Biomedical subjects

G Pagès

Publications and source records attributed to G Pagès.

At least 19 recordsLinked to original sources

PubMed2006

Vascular endothelial growth factor-A and Poly(A) binding protein-interacting protein 2 expression in human head and neck carcinomas: correlation and prognostic significance.

Vascular endothelial growth factor-A (VEGF-A) has been demonstrated to play an important role in tumour angiogenesis and to influence prognosis in many cancers. However its prognostic value in head and neck squamous cell carcinomas (HNSCCs) remains controversial. Therefore, we investigated the clinical relevance of VEGF-A expression in HNSCCs and analysed whether its expression was associated with PAIP2 protein levels, a VEGF-A mRNA-binding partner that strongly regulates VEGF-A expression in tissue culture. We determined the correlation of VEGF-A and PAIP2 protein levels, quantitatively evaluated in tumour tissue homogenates from 54 patients with HNSCC, to clinicopathological parameters. We showed that VEGF-A expression in HNSCC is correlated to the stage of tumour differentiation (P=0.050) and is an independent prognostic factor for progression-free survival (P=0.001) and overall survival (P=0.0004). In a pharynx carcinoma cell line, we demonstrated by RNA interference that VEGF-A expression is closely controlled by PAIP2. Moreover, in human HNSCCs, VEGF-A expression is significantly correlated to PAIP2 protein levels (P=0.0018). Nevertheless, PAIP2 expression is associated with neither clinicopathological factors nor patient's survival. Our data suggest that, in contrast to PAIP2 protein levels, which are unrelated to tumour prognosis, VEGF-A expression could serve as a prognostic marker in head and neck cancer and may be helpful for targeted therapies.

Adult↗

PubMed2006

Overexpression of Sp1 transcription factor induces apoptosis.

Transcription factor Sp1 has recently been shown to be overexpressed in a number of human cancers and its overexpression contributes to malignant transformation. Sp1 regulates the expression of a number of genes participating in multiple aspects of tumorigenesis such as angiogenesis, cell growth and apoptosis resistance. To better understand the role of increased Sp1 levels on apoptosis regulation we have used retroviruses to overexpress this protein in haematopoietic Baf-3 cells and in 3T3 fibroblasts. We have also used inducible expression systems to control ectopic Sp1 levels in different cell types. Surprisingly, Sp1 overexpression on its own induces apoptosis in all the cellular models tested. The apoptotic pathways induced by Sp1 overexpression are cell type specific. Finally, using a truncated form of Sp1, we show that Sp1-induced apoptosis requires its DNA-binding domain. Our results highlight that Sp1 levels in untransformed cells must be tightly regulated as Sp1 overexpression leads to the induction of apoptosis. Our results also suggest that cancer cells overexpressing Sp1 can avoid Sp1-induced apoptosis.

Animals↗

PubMed2001

pHi, aerobic glycolysis and vascular endothelial growth factor in tumour growth.

Signalling via growth factors, oncogenes and environmental stresses such as hypoxia, promotes the up-regulation of glycolysis, intracellular pH (pHi) and vascular endothelial growth factor (VEGF) via cooperative mechanisms. Somatic cell genetics was applied to a fibroblastic cell line (CCOL39) to disrupt either aerobic glycolysis, respiration, or a major pHi-regulating system, the Na-H exchanger (NHE1). We obtained stable mutants impaired either in phosphoglucose isomerase (pgi-), which produce virtually no lactic acid, or in respiration (res-), which over secrete lactic acid (three- to fourfold the wild-type rate). These mutations, which allowed us to analyse the incidence of lactic acid production on tumour development in nude mice, were analysed alone, or in combination, with the mutation nhe1- to evaluate in vivo the role of NHE1 on pHi control and cell proliferation. Ras-transformed pgi- cells (not forming lactic acid) form tumours like wild type transformed cells (100% incidence). The disruption of NHE1 however, strongly reduced tumour incidence to about 20%. In cells bearing both mutations, nhe1-, res-, and which therefore over-produce lactic acid, the situation is even more dramatic (0% incidence). In sharp contrast, association of nhe1- with pgi- restored 100% tumour incidence. We conclude that over-production of lactic acid is detrimental for tumour development and that NHE1, by controlling pHi, plays a key role in cell survival/proliferation and tumour growth. Finally we summarize our current knowledge on the signalling mechanisms leading to VEGF expression, another key component of tumour growth via neo-vascularization.

Aerobiosis↗

PubMed2001

RT-PCR method to quantify vascular endothelial growth factor expression.

The vascular endothelial growth factor (VEGF) is implicated in the progression of cancers. Its expression is well correlated with tumor growth and metastases. The availability of a rapid and sensitive method to detect the amounts of VEGF mRNA in biological samples of limited size, very small biopsies, or samples containing relatively few cells could provide an interesting prognostic tool for clinicians. We have developed an RT-PCR method that allows us to detect the VEGF mRNA from as little as 3 micrograms total mRNA. We have also shown that this protocol can be generalized to all cell lines tested. This method constitutes a very potent tool for the analysis of VEGF mRNA expression in different contexts.

Adenocarcinoma↗

PubMed2000

Signaling angiogenesis via p42/p44 MAP kinase and hypoxia.

Angiogenesis is associated with a number of pathological situations. In this study, we have focused our attention on the role of p42/p44 MAP (mitogen-activated protein) kinases and hypoxia in the control of angiogenesis. We demonstrate that p42/p44 MAP kinases play a pivotal role in angiogenesis by exerting a determinant action at three levels: i) persistent activation of p42/p44 MAP kinases abrogates apoptosis; ii) p42/p44 MAP kinase activity is critical for controlling proliferation and growth arrest of confluent endothelial cells; and iii) p42/p44 MAP kinases promote VEGF (vascular endothelial growth factor) expression by activating its transcription via recruitment of the AP-2/Sp1 (activator protein-2) complex on the proximal region (-88/-66) of the VEGF promoter and by direct phosphorylation of hypoxia-inducible factor 1 alpha (HIF-1 alpha). HIF-1 alpha plays a crucial role in the control of HIF-1 activity, which mediates hypoxia-induced VEGF expression. We show that oxygen-regulated HIF-1 alpha protein levels are not affected by intracellular localization (nucleus versus cytoplasm). Finally, we propose a model which suggests an autoregulatory feedback mechanism controlling HIF-1 alpha and therefore HIF-1-dependent gene expression.

Animals↗

PubMed2000

Stress-activated protein kinases (JNK and p38/HOG) are essential for vascular endothelial growth factor mRNA stability.

Stability of the vascular endothelial growth factor (VEGF) mRNA is tightly regulated through its 3'-untranslated region (3'-UTR). Here, we demonstrate that VEGF mRNA levels are increased by anisomycin, a strong activator of stress-activated protein kinases. Hence, VEGF mRNA induction is inhibited by SB202190, an inhibitor of JNK and p38/HOG kinase. Furthermore, VEGF mRNA expression is increased in cells that overexpress JNK and p38/HOG by an increase in its stability. We show by two different approaches that anisomycin exerts its effect on the VEGF mRNA 3'-UTR. First, by using an in vitro mRNA degradation assay, the half-life of the VEGF mRNA 3'-UTR region transcript was found to be increased when incubated with extracts from anisomycin-treated cells; and second, the 3'-UTR was also sufficient to confer mRNA instability to the Nhe3 (Na(+)/H(+) exchanger 3) heterologous reporter gene, and anisomycin treatment stabilized the chimeric mRNA (Nhe3 fused to the VEGF mRNA 3'-UTR). This chimeric mRNA is also more stable in cells overexpressing p38/HOG and JNK that have been stimulated by anisomycin. We show that such regulation is mediated through an AU-rich region of the 3'-UTR contained within a stable hairpin structure. By RNA electrophoretic mobility shift assays, we show that this region binds proteins specifically induced by anisomycin treatment. These findings clearly demonstrate a major role of stress-activated protein kinases in the post-transcriptional regulation of VEGF.

Anisomycin↗

PubMed2000

Identification of alternative spliced variants of human hypoxia-inducible factor-1alpha.

Mammalian cells are able to sense oxygen and regulate a number of genes in response to hypoxia. The transcription factor Hypoxia Inducible Factor-1 (HIF-1) was identified as an important key component of the hypoxia signaling pathway. HIF-1 is a heterodimer composed of two members of the basic helix-loop-helix transcription factor superfamily containing a PAS (PER-ARNT-SIM) domain: HIF-1alpha and HIF-1beta/ARNT. During the cloning by reverse transcriptase-polymerase chain reaction of the human HIF-1alpha subunit, we isolated two cDNA clones which corresponded to alternative splicing of the HIF-1alpha gene. Polymerase chain reaction analysis and sequencing revealed that both clones possessed three additional base pairs between exons 1 and 2. Also, one of them lacked 127 base pairs corresponding to exon 14. We demonstrate that the mRNA of this truncated form is expressed in several human cells lines and human skin but apparently not in rodents. When transfected in HEK 293 cells, the corresponding 736 amino acid protein (HIF-1alpha(736)) is regulated by hypoxia in a similar manner as the full-length HIF-1alpha (HIF-1alpha(FL)). In luciferase transfection assays, both recombinant proteins HIF-1alpha(736) and HIF-1alpha(FL) dimerize with HIF-1beta/ARNT and activate the VEGF promoter upon hypoxia. However, the shorter HIF-1alpha isoform is 3-fold less active than HIF-1alpha(FL), a result consistent with the lack of the C-terminal transactivation domain. As expected, this small isoform can compete with the endogenous and transfected full-length HIF-1alpha. Altogether, these results suggest that the HIF-1alpha(736) isoform modulates gene expression upon hypoxia.

Alternative Splicing↗

PubMed2000

MAP kinases and hypoxia in the control of VEGF expression.

Vascular endothelial growth factor (VEGF), a potent cytokine secreted by virtually all cells plays a key role in tumor angiogenesis. Disruption of one VEGF allele in mice has revealed a dramatic lethal effect in early embryogenesis, suggesting a very tight regulation of this gene. This commentary reviews the mechanisms whereby VEGF mRNA is controlled within the tumor environment by hypoxia and the MAP kinase signaling cascades. Using hamster fibroblasts as a cellular model, we demonstrated that the Ras-mediated activation of p42/p44 MAP kinases exerts a prominent action at the transcriptional level. In normoxic conditions, p42/p44 MAPKs activate the VEGF promoter at the proximal (-88/-66) region where Sp 1/AP-2 transcriptional factor complexes are recruited. At low O2 tension, the stabilized and nuclear hypoxia inducible factor- 1alpha (HIF-1alpha) is directly phosphorylated by p42/p44 MAPKs, an action which enhances HIF-1-dependent transcriptional activition of VEGF. In addition, MAPKs activated under various cellular stresses (p38MAPK and JNK), contribute to the increased expression of this angiogenic growth and survival factor by stabilizing the VEGF mRNA.

Animals↗

PubMed2000

Signaling angiogenesis via p42/p44 MAP kinase cascade.

Vascular endothelial growth factor (VEGF), a potent agonist secreted by virtually all cells, controls migration and division of vascular endothelial cells. Disruption of one VEGF allele in mice has revealed a dramatic lethal effect in early embryogenesis, suggesting a key role in vasculogenesis. We analyzed the regulation of VEGF mRNA in normal and transformed CCL39 fibroblasts and then dissected the VEGF promoter to identify the signaling pathway(s) controlling the activation of this promoter in response to growth factors, oncogenes, and hypoxic stress. We demonstrated that the p42/p44 MAP kinase signaling cascade controls VEGF expression at least at two levels. In normoxic conditions, MAPKs activate the VEGF promoter at the proximal (-88/-66) region where Sp-1/AP-2 factors bind. Activation of p42/p44 MAPKs is sufficient to turn on VEGF mRNA. At low O2 tension, hypoxia inducible factor-1 alpha (HIF-1 alpha), a limiting factor rapidly stabilized and phosphorylated, plays a key role in the expression of several genes including VEGF. We demonstrated that p42/p44MAPKs stoichiometrically phosphorylate HIF-1 alpha in vitro and that HIF-1-dependent VEGF gene expression is strongly enhanced by the exclusive activation of p42/p44MAPKs. Finally, we demonstrated that the regulation of p42/p44MAPK activity is critical for controlling proliferation and growth arrest of vascular endothelial cells at confluency. These results point to at least three major targets of angiogenesis where p42/p44 MAP kinases exert a determinant action.

Animals↗

PubMed2000

LPA as a paracrine mediator of adipocyte growth and function.

Adipogenesis corresponds to the recruitment of new adipocytes in adipose tissue, and results from the proliferation/differentiation of preadipocytes. Production of paracrine and autocrine factors by adipocytes plays an important role in adipogenesis. We recently demonstrated the existence of adipocyte production of lysophosphatidic acid (LPA) both in vitro and in situ. This production is modulated by catecholamines via alpha 2-adrenergic receptors. Adipocyte-LPA present in conditioned media increases the growth of a preadipose cell line in culture. This growth is associated with an activation of mitogen-activated protein kinases, and of the focal adhesion kinase. Because of the close proximity of preadipocytes and adipocytes within adipose tissue, adipocyte-LPA could play an important role in autocrine/paracrine control of adipogenesis.

Adipocytes↗

PubMed1999

Defective thymocyte maturation in p44 MAP kinase (Erk 1) knockout mice.

The p42 and p44 mitogen-activated protein kinases (MAPKs), also called Erk2 and Erk1, respectively, have been implicated in proliferation as well as in differentiation programs. The specific role of the p44 MAPK isoform in the whole animal was evaluated by generation of p44 MAPK-deficient mice by homologous recombination in embryonic stem cells. The p44 MAPK-/- mice were viable, fertile, and of normal size. Thus, p44 MAPK is apparently dispensable and p42 MAPK (Erk2) may compensate for its loss. However, in p44 MAPK-/- mice, thymocyte maturation beyond the CD4+CD8+ stage was reduced by half, with a similar diminution in the thymocyte subpopulation expressing high levels of T cell receptor (CD3high). In p44 MAPK-/- thymocytes, proliferation in response to activation with a monoclonal antibody to the T cell receptor in the presence of phorbol myristate acetate was severely reduced even though activation of p42 MAPK was more sustained in these cells. The p44 MAPK apparently has a specific role in thymocyte development.

Animals↗

PubMed1998

p42/p44 MAP kinase module plays a key role in the transcriptional regulation of the vascular endothelial growth factor gene in fibroblasts.

Vascular Endothelial Growth Factor (VEGF) is a potent mitogen for vascular endothelial cells that has been implicated in tumor neovascularization. We show that, in hamster fibroblasts (CCL39 cells), VEGF mRNAs are expressed at low levels in serum-deprived or exponentially growing cells, whereas it is rapidly induced after stimulation of quiescent cells with serum. CCL39 derivatives, transformed with Polyoma virus or with active members of the p42/p44 mitogen-activated protein (MAP) kinase pathway, Gly/Val point mutant of Ras at position 12 (Ras-Val12), MKK1 in which Ser218 and Ser222 were mutated to Asp (MKK1-SS/DD)), express very high levels of VEGF mRNA. To analyze the contribution of the p42/p44MAP kinase in this induction, we used the CCL39-derived cell line (Raf-1:ER) expressing an estradiol-activable Raf-1. We show a time and an estradiol dose-dependent up-regulation of VEGF mRNA clearly detectable after 2 h of stimulation. The induction of VEGF mRNA in response to conditioned activation of Raf-1 is reverted by an inhibitor of MKK1, PD 098059, highlighting a specific role for the p42/p44 MAP kinase pathway in VEGF expression. Interestingly, hypoxia has an additive effect on VEGF induction in CCL39 cells stimulated by serum or in Raf-1:ER cells stimulated by estradiol. In contrast to VEGF, the isoforms VEGF-B and VEGF-C are poorly regulated by growth and oncogenic factors. We have identified a GC-rich region of the VEGF promoter between -88 and -66 base pairs which contains all the elements responsible of its up-regulation by constitutive active Ras or MKK1-SS/DD. By mutation of the putative binding sites and electrophoretic mobility supershift experiments, we showed that the GC-rich region constitutively binds Sp1 and AP-2 transcription factors. Furthermore, following activation of the p42/p44 MAP kinase module, the binding of Sp1 and AP-2 is increased in the complexes formed in this region of the promoter. Altogether, these data suggest that hypoxia and p42/p44 MAP kinase independently play a key role in the regulation of the VEGF expression.

Animals↗

PubMed1998

Inhibition of the mitogen-activated protein kinase pathway triggers B16 melanoma cell differentiation.

In B16 melanoma cells, mitogen-activated protein (MAP) kinases are activated during cAMP-induced melanogenesis (Englaro, W., Rezzonico, R., Durand-Clément, M., Lallemand, D., Ortonne, J. P., and Ballotti, R. (1995) J. Biol. Chem. 270, 24315-24320). To establish the role of the MAP kinases in melanogenesis, we studied the effects of a specific MAP kinase kinase (MEK) inhibitor PD 98059 on different melanogenic parameters. We showed that PD 98059 inhibits the activation of MAP kinase extracellular signal-regulated kinase 1 by cAMP, but does not impair the effects of cAMP either on the morphological differentiation, characterized by an increase in dendrite outgrowth, or on the up-regulation of tyrosinase that is the key enzyme in melanogenesis. On the contrary, PD 98059 promotes by itself cell dendricity and increases the tyrosinase amount and activity. Moreover, down-regulation of the MAP kinase pathway by PD 98059, or with dominant negative mutants of p21(ras) and MEK, triggers a stimulation of the tyrosinase promoter activity and enhances the effect of cAMP on this parameter. Conversely, activation of the MAP kinase pathway, using constitutive active mutants of p21(ras) and MEK, leads to an inhibition of basal and cAMP-induced tyrosinase gene transcription. These results demonstrate that the MAP kinase pathway activation is not required for cAMP-induced melanogenesis. Furthermore, the inhibition of this pathway induces B16 melanoma cell differentiation, while a sustained activation impairs the melanogenic effect of cAMP-elevating agents.

Animals↗

PubMed1995

The mouse p44 mitogen-activated protein kinase (extracellular signal-regulated kinase 1) gene. Genomic organization and structure of the 5'-flanking regulatory region.

Mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase are ubiquitous kinases conserved from fungi to mammals. Their activity is regulated by phosphorylation on both threonine and tyrosine, and they play a crucial role in the regulation of proliferation and differentiation. We report here the cloning of the murine p44 MAP kinase (extracellular signal-regulated kinase 1) gene, the determination of its intron/exon boundaries, and the characterization of its promoter. The gene spans approximately eight kilobases (kb) and can be divided into nine exons and eight introns, each coding region exon containing from one to three of the highly conserved protein kinase domains. Primer extension analysis reveals the existence of two major start sites of transcription located at -183 and -186 base pairs (bp) as well as four discrete start sites for transcription located at -178, -192, -273, and -292 bp of the initiation of translation. However, the start site region lacks TATA-like sequences but does contain initiator-like sequences proximal to the major start sites obtained by primer extension. 1 kb of the promoter region has been sequenced. It contains three putative TATA boxes far upstream of the main start sites region, one AP-1 box, one AP-2 box, one Malt box, one GAGA box, one half serum-responsive element, and putative binding sites for Sp1 (five), GC-rich binding factor (five), CTF-NF1 (one), Myb (one), p53 (two), Ets-1 (one), NF-IL6 (two), MyoD (two), Zeste (one), and hepatocyte nuclear factor-5 (one). To determine the sites critical for the function of the p44 MAPK promoter, we constructed a series of chimeric genes containing variable regions of the 5'-flanking sequence of p44 MAPK gene and the coding region for luciferase. Activity of the promoter, measured by its capacity to direct expression of a luciferase reporter gene, is strong, being comparable with the activity of the Rous sarcoma virus promoter. Progressive deletions of the approximately 1 kb (-1200/-78) promoter region allowed us to define a minimal region of 186 bp (-284/-78) that has maximal promoter activity. Within this context, deletion of the AP-2 binding site reduces by 30-40% the activity of the promoter. Further deletion of this minimal promoter that removes the major start sites (-167/-78) surprisingly preserves promoter activity. This result implicates a major role of this region that contains the Sp1 sites.(ABSTRACT TRUNCATED AT 400 WORDS)

Alternative Splicing↗

PubMed1995

B-Raf protein isoforms interact with and phosphorylate Mek-1 on serine residues 218 and 222.

The B-raf/c-Rmil proto-oncogene belongs to the raf/mil family of serine/threonine protein kinases. It encodes multiple protein isoforms resulting from alternative splicing of two exons located upstream of the kinase domain. Recent studies suggested that B-Raf could be the intermediate molecule between Ras and Mek-1 (MAP Kinase Kinase) in signalling pathways specific of neural cells. However, there has been no evidence for a direct interaction between B-Raf and Mek-1. We report here that different B-Raf isoforms can be co-immunoprecipitated with anti-Mek-1 antisera in COS-1 cells and that the kinase activity of B-Raf is not required for its interaction with Mek-1. We also show that all B-Raf isoforms tested phosphorylate Mek-1 in a time-dependent manner, whereas kinase defective mutants fail to do so. Finally, we demonstrate that the constitutively activated S218D, S222D and S218D/S222D mutants of Mek-1 interact similarly with B-Raf. However, only the S218D and S222D mutants, and not the S218D/S222D double mutant, can be phosphorylated by B-Raf isoforms. Therefore, serine residues 218 and 222, previously shown to regulate Mek-1 activity, appear to be the major phosphorylation sites by B-Raf in vitro.

Animals↗

PubMed1995

[MAP kinase module: role in the control of cell proliferation].

A kinase cascade highly conserved throughout evolution, Raf/MAP kinase kinase kinase (MAPKKK)-->MAP kinase kinase (MAPKK)-->MAP kinase (MAPK)-->ribosomal S6 kinase (p90 RSK), is thought to play a crucial role in signal transduction from the membrane to the nucleus. In mammalian cells, this cascade is connected both to tyrosine kinase receptors and G protein-coupled receptors. Although the mode of activation at the receptor level differs, all mitogens activate the ubiquitously expressed isoforms of MAPK, p42 and p44. We have cloned, epitope tagged and expressed in fibroblasts, the Hamster MAPKK and p44 MAPK in order to analyze their time-course of activation, their subcellular localization, their regulatory phosphorylation sites and their role in cell cycle entry. We have demonstrated that MAPK activation was rapid, biphasic and persistent. The sustained phase of activation is only obtained with potent mitogenic agents, correlating with their ability to elicit cell cycle entry. Activation of MAPKK is also rapid and persistent but does not distinguish between mitogenic and non mitogenic factors, indicating that a distinction occurs at the MAPK level, probably by the action of specific phosphatases such as MAPK phosphatase MKP-1. Both isoforms of MAPK are translocated into the nucleus upon growth factor addition whereas the upstream activators (MAPKKK, Raf and MAPKK) remain cytoplasmic. MAPK translocation, together with the ability of MAPK to phosphorylate transcription factors, indicates that MAPK might constitute a relay between cytoplasmic and nuclear events. Finally we show that interfering with the MAP kinase cascade, by expressing either MAPK antisense, a MAPK dominant negative mutant or the MAPK specific phosphatase, MKP-1, suppresses the growth factor induced G0 to G1 transition. In addition, permanently activated versions of MAPKK reduce growth factor requirement, allow autonomous cell growth and induce tumor formation in nude mice. We therefore conclude that MAP kinase activation is both necessary and sufficient to trigger cell cycle entry.

Animals↗

PubMed1994

Enhanced phosphorylation of the C-terminal domain of RNA polymerase II upon serum stimulation of quiescent cells: possible involvement of MAP kinases.

The largest subunit of RNA polymerase (RNAP) II contains at it C-terminus an unusual domain comprising tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This C-terminal domain (CTD) can undergo phosphorylation at multiple sites giving rise to a form of the enzyme designated RNAP IIO. The unphosphorylated form is designated RNAP IIA. The largest subunits of RNAPs IIO and IIA are designated IIo and IIa, respectively. In quiescent NIH 3T3 fibroblasts, subunits IIo and IIa are present in comparable amounts. Upon serum stimulation, the amount of subunit IIo increases markedly and remains elevated for several hours. The increase of subunit IIo also occurs in transcription-inhibited cells and, therefore, is not a consequence of serum-activated transcription. This observation suggests that serum stimulation activates a CTD kinase and/or inhibits a CTD phosphatase. This hypothesis is supported by the finding that serum stimulates phosphorylation of a beta-galactosidase-CTD fusion protein expressed in these cells. Furthermore, an enhanced CTD kinase activity was discovered in lysates from serum-stimulated fibroblasts and was found to copurify with MAP kinases on a Mono Q column and to bind to anti-MAP kinase antibodies. The idea that MAP kinases phosphorylate the CTD in vivo is supported by the observation that subunit IIa, but not subunit IIb which lacks the CTD, is phosphorylated at multiple sites by purified MAP kinase. Consequently, the MAP kinases are a new class of CTD kinases which appear to be involved in the phosphorylation of RNAP II following serum stimulation. This phosphorylation may contribute to the transcriptional activation of serum-stimulated genes.

3T3 Cells↗

PubMed1994

Constitutive mutant and putative regulatory serine phosphorylation site of mammalian MAP kinase kinase (MEK1).

In response to various external stimuli, MAP kinases are activated by phosphorylation on tyrosine and threonine by MAP kinase kinase (MAPKK), a dual specificity kinase. This kinase is in turn activated via Raf-1 and MAPKK kinase (MAPKKK). To determine regulatory phosphorylation sites of MAPKK, we isolated a Chinese hamster cDNA, that we epitope-tagged and expressed in fibroblasts. This hamster MAPKK (MEK1 isoform) can reactivate recombinant p44mapk when immunoprecipitated from growth factor-stimulated cells or when incubated with an active form of MAPKKK. Mutations at either of two residues that are conserved among kinases, D208N or S222A, abolished MAPKK activity. However, only S222A/MAPKK showed a reduction in phosphorylation in response to active MAPKKK and exerted a dominant negative effect on the serum-stimulated endogenous MAPKK. Finally, replacing Ser222 with Asp, a negatively charged residue, restored MAPKK activity independently of the upstream kinase. These results strongly suggest that Ser222 represents one key MAPKKK-dependent phosphorylation site switching on and off the activity of MAPKK, an event crucial for growth control.

Amino Acid Sequence↗