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

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.     

Identification of a novel proline-arginine motif involved in CIN85-dependent clustering of Cbl and down-regulation of epidermal growth factor receptors

CIN85 is a multidomain adaptor protein implicated in Cbl-mediated down-regulation of receptor tyrosine kinases. CIN85 binding to Cbl is increased after growth factor stimulation and is critical for targeting receptor tyrosine kinases to clathrin-mediated endocytosis. Here we report the identification of a novel polyproline-arginine motif (PXXXPR), specifically recognized by the SH3 domains of CIN85 and its homologue CMS/CD2AP. This motif was indispensable for CIN85 binding to Cbl/Cbl-b, to other CIN85 SH3 domains' effectors, and for mediating an intramolecular interaction between the SH3-A domain and the proline-rich region of CIN85. Individual SH3 domains of CIN85 bound to PXXXPR peptides of Cbl/Cbl-b with micromolar affinities, whereas an extended structure of two or three SH3 domains bound with higher stoichiometry and increased affinity to the same peptides. This enabled full size CIN85 to simultaneously interact with multiple Cbl molecules, promoting their clustering in mammalian cells. The ability of CIN85 to cluster Cbl was important for ligand-induced stabilization of CIN85.Cbl.epidermal growth factor receptor complexes, as well as for epidermal growth factor receptor degradation in the lysosome. Thus, specific interactions of CIN85 SH3 domains with the PXXXPR motif in Cbl play multiple roles in down-regulation of receptor tyrosine kinases.     

Nucleolin, a shuttle protein promoting infection of human monocytes by Francisella tularensis

Background

Francisella tularensis is a highly virulent facultative intracellular bacterium, disseminating in vivo mainly within host mononuclear phagocytes. After entry into macrophages, F. tularensis initially resides in a phagosomal compartment, whose maturation is then arrested. Bacteria escape rapidly into the cytoplasm, where they replicate freely. We recently demonstrated that nucleolin, an eukaryotic protein able to traffic from the nucleus to the cell surface, acted as a surface receptor for F. tularensis LVS on human monocyte-like THP-1 cells.

Methodology/Principal Findings

Here, we followed the fate of nucleolin once F. tularensis has been endocytosed. We first confirmed by siRNA silencing experiments that expression of nucleolin protein was essential for binding of LVS on human macrophage-type THP-1 cells. We then showed that nucleolin co-localized with intracellular bacteria in the phagosomal compartment. Strikingly, in that compartment, nucleolin also co-localized with LAMP-1, a late endosomal marker. Co-immunoprecipation assays further demonstrated an interaction of nucleolin with LAMP-1. Co-localization of nucleolin with LVS was no longer detectable at 24 h when bacteria were multiplying in the cytoplasm. In contrast, with an iglC mutant of LVS, which remains trapped into the phagosomal compartment, or with inert particles, nucleolin/bacteria co-localization remained almost constant.

Conclusions/Significance

We herein confirm the importance of nucleolin expression for LVS binding and its specificity as nucleolin is not involved in binding of another intracellular pathogen as L. monocytogenes or an inert particle. Association of nucleolin with F. tularensis during infection continues intracellularly after endocytosis of the bacteria. The present work therefore unravels for the first time the presence of nucleolin in the phagosomal compartment of macrophages.     

MEK kinase 2 binds and activates protein kinase C-related kinase 2. Bifurcation of kinase regulatory pathways at the level of an MAPK kinase kinase

MEK kinase 2 (MEKK2) is a 70-kDa protein serine/threonine kinase that has been shown to function as a mitogen-activated protein kinase (MAPK) kinase kinase. MEKK2 has its kinase domain in the COOH-terminal moiety of the protein. The NH(2)-terminal moiety of MEKK2 has no signature motif that would suggest a defined regulatory function. Yeast two-hybrid screening was performed to identify proteins that bind MEKK2. Protein kinase C-related kinase 2 (PRK2) was found to bind MEKK2; PRK2 has been previously shown to bind RhoA and the Src homology 3 domain of Nck. PRK2 did not bind MEKK3, which is closely related to MEKK2. The MEKK2 binding site maps to amino acids 637-660 in PRK2, which is distinct from the binding sites for RhoA and Nck. This sequence is divergent in the closely related kinase PRK1, which did not bind MEKK2. In cells, MEKK2 and PRK2 are co-immunoprecipitated and PRK2 is activated by MEKK2. Similarly, purified recombinant MEKK2 activated PRK2 in vitro. MEKK2 activation of PRK2 is independent of MEKK2 regulation of the c-Jun NH(2)-terminal kinase pathway. MEKK2 activation of PRK2 results in a bifurcation of signaling for the dual control of MAPK pathways and PRK2 regulated responses.     

Inhibition of tristetraprolin deadenylation by poly(A) binding protein

Tristetraprolin (TTP) is the prototype for a family of RNA binding proteins that bind the tumor necrosis factor (TNF) messenger RNA AU-rich element (ARE), causing deadenylation of the TNF poly(A) tail, RNA decay, and silencing of TNF protein production. Using mass spectrometry sequencing we identified poly(A) binding proteins-1 and -4 (PABP1 and PABP4) in high abundance and good protein coverage from TTP immunoprecipitates. PABP1 significantly enhanced TNF ARE binding by RNA EMSA and prevented TTP-initiated deadenylation in an in vitro macrophage assay of TNF poly(A) stability. Neomycin inhibited TTP-promoted deadenylation at concentrations shown to inhibit the deadenylases poly(A) ribonuclease and CCR4. Stably transfected RAW264.7 macrophages overexpressing PABP1 do not oversecrete TNF; instead they upregulate TTP protein without increasing TNF protein production. The PABP1 inhibition of deadenylation initiated by TTP does not require the poly(A) binding regions in RRM1 and RRM2, suggesting a more complicated interaction than simple masking of the poly(A) tail from a 3'-exonuclease. Like TTP, PABP1 is a substrate for p38 MAP kinase. Finally, PABP1 stabilizes cotransfected TTP in 293T cells and prevents the decrease in TTP levels seen with p38 MAP kinase inhibition. These findings suggest several levels of functional antagonism between TTP and PABP1 that have implications for regulation of unstable mRNAs like TNF.     

Structure-Function Analysis of Nucleolin and ErbB Receptors Interactions

Background

The ErbB receptor tyrosine kinases and nucleolin are major contributors to malignant transformation. Recently we have found that cell-surface ErbB receptors interact with nucleolin via their cytoplasmic tail. Overexpression of ErbB1 and nucleolin leads to receptor phosphorylation, dimerization and anchorage independent growth.

Methodology/Principal Findings

In the present study we explored the regions of nucleolin and ErbB responsible for their interaction. Using mutational analyses, we addressed the structure–function relationship of the interaction between ErbB1 and nucleolin. We identified the ErbB1 nuclear localization domain as nucleolin interacting region. This region is important for nucleolin-associated receptor activation. Notably, though the tyrosine kinase domain is important for nucleolin-associated receptor activation, it is not involved in nucleolin/ErbB interactions. In addition, we demonstrated that the 212 c-terminal portion of nucleolin is imperative for the interaction with ErbB1 and ErbB4. This region of nucleolin is sufficient to induce ErbB1 dimerization, phosphorylation and growth in soft agar.

Conclusions/Significance

The oncogenic potential of ErbB depends on receptor levels and activation. Nucleolin affects ErbB dimerization and activation leading to enhanced cell growth. The C-terminal region of nucleolin and the ErbB1 NLS-domain mediate this interaction. Moreover, when the C-terminal 212 amino acids region of nucleolin is expressed with ErbB1, it can enhance anchorage independent cell growth. Taken together these results offer new insight into the role of ErbB1 and nucleolin interaction in malignant cells.     

Inhibition of CIN85-Mediated Invasion by a Novel SH3 Domain Binding Motif in the Lysyl Oxidase Propeptide

The lysyl oxidase gene inhibits Ras signaling in transformed fibroblasts and breast cancer cells. Its activity was mapped to the 162 amino acid propeptide domain (LOX-PP) of the lysyl oxidase precursor protein. LOX-PP inhibited the Her-2/Ras signaling axis in breast cancer cells, and reduced the Her-2-driven breast tumor burden in a xenograft model. Since its mechanism of action is largely unknown, co-affinity-purification/mass spectrometry was performed and the “Cbl-interacting protein of 85-kDa” (CIN85) identified as an associating protein. CIN85 is an SH3-containing adapter protein that is overexpressed in invasive breast cancers. The CIN85 SH3 domains interact with c-Cbl, an E3 ubiquitin ligase, via an unconventional PxxxPR ligand sequence, with the highest affinity displayed by the SH3-B domain. Interaction with CIN85 recruits c-Cbl to the AMAP1 complex where its ubiquitination activity is necessary for cancer cells to develop an invasive phenotype and to degrade the matrix. Direct interaction of LOX-PP with CIN85 was confirmed using co-immunoprecipitation analysis of lysates from breast cancer cells and of purified expressed proteins. CIN85 interaction with c-Cbl was reduced by LOX-PP. Domain specific CIN85 regions and deletion mutants of LOX-PP were prepared and used to map the sites of interaction to the SH3-B domain of CIN85 and to an epitope encompassing amino acids 111 to 116 of LOX-PP. Specific LOX-PP point mutant proteins P111A and R116A failed to interact with CIN85 or to compete for CIN85 binding with c-Cbl. Structural modeling identified a new atypical PxpxxRh SH3-binding motif in this region of LOX-PP. The LOX-PP interaction with CIN85 was shown to reduce the invasive phenotype of breast cancer cells, including their ability to degrade the surrounding extracellular matrix and for Matrigel outgrowth. Thus, LOX-PP interacts with CIN85 via a novel SH3-binding motif and this association reduces CIN85-promoted invasion by breast cancer cells.     

TRAIL/MEKK4/p38/HSP27/Akt survival network is biphasically modulated by the Src/CIN85/c-Cbl complex

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.     

A Screen for Selective Killing of Cells with Chromosomal Instability Induced by a Spindle Checkpoint Defect

Background

The spindle assembly checkpoint is crucial for the maintenance of a stable chromosome number. Defects in the checkpoint lead to Chromosomal INstability (CIN), which is linked to the progression of tumors with poor clinical outcomes such as drug resistance and metastasis. As CIN is not found in normal cells, it offers a cancer-specific target for therapy, which may be particularly valuable because CIN is common in advanced tumours that are resistant to conventional therapy.

Principal Findings

Here we identify genes that are required for the viability of cells with a CIN phenotype. We have used RNAi knockdown of the spindle assembly checkpoint to induce CIN in Drosophila and then screened the set of kinase and phosphatase genes by RNAi knockdown to identify those that induce apoptosis only in the CIN cells. Genes identified include those involved in JNK signaling pathways and mitotic cytoskeletal regulation.

Conclusions/Significance

The screen demonstrates that it is feasible to selectively kill cells with CIN induced by spindle checkpoint defects. It has identified candidates that are currently being pursued as cancer therapy targets (e.g. Nek2: NIMA related kinase 2), confirming that the screen is able to identify promising drug targets of clinical significance. In addition, several other candidates were identified that have no previous connection with mitosis or apoptosis. Further screening and detailed characterization of the candidates could potentially lead to the therapies that specifically target advanced cancers that exhibit CIN.     

Analysis of the Nse3/MAGE-binding domain of the Nse4/EID family proteins

Background

The Nse1, Nse3 and Nse4 proteins form a tight sub-complex of the large SMC5-6 protein complex. hNSE3/MAGEG1, the mammalian ortholog of Nse3, is the founding member of the MAGE (melanoma-associated antigen) protein family and the Nse4 kleisin subunit is related to the EID (E1A-like inhibitor of differentiation) family of proteins. We have recently shown that human MAGE proteins can interact with NSE4/EID proteins through their characteristic conserved hydrophobic pocket.

Methodology/Principal Findings

Using mutagenesis and protein-protein interaction analyses, we have identified a new Nse3/MAGE-binding domain (NMBD) of the Nse4/EID proteins. This short domain is located next to the Nse4 N-terminal kleisin motif and is conserved in all NSE4/EID proteins. The central amino acid residues of the human NSE4b/EID3 domain were essential for its binding to hNSE3/MAGEG1 in yeast two-hybrid assays suggesting they form the core of the binding domain. PEPSCAN ELISA measurements of the MAGEC2 binding affinity to EID2 mutant peptides showed that similar core residues contribute to the EID2-MAGEC2 interaction. In addition, the N-terminal extension of the EID2 binding domain took part in the EID2-MAGEC2 interaction. Finally, docking and molecular dynamic simulations enabled us to generate a structure model for EID2-MAGEC2. Combination of our experimental data and the structure modeling showed how the core helical region of the NSE4/EID domain binds into the conserved pocket characteristic of the MAGE protein family.

Conclusions/Significance

We have identified a new Nse4/EID conserved domain and characterized its binding to Nse3/MAGE proteins. The conservation and binding of the interacting surfaces suggest tight co-evolution of both Nse4/EID and Nse3/MAGE protein families.     

Deleted in Liver Cancer 1 (DLC1) Utilizes a Novel Binding Site for Tensin2 PTB Domain Interaction and Is Required for Tumor-Suppressive Function

Background

Deleted in liver cancer 1 (DLC1) is a Rho GTPase-activating protein (RhoGAP) frequently deleted and underexpressed in hepatocellular carcinoma (HCC) as well as in other cancers. Recent independent studies have shown interaction of DLC1 with members of the tensin focal adhesion protein family in a Src Homology 2 (SH2) domain-dependent mechanism. DLC1 and tensins interact and co-localize to punctate structures at focal adhesions. However, the mechanisms underlying the interaction between DLC1 and various tensins remain controversial.

Methodology/Principal Findings

We used a co-immunoprecipitation assay to identify a previously undocumented binding site at 375–385 of DLC1 that predominantly interacted with the phosphotyrosine binding (PTB) domain of tensin2. DLC1-tensin2 interaction is completely abolished in a DLC1 mutant lacking this novel PTB binding site (DLC1ΔPTB). However, as demonstrated by immunofluorescence and co-immunoprecipitation, neither the focal adhesion localization nor the interaction with tensin1 and C-terminal tensin-like (cten) were affected. Interestingly, the functional significance of this novel site was exhibited by the partial reduction of the RhoGAP activity, which, in turn, attenuated the growth-suppressive activity of DLC1 upon its removal from DLC1.

Conclusions/Significance

This study has provided new evidence that DLC1 also interacts with tensin2 in a PTB domain-dependent manner. In addition to properly localizing focal adhesions and preserving RhoGAP activity, DLC1 interaction with tensin2 through this novel focal adhesion binding site contributes to the growth-suppressive activity of DLC1.     

Pharmacological AMP Kinase Activators Target the Nucleolar Organization and Control Cell Proliferation

Aims

Phenformin, resveratrol and AICAR stimulate the energy sensor 5′-AMP activated kinase (AMPK) and inhibit the first step of ribosome biogenesis, de novo RNA synthesis in nucleoli. Nucleolar activities are relevant to human health, because ribosome production is crucial to the development of diabetic complications. Although the function of nucleoli relies on their organization, the impact of AMPK activators on nucleolar structures is not known. Here, we addressed this question by examining four nucleolar proteins that are essential for ribosome biogenesis.

Methods

Kidney cells were selected as model system, because diabetic nephropathy is one of the complications associated with diabetes mellitus. To determine the impact of pharmacological agents on nucleoli, we focused on the subcellular and subnuclear distribution of B23/nucleophosmin, fibrillarin, nucleolin and RPA194. This was achieved by quantitative confocal microscopy at the single-cell level in combination with cell fractionation and quantitative Western blotting.

Results

AMPK activators induced the re-organization of nucleoli, which was accompanied by changes in cell proliferation. Among the compounds tested, phenformin and resveratrol had the most pronounced impact on nucleolar organization. For B23, fibrillarin, nucleolin and RPA194, both agents (i) altered the nucleocytoplasmic distribution and nucleolar association and (ii) reduced significantly the retention in the nucleus. (iii) Phenformin and resveratrol also increased significantly the total concentration of B23 and nucleolin.

Conclusions

AMPK activators have unique effects on the subcellular localization, nuclear retention and abundance of nucleolar proteins. We propose that the combination of these events inhibits de novo ribosomal RNA synthesis and modulates cell proliferation. Our studies identified nucleolin as a target that is especially sensitive to pharmacological AMPK activators. Because of its response to pharmacological agents, nucleolin represents a potential biomarker for the development of drugs that diminish diabetic renal hypertrophy.     

TNNI3K,a Cardiac-Specific Kinase,Promotes Physiological Cardiac Hypertrophy in Transgenic Mice

Purpose

Protein kinase plays an essential role in controlling cardiac growth and hypertrophic remodeling. The cardiac troponin I-interacting kinase (TNNI3K), a novel cardiac specific kinase, is associated with cardiomyocyte hypertrophy. However, the precise function of TNNI3K in regulating cardiac remodeling has remained controversial.

Methods and Results

In a rat model of cardiac hypertrophy generated by transverse aortic constriction, myocardial TNNI3K expression was significantly increased by 1.62 folds (P<0.05) after constriction for 15 days. To investigate the role of TNNI3K in cardiac hypertrophy, we generated transgenic mouse lines with overexpression of human TNNI3K specifically in the heart. At the age of 3 months, the high-copy-number TNNI3K transgenic mice demonstrated a phenotype of concentric hypertrophy with increased heart weight normalized to body weight (1.31 fold, P<0.01). Echocardiography and non-invasive hemodynamic assessments showed enhanced cardiac function. No necrosis or myocyte disarray was observed in the heart of TNNI3K transgenic mice. This concentric hypertrophy maintained up to 12 months of age without cardiac dysfunction. The phospho amino acid analysis revealed that TNNI3K is a protein-tyrosine kinase. The yeast two-hybrid screen and co-immunoprecipitation assay identified cTnI as a target for TNNI3K. Moreover, TNNI3K overexpression induced cTnI phosphorylation at Ser22/Ser23 in vivo and in vitro, suggesting that TNNI3K is a novel upstream regulator for cTnI phosphorylation.

Conclusion

TNNI3K promotes a concentric hypertrophy with enhancement of cardiac function via regulating the phosphorylation of cTnI. TNNI3K could be a potential therapeutic target for preventing from heart failure.