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C-mip interacts with the p85 subunit of PI3 kinase and exerts a dual effect on ERK signaling via the recruitment of Dip1 and DAP kinase
FEBS Letters 584 (2010) 500–506
journal homepage: www.FEBSLetters.org
C-mip interacts with the p85 subunit of PI3 kinase and exerts a dual effect on ERK signaling via the recruitment of Dip1 and DAP kinase q Maud Kamal a,b, Andre Pawlak a,b, Fatima BenMohamed a,b, Asta Valanciuté a,b, Karine Dahan a,b, Marina Candelier a,b, Philippe Lang a,b,c,d, Georges Guellaën a,b, Djillali Sahali a,b,c,d,* a
INSERM, U 955, Equipe 21, Créteil F-94010, France Université Paris 12, Faculté de Médecine, UMRS 955, Equipe 21, Créteil, F-94010, France AP-HP, Groupe hospitalier Henri Mondor – Albert Chenevier, Service de Néphrologie, Créteil F-94010, France d Institut Francilien de Recheche en Nephrologie et Transplantation, Hopital Henri Mondor, France b c
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Article history: Received 21 September 2009 Revised 7 November 2009 Accepted 9 December 2009 Available online 16 December 2009 Edited by Zhijie Chang Keywords: C-mip PI3 kinase Lck DIP1 DAP-kinase ERK signaling
a b s t r a c t In naive T cells, Lck exerts a negative control on the ERK/MAPK pathway. We show that c-mip (c-maf inducing protein) interacts with the p85 subunit of PI3 kinase and inactivates Lck, which results in Erk1/2 and p38 MAPK activation. This effect is not enough to activate AP1 given the inability of ERK to migrate into the nucleus and to transactivate its target genes. We demonstrate that c-mip interacts with Dip1 and upregulates DAPK, which blocks the nuclear translocation of ERK1/2. This dual effect of c-mip is unique and might represent a potential mechanism to prevent the development of an immune response. Structured summary: MINT-7383650: p85 (uniprotkb:P27986) physically interacts (MI:0915) with c-Mip (uniprotkb:Q8IY22) by anti bait coimmunoprecipitation (MI:0006) MINT-7383661: c-Mip (uniprotkb:Q8IY22) physically interacts (MI:0915) with p85 (uniprotkb:P27986) by anti tag coimmunoprecipitation (MI:0007) MINT-7383676: p85 (uniprotkb:P27986) physically interacts (MI:0915) with p110 (uniprotkb:P42336) by anti bait coimmunoprecipitation (MI:0006) MINT-7383689, MINT-7383711: Dip-1 (uniprotkb:Q80SY4) physically interacts (MI:0915) with c-Mip (uniprotkb:Q8IY22) by anti tag coimmunoprecipitation (MI:0007) Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Cells respond to intra or extra cellular stimuli by engaging specific activation programs such as the signaling that leads to distinct mitogen-activated protein kinase (MAPK) cascades. Among these, the extra cellular signal regulated kinase (ERK1/2), c-jun N terminal kinase and p38 MAPK pathways are the best characterized. ERK1/2 are activated in response to growth factors via recruitment of membrane receptor tyrosine kinases, G protein-coupled receptors and integrins, whereas Jun N terminal kinase and p38 MAPK are sensitive to stress stimuli [1]. In T cells, PKCh functions as a regulator of JNK activation [2]. The ERK1/2 proteins are recruited at q This work was supported in part by an Avenir Program from INSERM, a grant from the French Kidney Foundation and Association pour l’Utilisation du Rein Artificiel (AURA). * Corresponding author. Address: INSERM, U 955, Equipe 21, Créteil F-94010, France. Fax: +33 149812539. E-mail address: [email protected] (D. Sahali).
most downstream in the signaling pathway where they play an important role in the transmission of signals involved in a wide range of cellular activities including proliferation, differentiation, survival and apoptosis [3]. These functions are mediated through sequential phosphorylation of diverse substrates such as phospholipases, transcription factors and cytoskeleton proteins. The subcellular localization of ERK1/2 determines their function: upon activation, ERK1/2 are phosphorylated and translocate into the nucleus while in unstimulated cells, they are sequestrated into the cytoplasm in inactive form. A major target of ERK activation is c-fos, a member of the AP1 transcription factor. It has been shown that sustained ERK1/2 activation influences the nature of AP1 complexes and upregulates the expression of cyclin D, allowing cells to progress in cell-cycle and to induce the upregulation of pro-proliferative genes [4]. In addition, recent studies show that ERK1/2 are involved in the regulation of innate and adaptive immunity [5]. The two isoforms of ERK, 42 and 44 kDa, are highly homologous and possess the same substrate specificity. Although
0014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2009.12.015
M. Kamal et al. / FEBS Letters 584 (2010) 500–506
most functions of ERK1 and ERK2 appear redundant, studies of isoform-specific invalidation in mice provides some differences. Thus, loss of ERK2 leads to implantation defects and embryonic lethality, whereas ERK1-deficient mice exhibit a bias toward Th1 type immune response and are susceptible to experimental autoimmune encephalomyelitis [6]. In the course of studies molecular mechanisms underlying the immunopathogenesis of idiopathic nephrotic syndrome, we isolated a new gene, c-mip (for c-maf inducing protein) [7]. The natural isoform, c-mip, encodes for an 86 kDa protein. The predicted structure of c-mip includes an N-terminal region containing a pleckstrin homology domain (PH), a middle region characterized by the presence of several interacting docking sites including, a 14-3-3 module, a PKC domain, an ERK domain, and an SH3 domain similar to the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K) and a C-terminal region containing a leucin-rich repeat (LRR) domain. The basal expression of c-mip transcript is very low in adult tissues and the protein is not detectable in physiological conditions. On the other hand, c-mip is upregulated at the mRNA and protein levels in two major sites, the podocytes and the peripheral lymphocytes during the active phase of the human disease (nephrotic syndrome). Our understanding about the role of cmip in cell signaling is far behind. We report here that c-mip interacts with PI3 kinase complex and displays a dual role in ERK signaling cascade. Although transient c-mip overexpression activates the MEK/ERK signaling, we found that active ERK did not translocate in nucleus but it was sequestrated in cytoplasmic compartment instead. Using two-hybrid system, we identified Dip1, a pro-apoptotic molecule, as a partner of c-mip. We demonstrate also that overexpression of cmip induces an upregulation of death-associated protein kinase DAP kinase, a known inhibitor of nuclear ERK translocation. These results suggest that c-mip induces cytoplasmic retention of active ERK, likely via a DAP kinase dependent mechanism.
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ml in complete RPMI medium and transfected with 1 lg DNA per 106 cells, using the Bio Rad gene pulser (Bio Rad, France), at 950 mF and 250 V. After electroporation, the cells were allowed to recover for 4 h in conditioned medium, then cultured 18 h in fresh medium containing Concanavalin A (Con A, 5 mg/ml) and Phytohemagglutinin (PHA, 2 mg/ml). At the end of the incubation, total RNA was isolated from cells and reverse transcribed as previously described [10]. 2.4. Identification of c-mip partners by yeast-two hybrid screening The yeast-two hybrid technique has been previously described [11]. Among proteins that we identified as interacting with cmip, we found the death-associated protein kinase (DAPK)-interacting protein 1 (DIP1). 2.5. Co-immunoprecipitations and Western blots All the primary antibodies used in this paper were purchased from Cell signaling except for the antibodies against pI3K, p110 and p85a, subunits (Santa Cruz), the monoclonal anti-V5 (Invitrogen), the monoclonal anti-DAPK and anti-FLAG antibodies, and the polyclonal ant-actin antibody (Sigma). The anti-c-mip polyclonal antibody production has previously been described [8]. The affinity purified anti-rabbit and anti-mouse secondary antibodies were purchased from Amersham and Santa Cruz, respectively. Immunoprecipitation and Western blot experiments were accomplished as previously described [8]. 2.6. Statistical analysis Statistical analysis of the data was performed using PRIZM 4 for Macintosh (Graphpad Software, Inc., USA). Unpaired or Paired Student t-tests were used. P value of less than 0.05 was considered significant.
2. Materials and methods 3. Results 2.1. Plasmid constructs The c-mip expression plasmid construct was previously described [8]. The Dip1 plasmid was kindly provided by Patricia J. Gallagher (Indiana University School of Medicine, Indianapolis, Indiana). The AP-1-luc, phRenilla-luc and pCRE-Luc vectors were provided from Promega and Stratagene, respectively. The c-jun cDNA was cloned in pcDNA3.1 expression vector (Invitrogen). The Ghrelin receptor (GHSR) cDNA was cloned in a pcDNA3.1 vector and was used as a positive control for cAMP activation. 2.2. Cell culture, transient transfections and dual luciferase reporter assay The preparation of peripheral blood mononuclear cells (PBMCs), cell culture conditions transients transfection experiments and dual luciferase reporter assays were performed as previously described [8]. 2.3. RT-qPCR The expression of cytokine mRNAs in the Jurkat cell line was assessed by quantitative reverse transcription-polymerase chain reaction (RT-qPCR), using the Light Cycler (Roche Diagnostics, Somerville, NJ), as previously described [9]. GAPDH were used for normalization of quantitative RT-PCR. The oligonucleotides used to amplify interferon c, IL-4 and GAPDH transcripts have been previously described [10]. Jurkat cells were cultured at 0.3 106 cells/
3.1. Overexpression of c-mip induces the upregulation of the PI3 kinase regulatory subunit, p85, but does not affect the expression of the catalytic subunit, p110 Since the structure of c-mip contains a PH domain, we studied the influence of c-mip on PI3 kinase, which plays a central role in proximal signaling. Immunoblotting of PBMC protein lysates showed that overexpression of c-mip induced an increased expression of p85a without affecting the expression of the catalytic subunit p110 (Fig. 1A). Similar results were obtained in c-miptransfected Jurkat T cells (Fig. 1B). The expression level of p85a in c-mip-overexpressing cells remains significantly upregulated upon activation with ConA/PHA (Fig. 1B), as compared with controls (P < 0.05). The presence of a PH domain in c-mip led us to look whether c-mip interacts with p85a. Protein lysates from c-mip and empty vector-transfected cells were immunoprecipitated with anti-p85a or anti-V5 and eluates were blotted with anti-V5 or anti-p85a, respectively. Cross-immunoprecipitations showed that c-mip interacts with endogenous p85a (Fig. 2, upper panel). We then analyzed the interaction of p85a with p110 in empty vector and c-mip-transfected cells. As shown in Fig. 2 (lower panel), p110 was clearly immunoprecipitated with p85a in c-mip overexpressing cells, while the level of p110 bound to p85a in empty vector-transfected cells was below the detection limits. This result suggests that c-mip increases the expression of p85a and interacts with PI3 kinase complex. We next analyzed the expression of Lck, an upstream signaling kinase. In resting T cells, Lck is maintained
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Fig. 2. C-mip interacts with the p85 subunit of PI3 kinase. Immunoprecipitation of protein lysates from Jurkat cells co-transfected with c-mip or empty vector (Ev) with anti-p85 or anti-V5. The eluates were blotted with anti-p110, anti-c-mip (V5) or anti-p85 antibody, as indicated. The heavy immunoglobulin chain (IgH) band is indicated.
Fig. 1. Overexpression of c-mip induces an upregulation of the subunit p85a of the PI3 kinase. (A) Expression of p85a and p110 in PBMCs transiently transfected with c-mip or empty vector (Ev). (B) Upper panel; Jurkat cells were transiently transfected with c-mip expression plasmid or empty vector (Ev); and then were or not stimulated for 1 h with Con A (5 lg/ml) and PHA (2 lg/ml); protein lysates were sequentially immunoblotted with anti-c-mip (V5), anti-p85a and anti-p110 antibodies. Reprobing with anti-actin antibody shows that equal amounts of proteins are loaded on the gel; lower panel, quantifications of p85a, using image J software. The bands were normalized over actin and plotted as fold induction. The data are expressed as the mean ± S.E.M and the two-tailed unpaired t-test is used for statistical analysis (*P < 0.05 vs. Ev; 1P < 0.05 vs. Ev 1 h).
in inactive conformation by Csk, a cytoplasmic protein tyrosine kinase that phosphorylates the major pool of Lck on tyrosine 505 (Lck-Y505) [12]. In unstimulated transfected cells, the expression level of inactive Lck is similar in empty vector- and c-mip overexpressing-cells. Upon activation by ConA/PHA, the level of LckY505 decreased significantly in empty vector but no significant change was observed in c-mip transfected cells (Fig. 3), suggesting that c-mip prevents the activation of Lck. 3.2. Overexpression of c-mip activates the MAPK-ERK1/2 pathway Since c-mip exhibits three putative docking sites for ERK, we assessed whether c-mip affects the MAPK signaling. In unstimulated cells, the pMEK1/2 proteins were significantly increased in c-mip overexpressing cells, while they were below detection limits in controls (Fig. 4A). Incubation with ConA/PHA increased the amount of pMEK1/2 basically and in c-mip-transfected cells after 1-h post stimulation, but the level declined at 6 h in empty vector-transfected cells, while it remained stable in c-mip overexpressing cells (Fig. 4A). We next analyzed the expression of pERK1/2 that are the immediate substrates of pMEK1/2. Overexpression of c-mip
Fig. 3. Overexpression of c-mip induces an inactivation of Lck. Jurkat cells were transiently transfected with c-mip or empty vector (Ev) then, cells were or not stimulated for 1 h with Con A (5 lg/ml) and PHA (2 lg/ml); protein lysates were immunobloted with anti-phopho-lck recognizing the inactive form (Lck-tyr505); then, the blots were sequentially reprobed with anti-total Lck and actin.
in Jurkat cells highly induced the expression of two 44 and 42 kDa-bands corresponding to phospho-ERK1 and phosphoERK2, respectively, while they were faintly detected in empty vector transfected cells (Fig. 4B). Further analysis of normal PBMC transfected with c-mip showed also an upregulation of pERK2 but we did not observe significant change of pERK1 (data not shown). We next determined the influence of c-mip on p38 MAPK. We found that Jurkat cells transiently transfected with c-mip expressed a higher level of phosphorylated p38, as compared with empty vector, while the total amount of protein did not change (Fig. 4C). By contrast overexpression of c-mip does not affect the phosphorylation status of the third MAPK pathway, JNK1/2 (Fig. 5A). This result led us to study the activation status of PKCh, a regulator of JNK1/2. As shown in Fig. 5B, we did not detect significant change in phosphorylation level of PKCh. We obtained identical results in transient transfections of PBMC (data not shown).
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Fig. 4. Overexpression of c-mip induces MEK1/2, ERK1/2 and p38 phosphorylation. (A) Jurkat cells were transiently transfected with c-mip or its empty vector (Ev), then stimulated for 1 h or not, with Con A (5 lg/ml) and PHA (2 lg/ml) and immunoblotted with antibodies against c-mip (V5), phospho-MEK1/2 and actin. (B) as (B) includes (C) Immunoblotting of protein lysates from Jurkat cells transiently transfected with c-mip or empty vector (ev), with antibodies against c-mip (V5), phospho-ERK1/2 and total ERK1/2, or phospho-p38 and total p38. Reprobing with anti-actin antibody shows equal amount in each lane.
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the AP1 signaling and blocks T cell activation [14]. The finding that c-mip induced ERK1/2 phosphorylation led us to assess whether AP-1 is activated following c-mip overexpression. Unexpectedly, we found that c-mip overexpression did not induce AP-1 activation, while it was highly induced by c-jun, which was used as positive control (Fig. 6A). To test whether c-mip influences the expression of cytokines, Jurkat cells were transfected with c-mip expression plasmid or empty vector and 4 h later, cells were incubated overnight with ConA/PHA. We then analyzed the expression of IFNg and IL-4 by quantitative PCR. As shown in Fig. 6B, overexpression of c-mip was associated with a downregulation of both cytokines. Since AP1 activation requires the nuclear translocation of ERK1/ 2, we sought to analyze the effect of c-mip on subcellular distribution of ERK1/2 by Western blotting of nuclear and cytosolic extracts. The lack of cytosolic protein contamination in the nuclear extracts was controlled by immunoblotting with calpain, a protein marker of the cytoplasm compartment (Fig. 7A). The expression of c-mip protein was detectable mainly in cytoplasmic and, at least degree, in nuclear fractions of c-mip overexpressing cells. The 42 and 44 kDa bands corresponding to pERK1/2 were only detected in the cytosol, with a higher intensity in c-mip overexpressing cells, as compared to empty vector overexpressing cells (Fig. 7A). On the other hand, in untransfected Jurkat cells stimulated with PMA, taken as control, pERK1/2 are detected in both, nuclear and cytosolic fractions (Fig. 7B). We conclude that overexpression of c-mip induces an upregulation of pERK1/2, which are confined to cytoplasmic compartment and thus precludes the AP1 activation. 3.4. C-mip interacts with DAP kinase-interacting protein-1 (Dip1) The full-length c-mip cDNA was used as bait in a yeast-two-hybrid interaction screen [11]. One of positive clones encodes for death associated protein kinase (DAPK)-interacting protein-1 (Dip-1), a 110 kDa protein that possesses an E3 ligase activity and it has been shown to interact in vitro and in vivo with DAPK [15]. We performed co-immunoprecipitation to confirm whether c-mip interacts with Dip1, using protein lysates from HEK cells transfected with c-mip alone, c-mip and Dip1, or Dip1 alone. As shown in Fig. 8A, Dip1 was clearly identified by immunoblotting of c-mip immunoprecipitates from protein lysates transfected with c-mip and Dip1 but not with c-mip alone. Conversely, we detected c-mip in Dip1 immunoprecipitates from HEK cells cotransfected with both expression plasmids but not with Dip1 alone. These results suggest that Dip 1 is a partner of c-mip. 3.5. Overexpression of c-mip induces an upregulation of DAP kinase
Fig. 5. Overexpression of c-mip does not induce PKCh and JNK activation. Jurkat cells were transiently transfected with c-mip expression plasmid or empty vector, then cells were or not stimulated 1 h with Con A (5 lg/ml) and PHA (2 lg/ml); protein lysates were immunoblotted with antibodies against c-mip (V5), phosphoJNK1/2 (A), or phospho-PKCh (B). Reprobing with anti-actin antibody shows equal amount in each lane.
The finding that c-mip activates ERK and interacts with Dip1, a regulator of DAPK, raised the possibility that the lack of ERK nuclear translocation may result from abnormal expression of DAP kinase. We tested the expression of DAPK in c-mip transfected Jurkat cells. As shown in Fig. 8B, c-mip induced a significant increase of endogenous DAPK, which was barely detectable in empty vector-transfected cells. The upregulation of DAPK by c-mip was also observed in other cell types in vitro and in vivo (data not shown), suggesting that the effect of c-mip on DAPK expression is cell type-independent. We conclude that c-mip induces an upregulation of DAPK, which might account for the retention of ERK in cytoplasmic compartment.
3.3. Overexpression of c-mip does not induce AP-1 activation 4. Discussion Upon activation by MEK1/2, phosphorylated ERK1/2 (pERK1/2) translocate to nucleus and activate their nuclear substrates such as Elk1 and c-fos [13]. Both c-fos and c-jun bind to AP1 sites and activate the AP1 target genes. Inhibition of ERK signaling switches off
C-mip is as new discovered gene of unknown function, which is initially identified in T lymphocytes of patients with minimal change nephrotic syndrome (MCNS) [7].
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Fig. 6. C-mip mediated-Erk activation is inefficient for AP1 activation. (A) Jurkat cells were transiently co-transfected with the c-mip or empty vector (Ev), and AP1-Luc reporter plasmid; the phRL-null vector was used as an internal control for transfection and the c-jun expression plasmid was used as a positive control. Transfected cells were cultured 24 h, then the luciferase activity was measured using the ‘‘Dual Luciferase reporter Assay”. The data are presented as relative luciferase activity (firefly luciferase/the renilla luciferase). (B) Relative expression of IFNg and Il-4 transcripts from Jurkat cells transiently transfected with the c-mip expression plasmid or empty vector and then treated with Con A and PHA as described in Section 2.
Fig. 7. Phospho-Erk does not translocate to nuclear compartment. (A) Protein lysates were immunoblotted with antibodies against c-mip (V5), phospho-ERK1/2 and calpain, which was used to control the purity of the nuclear extract. Western blot of nuclear and cytosolic fractions shows the exclusive cytoplasmic retention of phospho-ERK1/2 following c-mip overexpression. This experiment is representative of four independent experiments. NE = nuclear extracts, CE = cytosolic extracts, NT = not treated. (B) Control experiment showing the nuclear translocation of phospho-ERK1/2 in untransfected Jurkat cells treated with PMA for 30 min.
In the present study, we provide evidence that: (i) c-mip interacts with the PI3 kinase and increases the expression of p85 subunit. (ii) c-mip inhibits the activation of Lck induced by ConA/ PHA; (iii) c-mip induces the phosphorylation of the MEK/ERK signaling pathway but this activation is confined to cytoplasmic compartment and (vi) c-mip interacts with Dip1 and upregulates DAPK. The finding that c-mip maintains Lck in inactive conformation suggests that c-mip acts as a repressor in downstream steps of several signaling pathways. We demonstrate for the first time that c-
mip upregulates the subunit p85 of PI3K, without affecting the p110 levels. Previous studies have shown that overexpression of the wild-type p85 or the mutant p85 lacking the p110 binding site inhibits the PI3K signaling by competing with the binding of the p85–p110 dimer to insulin receptor substrate [16]. Conversely, mice-deficient in p85 display improved PI3K signaling [17]. In this setting, the increase of p85a may interfere with the active refolding of p110 and its subsequent binding to phosphoinositides. These data suggest that free p85 competes with the p85/p110 dimer for
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of an immune response since AP1 transcription factor is involved in T cell response and cytokine production [22]. Accordingly, we observed that overexpression of c-mip in activated Jurkat cells induces a downregulation of IFNg and Il-4. The results presented here for the first time show that c-mip interferes at different levels of cell signaling. The interaction with PI3 kinase and the inactivation of Lck suggest that c-mip exerts a negative regulatory role in proximal signaling. The fact that cmip interacts also with Dip1 suggest that c-mip is endowed with proapoptoic properties. Future investigations are required to explore the role of c-mip in several signaling pathways as well as its expression level in autoimmune diseases. Acknowledgments We are grateful to Dr. Patricia J. Gallagher (Indiana University School of Medicine, Indianapolis, Indiana, USA) for provide us the Dip1 plasmid. References
Fig. 8. C-mip interacts with Dip1 and upregulates death-associated protein kinase (DAPK). (A) HEK cells were co-transfected with c-mip and Dip1 expression plasmids, or empty vector. The upper panel shows inputs immunoblotted with anti-FLAG (Dip1), anti-c-mip (left) and anti-actin antibodies (right). Middle panel, protein lysates were immunoprecipitated with either anti-FLAG (left panel) or antic-mip (right panel) and eluates were probed with antibodies indicated in the figure. (B) Western-blot of DAPK in c-mip and empty vector-HEK transfected cells.
PIP2 binding and prevents activation of PI3K signaling. Recent experimental evidences challenge this hypothesis [18]. Indeed, it has been shown that monomeric p85, as p110, is unstable that p85 bound top110 [19]. Our results showing an inability of Con/ PHA to activate Lck in the presence of c-mip may suggest that increased expression of p85 is associated with an inhibition of PI3 kinase signaling. Inactivation of Lck in c-mip-overexpressing Jurkat cells induces indirectly a constitutive activation of the ERK/p38 MAPK pathways, such as reported in lck-deficient cells [20]. This effect is not enough to trigger AP1 activation given the inability of ERK to migrate in nuclear compartment and to transactivate its target genes, like cfos and c-jun. We demonstrate that c-mip interacts with Dip1 and upregulates DAPK, another partner of Dip1. This finding may explain how induction of c-mip leads to sequestrate ERK in the cytoplasm compartment. In fact, it has been shown that DAPK interacts with ERK and prevents its nuclear translocation, thus inhibiting the ERK signaling in the nucleus [21]. This phenomenon may represent a potential mechanism to prevent the development
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C-mip interacts with the p85 subunit of PI3 kinase and exerts a dual effect on ERK signaling via the recruitment of Dip1 and DAP kinase q Maud Kamal a,b, Andre Pawlak a,b, Fatima BenMohamed a,b, Asta Valanciuté a,b, Karine Dahan a,b, Marina Candelier a,b, Philippe Lang a,b,c,d, Georges Guellaën a,b, Djillali Sahali a,b,c,d,* a
INSERM, U 955, Equipe 21, Créteil F-94010, France Université Paris 12, Faculté de Médecine, UMRS 955, Equipe 21, Créteil, F-94010, France AP-HP, Groupe hospitalier Henri Mondor – Albert Chenevier, Service de Néphrologie, Créteil F-94010, France d Institut Francilien de Recheche en Nephrologie et Transplantation, Hopital Henri Mondor, France b c
a r t i c l e
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Article history: Received 21 September 2009 Revised 7 November 2009 Accepted 9 December 2009 Available online 16 December 2009 Edited by Zhijie Chang Keywords: C-mip PI3 kinase Lck DIP1 DAP-kinase ERK signaling
a b s t r a c t In naive T cells, Lck exerts a negative control on the ERK/MAPK pathway. We show that c-mip (c-maf inducing protein) interacts with the p85 subunit of PI3 kinase and inactivates Lck, which results in Erk1/2 and p38 MAPK activation. This effect is not enough to activate AP1 given the inability of ERK to migrate into the nucleus and to transactivate its target genes. We demonstrate that c-mip interacts with Dip1 and upregulates DAPK, which blocks the nuclear translocation of ERK1/2. This dual effect of c-mip is unique and might represent a potential mechanism to prevent the development of an immune response. Structured summary: MINT-7383650: p85 (uniprotkb:P27986) physically interacts (MI:0915) with c-Mip (uniprotkb:Q8IY22) by anti bait coimmunoprecipitation (MI:0006) MINT-7383661: c-Mip (uniprotkb:Q8IY22) physically interacts (MI:0915) with p85 (uniprotkb:P27986) by anti tag coimmunoprecipitation (MI:0007) MINT-7383676: p85 (uniprotkb:P27986) physically interacts (MI:0915) with p110 (uniprotkb:P42336) by anti bait coimmunoprecipitation (MI:0006) MINT-7383689, MINT-7383711: Dip-1 (uniprotkb:Q80SY4) physically interacts (MI:0915) with c-Mip (uniprotkb:Q8IY22) by anti tag coimmunoprecipitation (MI:0007) Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Cells respond to intra or extra cellular stimuli by engaging specific activation programs such as the signaling that leads to distinct mitogen-activated protein kinase (MAPK) cascades. Among these, the extra cellular signal regulated kinase (ERK1/2), c-jun N terminal kinase and p38 MAPK pathways are the best characterized. ERK1/2 are activated in response to growth factors via recruitment of membrane receptor tyrosine kinases, G protein-coupled receptors and integrins, whereas Jun N terminal kinase and p38 MAPK are sensitive to stress stimuli [1]. In T cells, PKCh functions as a regulator of JNK activation [2]. The ERK1/2 proteins are recruited at q This work was supported in part by an Avenir Program from INSERM, a grant from the French Kidney Foundation and Association pour l’Utilisation du Rein Artificiel (AURA). * Corresponding author. Address: INSERM, U 955, Equipe 21, Créteil F-94010, France. Fax: +33 149812539. E-mail address: [email protected] (D. Sahali).
most downstream in the signaling pathway where they play an important role in the transmission of signals involved in a wide range of cellular activities including proliferation, differentiation, survival and apoptosis [3]. These functions are mediated through sequential phosphorylation of diverse substrates such as phospholipases, transcription factors and cytoskeleton proteins. The subcellular localization of ERK1/2 determines their function: upon activation, ERK1/2 are phosphorylated and translocate into the nucleus while in unstimulated cells, they are sequestrated into the cytoplasm in inactive form. A major target of ERK activation is c-fos, a member of the AP1 transcription factor. It has been shown that sustained ERK1/2 activation influences the nature of AP1 complexes and upregulates the expression of cyclin D, allowing cells to progress in cell-cycle and to induce the upregulation of pro-proliferative genes [4]. In addition, recent studies show that ERK1/2 are involved in the regulation of innate and adaptive immunity [5]. The two isoforms of ERK, 42 and 44 kDa, are highly homologous and possess the same substrate specificity. Although
0014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2009.12.015
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most functions of ERK1 and ERK2 appear redundant, studies of isoform-specific invalidation in mice provides some differences. Thus, loss of ERK2 leads to implantation defects and embryonic lethality, whereas ERK1-deficient mice exhibit a bias toward Th1 type immune response and are susceptible to experimental autoimmune encephalomyelitis [6]. In the course of studies molecular mechanisms underlying the immunopathogenesis of idiopathic nephrotic syndrome, we isolated a new gene, c-mip (for c-maf inducing protein) [7]. The natural isoform, c-mip, encodes for an 86 kDa protein. The predicted structure of c-mip includes an N-terminal region containing a pleckstrin homology domain (PH), a middle region characterized by the presence of several interacting docking sites including, a 14-3-3 module, a PKC domain, an ERK domain, and an SH3 domain similar to the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K) and a C-terminal region containing a leucin-rich repeat (LRR) domain. The basal expression of c-mip transcript is very low in adult tissues and the protein is not detectable in physiological conditions. On the other hand, c-mip is upregulated at the mRNA and protein levels in two major sites, the podocytes and the peripheral lymphocytes during the active phase of the human disease (nephrotic syndrome). Our understanding about the role of cmip in cell signaling is far behind. We report here that c-mip interacts with PI3 kinase complex and displays a dual role in ERK signaling cascade. Although transient c-mip overexpression activates the MEK/ERK signaling, we found that active ERK did not translocate in nucleus but it was sequestrated in cytoplasmic compartment instead. Using two-hybrid system, we identified Dip1, a pro-apoptotic molecule, as a partner of c-mip. We demonstrate also that overexpression of cmip induces an upregulation of death-associated protein kinase DAP kinase, a known inhibitor of nuclear ERK translocation. These results suggest that c-mip induces cytoplasmic retention of active ERK, likely via a DAP kinase dependent mechanism.
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ml in complete RPMI medium and transfected with 1 lg DNA per 106 cells, using the Bio Rad gene pulser (Bio Rad, France), at 950 mF and 250 V. After electroporation, the cells were allowed to recover for 4 h in conditioned medium, then cultured 18 h in fresh medium containing Concanavalin A (Con A, 5 mg/ml) and Phytohemagglutinin (PHA, 2 mg/ml). At the end of the incubation, total RNA was isolated from cells and reverse transcribed as previously described [10]. 2.4. Identification of c-mip partners by yeast-two hybrid screening The yeast-two hybrid technique has been previously described [11]. Among proteins that we identified as interacting with cmip, we found the death-associated protein kinase (DAPK)-interacting protein 1 (DIP1). 2.5. Co-immunoprecipitations and Western blots All the primary antibodies used in this paper were purchased from Cell signaling except for the antibodies against pI3K, p110 and p85a, subunits (Santa Cruz), the monoclonal anti-V5 (Invitrogen), the monoclonal anti-DAPK and anti-FLAG antibodies, and the polyclonal ant-actin antibody (Sigma). The anti-c-mip polyclonal antibody production has previously been described [8]. The affinity purified anti-rabbit and anti-mouse secondary antibodies were purchased from Amersham and Santa Cruz, respectively. Immunoprecipitation and Western blot experiments were accomplished as previously described [8]. 2.6. Statistical analysis Statistical analysis of the data was performed using PRIZM 4 for Macintosh (Graphpad Software, Inc., USA). Unpaired or Paired Student t-tests were used. P value of less than 0.05 was considered significant.
2. Materials and methods 3. Results 2.1. Plasmid constructs The c-mip expression plasmid construct was previously described [8]. The Dip1 plasmid was kindly provided by Patricia J. Gallagher (Indiana University School of Medicine, Indianapolis, Indiana). The AP-1-luc, phRenilla-luc and pCRE-Luc vectors were provided from Promega and Stratagene, respectively. The c-jun cDNA was cloned in pcDNA3.1 expression vector (Invitrogen). The Ghrelin receptor (GHSR) cDNA was cloned in a pcDNA3.1 vector and was used as a positive control for cAMP activation. 2.2. Cell culture, transient transfections and dual luciferase reporter assay The preparation of peripheral blood mononuclear cells (PBMCs), cell culture conditions transients transfection experiments and dual luciferase reporter assays were performed as previously described [8]. 2.3. RT-qPCR The expression of cytokine mRNAs in the Jurkat cell line was assessed by quantitative reverse transcription-polymerase chain reaction (RT-qPCR), using the Light Cycler (Roche Diagnostics, Somerville, NJ), as previously described [9]. GAPDH were used for normalization of quantitative RT-PCR. The oligonucleotides used to amplify interferon c, IL-4 and GAPDH transcripts have been previously described [10]. Jurkat cells were cultured at 0.3 106 cells/
3.1. Overexpression of c-mip induces the upregulation of the PI3 kinase regulatory subunit, p85, but does not affect the expression of the catalytic subunit, p110 Since the structure of c-mip contains a PH domain, we studied the influence of c-mip on PI3 kinase, which plays a central role in proximal signaling. Immunoblotting of PBMC protein lysates showed that overexpression of c-mip induced an increased expression of p85a without affecting the expression of the catalytic subunit p110 (Fig. 1A). Similar results were obtained in c-miptransfected Jurkat T cells (Fig. 1B). The expression level of p85a in c-mip-overexpressing cells remains significantly upregulated upon activation with ConA/PHA (Fig. 1B), as compared with controls (P < 0.05). The presence of a PH domain in c-mip led us to look whether c-mip interacts with p85a. Protein lysates from c-mip and empty vector-transfected cells were immunoprecipitated with anti-p85a or anti-V5 and eluates were blotted with anti-V5 or anti-p85a, respectively. Cross-immunoprecipitations showed that c-mip interacts with endogenous p85a (Fig. 2, upper panel). We then analyzed the interaction of p85a with p110 in empty vector and c-mip-transfected cells. As shown in Fig. 2 (lower panel), p110 was clearly immunoprecipitated with p85a in c-mip overexpressing cells, while the level of p110 bound to p85a in empty vector-transfected cells was below the detection limits. This result suggests that c-mip increases the expression of p85a and interacts with PI3 kinase complex. We next analyzed the expression of Lck, an upstream signaling kinase. In resting T cells, Lck is maintained
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Fig. 2. C-mip interacts with the p85 subunit of PI3 kinase. Immunoprecipitation of protein lysates from Jurkat cells co-transfected with c-mip or empty vector (Ev) with anti-p85 or anti-V5. The eluates were blotted with anti-p110, anti-c-mip (V5) or anti-p85 antibody, as indicated. The heavy immunoglobulin chain (IgH) band is indicated.
Fig. 1. Overexpression of c-mip induces an upregulation of the subunit p85a of the PI3 kinase. (A) Expression of p85a and p110 in PBMCs transiently transfected with c-mip or empty vector (Ev). (B) Upper panel; Jurkat cells were transiently transfected with c-mip expression plasmid or empty vector (Ev); and then were or not stimulated for 1 h with Con A (5 lg/ml) and PHA (2 lg/ml); protein lysates were sequentially immunoblotted with anti-c-mip (V5), anti-p85a and anti-p110 antibodies. Reprobing with anti-actin antibody shows that equal amounts of proteins are loaded on the gel; lower panel, quantifications of p85a, using image J software. The bands were normalized over actin and plotted as fold induction. The data are expressed as the mean ± S.E.M and the two-tailed unpaired t-test is used for statistical analysis (*P < 0.05 vs. Ev; 1P < 0.05 vs. Ev 1 h).
in inactive conformation by Csk, a cytoplasmic protein tyrosine kinase that phosphorylates the major pool of Lck on tyrosine 505 (Lck-Y505) [12]. In unstimulated transfected cells, the expression level of inactive Lck is similar in empty vector- and c-mip overexpressing-cells. Upon activation by ConA/PHA, the level of LckY505 decreased significantly in empty vector but no significant change was observed in c-mip transfected cells (Fig. 3), suggesting that c-mip prevents the activation of Lck. 3.2. Overexpression of c-mip activates the MAPK-ERK1/2 pathway Since c-mip exhibits three putative docking sites for ERK, we assessed whether c-mip affects the MAPK signaling. In unstimulated cells, the pMEK1/2 proteins were significantly increased in c-mip overexpressing cells, while they were below detection limits in controls (Fig. 4A). Incubation with ConA/PHA increased the amount of pMEK1/2 basically and in c-mip-transfected cells after 1-h post stimulation, but the level declined at 6 h in empty vector-transfected cells, while it remained stable in c-mip overexpressing cells (Fig. 4A). We next analyzed the expression of pERK1/2 that are the immediate substrates of pMEK1/2. Overexpression of c-mip
Fig. 3. Overexpression of c-mip induces an inactivation of Lck. Jurkat cells were transiently transfected with c-mip or empty vector (Ev) then, cells were or not stimulated for 1 h with Con A (5 lg/ml) and PHA (2 lg/ml); protein lysates were immunobloted with anti-phopho-lck recognizing the inactive form (Lck-tyr505); then, the blots were sequentially reprobed with anti-total Lck and actin.
in Jurkat cells highly induced the expression of two 44 and 42 kDa-bands corresponding to phospho-ERK1 and phosphoERK2, respectively, while they were faintly detected in empty vector transfected cells (Fig. 4B). Further analysis of normal PBMC transfected with c-mip showed also an upregulation of pERK2 but we did not observe significant change of pERK1 (data not shown). We next determined the influence of c-mip on p38 MAPK. We found that Jurkat cells transiently transfected with c-mip expressed a higher level of phosphorylated p38, as compared with empty vector, while the total amount of protein did not change (Fig. 4C). By contrast overexpression of c-mip does not affect the phosphorylation status of the third MAPK pathway, JNK1/2 (Fig. 5A). This result led us to study the activation status of PKCh, a regulator of JNK1/2. As shown in Fig. 5B, we did not detect significant change in phosphorylation level of PKCh. We obtained identical results in transient transfections of PBMC (data not shown).
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Fig. 4. Overexpression of c-mip induces MEK1/2, ERK1/2 and p38 phosphorylation. (A) Jurkat cells were transiently transfected with c-mip or its empty vector (Ev), then stimulated for 1 h or not, with Con A (5 lg/ml) and PHA (2 lg/ml) and immunoblotted with antibodies against c-mip (V5), phospho-MEK1/2 and actin. (B) as (B) includes (C) Immunoblotting of protein lysates from Jurkat cells transiently transfected with c-mip or empty vector (ev), with antibodies against c-mip (V5), phospho-ERK1/2 and total ERK1/2, or phospho-p38 and total p38. Reprobing with anti-actin antibody shows equal amount in each lane.
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the AP1 signaling and blocks T cell activation [14]. The finding that c-mip induced ERK1/2 phosphorylation led us to assess whether AP-1 is activated following c-mip overexpression. Unexpectedly, we found that c-mip overexpression did not induce AP-1 activation, while it was highly induced by c-jun, which was used as positive control (Fig. 6A). To test whether c-mip influences the expression of cytokines, Jurkat cells were transfected with c-mip expression plasmid or empty vector and 4 h later, cells were incubated overnight with ConA/PHA. We then analyzed the expression of IFNg and IL-4 by quantitative PCR. As shown in Fig. 6B, overexpression of c-mip was associated with a downregulation of both cytokines. Since AP1 activation requires the nuclear translocation of ERK1/ 2, we sought to analyze the effect of c-mip on subcellular distribution of ERK1/2 by Western blotting of nuclear and cytosolic extracts. The lack of cytosolic protein contamination in the nuclear extracts was controlled by immunoblotting with calpain, a protein marker of the cytoplasm compartment (Fig. 7A). The expression of c-mip protein was detectable mainly in cytoplasmic and, at least degree, in nuclear fractions of c-mip overexpressing cells. The 42 and 44 kDa bands corresponding to pERK1/2 were only detected in the cytosol, with a higher intensity in c-mip overexpressing cells, as compared to empty vector overexpressing cells (Fig. 7A). On the other hand, in untransfected Jurkat cells stimulated with PMA, taken as control, pERK1/2 are detected in both, nuclear and cytosolic fractions (Fig. 7B). We conclude that overexpression of c-mip induces an upregulation of pERK1/2, which are confined to cytoplasmic compartment and thus precludes the AP1 activation. 3.4. C-mip interacts with DAP kinase-interacting protein-1 (Dip1) The full-length c-mip cDNA was used as bait in a yeast-two-hybrid interaction screen [11]. One of positive clones encodes for death associated protein kinase (DAPK)-interacting protein-1 (Dip-1), a 110 kDa protein that possesses an E3 ligase activity and it has been shown to interact in vitro and in vivo with DAPK [15]. We performed co-immunoprecipitation to confirm whether c-mip interacts with Dip1, using protein lysates from HEK cells transfected with c-mip alone, c-mip and Dip1, or Dip1 alone. As shown in Fig. 8A, Dip1 was clearly identified by immunoblotting of c-mip immunoprecipitates from protein lysates transfected with c-mip and Dip1 but not with c-mip alone. Conversely, we detected c-mip in Dip1 immunoprecipitates from HEK cells cotransfected with both expression plasmids but not with Dip1 alone. These results suggest that Dip 1 is a partner of c-mip. 3.5. Overexpression of c-mip induces an upregulation of DAP kinase
Fig. 5. Overexpression of c-mip does not induce PKCh and JNK activation. Jurkat cells were transiently transfected with c-mip expression plasmid or empty vector, then cells were or not stimulated 1 h with Con A (5 lg/ml) and PHA (2 lg/ml); protein lysates were immunoblotted with antibodies against c-mip (V5), phosphoJNK1/2 (A), or phospho-PKCh (B). Reprobing with anti-actin antibody shows equal amount in each lane.
The finding that c-mip activates ERK and interacts with Dip1, a regulator of DAPK, raised the possibility that the lack of ERK nuclear translocation may result from abnormal expression of DAP kinase. We tested the expression of DAPK in c-mip transfected Jurkat cells. As shown in Fig. 8B, c-mip induced a significant increase of endogenous DAPK, which was barely detectable in empty vector-transfected cells. The upregulation of DAPK by c-mip was also observed in other cell types in vitro and in vivo (data not shown), suggesting that the effect of c-mip on DAPK expression is cell type-independent. We conclude that c-mip induces an upregulation of DAPK, which might account for the retention of ERK in cytoplasmic compartment.
3.3. Overexpression of c-mip does not induce AP-1 activation 4. Discussion Upon activation by MEK1/2, phosphorylated ERK1/2 (pERK1/2) translocate to nucleus and activate their nuclear substrates such as Elk1 and c-fos [13]. Both c-fos and c-jun bind to AP1 sites and activate the AP1 target genes. Inhibition of ERK signaling switches off
C-mip is as new discovered gene of unknown function, which is initially identified in T lymphocytes of patients with minimal change nephrotic syndrome (MCNS) [7].
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Fig. 6. C-mip mediated-Erk activation is inefficient for AP1 activation. (A) Jurkat cells were transiently co-transfected with the c-mip or empty vector (Ev), and AP1-Luc reporter plasmid; the phRL-null vector was used as an internal control for transfection and the c-jun expression plasmid was used as a positive control. Transfected cells were cultured 24 h, then the luciferase activity was measured using the ‘‘Dual Luciferase reporter Assay”. The data are presented as relative luciferase activity (firefly luciferase/the renilla luciferase). (B) Relative expression of IFNg and Il-4 transcripts from Jurkat cells transiently transfected with the c-mip expression plasmid or empty vector and then treated with Con A and PHA as described in Section 2.
Fig. 7. Phospho-Erk does not translocate to nuclear compartment. (A) Protein lysates were immunoblotted with antibodies against c-mip (V5), phospho-ERK1/2 and calpain, which was used to control the purity of the nuclear extract. Western blot of nuclear and cytosolic fractions shows the exclusive cytoplasmic retention of phospho-ERK1/2 following c-mip overexpression. This experiment is representative of four independent experiments. NE = nuclear extracts, CE = cytosolic extracts, NT = not treated. (B) Control experiment showing the nuclear translocation of phospho-ERK1/2 in untransfected Jurkat cells treated with PMA for 30 min.
In the present study, we provide evidence that: (i) c-mip interacts with the PI3 kinase and increases the expression of p85 subunit. (ii) c-mip inhibits the activation of Lck induced by ConA/ PHA; (iii) c-mip induces the phosphorylation of the MEK/ERK signaling pathway but this activation is confined to cytoplasmic compartment and (vi) c-mip interacts with Dip1 and upregulates DAPK. The finding that c-mip maintains Lck in inactive conformation suggests that c-mip acts as a repressor in downstream steps of several signaling pathways. We demonstrate for the first time that c-
mip upregulates the subunit p85 of PI3K, without affecting the p110 levels. Previous studies have shown that overexpression of the wild-type p85 or the mutant p85 lacking the p110 binding site inhibits the PI3K signaling by competing with the binding of the p85–p110 dimer to insulin receptor substrate [16]. Conversely, mice-deficient in p85 display improved PI3K signaling [17]. In this setting, the increase of p85a may interfere with the active refolding of p110 and its subsequent binding to phosphoinositides. These data suggest that free p85 competes with the p85/p110 dimer for
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of an immune response since AP1 transcription factor is involved in T cell response and cytokine production [22]. Accordingly, we observed that overexpression of c-mip in activated Jurkat cells induces a downregulation of IFNg and Il-4. The results presented here for the first time show that c-mip interferes at different levels of cell signaling. The interaction with PI3 kinase and the inactivation of Lck suggest that c-mip exerts a negative regulatory role in proximal signaling. The fact that cmip interacts also with Dip1 suggest that c-mip is endowed with proapoptoic properties. Future investigations are required to explore the role of c-mip in several signaling pathways as well as its expression level in autoimmune diseases. Acknowledgments We are grateful to Dr. Patricia J. Gallagher (Indiana University School of Medicine, Indianapolis, Indiana, USA) for provide us the Dip1 plasmid. References
Fig. 8. C-mip interacts with Dip1 and upregulates death-associated protein kinase (DAPK). (A) HEK cells were co-transfected with c-mip and Dip1 expression plasmids, or empty vector. The upper panel shows inputs immunoblotted with anti-FLAG (Dip1), anti-c-mip (left) and anti-actin antibodies (right). Middle panel, protein lysates were immunoprecipitated with either anti-FLAG (left panel) or antic-mip (right panel) and eluates were probed with antibodies indicated in the figure. (B) Western-blot of DAPK in c-mip and empty vector-HEK transfected cells.
PIP2 binding and prevents activation of PI3K signaling. Recent experimental evidences challenge this hypothesis [18]. Indeed, it has been shown that monomeric p85, as p110, is unstable that p85 bound top110 [19]. Our results showing an inability of Con/ PHA to activate Lck in the presence of c-mip may suggest that increased expression of p85 is associated with an inhibition of PI3 kinase signaling. Inactivation of Lck in c-mip-overexpressing Jurkat cells induces indirectly a constitutive activation of the ERK/p38 MAPK pathways, such as reported in lck-deficient cells [20]. This effect is not enough to trigger AP1 activation given the inability of ERK to migrate in nuclear compartment and to transactivate its target genes, like cfos and c-jun. We demonstrate that c-mip interacts with Dip1 and upregulates DAPK, another partner of Dip1. This finding may explain how induction of c-mip leads to sequestrate ERK in the cytoplasm compartment. In fact, it has been shown that DAPK interacts with ERK and prevents its nuclear translocation, thus inhibiting the ERK signaling in the nucleus [21]. This phenomenon may represent a potential mechanism to prevent the development
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