CIN85 regulates the ability of MEKK4 to activate the p38 MAP kinase pathway

doi:10.1016/j.bbrc.2005.10.032

Biochemical and Biophysical Research Communications

Volume 338, Issue 2, 16 December 2005, Pages 808-814

https://doi.org/10.1016/j.bbrc.2005.10.032 Get rights and content

Abstract

CIN85 is a multi-adaptor protein involved in different cellular functions including the down-regulation of activated receptor tyrosine kinases and survival of neuronal cells. CIN85 contains three SH3 domains that specifically bind a unique proline-arginine motif (PxxxPR) found in several CIN85 effectors. In this report, we show that the MAP kinase kinase kinase MEKK4 is a new CIN85-interacting partner. This interaction is mediated by the engagement of the SH3 domains of CIN85 to three PxxxPR motifs located within MEKK4 sequence. By disrupting this interaction we demonstrated that CIN85 binding to MEKK4 enhances the activation of MKK6 and of the downstream p38 MAP kinase following oxidative stress and growth factor stimulation. CIN85 was also shown to regulate the activation of MEKK4 by GADD45 proteins and promote multi-ubiquitination of MEKK4. Taken together, these results indicate a novel role for CIN85 in the regulation of cellular stress response via the MAPK pathways.

Section snippets

Materials and methods

Antibodies and reagents. EGF was purchased from Intergen. Mouse anti-HA antibody (12CA5) was from Roche Diagnostic, rabbit anti-FLAG antibody was from Sigma–Aldrich. Anti-MEKK4 and anti-p38 rabbit sera were kindly provided by P. Gerwins. Anti-MKK3/6 rabbit antibody (sc-13069) and goat anti-MEKK4 antibody (sc-6846) were from Santa Cruz. Anti-Cbl (RF) and anti-CIN85 (CT) antibodies were previously described [6], [8]. Anti-phospho-p38^{Thr180/Tyr182} rabbit polyclonal antibody, anti-phospho-JNK

CIN85 interacts with MEKK4

The search for PxxxPR motif containing proteins led to the identification of MEKK4, which contains three of the CIN85 putative binding motifs (Fig. 1A). In order to confirm that CIN85 really binds to MEKK4, we first performed GST pull-down analysis with the SH3 domains fused individually or in tandem to GST. As shown in Fig. 1B, each SH3 domain of CIN85 binds individually to MEKK4 and all three SH3s expressed in tandem (GST-SH3ABC) bind MEKK4 even more potently. These interactions are specific

Discussion

Since its identification as a Cbl-interacting protein [20], CIN85 has been found to interact with many molecular partners through its SH3 domains and proline-rich region. The identification of the particular proline motif recognized by CIN85’s SH3 domains led to the discovery of numerous CIN85-interacting proteins [9], [10], [11]. Many of these proteins are involved in the formation and trafficking of endocytic vesicles, an essential mechanism for the down-modulation of RTKs by CIN85, as well

Acknowledgments

We thank K. Kowanetz for the initial identification of MEKK4 as a putative CIN85 binding partner. We are also thankful to P. Gerwins, A. Fornace, and M. Landstrom for providing us with expression vectors. This work was partially supported by the Swedish Strategic Foundation, Boehringer Ingelheim Foundation and Deutsche Forschungsgemeinschaft to I.D.). Philippe Soubeyran is a research fellow from the ARC (Association pour la Recherche sur le Cancer).

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ASK1 oligomerization is necessary for the initiation of the MAPK signaling cascade [16]. Interestingly, other MAP3Ks, MEKK4 and TAK1, have also been proposed to be activated by H2O2 [17,18]. This suggests the possibility that multiple MAP3Ks are redox-regulated and subject to peroxiredoxin-mediated oxidation.
The p38 mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in the cellular response to various stresses and its deregulation accompanies pathological conditions such as cancer and chronic inflammation. Hydrogen peroxide (H₂O₂) is a well-established activator of the p38 MAPK signaling pathway. However, the mechanisms of H₂O₂-induced p38 activation are not yet fully understood. In Drosophila cells, we find that H₂O₂-induced activation of p38 depends on the MAPK kinase kinase (MAP3K) Mekk1. In line with the emerging role of peroxiredoxins as H₂O₂ sensors and signal transmitters we observe an H₂O₂-dependent interaction between Mekk1 and the cytosolic peroxiredoxin of Drosophila, Jafrac1. In human cells, MEKK4 (the homologue of Mekk1) and peroxiredoxin-2 (Prx2) interact in a similar manner, suggesting an evolutionarily conserved mechanism. In both organisms, H₂O₂ induces transient disulfide-linked conjugates between the MAP3K and a typical 2-Cys peroxiredoxin. We propose that these conjugates represent the relaying of oxidative equivalents from H₂O₂ to the MAP3K and that the oxidation of Mekk1/MEKK4 leads to the downstream activation of p38 MAPK. Indeed, the depletion of cytosolic 2-Cys peroxiredoxins in human cells diminished H₂O₂-induced activation of p38 MAPK.
HER2/HER3 regulates extracellular acidification and cell migration through MTK1 (MEKK4)
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MCF-7 or T-47D cells were stimulated with HRG or sorbitol. Sorbitol was used to induce osmotic stress and activate MTK1, since MTK1 is known to function in the p38 MAP kinase pathway [11,14,58]. After stimulation, MTK1 was immunoprecipitated with MTK1 antibody that recognizes the amino-terminal proline-rich region of MTK1 (Fig. 1A).
Human MAP3K4 (MTK1) functions upstream of mitogen activated protein kinases (MAPKs). In this study we show MTK1 is required for human epidermal growth factor receptor 2/3 (HER2/HER3)-heregulin beta1 (HRG) induced cell migration in MCF-7 breast cancer cells. We demonstrate that HRG stimulation leads to association of MTK1 with activated HER3 in MCF-7 and T-47D breast cancer cells. Activated HER3 association with MTK1 is dependent on HER2 activation and is decreased by pre-treatment with the HER2 inhibitor, lapatinib. Moreover, we also identify the actin interacting region (AIR) on MTK1. Disruption of actin cytoskeletal polymerization with cytochalasin D inhibited HRG induced MTK1/HER3 association. Additionally, HRG stimulation leads to extracellular acidification that is independent of cellular proliferation. HRG induced extracellular acidification is significantly inhibited when MTK1 is knocked down in MCF-7 cells. Similarly, pre-treatment with lapatinib significantly decreased HRG induced extracellular acidification. Extracellular acidification is linked with cancer cell migration. We performed scratch assays that show HRG induced cell migration in MCF-7 cells. Knockdown of MTK1 significantly inhibited HRG induced cell migration. Furthermore, pre-treatment with lapatinib also significantly decreased cell migration. Cell migration is required for cancer cell metastasis, which is the major cause of cancer patient mortality. We identify MTK1 in the HER2/HER3-HRG mediated extracellular acidification and cell migration pathway in breast cancer cells.
TRAIL/MEKK4/p38/HSP27/Akt survival network is biphasically modulated by the Src/CIN85/c-Cbl complex
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This in turn increases HSP27 phosphorylation to intensify the decrease in Src activity that is mediated by pHSP27/TRAF6/Src. Aissouni et al. [6] showed that the interaction of CIN85 with MEKK4 increases MAPKs activation. In contrast with their results, p38 phosphorylation increased during TRAIL treatment when CIN85 was downregulated.
Previously, we showed that mitogen-activated protein kinase/extracellular signal-related kinase 4 (MEKK4) is responsible for p38 activation and that its activation during tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment also increases the catalytic activity of Akt. Here, we further investigated how the TRAIL-induced MEKK4/p38/heat shock protein (HSP27)/Akt survival network is modulated by the Src/c-Cbl interacting protein of 85 kDa (CIN85)/c-Cbl complex. TRAIL-induced activation of Akt catalytic activity and phosphorylation were highly correlated with p38/HSP27 phosphorylation, whereas the phosphorylation of p38/HSP27 increased further during incubation with curcumin and TRAIL, which caused significant apoptotic cell death. CIN85, a c-Cbl-binding protein, plays an essential role in connecting cell survival to cell death. The interaction of CIN85 with MEKK4 was increased during the late phase of TRAIL incubation, suggesting that sustained p38 and HSP27 phosphorylation protects cells by preventing further cell death. However, further increases in p38/HSP27 phosphorylation induced by cotreatment with curcumin and TRAIL converted cell fate to death. Taken together, these data demonstrate that phosphorylated p38/HSP27 as biphasic modulators act in conjunction with CIN85 to determine whether cells survive or die in response to apoptotic stress.
Intersectin 1 forms a complex with adaptor protein Ruk/CIN85 in vivo independently of epidermal growth factor stimulation
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Intersectin 1 (ITSN1) is an adaptor protein involved in clathrin-mediated endocytosis, apoptosis, signal transduction and cytoskeleton organization. Here, we show that ITSN1 forms a complex with adaptor protein Ruk/CIN85, implicated in downregulation of receptor tyrosine kinases. The interaction is mediated by the SH3A domain of ITSN1 and the third or fourth proline-rich blocks of Ruk/CIN85, and does not depend on epidermal growth factor stimulation, suggesting a constitutive association of ITSN1 with Ruk/CIN85. Moreover, both proteins colocalize in MCF-7 cells with their common binding partner, the ubiquitin ligase c-Cbl. The possible biological role of the interaction between ITSN1 and Ruk/CIN85 is discussed.
Novel Insights into the Mechanisms of CIN85 SH3 Domains Binding to Cbl Proteins: Solution-Based Investigations and In Vivo Implications
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CIN85 is a multifunctional protein that plays key roles in endocytic down-regulation of receptor tyrosine kinases, apoptosis, cell adhesion, and cytoskeleton rearrangement. Its three SH3 domains (CIN85A, CIN85B, and CIN85C) allow it to recruit multiple binding partners. To understand the manifold interactions of CIN85, we present a detailed high-resolution solution structural study of CIN85A and CIN85B binding to proline–arginine peptides derived from the cognate ligands Cbl and Cbl-b. We report the structure of CIN85B and provide evidence that both CIN85A and CIN85B, in isolation or when linked, form heterodimeric complexes with the peptides. We report unusual curved chemical shift changes for several residues of CIN85A when titrated with Cbl-b peptide, indicating the existence of more than one complex form. Here we demonstrate that CIN85A and CIN85B use different mechanisms for peptide binding.
CD2AP/CIN85 balance determines receptor tyrosine kinase signaling response in podocytes
2007, Journal of Biological Chemistry
Citation Excerpt :
Interestingly, Grb2 was identified earlier in association with CIN85 complexes in other cells; however, a time-dependent dynamic of this interaction was not previously described (23). In other epithelial cells, CIN85 is a well known mediator of ligand-dependent ubiquitination and down-regulation of RTKs (12, 24, 25), and is also described as a regulator of MEKK activity (26). Therefore we assume that the dysregulated expression of CIN85 in podocytes accounts for the detected signaling effects on different levels.
Defects in podocyte signaling are the basis of many inherited glomerular diseases leading to glomerulosclerosis. CD2-associated protein (CD2AP) is highly expressed in podocytes and is considered to play an important role in the maintenance of the glomerular slit diaphragm. Mice deficient for CD2AP (CD2AP^-/-) appear normal at birth but develop a rapid onset nephrotic syndrome at 3 weeks of age. We demonstrate that impaired intracellular signaling with subsequent podocyte damage is the reason for this delayed podocyte injury in CD2AP^-/- mice. We document that CD2AP deficiency in podocytes leads to diminished signal initiation and termination of signaling pathways mediated by receptor tyrosine kinases (RTKs). In addition, we demonstrate that CIN85, a paralog of CD2AP, is involved in termination of RTK signaling in podocytes. CIN85 protein expression is increased in CD2AP^-/- podocytes in vitro. Stimulation of CD2AP^-/- podocytes with various growth factors, including insulin-like growth factor 1, vascular endothelial growth factor, and fibroblast growth factor, resulted in a significantly decreased phosphatidylinositol 3-kinase/AKT and ERK signaling response. Moreover, increased CIN85 protein is detectable in podocytes in diseased CD2AP^-/- mice, leading to decreased base-line activation of ERK and decreased phosphorylation after growth factor stimulation in vivo. Because repression of CIN85 protein leads to a restored RTK signaling response, our results support an important role of CD2AP/CIN85 protein balance in the normal signaling response of podocytes.

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CIN85 regulates the ability of MEKK4 to activate the p38 MAP kinase pathway

Abstract

Section snippets

Materials and methods

CIN85 interacts with MEKK4

Discussion

Acknowledgments

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