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Upon their interaction with cognate antigen, T cells integrate different extracellular and intracellular signals involving basal and induced protein-protein interactions, as well as the binding of proteins to lipids, which can lead to either cell activation or inhibition. Here, we show that the selective T cell expression of CMIP, a new adapter protein, by targeted transgenesis drives T cells toward a naïve phenotype. We found that CMIP inhibits activation of the Src kinases Fyn and Lck after CD3/CD28 costimulation and the subsequent localization of Fyn and Lck to LRs. Video microscopy analysis showed that CMIP blocks the recruitment of LAT and the lipid raft marker cholera toxin B at the site of TCR engagement. Proteomic analysis identified several protein clusters differentially modulated by CMIP and, notably, Cofilin-1, which is inactivated in CMIP-expressing T cells. Moreover, transgenic T cells exhibited the downregulation of GM3 synthase, a key enzyme involved in the biosynthesis of gangliosides. These results suggest that CMIP negatively impacts proximal signaling and cytoskeletal rearrangement and defines a new mechanism for the negative regulation of T cells that could be a therapeutic target.

Anti-cancer therapeutic approaches targeting the vascular endothelial growth factor (VEGF) ligand (anti-VEGF) or inhibiting its receptors (RTKI) have recently been developed. In spite of the promising results achieved, a serious drawback and dose-limiting side effect is the development, among others, of renal complications. This encompasses two glomerular pathological entities, namely minimal change/focal segmental glomerulosclerosis and thrombotic micro-angiopathy, involving two distinct cell types, podocytes and endothelial cells, respectively. The mechanisms that link anti-cancer therapy by RTKI to podocyte dysfunction and nephrotic level proteinuria are still poorly understood. Nevertheless, recent findings strongly suggest a central role of RelA, the master subunit of NF-κB and c-mip, an active player in podocyte disorders. RelA, which is up-regulated following anti-VEGF therapy, is inactivated by RTKI, leading to c-mip over-expression in the podocyte. This results in severe alterations in the architecture of podocyte actin cytoskeleton and subsequent severe proteinuria. Hence, clarifying the mechanisms linking c-mip and RelA as key pathogenic factors represents a critical goal in the understanding of different glomerulopathies. In the context of VEGF-targeted anti-cancer therapy, the study of these mechanisms along with the molecular cross-talk between podocyte and endothelial cell constitutes the basis for the emerging field of onconephrology., (© The Author 2014. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

Idiopathic nephrotic syndrome comprises several podocyte diseases of unknown origin that affect the glomerular podocyte, which controls the permeability of the filtration barrier in the kidney to proteins. It is characterized by the daily loss of more than 3 g of protein in urine and the lack of inflammatory lesions or cell infiltration. We found that the abundance of c-mip (c-maf inducing protein) was increased in the podocytes of patients with various acquired idiopathic nephrotic syndromes in which the podocyte is the main target of injury. Mice engineered to have excessive c-mip in podocytes developed proteinuria without morphological alterations, inflammatory lesions, or cell infiltration. Excessive c-mip blocked podocyte signaling by preventing the interaction of the slit diaphragm transmembrane protein nephrin with the tyrosine kinase Fyn, thereby decreasing phosphorylation of nephrin in vitro and in vivo. Moreover, c-mip inhibited interactions between Fyn and the cytoskeletal regulator N-WASP (neural Wiskott-Aldrich syndrome protein) and between the adaptor protein Nck and nephrin, potentially accounting for cytoskeletal disorganization and the effacement of foot processes seen in idiopathic nephrotic syndromes. The intravenous injection of small interfering RNA targeting c-mip prevented lipopolysaccharide-induced proteinuria in mice. Together, these results identify c-mip as a key component in the molecular pathogenesis of acquired podocyte diseases.

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., (2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The fine regulation of NF-kappaB activity is crucial for both resting and stimulated cells and relies on complex balance between multiple activators and inhibitors. We report here that c-mip, a recently identified pleckstrin homology (PH) and leucine-rich repeat (LRR)-domain-containing protein, inactivates GSKbeta and interacts with RelA, a key member of the NF-kappaB family. We show that c-mip inhibits the degradation of I-kappaBalpha and impedes the dissociation of the NF-kappaB/I-kappaBalpha complexes. C-mip acts downstream signaling of classical NF-kappaB pathway and may represent one of the missing links in the control of NF-kappaB activity.

Using a yeast two-hybrid screen, we identified Filamin-A as a binding partner of the new adapter protein c-mip (c-maf inducing protein) and it's splice variant Tc-mip (truncated c-maf inducing protein). We have previously shown that Tc-mip is involved in Th2 signaling pathway and cytoskeletal reorganization in patients with minimal change nephrotic syndrome (MCNS), the most frequent glomerular disease in children. We showed that Filamin-A and c-mip or Tc-mip co-immunoprecipitate from c-mip or Tc-mip Jurkat transfected cells using antibodies directed against both types of proteins. In co-immunoprecipitate Jurkat cells, Filamin-A and c-mip were distributed evenly in the cytoplasm, whereas in Tc-mip-transfected Jurkat cells, Filamin-A was expressed in zones facing the cell contact. Moreover, we found that Filamin-A was upregulated in T lymphocytes of MCNS patients, as compared to normal subjects. These findings suggest that Filamin-A interacts with c-mip/Tc-mip in this new T-cell signaling pathway.

Several arguments suggest that minimal change nephrotic syndrome (MCNS) results from yet unknown systemic disorder of T cell function. By screening a cDNA library from T cell relapse, we identified a new pleckstrin homology (PH) domain-containing protein encoded by a gene located on chromosome 16q24. Two alternative transcripts were identified. The first species (c-mip) was expressed in fetal liver, kidney, and peripheral blood mononuclear cells (PBMCs), but weakly detected in PBMCs from MCNS patients. The second form (Tc-mip, standing for truncated c-maf inducing protein), corresponds to subtracted transcript and lacks the NH2-terminal PH domain. The expression of Tc-mip was restricted to fetal liver, thymus, and MCNS PBMCs where it was specifically recruited in CD4+ T cells subset. Overexpression of Tc-mip in T cell Jurkat induced c-maf, transactivated the interleukin 4 gene and down-regulated the interferon gamma expression, characteristic of a Th2 commitment. Moreover, the overexpression of Tc-mip induced Src phosphorylation, T cell clustering, and a cellular redistribution of the cytoskeleton-associated L-plastin, by a PI3 kinase independent pathway. Tc-mip represents therefore the first identified protein, which links proximal signaling to c-maf induction.