Joe W. Ramos

The regulation of extracellular signal-regulated kinase (ERK) in mammalian cells

The mitogen-activated protein (MAP) kinase extracellular-signal-regulated kinases (ERKs) are activated by diverse mechanisms. These include ligation of receptor tyrosine kinases such as epidermal growth factor (EGF) and cell adhesion receptors such as the integrins. In general, ligand binding of these receptors leads to GTP loading and activation of the small GTPase Ras, which recruits Raf to the membrane where it is activated. Raf subsequently phosphorylates the dual specificity MAP/ERK kinase (MEK1/2) which in turn phosphorylates and thereby activates ERK. ERK is a promiscuous kinase and can phosphorylate more than 100 different substrates. Therefore activation of ERK can affect a broad array of cellular functions including proliferation, survival, apoptosis, motility, transcription, metabolism and differentiation. ERK activity is controlled by many distinct mechanisms. Scaffold proteins control when and where ERK is activated while anchoring proteins can restrain ERK localization to specific subcellular compartments. Meanwhile, phosphatases dephosphorylate and inactivate ERK thereby shutting off the pathway. Finally, several feedback mechanisms have been identified downstream of ERK activation. Here we will focus on the diverse mechanisms of ERK regulation in mammalian cells.

PEA-15 mediates cytoplasmic sequestration of ERK MAP kinase.

The ERK 1/2 MAP kinase pathway controls cell growth and survival and modulates integrin function. Here, we report that PEA-15, a protein variably expressed in multiple cell types, blocks ERK-dependent transcription and proliferation by binding ERKs and preventing their localization in the nucleus. PEA-15 contains a nuclear export sequence required for its capacity to anchor ERK in the cytoplasm. Genetic deletion of PEA-15 results in increased ERK nuclear localization with consequent increased cFos transcription and cell proliferation. Thus, PEA-15 can redirect the biological outcome of MAP kinase signaling by regulating the subcellular localization of ERK MAP kinase.

Complementation of dominant suppression implicates CD98 in integrin activation

The integrin family of adhesion receptors are involved in cell growth, migration and tumour metastasis. Integrins are heterodimeric proteins composed of an α and a β subunit, each with a large extracellular, a single transmembrane, and a short cytoplasmic domain. The dynamic regulation of integrin affinity for ligands in response to cellular signals is central to integrin function. This process is energy dependent and is mediated through integrin cytoplasmic domains. However, the cellular machinery regulating integrin affinity remains poorly understood. Here we describe a genetic strategy to disentangle integrin signalling pathways. Dominant suppression occurs when overexpression of isolated integrin β1 cytoplasmic domains blocks integrin activation. Proteins involved in integrin signalling were identified by their capacity to complement dominant suppression in an expression cloning scheme. CD98, an early T-cell activation antigen that associates with functional integrins, was found to regulate integrin activation. Furthermore, antibody-mediated crosslinking of CD98 stimulated β1 integrin-dependent cell adhesion. These data indicate that CD98 is involved in regulating integrin affinity, and validate an unbiased genetic approach to analysing integrin signalling pathways.

Recognition of ERK MAP kinase by PEA-15 reveals a common docking site within the death domain and death effector domain

PEA‐15 is a multifunctional protein that modulates signaling pathways which control cell proliferation and cell death. In particular, PEA‐15 regulates the actions of the ERK MAP kinase cascade by binding to ERK and altering its subcellular localization. The three‐dimensional structure of PEA‐15 has been determined using NMR spectroscopy and its interaction with ERK defined by characterization of mutants that modulate ERK function. PEA‐15 is composed of an N‐terminal death effector domain (DED) and a C‐terminal tail of irregular structure. NMR ‘footprinting’ and mutagenesis identified elements of both the DED and tail that are required for ERK binding. Comparison of the DED‐binding surface for ERK2 with the death domain (DD)‐binding surface of Drosophila Tube revealed an unexpected similarity between the interaction modes of the DD and DED motifs in these proteins. Despite a lack of functional or sequence similarity between PEA‐15 and Tube, these proteins utilize a common surface of the structurally similar DD and DED to recognize functionally diverse targets.

Phosphorylation of PEA-15 switches its binding specificity from ERK/MAPK to FADD

Cell signalling pathways that regulate proliferation and those that regulate programmed cell death (apoptosis) are co-ordinated. The proteins and mechanisms that mediate the integration of these pathways are not yet fully described. The phosphoprotein PEA-15 (phosphoprotein enriched in astrocytes) can regulate both the ERK (extracellular-signal-regulated kinase)/MAPK (mitogen-activated protein kinase) pathway and the death receptor-initiated apoptosis pathway. This is the result of PEA-15 binding to the ERK/MAPK or the proapoptotic protein FADD (Fas-activated death domain protein) respectively. The mechanism by which binding of PEA-15 to these proteins is controlled has not been elucidated. PEA-15 is a phosphoprotein containing a Ser-104 phosphorylated by protein kinase C and a Ser-116 phosphorylated by CamKII (calcium/calmodulin-dependent protein kinase II) or AKT. Phosphorylation of Ser-104 is implicated in the regulation of glucose metabolism, while phosphorylation at Ser-116 is required for PEA-15 recruitment to the DISC (death-initiation signalling complex). Moreover, PEA-15 must be phosphorylated at Ser-116 to inhibit apoptosis. In the present study, we report that phosphorylation at Ser-104 blocks ERK binding to PEA-15 in vitro and in vivo, whereas phosphorylation at Ser-116 promotes its binding to FADD. We further characterize phospho-epitope-binding antibodies to these sites. We report that phosphorylation does not influence the distribution of PEA-15 between the cytoplasm and nucleus of the cell since all phosphorylated states are found predominantly in the cytoplasm. We propose that phosphorylation of PEA-15 acts as the switch that controls whether PEA-15 influences proliferation or apoptosis.

Death effector domain-containing proteins.

Abstract.Death effector domains (DEDs) are protein-protein interaction structures that are found in proteins that regulate a variety of signal transduction pathways. DEDs are a part of the larger family of Death Domain structures that have been primarily described in the control of programmed cell death. The seven standard DED-containing proteins are fas associated death domain protein (FADD), Caspase-8 and 10, cellular FLICE-like inhibitory protein (c-FLIP), death effector domain containing DNA binding (DEDD), DEDD2 and phosphoprotein enriched in astrocytes 15-Kda (PEA-15). These proteins are particularly associated with the regulation of apoptosis and proliferation mediated by the tumor necrosis factor α (TNFα) receptor family. Consequently DED-containing proteins are reported to regulate transcription, migration, and proliferation, in addition to both pro and anti-apoptotic functions. Moreover, DED proteins are essential in embryonic development and homeostasis of the immune system. Here we focus on the role of DED-containing proteins in development and the pathologies arising from abnormal expression of these proteins.

PEA-15 Binding to ERK1/2 MAPKs Is Required for Its Modulation of Integrin Activation

Activation of Raf-1 suppresses integrin activation, potentially through the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). However, bulk ERK1/2 activation does not correlate with suppression. PEA-15 reverses suppression of integrin activation and binds ERK1/2. Here we report that PEA-15 reversal of integrin suppression depends on its capacity to bind ERK1/2, indicating that ERK1/2 function is indeed required for suppression. Mutations in either the death effector domain or C-terminal tail of PEA-15 that block ERK1/2 binding abrogated the reversal of integrin suppression. Furthermore, we used ERK/p38 chimeras and site-directed mutagenesis to identify ERK1/2 residues required for binding PEA-15. Mutations of residues that precede the αG helix and within the mitogen-activated protein kinase insert blocked ERK2 binding to PEA-15, but not activation of ERK2. These ERK2 mutants blocked the ability of PEA-15 to reverse suppression of integrin activation. Thus, PEA-15 regulation of integrin activation depends on its binding to ERK1/2. To directly test the role of ERK1/2 localization in suppression, we enforced membrane association of ERK1 and 2 by joining a membrane-targeting CAAX box sequence to them. Both ERK1-CAAX and ERK2-CAAX were membrane-localized and suppressed integrin activation. In contrast to suppression by membrane-targeted Raf-CAAX, suppression by ERK1/2-CAAX was not reversed by PEA-15. Thus, ERK1/2 are the Raf effectors for suppression of integrin activation, and PEA-15 reverses suppression by binding ERK1/2.

ERK MAP kinase is targeted to RSK2 by the phosphoprotein PEA-15.

The ERK pathway responds to extracellular stimuli and oncogenes by modulating cellular processes, including transcription, adhesion, survival, and proliferation. ERK has diverse substrates that carry out these functions. The processes that are modulated are determined in part by the substrates that ERK phosphorylates. We demonstrate that PEA-15 (phosphoprotein enriched in astrocytes, 15 kDa) targets ERK to RSK2 and thereby enhances RSK2 activation. PEA-15 independently bound ERK and RSK2 and increased ERK association with RSK2 in a concentration-dependent manner. PEA-15 increased RSK2 activity and CREB-mediated transcription, and this process was regulated by phosphorylation of PEA-15. Finally, phorbol ester stimulation of PEA-15-null lymphocytes resulted in impaired RSK2 activation that was rescued by exogenous PEA-15 expression. Therefore, PEA-15 functions as a scaffold to enhance ERK activation of RSK2, and this activity is regulated by phosphorylation. Thus, PEA-15 can integrate signal transduction to provide a specific physiological outcome from activation of the multipotent ERK MAP kinase pathway.

R-Ras Regulates Migration through an Interaction with Filamin A in Melanoma Cells

Background Changes in cell adhesion and migration in the tumor microenvironment are key in the initiation and progression of metastasis. R-Ras is one of several small GTPases that regulate cell adhesion and migration on the extracellular matrix, however the mechanism has not been completely elucidated. Using a yeast two-hybrid approach we sought to identify novel R-Ras binding proteins that might mediate its effects on integrins. Methods and Findings We identified Filamin A (FLNa) as a candidate interacting protein. FLNa is an actin-binding scaffold protein that also binds to integrin β1, β2 and β7 tails and is associated with diverse cell processes including cell migration. Indeed, M2 melanoma cells require FLNa for motility. We further show that R-Ras and FLNa interact in co-immunoprecipitations and pull-down assays. Deletion of FLNa repeat 3 (FLNaΔ3) abrogated this interaction. In M2 melanoma cells active R-Ras co-localized with FLNa but did not co-localize with FLNa lacking repeat 3. Thus, activated R-Ras binds repeat 3 of FLNa. The functional consequence of this interaction was that active R-Ras and FLNa coordinately increased cell migration. In contrast, co-expression of R-Ras and FLNaΔ3 had a significantly reduced effect on migration. While there was enhancement of integrin activation and fibronectin matrix assembly, cell adhesion was not altered. Finally, siRNA knockdown of endogenous R-Ras impaired FLNa-dependent fibronectin matrix assembly. Conclusions These data support a model in which R-Ras functionally associates with FLNa and thereby regulates integrin-dependent migration. Thus in melanoma cells R-Ras and FLNa may cooperatively promote metastasis by enhancing cell migration.

In vitro and in vivo evaluation of water-soluble iminophosphorane ruthenium(II) compounds. A potential chemotherapeutic agent for triple negative breast cancer.

A series of organometallic ruthenium(II) complexes containing iminophosphorane ligands have been synthesized and characterized. Cationic compounds with chloride as counterion are soluble in water (70–100 mg/mL). Most compounds (especially highly water-soluble 2) are more cytotoxic to a number of human cancer cell lines than cisplatin. Initial mechanistic studies indicate that the cell death type for these compounds is mainly through canonical or caspase-dependent apoptosis, nondependent on p53, and that the compounds do not interact with DNA or inhibit protease cathepsin B. In vivo experiments of 2 on MDA-MB-231 xenografts in NOD.CB17-Prkdc SCID/J mice showed an impressive tumor reduction (shrinkage) of 56% after 28 days of treatment (14 doses of 5 mg/kg every other day) with low systemic toxicity. Pharmacokinetic studies showed a quick absorption of 2 in plasma with preferential accumulation in the breast tumor tissues when compared to kidney and liver, which may explain its high efficacy in vivo.