Proteomic Analysis Reveals a Role for RSK in p120-catenin Phosphorylation and Melanoma Cell-Cell Adhesion.

The RAS/mitogen-activated protein kinase (MAPK) signaling pathway regulates various biological functions, including cell survival, proliferation and migration. This pathway is frequently deregulated in cancer, including melanoma, which is the most aggressive form of skin cancer. RSK (p90 ribosomal S6 kinase) is a MAPK-activated protein kinase required for melanoma growth and proliferation, but relatively little is known about its function and the nature of its cellular partners. In this study, we used a proximity-based labeling approach to identify RSK proximity partners in cells. We identified many potential RSK-interacting proteins, including p120ctn (p120-catenin), which is an essential component of adherens junction (AJ). We found that RSK phosphorylates p120ctn on Ser320, which appears to be constitutively phosphorylated in melanoma cells. We also found that RSK inhibition increases melanoma cell-cell adhesion, suggesting that constitutive RAS/MAPK signaling negatively regulates AJ integrity. Together, our results indicate that RSK plays an important role in the regulation of melanoma cell-cell adhesion.

The synthetic diazonamide DZ-2384 has distinct effects on microtubule curvature and dynamics without neurotoxicity.

Microtubule-targeting agents (MTAs) are widely used anticancer agents, but toxicities such as neuropathy limit their clinical use. MTAs bind to and alter the stability of microtubules, causing cell death in mitosis. We describe DZ-2384, a preclinical compound that exhibits potent antitumor activity in models of multiple cancer types. It has an unusually high safety margin and lacks neurotoxicity in rats at effective plasma concentrations. DZ-2384 binds the vinca domain of tubulin in a distinct way, imparting structurally and functionally different effects on microtubule dynamics compared to other vinca-binding compounds. X-ray crystallography and electron microscopy studies demonstrate that DZ-2384 causes straightening of curved protofilaments, an effect proposed to favor polymerization of tubulin. Both DZ-2384 and the vinca alkaloid vinorelbine inhibit microtubule growth rate; however, DZ-2384 increases the rescue frequency and preserves the microtubule network in nonmitotic cells and in primary neurons. This differential modulation of tubulin results in a potent MTA therapeutic with enhanced safety.

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.

APC is an RNA-binding protein, and its interactome provides a link to neural development and microtubule assembly.

Adenomatous polyposis coli (APC) is a microtubule plus-end scaffolding protein important in biology and disease. APC is implicated in RNA localization, although the mechanisms and functional significance remain unclear. We show APC is an RNA-binding protein and identify an RNA interactome by HITS-CLIP. Targets were highly enriched for APC-related functions, including microtubule organization, cell motility, cancer, and neurologic disease. Among the targets is ß2B-tubulin, known to be required in human neuron and axon migration. We show ß2B-tubulin is synthesized in axons and localizes preferentially to dynamic microtubules in the growth cone periphery. APC binds the ß2B-tubulin 3' UTR; experiments interfering with this interaction reduced ß2B-tubulin mRNA axonal localization and expression, depleted dynamic microtubules and the growth cone periphery, and impaired neuron migration. These results identify APC as a platform binding functionally related protein and RNA networks, and suggest a self-organizing model for the microtubule to localize synthesis of its own subunits.

Phosphoproteomic analysis identifies the tumor suppressor PDCD4 as a RSK substrate negatively regulated by 14-3-3.

The Ras/MAPK signaling cascade regulates various biological functions, including cell growth and proliferation. As such, this pathway is frequently deregulated in several types of cancer, including most cases of melanoma. RSK (p90 ribosomal S6 kinase) is a MAPK-activated protein kinase required for melanoma growth and proliferation, but relatively little is known about its exact function and the nature of its substrates. Herein, we used a quantitative phosphoproteomics approach to define the signaling networks regulated by RSK in melanoma. To more accurately predict direct phosphorylation substrates, we defined the RSK consensus phosphorylation motif and found significant overlap with the binding consensus of 14-3-3 proteins. We thus characterized the phospho-dependent 14-3-3 interactome in melanoma cells and found that a large proportion of 14-3-3 binding proteins are also potential RSK substrates. Our results show that RSK phosphorylates the tumor suppressor PDCD4 (programmed cell death protein 4) on two serine residues (Ser76 and Ser457) that regulate its subcellular localization and interaction with 14-3-3 proteins. We found that 14-3-3 binding promotes PDCD4 degradation, suggesting an important role for RSK in the inactivation of PDCD4 in melanoma. In addition to this tumor suppressor, our results suggest the involvement of RSK in a vast array of unexplored biological functions with relevance in oncogenesis.

Proteomic analysis of cap-dependent translation identifies LARP1 as a key regulator of 5'TOP mRNA translation.

The mammalian target of rapamycin (mTOR) promotes cell growth and proliferation by promoting mRNA translation and increasing the protein synthetic capacity of the cell. Although mTOR globally promotes translation by regulating the mRNA 5' cap-binding protein eIF4E (eukaryotic initiation factor 4E), it also preferentially regulates the translation of certain classes of mRNA via unclear mechanisms. To help fill this gap in knowledge, we performed a quantitative proteomic screen to identify proteins that associate with the mRNA 5' cap in an mTOR-dependent manner. Using this approach, we identified many potential regulatory factors, including the putative RNA-binding protein LARP1 (La-related protein 1). Our results indicate that LARP1 associates with actively translating ribosomes via PABP and that LARP1 stimulates the translation of mRNAs containing a 5' terminal oligopyrimidine (TOP) motif, encoding for components of the translational machinery. We found that LARP1 associates with the mTOR complex 1 (mTORC1) and is required for global protein synthesis as well as cell growth and proliferation. Together, these data reveal important molecular mechanisms involved in TOP mRNA translation and implicate LARP1 as an important regulator of cell growth and proliferation.

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.

Gab2 phosphorylation by RSK inhibits Shp2 recruitment and cell motility.

The scaffolding adapter protein Gab2 (Grb2-associated binder) participates in the signaling response evoked by various growth factors and cytokines. Gab2 is overexpressed in several human malignancies, including breast cancer, and was shown to promote mammary epithelial cell migration. The role of Gab2 in the activation of different signaling pathways is well documented, but less is known regarding the feedback mechanisms responsible for its inactivation. We now demonstrate that activation of the Ras/mitogen-activated protein kinase (MAPK) pathway promotes Gab2 phosphorylation on basic consensus motifs. More specifically, we show that RSK (p90 ribosomal S6 kinase) phosphorylates Gab2 on three conserved residues, both in vivo and in vitro. Mutation of these phosphorylation sites does not alter Gab2 binding to Grb2, but instead, we show that Gab2 phosphorylation inhibits the recruitment of the tyrosine phosphatase Shp2 in response to growth factors. Expression of an unphosphorylatable Gab2 mutant in mammary epithelial cells promotes an invasion-like phenotype and increases cell motility. Taken together, these results suggest that RSK is part of a negative-feedback loop that restricts Gab2-dependent epithelial cell motility. On the basis of the widespread role of Gab2 in receptor signaling, these findings also suggest that RSK plays a regulatory function in diverse receptor systems.

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.

Glycogen synthase kinase-3 phosphorylates CdGAP at a consensus ERK 1 regulatory site.

Rho GTPases regulate a multitude of cellular processes from cytoskeletal reorganization to gene transcription and are negatively regulated by GTPase-activating proteins (GAPs). Cdc42 GTPase-activating protein (CdGAP) is a ubiquitously expressed GAP for Rac1 and Cdc42. In this study, we set out to identify CdGAP-binding partners and, using a yeast two-hybrid approach, glycogen synthase kinase 3alpha (GSK-3alpha) was identified as a partner for CdGAP. GSK-3 exists in two isoforms, alpha and beta, and is involved in regulating many cellular functions from insulin response to tumorigenesis. We show that GSK-3alpha and -beta interact with CdGAP in mammalian cells. We also demonstrate that GSK-3 phosphorylates CdGAP both in vitro and in vivo on Thr-776, which we have previously shown to be an ERK 1/2 phosphorylation site involved in CdGAP regulation. We report that the mRNA and protein levels of CdGAP are increased upon serum stimulation and that GSK-3 activity is necessary for the up-regulation of the protein levels of CdGAP but not for the increase in mRNA. We conclude that GSK-3 is an important regulator of CdGAP and that regulation of CdGAP protein levels by serum presents a novel mechanism for cells to control Cdc42/Rac1 GTPase signaling pathways.

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.