共查询到20条相似文献,搜索用时 15 毫秒
1.
L McKendrick S J Morley V M Pain R Jagus B Joshi 《European journal of biochemistry》2001,268(20):5375-5385
Eukaryotic translation initiation factor 4E (eIF4E) is essential for efficient translation of the vast majority of capped cellular mRNAs; it binds the 5'-methylated guanosine cap of mRNA and serves as a nucleation point for the assembly of the 48S preinitiation complex. eIF4E is phosphorylated in vivo at residue 209 of the human sequence. The phosphorylated form is often regarded as the active state of the protein, with ribosome-associated eIF4E enriched for the phosphorylated form and increased phosphorylation often correlated with upregulation of rates of protein synthesis. However, the only reported measured effect attributable to phosphorylation at the physiological site has been a relatively small increase in the affinity of eIF4E for the mRNA m7GTP cap structure. Here, we provide data to suggest that phosphorylation of eIF4E at Ser209 is not required for translation. eIF4E that is modified such that it cannot be phosphorylated (Ser209-->Ala), is unimpaired in its ability to restore translation to an eIF4E-dependent in vitro translation system. In addition, both the wild-type and mutant forms of eIF4E interact equally well with eIF4G, with the phosphorylation of eIF4E not required to effect the change in conformation of eIF4G that is required for efficient cleavage of eIF4G by L-protease. Furthermore, we show that wild-type and phosphorylation-site variants of eIF4E protein are equally able to rescue the lethal phenotype of eIF4E deletion in S. cerevisiae. 相似文献
2.
3.
Callie A. Norris Mu He Liang-I Kang Michael Qi Ding Josiah E. Radder Meagan M. Haynes Yu Yang Shirish Paranjpe William C. Bowen Anne Orr George K. Michalopoulos Donna B. Stolz Wendy M. Mars 《PloS one》2014,9(4)
Exogenous interleukin 6 (IL-6), synthesized at the initiation of the acute phase response, is considered responsible for signaling hepatocytes to produce acute phase proteins. It is widely posited that IL-6 is either delivered to the liver in an endocrine fashion from immune cells at the site of injury, or alternatively, in a paracrine manner by hepatic immune cells within the liver. A recent publication showed there was a muted IL-6 response in lipopolysaccharide (LPS)-injured mice when nuclear NFκB was specifically inactivated in the hepatocytes. This indicates hepatocellular signaling is also involved in regulating the acute phase production of IL-6. Herein, we present extensive in vitro and in vivo evidence that normal hepatocytes are directly induced to synthesize IL-6 mRNAs and protein by challenge with LPS, a bacterial hepatotoxin, and by HGF, an important regulator of hepatic homeostasis. As the IL-6 receptor is found on the hepatocyte, these results reveal that induction of the acute phase response can be regulated in an autocrine as well as endocrine/paracrine fashion. Further, herein we provide data indicating that following partial hepatectomy (PHx), HGF differentially regulates IL-6 production in hepatocytes (induces) versus immune cells (suppresses), signifying disparate regulation of the cell sources involved in IL-6 production is a biologically relevant mechanism that has previously been overlooked. These findings have wide ranging ramifications regarding how we currently interpret a variety of in vivo and in vitro biological models involving elements of IL-6 signaling and the hepatic acute phase response. 相似文献
4.
Zheng Tan Na Xie Sami Banerjee Huachun Cui Mingui Fu Victor J. Thannickal Gang Liu 《The Journal of biological chemistry》2015,290(1):46-55
There has been fast growing evidence showing that glycolysis plays a critical role in the activation of immune cells. Enhanced glycolysis leads to increased formation of intracellular lactate that is exported to the extracellular environment by monocarboxylate transporter 4 (MCT4). Although the biological activities of extracellular lactate have been well studied, it is less understood how the lactate export is regulated or whether lactate export affects glycolysis during inflammatory activation. In this study, we found that MCT4 is up-regulated by TLR2 and TLR4, but not TLR3 agonists in a variety of macrophages. The increased expression of MCT4 was mediated by MYD88 in a NF-κB-dependent manner. Furthermore, we found that MCT4 is required for macrophage activation upon TLR2 and TLR4 stimulations, as evidenced by attenuated expression of proinflammatory mediators in macrophages with MCT4 knockdown. Mechanistically, we found that MCT4 knockdown leads to enhanced intracellular accumulation of lactate and decreased glycolysis in LPS-treated macrophages. We found that LPS-induced expression of key glycolytic enzymes hexokinase 2 and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 is diminished in macrophages with MCT4 knockdown. Our data suggest that MCT4 up-regulation represents a positive feedback mechanism in macrophages to maintain a high glycolytic rate that is essential to a fully activated inflammatory response. 相似文献
5.
Tram Thu Vuong Trine M. Reine Amanda Sudworth Trond G. Jenssen Svein O. Kolset 《The journal of histochemistry and cytochemistry》2015,63(4):280-292
Syndecans are important cell surface proteoglycans with many functions; yet, they have not been studied to a very large extent in primary human endothelial cells. The purpose of this study was to investigate syndecan-4 expression in cultured human umbilical vein endothelial cells (HUVECs) and assess its role in inflammatory reactions and experimental wound healing. qRT-PCR analysis revealed that syndecan-3 and syndecan-4 were highly expressed in HUVECs, whereas the expression of syndecan-1 and -2 was low. HUVECs were cultured with the inflammatory mediators lipopolysaccharide (LPS) and interleukin 1β (IL-1β). As a result, syndecan-4 expression showed a rapid and strong increase. Syndecan-1 and -2 expressions decreased, whereas syndecan-3 was unaffected. Knockdown of syndecan-4 using siRNA resulted in changes in cellular morphology and focal adhesion sites, delayed wound healing and tube formation, and increased secretion of the pro-inflammatory and angiogenic chemokine, CXCL8. These data suggest functions for syndecan-4 in inflammatory reactions, wound healing and angiogenesis in primary human endothelial cells. 相似文献
6.
7.
The translation initiation factor eIF-4E binds to a common motif shared by the translation factor eIF-4 gamma and the translational repressors 4E-binding proteins. 总被引:23,自引:14,他引:23 下载免费PDF全文
Eukaryotic translation initiation factor 4E (eIF-4E), which possesses cap-binding activity, functions in the recruitment of mRNA to polysomes as part of a three-subunit complex, eIF-4F (cap-binding complex). eIF-4E is the least abundant of all translation initiation factors and a target of growth regulatory pathways. Recently, two human cDNAs encoding novel eIF-4E-binding proteins (4E-BPs) which function as repressors of cap-dependent translation have been cloned. Their interaction with eIF-4E is negatively regulated by phosphorylation in response to cell treatment with insulin or growth factors. The present study aimed to characterize the molecular interactions between eIF-4E and the other subunits of eIF-4F and to similarly characterize the molecular interactions between eIF-4E and the 4E-BPs. A 49-amino-acid region of eIF-4 gamma, located in the N-terminal side of the site of cleavage by Picornaviridae protease 2A, was found to be sufficient for interacting with eIF-4E. Analysis of deletion mutants in this region led to the identification of a 12-amino-acid sequence conserved between mammals and Saccharomyces cerevisiae that is critical for the interaction with eIF-4E. A similar motif is found in the amino acid sequence of the 4E-BPs, and point mutations in this motif abolish the interaction with eIF-4E. These results shed light on the mechanisms of eIF-4F assembly and on the translational regulation by insulin and growth factors. 相似文献
8.
James Sinnett-Smith Rodrigo Jacamo Robert Kui YunZu M. Wang Steven H. Young Osvaldo Rey Richard T. Waldron Enrique Rozengurt 《The Journal of biological chemistry》2009,284(20):13434-13445
Rapid protein kinase D (PKD) activation and phosphorylation via protein
kinase C (PKC) have been extensively documented in many cell types cells
stimulated by multiple stimuli. In contrast, little is known about the role
and mechanism(s) of a recently identified sustained phase of PKD activation in
response to G protein-coupled receptor agonists. To elucidate the role of
biphasic PKD activation, we used Swiss 3T3 cells because PKD expression in
these cells potently enhanced duration of ERK activation and DNA synthesis in
response to Gq-coupled receptor agonists. Cell treatment with the
preferential PKC inhibitors GF109203X or Gö6983 profoundly inhibited PKD
activation induced by bombesin stimulation for <15 min but did not prevent
PKD catalytic activation induced by bombesin stimulation for longer times
(>60 min). The existence of sequential PKC-dependent and PKC-independent
PKD activation was demonstrated in 3T3 cells stimulated with various
concentrations of bombesin (0.3–10 nm) or with vasopressin, a
different Gq-coupled receptor agonist. To gain insight into the
mechanisms involved, we determined the phosphorylation state of the activation
loop residues Ser744 and Ser748. Transphosphorylation
targeted Ser744, whereas autophosphorylation was the predominant
mechanism for Ser748 in cells stimulated with Gq-coupled
receptor agonists. We next determined which phase of PKD activation is
responsible for promoting enhanced ERK activation and DNA synthesis in
response to Gq-coupled receptor agonists. We show, for the first
time, that the PKC-independent phase of PKD activation mediates prolonged ERK
signaling and progression to DNA synthesis in response to bombesin or
vasopressin through a pathway that requires epidermal growth factor
receptor-tyrosine kinase activity. Thus, our results identify a novel
mechanism of Gq-coupled receptor-induced mitogenesis mediated by
sustained PKD activation through a PKC-independent pathway.The understanding of the mechanisms that control cell proliferation
requires the identification of the molecular pathways that govern the
transition of quiescent cells into the S phase of the cell cycle. In this
context the activation and phosphorylation of protein kinase D
(PKD),4 the founding
member of a new protein kinase family within the
Ca2+/calmodulin-dependent protein kinase (CAMK) group and separate
from the previously identified PKCs (for review, see Ref.
1), are attracting intense
attention. In unstimulated cells, PKD is in a state of low catalytic (kinase)
activity maintained by autoinhibition mediated by the N-terminal domain, a
region containing a repeat of cysteinerich zinc finger-like motifs and a
pleckstrin homology (PH) domain
(1–4).
Physiological activation of PKD within cells occurs via a
phosphorylation-dependent mechanism first identified in our laboratory
(5–7).
In response to cellular stimuli
(1), including phorbol esters,
growth factors (e.g. PDGF), and G protein-coupled receptor (GPCR)
agonists (6,
8–16)
that signal through Gq, G12, Gi, and Rho
(11,
15–19),
PKD is converted into a form with high catalytic activity, as shown by in
vitro kinase assays performed in the absence of lipid co-activators
(5,
20).During these studies multiple lines of evidence indicated that PKC activity
is necessary for rapid PKD activation within intact cells. For example, rapid
PKD activation was selectively and potently blocked by cell treatment with
preferential PKC inhibitors (e.g. GF109203X or Gö6983) that do
not directly inhibit PKD catalytic activity
(5,
20), implying that PKD
activation in intact cells is mediated directly or indirectly through PKCs.
Many reports demonstrated the operation of a rapid PKC/PKD signaling cascade
induced by multiple GPCR agonists and other receptor ligands in a range of
cell types (for review, see Ref.
1). Our previous studies
identified Ser744 and Ser748 in the PKD activation loop
(also referred as activation segment or T-loop) as phosphorylation sites
critical for PKC-mediated PKD activation
(1,
4,
7,
17,
21). Collectively, these
findings demonstrated the existence of a rapidly activated PKC-PKD protein
kinase cascade(s). In a recent study we found that the rapid PKC-dependent PKD
activation was followed by a late, PKC-independent phase of catalytic
activation and phosphorylation induced by stimulation of the bombesin
Gq-coupled receptor ectopically expressed in COS-7 cells
(22). This study raised the
possibility that PKD mediates rapid biological responses downstream of PKCs,
whereas, in striking contrast, PKD could mediate long term responses through
PKC-independent pathways. Despite its potential importance for defining the
role of PKC and PKD in signal transduction, this hypothesis has not been
tested in any cell type.Accumulating evidence demonstrates that PKD plays an important role in
several cellular processes and activities, including signal transduction
(14,
23–25),
chromatin organization (26),
Golgi function (27,
28), gene expression
(29–31),
immune regulation (26), and
cell survival, adhesion, motility, differentiation, DNA synthesis, and
proliferation (for review, see Ref.
1). In Swiss 3T3 fibroblasts, a
cell line used extensively as a model system to elucidate mechanisms of
mitogenic signaling
(32–34),
PKD expression potently enhances ERK activation, DNA synthesis, and cell
proliferation induced by Gq-coupled receptor agonists
(8,
14). Here, we used this model
system to elucidate the role and mechanism(s) of biphasic PKD activation.
First, we show that the Gq-coupled receptor agonists bombesin and
vasopressin, in contrast to phorbol esters, specifically induce PKD activation
through early PKC-dependent and late PKC-independent mechanisms in Swiss 3T3
cells. Subsequently, we demonstrate for the first time that the
PKC-independent phase of PKD activation is responsible for promoting ERK
signaling and progression to DNA synthesis through an epidermal growth factor
receptor (EGFR)-dependent pathway. Thus, our results identify a novel
mechanism of Gq-coupled receptor-induced mitogenesis mediated by
sustained PKD activation through a PKC-independent pathway. 相似文献
9.
S Joshi-Barve A De Benedetti R E Rhoads 《The Journal of biological chemistry》1992,267(29):21038-21043
Expression of antisense RNA against eukaryotic translation initiation factor 4E (eIF-4E) in HeLa cells causes a reduction in the levels of both eIF-4E and eIF-4 gamma (p220) and a concomitant decrease in the rates of both cell growth and protein synthesis (De Benedetti, A., Joshi-Barve, S., Rinker-Schaffer, C., and Rhoads, R. E. (1991) Mol. Cell Biol. 11, 5435-5445). The synthesis of most proteins in the antisense RNA-expressing cells (AS cells) is decreased, but certain proteins continue to be synthesized. In the present study, we identified many of these as stress-inducible or heat shock proteins (HSPs). By mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by reactivity with monoclonal antibodies generated against human HSPs, four of these were shown to be HSP 90, HSP 70, HSP 65, and HSP 27. The steady-state levels of HSP 90, 70, and 27 were elevated in relation to total protein in AS cells. Pulse labeling and immunoprecipitation indicated that HSP 90 and HSP 70 were synthesized more rapidly in AS cells than in control cells. The accelerated synthesis of HSPs in the AS cells was not due, however, to increased mRNA levels; the levels of HSP 90 and 70 mRNAs either remained the same or decreased after induction of antisense RNA expression. Actin mRNA, a typical cellular mRNA, was found on high polysomes in control cells but shifted to smaller polysomes in AS cells, as expected from the general decrease in translational initiation caused by eIF-4E and eIF-4 gamma depletion. HSP 90 and 70 mRNAs showed the opposite behavior; they were associated with small polysomes in control cells but shifted to higher polysomes in AS cells. These results demonstrate that HSP mRNAs have little or no requirement in vivo for the cap-recognition machinery and suggest that these mRNAs may utilize an alternative, cap-independent mechanism of translational initiation. 相似文献
10.
11.
目的:探讨真核翻译起始因子4E(e IF-4E)和基质金属蛋白酶9(MMP9)在子宫内膜癌组织中的表达及其临床意义。方法:应用免疫组化法检测e IF-4E和MMP9在16例正常子宫内膜组织、10例增生子宫内膜组织及42例子宫内膜癌组织中的表达,分析其与子宫内膜癌临床病理之间的关系,并研究二者的相关性。结果:e IF-4E与MMP9在三组组织中的表达阳性率逐渐增加,三组间的差异有统计学意义(P0.05),两两比较,正常内膜和内膜癌之间的差异有统计学意义(P0.0167)。e IF-4E和MMP9的表达与子宫内膜癌的FIGO分期、组织学分级及淋巴结转移具有显著相关性(P0.05);子宫内膜癌组织中e IF-4E与MMP9的阳性表达呈正相关(r=0.717,P0.05)。结论:e IF-4E和MMP9在子宫内膜癌组织中表达显著上调,随FIGO分期的增加、组织学分级的增高及有淋巴结转移而增高,可能与子宫内膜癌的发生发展相关,二者的联合检测对于评估子宫内膜癌患者的病情及预后有重要的参考意义。 相似文献
12.
Tyrosine Phosphorylation of Protein Kinase Cδ Is Essential for Its Apoptotic Effect in Response to Etoposide 下载免费PDF全文
Michal Blass Ilana Kronfeld Gila Kazimirsky Peter M. Blumberg Chaya Brodie 《Molecular and cellular biology》2002,22(1):182-195
Protein kinase Cdelta (PKCdelta) is involved in the apoptosis of various cells in response to diverse stimuli. In this study, we characterized the role of PKCdelta in the apoptosis of C6 glioma cells in response to etoposide. We found that etoposide induced apoptosis in the C6 cells within 24 to 48 h and arrested the cells in the G(1)/S phase of the cell cycle. Overexpression of PKCdelta increased the apoptotic effect induced by etoposide, whereas the PKCdelta selective inhibitor rottlerin and the PKCdelta dominant-negative mutant K376R reduced this effect compared to control cells. Etoposide-induced tyrosine phosphorylation of PKCdelta and its translocation to the nucleus within 3 h was followed by caspase-dependent cleavage of the enzyme. Using PKC chimeras, we found that both the regulatory and catalytic domains of PKCdelta were necessary for its apoptotic effect. The role of tyrosine phosphorylation of PKCdelta in the effects of etoposide was examined using cells overexpressing a PKCdelta mutant in which five tyrosine residues were mutated to phenylalanine (PKCdelta5). These cells exhibited decreased apoptosis in response to etoposide compared to cells overexpressing PKCdelta. Likewise, activation of caspase 3 and the cleavage of the PKCdelta5 mutant were significantly lower in cells overexpressing PKCdelta5. Using mutants of PKCdelta altered at individual tyrosine residues, we identified tyrosine 64 and tyrosine 187 as important phosphorylation sites in the apoptotic effect induced by etoposide. Our results suggest a role of PKCdelta in the apoptosis induced by etoposide and implicate tyrosine phosphorylation of PKCdelta as an important regulator of this effect. 相似文献
13.
14.
Expression of antisense RNA against initiation factor eIF-4E mRNA in HeLa cells results in lengthened cell division times, diminished translation rates, and reduced levels of both eIF-4E and the p220 component of eIF-4F. 总被引:16,自引:2,他引:16 下载免费PDF全文
A De Benedetti S Joshi-Barve C Rinker-Schaeffer R E Rhoads 《Molecular and cellular biology》1991,11(11):5435-5445
HeLa cells were transformed to express antisense RNA against initiation factor eIF-4E mRNA from an inducible promoter. In the absence of inducer, these cells (AS cells) were morphologically similar to control cells but grew four- to sevenfold more slowly. Induction of antisense RNA production was lethal. Both eIF-4E mRNA and protein levels were reduced in proportion to the degree of antisense RNA expression, as were the rates of protein synthesis in vivo and in vitro. Polysomes were disaggregated with a concomitant increase in ribosomal subunits. Translation in vitro was restored by addition of the initiation factor complex eIF-4F but not by eIF-4E alone. Immunological analysis revealed that the p220 component of eIF-4F was decreased in extracts of AS cells and undetectable in AS cells treated with inducer, suggesting that p220 and eIF-4E levels are coordinately regulated. eIF-4A, another component of eIF-4F, was unaltered. 相似文献
15.
Phosphorylation of translation initiation factor eIF-4E is induced in a ras-dependent manner during nerve growth factor-mediated PC12 cell differentiation. 下载免费PDF全文
Translation initiation factor eIF-4E, which binds to the 5' cap structure of eukaryotic mRNAs, is believed to play an important role in the control of cell growth. Consistent with this, overexpression of eIF-4E in fibroblasts results in their malignant transformation. The activity of eIF-4E is thought to be regulated by phosphorylation on a single serine residue (Ser-53). Treatment of rat pheochromocytoma (PC12) cells with nerve growth factor (NGF) strongly curtails their growth and causes their differentiation into cells that resemble sympathetic neurons. The present study shows that eIF-4E is rapidly phosphorylated in PC12 cells upon NGF treatment, resulting in a significant increase in the steady-state levels of the phosphorylated protein. In contrast, epidermal growth factor, a factor which elicits a weak mitogenic response in PC12 cells, did not significantly enhance eIF-4E phosphorylation. We also show that although the mitogen and tumor promoter, phorbol 12-myristate-13-acetate, is able to induce phosphorylation of eIF-4E in PC12 cells, the NGF-mediated increase is primarily a protein kinase C-independent response. The NGF-induced enhancement of eIF-4E phosphorylation is abrogated in PC12 cells expressing a dominant inhibitory ras mutant (Ser-17 replaced by Asn), indicating that eIF-4E phosphorylation is dependent on a ras signalling pathway. As phosphorylation of eIF-4E effects translation initiation, these results suggest that NGF-mediated and ras-dependent eIF-4E phosphorylation may play a role in switching the pattern of gene expression during the differentiation of PC12 cells. 相似文献
16.
P T Tuazon S J Morley T E Dever W C Merrick R E Rhoads J A Traugh 《The Journal of biological chemistry》1990,265(18):10617-10621
Phosphorylation by protein kinase C of the mRNA cap binding protein purified as part of a cap binding protein complex (eIF-4F) or as a single protein (eIF-4E), has been examined. Significant phosphorylation (up to 1 mol of phosphate/mol of p25 subunit) occurs only when the protein is part of the eIF-4F complex. With purified eIF-4E, using the same conditions, up to 0.1 mol of phosphate can be incorporated. Tryptic phosphopeptide maps show that the site phosphorylated in the Mr 25,000 subunit of eIF-4F (eIF-4F p25) is the same as that modified in purified eIF-4E. Kinetic measurements obtained from initial rates indicate that the Km values for eIF-4F and eIF-4E are similar, although the Vmax is 5-6 times higher for the complex. Dephosphorylation of eIF-4F p25, previously phosphorylated with protein kinase C, occurs in reticulocyte lysate with a half-life of 15-20 min, whereas little dephosphorylation is observed after 15 min with the purified phosphorylated eIF-4E. Phosphorylation of eIF-4F on the p220 and p25 subunits does not affect the stability of the complex as indicated by gel filtration on Sephacryl S-300. However, addition of non-phosphorylated eIF-4E to the phosphorylated complex results in the dissociation of the complex. These results suggest that interaction of p25 with other subunits in the complex greatly affects phosphorylation/dephosphorylation of p25. Since the rate of phosphorylation/dephosphorylation is significantly greater in the complex, regulation of the cap binding protein by phosphorylation appears to occur primarily on eIF-4F. 相似文献
17.
Addition of L-pyrroline-5-carboxylic acid to reticulocyte lysates inhibits protein synthesis and induced phosphoproteins of 25 and 14 kDa. The 25 kDa phosphoprotein had the same Mr and pI as phosphorylated eIF-4E. Incubation of lysates with L-pyrroline-5-carboxylic acid did not alter the crosslinking of eIF-4E to reovirus mRNA caps. These results suggest that modifications of the translational apparatus other than eIF-4E phosphorylation may mediate the inhibitory effect seen with L-pyrroline-5-carboxylic acid and/or that phosphorylation of eIF-4E may effect functions subsequent to its interaction with the mRNA cap such as protein-protein interactions with other cap-specific translation factors. 相似文献
18.
E L McMullin W E Hogancamp R D Abramson W C Merrick C H Hagedorn 《Biochemical and biophysical research communications》1988,153(3):925-932
Changes in the extent of phosphorylation of the 25 kDa subunit of eIF-4F occur during several major biological events including mitosis and heat shock in mammalian cells and shortly after fertilization of sea urchin (Lytechinus pictus) eggs. In vitro phosphorylation studies using highly purified protein kinases demonstrated that the 220 kDa subunit of eIF-4F was phosphorylated by cAMP dependent protein kinase, protein kinase C and probably to a lesser extent by cGMP dependent protein kinase. In addition, eIF-4A was readily phosphorylated by cAMP and cGMP dependent protein kinases whereas p48 of eIF-4F was not. The effect of these phosphorylation events on eIF-4F function, its assembly or disassembly, susceptibility to viral initiated proteolysis or the ability of p25 to be phosphorylated at serine-53 remain to be investigated. 相似文献
19.
Tyrosine Phosphorylation of Extracellular Signal-Regulated Protein Kinase 4 in Response to Growth Factors 总被引:2,自引:0,他引:2
†‡Xing Peng †‡James M. Angelastro †‡ Lloyd A. Greene 《Journal of neurochemistry》1996,66(3):1191-1197
Abstract: Extracellular signal-regulated protein kinases (ERKs) are members of the mitogen-activated protein kinase family that are rapidly phosphorylated and activated in response to various extracellular stimuli, including growth factors. Of these, the ERK1 and ERK2 forms are by far the most abundant and the most studied. Much less is known about other ERK forms, including one previously designated ERK4 on the basis of its cross-reactivity with ERK1 and ERK2. We report here that ERK4 in rat PC12 pheochromocytoma cells can be immunoprecipitated by anti-ERK antiserum R2 and have used this reagent to characterize this species further. We find that ERK4 rapidly becomes tyrosine-phosphorylated in response to nerve growth factor (NGF) and epidermal growth factor (EGF) and, to a lesser degree, in response to insulin and a permeant cyclic AMP analogue. As in the case of ERK1 and ERK2, tyrosine phosphorylation of ERK4 occurs by a ras-dependent pathway in response to NGF and EGF and shows prolonged kinetics for NGF but not EGF treatment. Recognition by multiple antisera directed against various domains of ERK1 supports classification of ERK4 within the ERK family; however, two-dimensional gel analysis clearly distinguishes ERK4 from isoforms of ERK1. These findings thus reveal an additional member of the ERK family that is responsive to growth factors and that could play a distinct role in intracellular signaling. 相似文献