Joseph Tcherkezian

Current knowledge of the large RhoGAP family of proteins

The Rho GTPases are implicated in almost every fundamental cellular process. They act as molecular switches that cycle between an active GTP‐bound and an inactive GDP‐bound state. Their slow intrinsic GTPase activity is greatly enhanced by RhoGAPs (Rho GTPase‐activating proteins), thus causing their inactivation. To date, more than 70 RhoGAPs have been identified in eukaryotes, ranging from yeast to human, and based on sequence homology of their RhoGAP domain, we have grouped them into subfamilies. In the present Review, we discuss their regulation, biological functions and implication in human diseases.

Transmembrane Receptor DCC Associates with Protein Synthesis Machinery and Regulates Translation

Extracellular signals regulate protein translation in many cell functions. A key advantage of control at the translational level is the opportunity to regulate protein synthesis within specific cellular subregions. However, little is known about mechanisms that may link extracellular cues to translation with spatial precision. Here, we show that a transmembrane receptor, DCC, forms a binding complex containing multiple translation components, including eukaryotic initiation factors, ribosomal large and small subunits, and monosomes. In neuronal axons and dendrites DCC colocalizes in particles with translation machinery, and newly synthesized protein. The extracellular ligand netrin promoted DCC-mediated translation and disassociation of translation components. The functional and physical association of a cell surface receptor with the translation machinery leads to a generalizable model for localization and extracellular regulation of protein synthesis, based on a transmembrane translation regulation complex.

Phosphorylation of DCC by Fyn mediates Netrin-1 signaling in growth cone guidance

Netrin-1 acts as a chemoattractant molecule to guide commissural neurons (CN) toward the floor plate by interacting with the receptor deleted in colorectal cancer (DCC). The molecular mechanisms underlying Netrin-1–DCC signaling are still poorly characterized. Here, we show that DCC is phosphorylated in vivo on tyrosine residues in response to Netrin-1 stimulation of CN and that the Src family kinase inhibitors PP2 and SU6656 block both Netrin-1–dependent phosphorylation of DCC and axon outgrowth. PP2 also blocks the reorientation of Xenopus laevis retinal ganglion cells that occurs in response to Netrin-1, which suggests an essential role of the Src kinases in Netrin-1–dependent orientation. Fyn, but not Src, is able to phosphorylate the intracellular domain of DCC in vitro, and we demonstrate that Y1418 is crucial for DCC axon outgrowth function. Both DCC phosphorylation and Netrin-1–induced axon outgrowth are impaired in Fyn−/− CN and spinal cord explants. We propose that DCC is regulated by tyrosine phosphorylation and that Fyn is essential for the response of axons to Netrin-1.

The expanding role of mTOR in cancer cell growth and proliferation

The mechanistic/mammalian target of rapamycin (mTOR) is a conserved protein kinase that controls several anabolic processes required for cell growth and proliferation. As such, mTOR has been implicated in an increasing number of pathological conditions, including cancer, obesity, type 2 diabetes and neurodegeneration. As part of the mTOR complex 1 (mTORC1), mTOR regulates cell growth by promoting the biosynthesis of proteins, lipids and nucleic acids. Several mTORC1 substrates have been shown to regulate protein synthesis, including the eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BPs) and the ribosomal S6 kinases (S6Ks) 1 and 2. In this work, we focus on the signalling pathways that lie both upstream and downstream of mTORC1, as well as their relevance to human pathologies. We further discuss pharmacological approaches that target mTOR and their applications for the treatment of cancer.

Glycogen synthase kinase-3β positively regulates protein synthesis and cell proliferation through the regulation of translation initiation factor 4E-binding protein 1

Protein synthesis has a key role in the control of cell proliferation, and its deregulation is associated with pathological conditions, notably cancer. Rapamycin, an inhibitor of mammalian target of rapamycin complex 1 (mTORC1), was known to inhibit protein synthesis. However, it does not substantially inhibit protein synthesis and cell proliferation in many cancer types. We were interested in finding a novel target in rapamycin-resistant cancer. The rate-limiting factor for translation is eukaryotic translation initiation factor 4E (eIF4E), which is negatively regulated by eIF4E-binding protein 1 (4E-BP1). Here, we provide evidence that glycogen synthase kinase (GSK)-3β promotes cell proliferation through positive regulation of protein synthesis. We found that GSK-3β phosphorylates and inactivates 4E-BP1, thereby increasing eIF4E-dependent protein synthesis. Considering the clinical relevance of pathways regulating protein synthesis, our study provides a promising new strategy and target for cancer therapy.

Extracellular Signal-Regulated Kinase 1 Interacts with and Phosphorylates CdGAP at an Important Regulatory Site

ABSTRACT Rho GTPases regulate multiple cellular processes affecting both cell proliferation and cytoskeletal dynamics. Their cycling between inactive GDP- and active GTP-bound states is tightly regulated by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs). We have previously identified CdGAP (for Cdc42 GTPase-activating protein) as a specific GAP for Rac1 and Cdc42. CdGAP consists of an N-terminal RhoGAP domain and a C-terminal proline-rich region. In addition, CdGAP is a member of the impressively large number of mammalian RhoGAP proteins that is well conserved among both vertebrates and invertebrates. In mice, we find two predominant isoforms of CdGAP differentially expressed in specific tissues. We report here that CdGAP is highly phosphorylated in vivo on serine and threonine residues. We find that CdGAP is phosphorylated downstream of the MEK-extracellular signal-regulated kinase (ERK) pathway in response to serum or platelet-derived growth factor stimulation. Furthermore, CdGAP interacts with and is phosphorylated by ERK-1 and RSK-1 in vitro. A putative DEF (docking for ERK FXFP) domain located in the proline-rich region of CdGAP is required for efficient binding and phosphorylation by ERK1/2. We identify Thr776 as an in vivo target site of ERK1/2 and as an important regulatory site of CdGAP activity. Together, these data suggest that CdGAP is a novel substrate of ERK1/2 and mediates cross talk between the Ras/mitogen-activated protein kinase pathway and regulation of Rac1 activity.

Cell cortex composition and homeostasis resolved by integrating proteomics and quantitative imaging.

The cellular actin cortex is the cytoskeletal structure primarily responsible for the control of animal cell shape and as such plays a central role in cell division, migration, and tissue morphogenesis. Due to the lack of experimental systems where the cortex can be investigated independently from other organelles, little is known about its composition, assembly, and homeostasis. Here, we describe novel tools to resolve the composition and regulation of the cortex. We report and validate a protocol for cortex purification based on the separation of cellular blebs. Mass spectrometry analysis of purified cortices provides a first extensive list of cortical components. To assess the function of identified proteins, we design an automated imaging assay for precise quantification of cortical actomyosin assembly dynamics. We show subtle changes in cortex assembly dynamics upon depletion of the identified cortical component profilin. Our widely applicable integrated method paves the way for systems‐level investigations of the actomyosin cortex and its regulation during morphogenesis.

RSK promotes G2 DNA damage checkpoint silencing and participates in melanoma chemoresistance

The incidence of malignant melanoma is growing rapidly worldwide and there is still no effective therapy for metastatic disease. This type of cancer is highly resistant to conventional DNA-damaging chemotherapeutics, and intense research has been dedicated for understanding the molecular pathways underlying chemoresistance. The Ras/mitogen-activated protein kinase (MAPK) signalling pathway is often deregulated in melanoma, which frequently harbours activating mutations in NRAS or BRAF. Herein, we demonstrate that the MAPK-activated protein kinase RSK (p90 ribosomal S6 kinase) contributes to melanoma chemoresistance by altering their response to chemotherapeutic agents. We find that RSK phosphorylates checkpoint kinase 1 (Chk1) at an inhibitory site, Ser280, both in vitro and in vivo. Our results indicate that RSK is the predominant protein kinase operating downstream of mitogens and oncogenes of the Ras/MAPK pathway, and consistent with this, we find that RSK constitutively phosphorylates Chk1 in melanoma. We show that RSK inhibition increases Chk1 activity in response to DNA-damaging agents, suggesting that the Ras/MAPK pathway modulates Chk1 function and the response to DNA damage. Accordingly, we demonstrate that RSK promotes G2 DNA damage checkpoint silencing in a Chk1-dependent manner, and find that RSK inhibitors sensitize melanoma cells to DNA-damaging agents. Together, our results identify a novel link between the Ras/MAPK pathway and the DNA damage response, and suggest that RSK inhibitors may be used to modulate chemosensitivity, which is one of the major obstacles to melanoma treatment.

Disruption of TBC1D7, a subunit of the TSC1-TSC2 protein complex, in intellectual disability and megalencephaly

Background Mutations in TSC1 or TSC2 cause the tuberous sclerosis complex (TSC), a disorder characterised by the development of hamartomas or benign tumours in various organs as well as the variable presence of epilepsy, intellectual disability (ID) and autism. TSC1, TSC2 and the recently described protein TBC1D7 form a complex that inhibits mTORC1 signalling and limits cell growth. Although it has been proposed that mutations in TBC1D7 might also cause TSC, loss of its function has not yet been documented in humans. Methods and Results We used homozygosity mapping and exome sequencing to study a consanguineous family with ID and megalencephaly but without any specific features of TSC. We identified only one rare coding variant, c.538delT:p.Y180fsX1 in TBC1D7, in the regions of homozygosity shared by the affected siblings. We show that this mutation abolishes TBC1D7 expression and is associated with increased mTORC1 signalling in cells of the affected individuals. Conclusions Our study suggests that disruption of TBC1D7 causes ID but without the other typical features found in TSC. Although megalencephaly is not commonly observed in TSC, it has been associated with mTORC1 activation. Our observation thus reinforces the relationship between this pathway and the development of megalencephaly.

Phosphorylation of the Eukaryotic Translation Initiation Factor 4E- Transporter (4E-T) by c-Jun N-Terminal Kinase Promotes Stress- Dependent P-Body Assembly

ABSTRACT Processing bodies (PBs, or P bodies) are cytoplasmic granules involved in mRNA storage and degradation that participate in the regulation of gene expression. PBs concentrate nontranslated mRNAs and several factors involved in mRNA decay and translational repression, including the eukaryotic translation initiation factor 4E-transporter (4E-T). 4E-T is required for PB assembly, but little is known about the molecular mechanisms that regulate its function. Here, we demonstrate that oxidative stress promotes multisite 4E-T phosphorylation. We show that the c-Jun N-terminal kinase (JNK) is targeted to PBs in response to oxidative stress and promotes the phosphorylation of 4E-T. Quantitative mass spectrometry analysis reveals that JNK phosphorylates 4E-T on six proline-directed sites that are required for the formation of the 4E-T complex upon stress. We have developed an image-based computational method to quantify the size, number, and density of PBs in cells, and we find that while 4E-T is required for steady-state PB assembly, its phosphorylation facilitates the formation of larger PBs upon oxidative stress. Using polysomal mRNA profiling, we assessed global and specific mRNA translation but did not find that 4E-T phosphorylation impacts translational control. Collectively, these data support a model whereby PB assembly is regulated by a two-step mechanism involving a 4E-T-dependent assembly stage in unstressed cells and a 4E-T phosphorylation-dependent aggregation stage in response to stress stimuli.