause pseudohypoaldosteronism type II featuring hypertension, hyperkalemia, and metabolic acidosis. WNK1 and WNK4 were shown to regulate sodium chloride cotransporter (NCC) activity through phosphorylating SPAK and OSR1. Previous studies including ours have also shown that WNK4 inhibits NCC function and its protein expression. A recent study reported that a phorbol ester inhibits NCC function via activation of extracellular signal-regulated kinase (ERK) 1/2 kinase. In the current study, we investigated whether WNK4 affects NCC via the MAPK ERK1/2 signaling pathway. We found that WNK4 increased ERK1/2 phosphorylation in a dose-dependent manner in mouse distal convoluted tubule (mDCT) cells, whereas WNK4 mutants with the PHA II mutations (E562K and R1185C) lost the ability to increase the ERK1/2 phosphorylation. Hypertonicity significantly increased ERK1/2 phosphorylation in mDCT cells. Knock-down of WNK4 expression by siRNA resulted in a decrease of ERK1/2 phosphorylation. We further showed that WNK4 knock-down significantly increases the cell surface and total NCC protein expressions and ERK1/2 knock-down also significantly increases cell surface and total NCC expression. These data suggest that WNK4 inhibits NCC through activating the MAPK ERK1/2 signaling pathway.