Photoaffinity labelling-based chemoproteomic strategy identifies PEBP1 as the target of ethyl gallate against macrophage activation.

Ulcerative colitis (UC) is an inflammatory disease of the colon with an unmet need for therapeutic targets. Ethyl gallate (EG) is a natural small molecule for UC treatment, but its cellular target is unknown. By labelling EG with a diazirine photocrosslinker and a click chemistry handle, we identified phosphatidyl-ethanolamine binding protein1 (PEBP1) as a direct cellular target of EG by forming hydrogen bonds with Asp70 and Tyr120. In particular, hydrogen/deuterium exchange mass spectrometry indicated that EG induced the sequence (residues 141-153) embedding to inhibit S153 phosphorylation of PEBP1. Additionally, the EG-mediated sequence (residues 108-122) exposure significantly enhanced PEBP1-Raf-1 interaction to block the downstream NF-κB inflammatory pathway in macrophages. Moreover, PEBP1 siRNA substantially reversed the EG-dependent down-regulation of the phosphorylation of IKKß, IκBα and NF-κB, demonstrating that the NF-κB signal functioned as an essential anti-inflammation mechanism of PEBP1. Collectively, we revealed PEBP1 as a previously undescribed cellular target in macrophages for UC therapy and identified a new allosteric site for PEBP1 biology study using EG as a chemical probe.

Resveratrol binds and activates RKIP protein in colorectal cancer.

Raf-1 kinase inhibitory protein (RKIP) acts as a tumor cell metastasis suppressor and prognostic indicator for survival in various cancers. Its use is predicted to improve therapy for various malignancies, including colorectal cancer (CRC). RKIP, frequently denoted as phosphatidylethanolamine-binding protein 1, is expressed in all normal mammalian tissues. RKIP functions as an inhibitor of the Raf-1, PI-3K, and MAP kinase (MAPK) pathways. In this study, we found that resveratrol induced the expression of RKIP at protein levels. To elucidate the structural basis of the interaction between resveratrol and RKIP, we performed computational studies that explore the binding affinity and ligand efficacy of resveratrol against RKIP. This study reveals the prognostic significance of RKIP metastasis suppressor activity against CRC and its structural arrangements during drug-target interactions.

HIF-1α and RKIP: a computational approach for pancreatic cancer therapy.

Protein-protein interactions (PPIs) are important biochemical processes that represent a major challenge in modern biology. Current approaches, which include high-throughput screening and computer aided ligand design, have limitations regarding the identification of hit matter. This current investigation focuses on computational study for protein-protein docking of hypoxia inducible factor-1α (HIF-1α), a tumor inducible factor, and Raf-1 kinase inhibitory protein (RKIP), a tumor metastasis suppressor. These are individually crystallized structures of interacting proteins, which interact to generate a conformational space. HIF activity in pancreatic tumors is determined by hypoxia and HIF-1α subunit availability. RKIP can be used as a prognostic indicator in a number of tumors. The interaction of RKIP with HIF-1α protects against pancreatic cancer (PC) metastasis by inhibiting its hypoxia function. We have explored the binding affinity between both the proteins with the HADDOCK (high ambiguity driven protein-protein docking) server, which determined that 158 structures in 11 clusters represent 79.0% of water-refined models. Of the best 10 clusters, the structures of cluster 2 were found to be better, as they had the lowest Z-score. Further supporting HIF-1α-RKIP interaction, pulldown assay has shown dissociation of RKIP from HIF-1α after CoCl2 treatment in both PC cell lines.

RAF Kinase Inhibitor Protein in Myeloid Leukemogenesis.

RAF kinase inhibitor protein (RKIP) is an essential regulator of intracellular signaling. A somatic loss of RKIP expression is a frequent event in solid human cancers, and a role of RKIP as metastasis-suppressor is widely accepted nowadays. Recently, RKIP loss has been described in acute myeloid leukemia (AML) and a series of other myeloid neoplasias (MNs). Functional in vitro and in vivo experiments revealed that RKIP is an essential player within the development of these liquid tumors; however, the respective role of RKIP seems to be complex and multi-faceted. In this review, we will summarize the current knowledge about RKIP in myeloid leukemogenesis. We will initially describe its involvement in physiologic hematopoiesis, and will then proceed to discuss its role in the development of AML and other MNs. Finally, we will discuss potential therapeutic implications arising thereof.

Functional analysis and characterization of antimicrobial phosphatidylethanolamine-binding protein BmPEBP in the silkworm Bombyx mori.

Phosphatidylethanolamine-binding proteins (PEBPs) are a class of highly conserved, biologically diverse proteins, which are widely distributed in plants, insects, and mammals. In this study, a Bombyx mori PEBP (BmPEBP) gene was reported, which encodes a protein composed of 209 amino acid residues. BmPEBP includes a predicted signal peptide, indicating that it is an extracellular protein, which differs from the cytoplasmic PEBPs of plants and mammals. Recombinant soluble BmPEBP was successfully synthesized using a prokaryotic expression system and was then purified effectively by Ni2+-NTA affinity chromatography and gel filtration. Far-ultraviolet circular dichroism spectra indicated that BmPEBP had a well-defined ß-sheet structure, with the ß-sheet content accounting for about 41% of the protein. BmPEBP had a relatively stable structure at temperatures ranging from 15 °C to 57.5 °C. The Tm, ΔH, and ΔS of BmPEBP were 62.27 °C ±â€¯0.14 °C, 570.10 ±â€¯0.17 kJ/mol, and 1.70 ±â€¯0.03 KJ/(mol·K), respectively. Homology modeling analysis suggested that the active sites of BmPEBP were conserved, comprising Pro96, His111, and His143. Quantitative real-time PCR showed that BmPEBP was highly expressed in the silk gland and had very low expression in other tissues. However, BmPEBP expression was significantly upregulated in the larval fat body after infection with two kinds of fungi, Beauveria bassiana and Candida albicans. Moreover, in vitro fungal inhibition tests showed that BmPEBP could significantly inhibit the sporular growth of Saccharomyces cerevisiae, C. albicans, B. bassiana, and Aspergillus fumigatus. To our knowledge, this is the first report to reveal the antifungal role of a PEBP in insects.

Anti-leprosy drug Clofazimine binds to human Raf1 kinase inhibitory protein and enhances ERK phosphorylation.

Human Raf1 kinase inhibitory protein (hRKIP) is an important modulator of the Ras/Raf1/MEK/ERK signaling pathway. Here, we demonstrated that anti-leprosy drug Clofazimine can bind to hRKIP with a significantly stronger affinity than the endogenous substrate phosphatidylethanolamine (PE) by using Biolayer interference technology. Moreover, we identified that residues P74, S75, K80, P111, P112, V177, and P178 play crucial roles in the binding of hRKIP to Clofazimine by using a combination of Nuclear Magnetic Resonance spectroscopy and molecular docking approach. These residues are located at the conserved ligand-binding pocket of hRKIP. Furthermore, we found that 3.2 µM Clofazimine could significantly increase the ERK phosphorylation level by about 37%. Our results indicate that Clofazimine can enhance Ras/Raf1/MEK/ERK signaling transduction pathway via binding to hRKIP. This work provides valuable hints for exploiting Clofazimine as a potential lead compound to efficiently treat the diseases related to RKIP or the Ras/Raf/MEK/ERK pathway.

A modified HLA-A*0201-restricted CTL epitope from human oncoprotein (hPEBP4) induces more efficient antitumor responses.

We previously identified human phosphatidylethanolamine-binding protein 4 (hPEBP4) as an antiapoptotic protein with increased expression levels in breast, ovarian and prostate cancer cells, but low expression levels in normal tissues, which makes hPEBP4 an attractive target for immunotherapy. Here, we developed hPEBP4-derived immunogenic peptides for inducing antigen-specific cytotoxic T lymphocytes (CTLs) targeting breast cancer. A panel of hPEBP4-derived peptides predicted by peptide-MHC-binding algorithms was evaluated to characterize their HLA-A2.1 affinity and immunogenicity. We identified a novel immunogenic peptide, P40-48 (TLFCQGLEV), that was capable of eliciting specific CTL responses in HLA-A2.1/Kb transgenic mice, as well as in peripheral blood lymphocytes from breast cancer patients. Furthermore, amino-acid substitutions in the P40-48 sequence improved its immunogenicity against hPEBP4, a self-antigen, thus circumventing tolerance. We designed peptide analogs by preferred auxiliary HLA-A*0201 anchor residue replacement, which induced CTLs that were crossreactive to the native peptide. Several analogs were able to stably bind to HLA-A*0201 and elicit specific CTL responses better than the native sequence. Importantly, adoptive transfer of CTLs induced by vaccination with two analogs more effectively inhibited tumor growth than the native peptide. These data indicate that peptide analogs with high immunogenicity represent promising candidates for peptide-mediated therapeutic cancer vaccines.

Conserved salt-bridge competition triggered by phosphorylation regulates the protein interactome.

Phosphorylation is a major regulator of protein interactions; however, the mechanisms by which regulation occurs are not well understood. Here we identify a salt-bridge competition or "theft" mechanism that enables a phospho-triggered swap of protein partners by Raf Kinase Inhibitory Protein (RKIP). RKIP transitions from inhibiting Raf-1 to inhibiting G-protein-coupled receptor kinase 2 upon phosphorylation, thereby bridging MAP kinase and G-Protein-Coupled Receptor signaling. NMR and crystallography indicate that a phosphoserine, but not a phosphomimetic, competes for a lysine from a preexisting salt bridge, initiating a partial unfolding event and promoting new protein interactions. Structural elements underlying the theft occurred early in evolution and are found in 10% of homo-oligomers and 30% of hetero-oligomers including Bax, Troponin C, and Early Endosome Antigen 1. In contrast to a direct recognition of phosphorylated residues by binding partners, the salt-bridge theft mechanism represents a facile strategy for promoting or disrupting protein interactions using solvent-accessible residues, and it can provide additional specificity at protein interfaces through local unfolding or conformational change.

PEBP1 Wardens Ferroptosis by Enabling Lipoxygenase Generation of Lipid Death Signals.

Ferroptosis is a form of programmed cell death that is pathogenic to several acute and chronic diseases and executed via oxygenation of polyunsaturated phosphatidylethanolamines (PE) by 15-lipoxygenases (15-LO) that normally use free polyunsaturated fatty acids as substrates. Mechanisms of the altered 15-LO substrate specificity are enigmatic. We sought a common ferroptosis regulator for 15LO. We discovered that PEBP1, a scaffold protein inhibitor of protein kinase cascades, complexes with two 15LO isoforms, 15LO1 and 15LO2, and changes their substrate competence to generate hydroperoxy-PE. Inadequate reduction of hydroperoxy-PE due to insufficiency or dysfunction of a selenoperoxidase, GPX4, leads to ferroptosis. We demonstrated the importance of PEBP1-dependent regulatory mechanisms of ferroptotic death in airway epithelial cells in asthma, kidney epithelial cells in renal failure, and cortical and hippocampal neurons in brain trauma. As master regulators of ferroptotic cell death with profound implications for human disease, PEBP1/15LO complexes represent a new target for drug discovery.

Three FT and multiple CEN and BFT genes regulate maturity, flowering, and vegetative phenology in kiwifruit.

Kiwifruit is a woody perennial horticultural crop, characterized by excessive vegetative vigor, prolonged juvenility, and low productivity. To understand the molecular factors controlling flowering and winter dormancy, here we identify and characterize the kiwifruit PEBP (phosphatidylethanolamine-binding protein) gene family. Five CEN-like and three BFT-like genes are differentially expressed and act as functionally conserved floral repressors, while two MFT-like genes have no impact on flowering time. FT-like genes are differentially expressed, with AcFT1 confined to shoot tip and AcFT2 to mature leaves. Both act as potent activators of flowering, but expression of AcFT2 in Arabidopsis resulted in a greater impact on plant morphology than that of AcFT1. Constitutive expression of either construct in kiwifruit promoted in vitro flowering, but AcFT2 displayed a greater flowering activation efficiency than AcFT1, leading to immediate floral transition and restriction of leaf development. Both leaf and flower differentiation were observed in AcFT1 kiwifruit lines. Sequential activation of specific PEBP genes in axillary shoot buds during growth and dormancy cycles indicated specific roles in regulation of kiwifruit vegetative and reproductive phenologies. AcCEN and AcCEN4 marked active growth, AcBFT2 was associated with suppression of latent bud growth during winter, and only AcFT was activated after cold accumulation and dormancy release.

Crosstalks between Raf-kinase inhibitor protein and cancer stem cell transcription factors (Oct4, KLF4, Sox2, Nanog).

Raf-kinase inhibitor protein has been reported to inhibit both the Raf/mitogen extracellular signal-regulated kinase/extracellular signal-regulated kinase and nuclear factor kappa-light-chain of activated B cells pathways. It has also been reported in cancers that Raf-kinase inhibitor protein behaves as a metastatic suppressor as well as a chemo-immunosensitizing factor to drug/immune-mediated apoptosis. The majority of cancers exhibit low or no levels of Raf-kinase inhibitor protein. Hence, the activities of Raf-kinase inhibitor protein contrast, in part, to those mediated by several cancer stem cell transcription factors for their roles in resistance and metastasis. In this review, the existence of crosstalks in the signaling pathways between Raf-kinase inhibitor protein and several cancer stem cell transcription factors (Oct4, KLF4, Sox2 and Nanog) was assembled. Oct4 is induced by Lin28, and Raf-kinase inhibitor protein inhibits the microRNA binding protein Lin28. The expression of Raf-kinase inhibitor protein inversely correlates with the expression of Oct4. KLF4 does not interact directly with Raf-kinase inhibitor protein, but rather interacts indirectly via Raf-kinase inhibitor protein's regulation of the Oct4/Sox2/KLF4 complex through the mitogen-activated protein kinase pathway. The mechanism by which Raf-kinase inhibitor protein inhibits Sox2 is via the inhibition of the mitogen-activated protein kinase pathway by Raf-kinase inhibitor protein. Thus, Raf-kinase inhibitor protein's relationship with Sox2 is via its regulation of Oct4. Inhibition of extracellular signal-regulated kinase by Raf-kinase inhibitor protein results in the upregulation of Nanog. The inhibition of Oct4 by Raf-kinase inhibitor protein results in the failure of the heterodimer formation of Oct4 and Sox2 that is necessary to bind to the Nanog promoter for the transcription of Nanog. The findings revealed that there exists a direct correlation between the expression of Raf-kinase inhibitor protein and the expression of each of the above transcription factors. Based on these analyses, we suggest that the expression level of Raf-kinase inhibitor protein may be involved in the regulation of the cancer stem cell phenotype.

Integrated bioinformatics, computational and experimental methods to discover novel Raf/extracellular-signal regulated kinase (ERK) dual inhibitors against breast cancer cells.

Beginning with our previously reported ERK inhibitor BL-EI001, we found Raf1 to be an important regulator in the ERK interactive network, and then we designed and synthesized a novel series of Raf1/ERK dual inhibitors against human breast cancers through integrative computational, synthetic and biological screening methods. Moreover, we found that compound 9d suppressed the proliferation of breast cancer cell lines and induced cellular apoptosis via a mitochondrial pathway with only partial dependence on Raf1 and ERK. Our results suggest that an integrative method including in silico design, chemical synthesis, biological screening and bioinformatics analysis could be an attractive strategy for the discovery of multi-target inhibitors against breast cancer.

PEBP1, a RAF kinase inhibitory protein, negatively regulates starvation-induced autophagy by direct interaction with LC3.

Autophagy plays a critical role in maintaining cell homeostasis in response to various stressors through protein conjugation and activation of lysosome-dependent degradation. MAP1LC3B/LC3B (microtubule- associated protein 1 light chain 3 ß) is conjugated with phosphatidylethanolamine (PE) in the membranes and regulates initiation of autophagy through interaction with many autophagy-related proteins possessing an LC3-interacting region (LIR) motif, which is composed of 2 hydrophobic amino acids (tryptophan and leucine) separated by 2 non-conserved amino acids (WXXL). In this study, we identified a new putative LIR motif in PEBP1/RKIP (phosphatidylethanolamine binding protein 1) that was originally isolated as a PE-binding protein and also a cellular inhibitor of MAPK/ERK signaling. PEBP1 was specifically bound to PE-unconjugated LC3 in cells, and mutation (WXXL mutated to AXXA) of this LIR motif disrupted its interaction with LC3 proteins. Interestingly, overexpression of PEBP1 significantly inhibited starvation-induced autophagy by activating the AKT and MTORC1 (mechanistic target of rapamycin [serine/threonine kinase] complex 1) signaling pathway and consequently suppressing the ULK1 (unc-51 like autophagy activating kinase 1) activity. In contrast, ablation of PEBP1 expression dramatically promoted the autophagic process under starvation conditions. Furthermore, PEBP1 lacking the LIR motif highly stimulated starvation-induced autophagy through the AKT-MTORC1-dependent pathway. PEBP1 phosphorylation at Ser153 caused dissociation of LC3 from the PEBP1-LC3 complex for autophagy induction. PEBP1-dependent suppression of autophagy was not associated with the MAPK pathway. These findings suggest that PEBP1 can act as a negative mediator in autophagy through stimulation of the AKT-MTORC1 pathway and direct interaction with LC3.

Revisiting the phosphatidylethanolamine-binding protein (PEBP) gene family reveals cryptic FLOWERING LOCUS T gene homologs in gymnosperms and sheds new light on functional evolution.

Angiosperms and gymnosperms are two major groups of extant seed plants. It has been suggested that gymnosperms lack FLOWERING LOCUS T (FT), a key integrator at the core of flowering pathways in angiosperms. Taking advantage of newly released gymnosperm genomes, we revisited the evolutionary history of the plant phosphatidylethanolamine-binding protein (PEBP) gene family through phylogenetic reconstruction. Expression patterns in three gymnosperm taxa and heterologous expression in Arabidopsis were studied to investigate the functions of gymnosperm FT-like and TERMINAL FLOWER 1 (TFL1)-like genes. Phylogenetic reconstruction suggests that an ancient gene duplication predating the divergence of seed plants gave rise to the FT and TFL1 genes. Expression patterns indicate that gymnosperm TFL1-like genes play a role in the reproductive development process, while GymFT1 and GymFT2, the FT-like genes resulting from a duplication event in the common ancestor of gymnosperms, function in both growth rhythm and sexual development pathways. When expressed in Arabidopsis, both spruce FT-like and TFL1-like genes repressed flowering. Our study demonstrates that gymnosperms do have FT-like and TFL1-like genes. Frequent gene and genome duplications contributed significantly to the expansion of the plant PEBP gene family. The expression patterns of gymnosperm PEBP genes provide novel insight into the functional evolution of this gene family.

Understanding perspectives of signalling mechanisms regulating PEBP1 function.

UNLABELLED: Phosphatidylethanolamine-binding protein 1 (PEBP1), also known as Raf kinase inhibitor protein, belongs to PEBP family of proteins. It is known to interact with many proteins that are mainly involved in pathways that monitor cell proliferation and differentiation. PEBP1 in many cells interacts with several pathways, namely MAPK, GRK2, NF-кB, etc. that keeps the cell proliferation and differentiation in check. This protein is expressed by many cells in humans, including neurons where it is predominantly involved in production of choline acetyltransferase. Deregulated PEBP1 is known to cause cancer, diabetic nephropathy and neurodegenerative diseases like Alzheimer's and dementia. Recent research led to the discovery of many drugs that mainly target the interaction of PEBP1 with its partners. These compounds are known to bind PEBP1 in its conserved domain which abrogate its association with interacting partners in several different pathways. We outline here the latest developments in understanding of PEBP1 function in maintaining cell integrity. Copyright © 2016 John Wiley & Sons, Ltd. SIGNIFICANCE OF THE STUDY: Phosphatidylethanolamine-binding protein is crucial in regulation of MAPK and PKC pathways. Its diverse roles, including regulating these pathways keep cell differentiation and proliferation in check. This review outlines some latest findings which greatly add to our current knowledge of phosphatidylethanolamine-binding protein.

Phosphatidylethanolamine binding protein 4 (PEBP4) is a secreted protein and has multiple functions.

Phosphatidylethanolamine binding proteins (PEBP) represent a superfamily of proteins that are conserved from bacteria to humans. In mammals, four members have been identified, PEBP1-4. To determine the functional differences among PEBP1-4 and the underlying mechanism for their actions, we performed a sequence alignment and found that PEBP4 contains a signal peptide and potential glycosylation sites, whereas PEBP1-3 are intracellular proteins. To test if PEBP4 is secreted, we made constructs with Myc epitope at the amino (N) terminus or carboxyl (C) terminus to mask the signal sequence or keep it free, respectively. Our data revealed that both mouse and human PEBP4 were secreted when the epitope was tagged at their C-terminus. To our surprise, secretion was dependent upon the C-terminal conserved domain in addition to the N-terminal signal sequence. When the epitope was placed to the N-terminus, the recombinant protein failed to secrete and instead, was retained in the cytoplasm. Mass spectrometry detected asparagine (N)-glycosylation on the secreted PEBP4. Although overexpression of N-terminal tagged PEBP4 resulted in an inhibition of ERK activation by EGF, that with a C-terminal epitope tag did not have such an effect. Likewise, transfection of PEBP4 shRNA did not appear to affect ERK activation, suggesting that PEBP4 does not participate in the regulation of this pathway. In contrast, PEBP4 siRNA suppressed phosphorylation of Act at S473. Therefore, our results suggest that PEBP4 is a multifunctional protein and can be secreted. It will be important to investigate the mechanism by which PEBP4 is secreted and regulates cellular events.

Three FLOWERING LOCUS T-like genes function as potential florigens and mediate photoperiod response in sorghum.

Sorghum is a typical short-day (SD) plant and its use in grain or biomass production in temperate regions depends on its flowering time control, but the underlying molecular mechanism of floral transition in sorghum is poorly understood. Here we characterized sorghum FLOWERING LOCUS T (SbFT) genes to establish a molecular road map for mechanistic understanding. Out of 19 PEBP genes, SbFT1, SbFT8 and SbFT10 were identified as potential candidates for encoding florigens using multiple approaches. Phylogenetic analysis revealed that SbFT1 clusters with the rice Hd3a subclade, while SbFT8 and SbFT10 cluster with the maize ZCN8 subclade. These three genes are expressed in the leaf at the floral transition initiation stage, expressed early in grain sorghum genotypes but late in sweet and forage sorghum genotypes, induced by SD treatment in photoperiod-sensitive genotypes, cooperatively repressed by the classical sorghum maturity loci, interact with sorghum 14-3-3 proteins and activate flowering in transgenic Arabidopsis plants, suggesting florigenic potential in sorghum. SD induction of these three genes in sensitive genotypes is fully reversed by 1 wk of long-day treatment, and yet, some aspects of the SD treatment may still make a small contribution to flowering in long days, indicating a complex photoperiod response mediated by SbFT genes.

Functional evolution of phosphatidylethanolamine binding proteins in soybean and Arabidopsis.

Gene duplication provides resources for novel gene functions. Identification of the amino acids responsible for functional conservation and divergence of duplicated genes will strengthen our understanding of their evolutionary course. Here, we conducted a systemic functional investigation of phosphatidylethanolamine binding proteins (PEBPs) in soybean (Glycine max) and Arabidopsis thaliana. Our results demonstrated that after the ancestral duplication, the lineage of the common ancestor of the FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1) subfamilies functionally diverged from the MOTHER OF FT AND TFL1 (MFT) subfamily to activate flowering and repress flowering, respectively. They also underwent further specialization after subsequent duplications. Although the functional divergence increased with duplication age, we observed rapid functional divergence for a few pairs of young duplicates in soybean. Association analysis between amino acids and functional variations identified critical amino acid residues that led to functional differences in PEBP members. Using transgenic analysis, we validated a subset of these differences. We report clear experimental evidence for the functional evolution of the PEBPs in the MFT, FT, and TFL1 subfamilies, which predate the origin of angiosperms. Our results highlight the role of amino acid divergence in driving evolutionary novelty after duplication.

Structural basis for RKIP binding with its substrate Raf1 kinase.

Raf1 kinase inhibitor protein (RKIP) negatively regulates the Raf1/MEK/ERK pathway which is vital for cell growth and differentiation. It is also a biomarker in clinical cancer diagnosis. RKIP binds to the N-terminus of Raf1 kinase but little is known about the structural basis of RKIP binding with Raf1. Here, we demonstrate that the N-terminus of human Raf1 kinase (hRaf11-147aa) binds with human RKIP (hRKIP) at its ligand-binding pocket, loop "127-149", and the C-terminal helix by NMR experiments. D70, D72, E83, Y120, and Y181 were further verified as the key residues participating in the interaction of hRKIP and hRaf11-147aa. G143-R146 fragment was also critical for hRKIP binding with hRaf11-147aa, for its deletion decreased the binding affinity around 300 times, from 154 to 0.46 mM(-1). Our results provide important structural clues for designing the lead compound that disrupts RKIP-Raf1 interaction.

Regulation of the MAPK pathway by raf kinase inhibitory protein.

The Raf kinase inhibitor protein 1 (RKIP-1) was the first reported endogenous inhibitor of Raf-1-MEK-ERK/MAPK cascade, by interfering with the phosphorylation of MEK by Raf-1. However, RKIP's functions related to the MAPK signaling are far more complex. Newer data indicate that by modulating different protein-protein interactions, RKIP is involved in fine-tuning cell signaling, modulating ERK dynamics, and regulating cross talk between different pathways. Here, we describe the molecular mechanisms by which RKIP controls MAPK signaling at different levels and vice versa and its regulation via feedback phosphorylation. We also focus on several discrepancies and questions that remain, such as the RKIP binding regulation by Raf-1 N-region phosphorylation, the possible B-Raf inhibition, and the effects of RKIP-lipid binding. We also describe how RKIP's role as key signaling modulator of many cell fate decisions leads to the fact that fine control of RKIP activity and regulation is crucial to avoid pathological processes, such as metastasis, pulmonary arterial hypertension, and heart failure.