Angiogenin activates Erk1/2 in human umbilical vein endothelial cells

Angiogenin is a potent angiogenic factor that binds to endothelial cells and is endocytosed and rapidly translocated to the nucleus where it is concentrated in the nucleolus and binds to DNA. Angiogenin also activates cell-associated proteases, induces cell invasion and migration, stimulates cell proliferation, and organizes cultured cells to form tubular structures. The intracellular signaling pathways that mediate these various cellular responses are not well understood. Here we report that angiogenin induces transient phosphorylation of extracellular signal-related kinase1/2 (Erk1/2) in cultured human umbilical vein endothelial cells. Angiogenin does not affect the phosphorylation status of stress-associated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p38 mitogen-activated protein (MAP) kinases. PD98059--a specific inhibitor of MAP or Erk kinase 1 (MEK 1), the upstream kinase that phosphorylates Erk1/2--abolishes angiogenin-induced Erk phosphorylation and cell proliferation without affecting nuclear translocation of angiogenin. In contrast, neomycin, a known inhibitor of nuclear translocation and cell proliferation, does not interfere with angiogenin-induced Erk1/2 phosphorylation. These data indicate that both intracellular signaling pathways and direct nuclear functions of angiogenin are required for angiogenin-induced cell proliferation and angiogenesis.     

Molecular cloning of mammalian Spred-3 which suppresses tyrosine kinase-mediated Erk activation

We have reported on Spred-1 and Spred-2, which inhibit MAP kinase activation by interacting with c-kit and ras/raf. Here, we report the cloning of a third member in this family, Spred-3. Spred-3 is expressed exclusively in the brain and its gene locates in chromosome 19q13.13 in human. Like Spred-1 and -2, Spred-3 contains an EVH1 domain in the N-terminus and a Sprouty-related cysteine-rich region (SPR domain) in the C-terminus that is necessary for membrane localization. However, Spred-3 does not possess a functional c-kit binding domain (KBD), since the critical amino acid Arg residue in this region was replaced with Gly in Spred-3. Although Spred-3 suppressed growth factor-induced MAP kinase (Erk) activation, inhibitory activity of Spred-3 was lower than that of Spred-1 or Spred-2. By the analysis of chimeric molecules between Spred-3 and Spred-1, we found that the SPR domain, rather than KBD, is responsible for efficient Erk suppression. The finding of Spred-3 revealed the presence of a novel family of regulators for the Ras/MAP kinase pathway, each member of which may have different specificities for extracellular signals.     

Examining the mechanism of Erk nuclear translocation using green fluorescent protein

In neuronal cells, the mitogen-activated protein kinase (MAP kinase) cascade is an important mediator of neurotrophin signaling from cell surface receptors to the nucleus, resulting in changes in gene expression. Nuclear localization of Erk is thought to be required for these effects. To examine the mechanism and regulation of Erk nuclear translocation, we have created a green fluorescent protein (GFP)-labeled Erk2 construct, which provides a sensitive means to follow the movement of Erk from the cytoplasm to the nucleus following receptor-mediated MAP kinase activation. Using this system in PC12 cells, we have examined a number of mechanisms that have been implicated in regulating the translocation of Erk. In PC12 cells, NGF and EGF induce a rapid translocation of GFP-Erk that requires Ras and Mek. We have found that prolonged phosphorylation of Erk is not required for the rapid and early influx of Erk into the nucleus following growth factor stimulation. Furthermore, following influx, GFP-Erk rapidly returned to the cytoplasm regardless of its phosphorylation state. The release of Erk from its cytoplasmic activator, Mek, followed by the dimerization of Erk, was sufficient to stimulate nuclear uptake, whereas Erk kinase activity was dispensable. PKA activity has been reported to be required for Erk translocation in PC12 cells. However, PKA activity was also not necessary for the early translocation of Erk into the nucleus by NGF or Ras, but it was able to induce a small influx of Erk that could be measured with GFP-Erk2.     

Activation of mitogen-activated protein kinases (Erk1 and Erk2) cascade results in phosphorylation of NF-M tail domains in transfected NIH 3T3 cells.

Neurofilaments (NFs) are neuron-specific intermediate filaments, and are the major cytoskeletal component in large myelinated axons. Lysine-serine-proline (KSP) repeats in the tail domains of high molecular weight NF proteins (NF-M and NF-H) are extensively phosphorylated in vivo in the axon. This phosphorylation in the tail domain has been postulated to play an important role in mediating neuron-specific properties, including axonal caliber and conduction velocity. Recent studies have shown that the mitogen-activated protein kinases (extracellular signal-regulated kinases, Erk1 and Erk2) phosphorylate KSP motifs in peptide substrates derived from the NF-M and NF-H tail domains in vitro. However, it is not clear whether activation of the mitogen activated protein (MAP) kinase pathway is able to phosphorylate these domains in vivo. To answer this question, a constitutively active form of mitogen-activated Erk activating kinase (MEK1) was cotransfected with an NF-M expression construct into NIH 3T3 cells. The activated mutant, but not the dominant negative mutant, induced phosphorylation of NF-M. In addition, it was shown that epidermal growth factor, which induces the MAP kinase cascade in NIH 3T3 cells, also activated endogenous Erk1 and Erk2 and NF-M tail domain phosphorylation in the transfected cells. These results present direct evidence that in-vivo activation of Erk1 and Erk 2 is sufficient for NF-M tail domain phosphorylation in transfected cells.     

Phosphatidylinositol 3-kinase contributes to Erk1/Erk2 MAP kinase activation associated with hepatocyte growth factor-induced cell scattering

MAP kinase cascade-dependent responses were investigated during scattering of HepG2 human hepatoma cells stimulated by HGF or phorbol ester. Inhibition of phosphatidylinositol 3-kinase with LY294002 prevented completely the dissociation of cells. Inhibition of MAP kinase kinase (MEK) with PD98059 prevented the development of characteristic morphological changes associated with cell migration. EGF, which failed to induce cell scattering, caused a short-term increase in the phosphorylation of Erk1/Erk2 MAP kinases. On the contrary, HGF or phorbol ester stimulated the phosphorylation of MAP kinases for a long time. Experiments performed with LY294002 indicated that phosphatidylinositol 3-kinase contributed to the HGF-stimulated phosphorylation of Erk1/Erk2. This finding was confirmed by the demonstration that the MAP kinase cascade-dependent expression of a high-Mr (>300 kDa) protein pair appearing in the course of cell scattering was inhibited by LY294002 in HGF-induced cells but was not inhibited in phorbol ester-treated cells.     

Activation of the Erk8 mitogen-activated protein (MAP) kinase by RET/PTC3, a constitutively active form of the RET proto-oncogene

Mitogen-activated protein (MAP) kinases have a central role in several biological functions, including cell adhesion and spreading, chemotaxis, cell cycle progression, differentiation, and apoptosis. Extracellular signal-regulated kinase 8 (Erk8) is a large MAP kinase whose activity is controlled by serum and the c-Src non-receptor tyrosine kinase. Here, we show that RET/PTC3, an activated form of the RET proto-oncogene, was able to activate Erk8, and we demonstrate that such MAP kinase participated in RET/PTC3-dependent stimulation of the c-jun promoter. By using RET/PTC3 molecules mutated in specific tyrosine autophosphorylation sites, we characterized Tyr(981), a known binding site for c-Src, as a major determinant of RET/PTC3-induced Erk8 activation, although, surprisingly, the underlying mechanism did not strictly depend on the activity of Src. In contrast, we present evidence that RET/PTC3 acts on Erk8 through Tyr(981)-mediated activation of c-Abl. Furthermore, we localized the region responsible for the modulation of Erk8 activity by the RET/PTC3 and Abl oncogenes in the Erk8 C-terminal domain. Altogether, these results support a role for Erk8 as a novel effector of RET/PTC3 and, therefore, RET biological functions.     

Protein kinase C decreases the hepatocyte growth factor-induced activation of Erk1/Erk2 MAP kinases

HGF and phorbol ester induce the scattering of HepG2 cells. Recently, we have reported that the motility and morphological responses that accompany this process require the activation of Erk1/Erk2 MAP kinases, and phosphatidylinositol 3-kinase contributes to the activation of Erk1/Erk2 in HGF-induced cells. The cell scattering-associated appearance of a high-M(r) (>300 kDa) protein pair has also been observed, and has been proven to be a sensitive marker of the intensity of Erk1/Erk2 activation. Our present study demonstrates that in HGF-induced cells protein kinase C and phosphatidylinositol 3-kinase regulate oppositely the expression of these cell scattering-associated proteins. While in phorbol ester-treated cells the sustained activation of protein kinase C is essential for this expression, in HGF-induced cells the inhibition of protein kinase C with bisindolylmaleimide I stimulates the expression. Protein kinase C reduces the HGF-induced phosphorylation of Erk1/Erk2, and in this way it can limit the intensity of Erk1/Erk2-dependent gene-expression     

Non-transactivational, dual pathways for LPA-induced Erk1/2 activation in primary cultures of brown pre-adipocytes

In many cell types, G-protein-coupled receptor (GPCR)-induced Erk1/2 MAP kinase activation is mediated via receptor tyrosine kinase (RTK) transactivation, in particular via the epidermal growth factor (EGF) receptor. Lysophosphatidic acid (LPA), acting via GPCRs, is a mitogen and MAP kinase activator in many systems, and LPA can regulate adipocyte proliferation. The mechanism by which LPA activates the Erk1/2 MAP kinase is generally accepted to be via EGF receptor transactivation. In primary cultures of brown pre-adipocytes, EGF can induce Erk1/2 activation, which is obligatory and determinant for EGF-induced proliferation of these cells. Therefore, we have here examined whether LPA, via EGF transactivation, can activate Erk1/2 in brown pre-adipocytes. We found that LPA could induce Erk1/2 activation. However, the LPA-induced Erk1/2 activation was independent of transactivation of EGF receptors (or PDGF receptors) in these cells (whereas in transformed HIB-1B brown adipocytes, the LPA-induced Erk1/2 activation indeed proceeded via EGF receptor transactivation). In the brown pre-adipocytes, LPA instead induced Erk1/2 activation via two distinct non-transactivational pathways, one G_i-protein dependent, involving PKC and Src activation, the other, a PTX-insensitive pathway, involving PI3K (but not Akt) activation. Earlier studies showing LPA-induced Erk1/2 activation being fully dependent on RTK transactivation have all been performed in cell lines and transfected cells. The present study implies that in non-transformed systems, RTK transactivation may not be involved in the mediation of GPCR-induced Erk1/2 MAP kinase activation.     

Erk MAP kinase regulates branching morphogenesis in the developing mouse kidney.

Branching morphogenesis of epithelium is a common and important feature of organogenesis; it is, for example, responsible for development of renal collecting ducts, lung airways, milk ducts of mammary glands and seminal ducts of the prostate. In each case, epithelial development is controlled by a variety of mesenchyme-derived molecules, both soluble (e.g. growth factors) and insoluble (e.g. extracellular matrix). Little is known about how these varied influences are integrated to produce a coherent morphogenetic response, but integration is likely to be achieved at least partly by cytoplasmic signal transduction networks. Work in other systems (Drosophila tracheae, MDCK models) suggests that the mitogen-activated protein (MAP) kinase pathway might be important to epithelial branching. We have investigated the role of the MAP kinase pathway in one of the best characterised mammalian examples of branching morphogenesis, the ureteric bud of the metanephric kidney. We find that Erk MAP kinase is normally active in ureteric bud, and that inhibiting Erk activation with the MAP kinase kinase inhibitor, PD98059, reversibly inhibits branching in a dose-dependent manner, while allowing tubule elongation to continue. When Erk activation is inhibited, ureteric bud tips show less cell proliferation than controls and they also produce fewer laminin-rich processes penetrating the mesenchyme and fail to show the strong concentration of apical actin filaments typical of controls; apoptosis and expression of Ret and Ros, are, however, normal. The activity of the Erk MAP kinase pathway is dependent on at least two known regulators of ureteric bud branching; the GDNF-Ret signalling system and sulphated glycosaminoglycans. MAP kinase is therefore essential for normal branching morphogenesis of the ureteric bud, and lies downstream of significant extracellular regulators of ureteric bud development.     

Erk phosphorylates threonine 42 residue of ribosomal protein S3

The ribosomal protein S3 (rpS3) is involved in ribosome biogenesis as a member of ribosomal small subunit and also plays a role in the repair of damaged DNA. Extracellular signal-regulated kinase (Erk), a MAP kinase, is known to play important roles in the regulation of cell growth, differentiation, and apoptosis. In this study, the sequence analysis of rpS3 protein revealed that this protein has a putative FXFP motif which is believed to be an Erk binding site. Indeed, the motif was demonstrated as an Erk binding site by co-immunoprecipitation. In addition to this, it was revealed that Erk specifically phosphorylated Thr 42 residue of rpS3 in vitro and in vivo using the various mutants of rpS3. Taken together, rpS3 appears to be phosphorylated by activated Erk in proliferating cells, resulting in the decreased interaction between two proteins.     

Erk 5 is necessary for sustained PDGF-induced Akt phosphorylation and inhibition of apoptosis

Extracellular regulated kinase (Erk) 5 is a member of the mitogen activated protein (MAP) kinase family that has been implicated in both cell proliferation and survival. In the present study, we found that stimulation with platelet-derived growth factor (PDGF)-BB leads to a transient activation of Erk5, which was shown to be dependent on recruitment of both Src kinases and the tyrosine phosphatase Shp2 to the activated PDGF receptor β (PDGFRβ). We could also demonstrate that Shp2 docking to the receptor is critical for Src kinase activation, suggesting that Shp2 may contribute to Erk5 activation through its involvement in Src kinase activation. Under control conditions, PDGF-BB promoted a sustained Akt phosphorylation. However, reduction of the expression of Erk5 by siRNA resulted in only a transient Akt phosphorylation, and an inability of PDGF-BB to suppress caspase 3 activation and inhibit apoptotic nuclear morphological changes such as condensed or fragmented chromatin under serum-free conditions.     

Nek2A specifies the centrosomal localization of Erk2

Nek2A is a cell-cycle-regulated protein kinase that localizes to the centrosome and kinetochore. Our recent studies provide a link between Nek2A and spindle checkpoint signaling [J. Biol. Chem. 279 (2004) 20049]. Extracellular signal-regulated kinase 2 (Erk2) is an important kinase, which belongs to mitogen activating protein (MAP) kinase family. Here we demonstrated that Nek2A binds specifically to Erk2. Erk2 interacts with Nek2A via a conserved Erk2 docking site located to the C-terminus of Nek2A. Our studies indicate this docking site is essential and sufficient for a direct Nek2A-Erk2 interaction. In addition, our immunocytochemical studies show that Nek2A and Erk2 are co-localized to centrosome. Significantly, elimination of Nek2A by RNA interference delocalized Erk2 from its centrosomal location, while inhibition of Erk2 kinase activity did not affect the localization of Nek2A in centrosome. We propose that Erk2 links extracellular signaling to centrosome dynamics by Nek2A.     

TGF-beta activates Erk MAP kinase signalling through direct phosphorylation of ShcA

Erk1/Erk2 MAP kinases are key regulators of cell behaviour and their activation is generally associated with tyrosine kinase signalling. However, TGF-beta stimulation also activates Erk MAP kinases through an undefined mechanism, albeit to a much lower level than receptor tyrosine kinase stimulation. We report that upon TGF-beta stimulation, the activated TGF-beta type I receptor (TbetaRI) recruits and directly phosphorylates ShcA proteins on tyrosine and serine. This dual phosphorylation results from an intrinsic TbetaRI tyrosine kinase activity that complements its well-defined serine-threonine kinase function. TGF-beta-induced ShcA phosphorylation induces ShcA association with Grb2 and Sos, thereby initiating the well-characterised pathway linking receptor tyrosine kinases with Erk MAP kinases. We also found that TbetaRI is tyrosine phosphorylated in response to TGF-beta. Thus, TbetaRI, like the TGF-beta type II receptor, is a dual-specificity kinase. Recruitment of tyrosine kinase signalling pathways may account for aspects of TGF-beta biology that are independent of Smad signalling.     

Apical expression of human full-length hCEACAM1-4L protein renders the Madin Darby Canine Kidney cells responsive to lipopolysaccharide leading to TLR4-dependent Erk1/2 and p38 MAPK signalling

CEACAM1 expressed by granulocytes and epithelial cells is recognized as a membrane-associated receptor by some Gram-negative pathogens. Here we report a previously unsuspected role of human CEACAM1-4L (hCEACAM1-4L) in polarized epithelial cells. We find that in contrast with non-transfected cells, Madin Darby Canine Kidney strain II (MDCK) engineered for the apical expression of the long cytoplasmic chain protein hCEACAM1-4L showed a serum-independent increase in the phosphorylation of the extracellular signal-regulated kinase 1/2 (Erk1/2) and p38 mitogen-activated protein kinases (MAPKs) after treatment with lipopolysaccharide (LPS) of wild-type, diffusely adhering Afa/Dr Escherichia coli (Afa/Dr DAEC) strain IH11128. Aggregates of FITC-LPS bind the apical domain of MDCK-hCEACAM1-4L cells colocalizing with the apically expressed hCEACAM1-4L protein and do not bind MDCK-pCEP cells, and surface plasmon resonance analysis shows that LPS binds to the extracellular domain of the CEACAM1-4L protein. We showed that cell polarization and lipid rafts positively control the LPS-IH11128-induced phosphorylation of Erk1/2 in MDCK-hCEACAM1-4L cells. Structure-function analysis using mutated hCEACAM1-4L protein shows that the cytoplasmic domain of the protein is needed for LPS-induced MAPK signalling, and that phosphorylation of Tyr-residues is not increased in association with MAPK signalling. The hCEACAM1-4L-dependent Erk1/2 phosphorylation develops in the presence of lipid A and does not develop in the presence of penta-acylated LPS. Finally, small interfering RNA (siRNA) silencing of canine TLR4 abolishes the hCEACAM1-4L-dependent, LPS-induced phosphorylation of Erk1/2. Collectively, our results support the notion that the apically expressed, full-length hCEACAM1-4L protein functions as a novel LPS-conveying molecule at the mucosal surface of polarized epithelial cells for subsequent MD-2/TLR4 receptor-dependent MAPK Erk1/2 and p38 signalling.     

Bisindolylmaleimide I suppresses fibroblast growth factor-mediated activation of Erk MAP kinase in chondrocytes by preventing Shp2 association with the Frs2 and Gab1 adaptor proteins

Fibroblast growth factors (FGFs) inhibit chondrocyte proliferation via the Erk MAP kinase pathway. Here, we explored the role of protein kinase C in FGF signaling in chondrocytes. Erk activity in FGF2-treated RCS (rat chondrosarcoma) chondrocytes or human primary chondrocytes was abolished by the protein kinase C inhibitor bisindolylmaleimide I (Bis I). Bis I inhibited FGF2-induced activation of MEK, Raf-1, and Ras members of Erk signaling module but not the FGF2-induced tyrosine phosphorylation of Frs2 or the kinase activity of FGFR3, demonstrating that it targets the Erk cascade immediately upstream of Ras. Indeed, Bis I abolished the FGF2-mediated association of Shp2 tyrosine phosphatase with Frs2 and Gab1 adaptor proteins necessary for proper Ras activation. We also determined which PKC isoform is involved in FGF2-mediated activation of Erk. When both conventional and novel PKCs expressed by RCS chondrocytes (PKCalpha, -gamma, -delta, and -epsilon) were down-regulated by phorbol ester, cells remained responsive to FGF2 with Erk activation, and this activation was sensitive to Bis I. Moreover, treatment with PKClambda/zeta pseudosubstrate lead to significant reduction of FGF2-mediated activation of Erk, suggesting involvement of an atypical PKC.     

Erk1/2-dependent phosphorylation of Galpha-interacting protein stimulates its GTPase accelerating activity and autophagy in human colon cancer cells

Galpha-interacting protein (GAIP) is a regulator of G protein signaling (RGS) that accelerates the rate of GTP hydrolysis by the alpha-subunit of the trimeric G(i3) protein. Both proteins are part of a signaling pathway that controls lysosomal-autophagic catabolism in human colon cancer HT-29 cells. Here we show that GAIP is phosphorylated by an extracellular signal-regulated (Erk1/2) MAP kinase-dependent pathway sensitive to amino acids, MEK1/2 (PD098059), and protein kinase C (GF109203X) inhibitors. An in vitro phosphorylation assay demonstrates that Erk2-dependent phosphorylation of GAIP stimulates its GTPase-activating protein activity toward the Galpha(i3) protein (k = 0.187 +/- 0.001 s(-)(1), EC(50) = 1.12 +/- 0.10 microm) when compared with unphosphorylated recombinant GAIP (k = 0.145 +/- 0.003 s(-)(1), EC(50) = 3.16 +/- 0. 12 microm) or to GAIP phosphorylated by other Ser/Thr protein kinases (protein kinase C, casein kinase II). This stimulation and the phosphorylation of GAIP by Erk2 were abrogated when serine at position 151 in the RGS domain was substituted by an alanine residue using site-directed mutagenesis. Furthermore, the lysosomal-autophagic pathway was not stimulated in S151A-GAIP mutant-expressing cells when compared with wild-type GAIP-expressing cells. These results demonstrate that the GTPase-activating protein activity of GAIP is stimulated by Erk2 phosphorylation. They also suggested that Erk1/2 and GAIP are engaged in the signaling control of a major catabolic pathway in intestinal derived cells.     

Mitogen-activated protein kinases activate the serine/threonine kinases Mnk1 and Mnk2.

Mitogen-activated protein (MAP) kinases bind tightly to many of their physiologically relevant substrates. We have identified a new subfamily of murine serine/threonine kinases, whose members, MAP kinase-interacting kinase 1 (Mnk1) and Mnk2, bind tightly to the growth factor-regulated MAP kinases, Erk1 and Erk2. MNK1, but not Mnk2, also binds strongly to the stress-activated kinase, p38. MNK1 complexes more strongly with inactive than active Erk, implying that Mnk and Erk may dissociate after mitogen stimulation. Erk and p38 phosphorylate MNK1 and Mnk2, which stimulates their in vitro kinase activity toward a substrate, eukaryotic initiation factor-4E (eIF-4E). Initiation factor eIF-4E is a regulatory phosphoprotein whose phosphorylation is increased by insulin in an Erk-dependent manner. In vitro, MNK1 rapidly phosphorylates eIF-4E at the physiologically relevant site, Ser209. In cells, Mnk1 is post-translationally modified and enzymatically activated in response to treatment with either peptide growth factors, phorbol esters, anisomycin or UV. Mitogen- and stress-mediated MNK1 activation is blocked by inhibitors of MAP kinase kinase 1 (Mkk1) and p38, demonstrating that Mnk1 is downstream of multiple MAP kinases. MNK1 may define a convergence point between the growth factor-activated and one of the stress-activated protein kinase cascades and is a candidate to phosphorylate eIF-4E in cells.     

MKP3 negatively modulates PDGF-induced Akt and Erk5 phosphorylation as well as chemotaxis

MAP kinase phosphatase-3 (MKP3), also known as DUSP6 or Pyst1, is a dual specificity phosphatase considered to selectively dephosphorylate extracellular-signal-regulated kinase 1/2 (Erk1/2). Here, we report that in NIH3T3 cells, MKP3 is induced in response to platelet-derived growth factor (PDGF)-BB treatment in an Erk1/2- and phosphatidylinositol 3-kinase (PI3K)-dependent manner, but independently of Erk5 expression. Silencing of MKP3 expression did not affect PDGF-BB-induced Erk1/2 or p38 phosphorylation; however, their basal level of phosphorylation was elevated. Furthermore, we found that PDGF-BB-mediated activation of Erk5 and Akt was enhanced when the MKP3 expression was reduced. Interfering with Mek1/2 or PI3K using the inhibitors CI-1040 and LY-294002, respectively, inhibited PDGF-BB-induced MKP3 expression. Functionally, we found that MKP3 silencing did not affect cell proliferation, but enhanced the chemotactic response toward PDGF-BB. Although both Akt and Erk5 have been linked to increased cell survival, downregulation of MKP3 did not alter the ability of PDGF-BB to protect NIH3T3 cells from starvation-induced apoptosis. However, we observed an increased apoptosis in untreated cells with reduced MKP3 expression. In summary, our data indicate that there is negative cross-talk between Erk1/2 and Erk5 that involves regulation of MKP3 expression, and that PI3K in addition to promoting Akt phosphorylation also negatively modulates Akt, through MKP3 expression.