The RKIP (Raf-1 Kinase Inhibitor Protein) conserved pocket binds to the phosphorylated N-region of Raf-1 and inhibits the Raf-1-mediated activated phosphorylation of MEK

The Raf-MEK-ERK pathway regulates many fundamental biological processes, and its activity is finely tuned at multiple levels. The Raf kinase inhibitory protein (RKIP) is a widely expressed negative modulator of the Raf-MEK-ERK signaling pathway. We have previously shown that RKIP inhibits the phosphorylation of MEK by Raf-1 through interfering with the formation of a kinase-substrate complex by direct binding to both Raf-1 and MEK. Here, we show that the evolutionarily conserved ligand-binding pocket of RKIP is required for its inhibitory activity towards the Raf-1 kinase mediated activation of MEK. Single amino acid substitutions of two of the conserved residues form the base and the wall of the pocket confers a loss-of-function phenotype on RKIP. Loss-of-function RKIP mutants still appear to bind to Raf-1. However the stability of the complexes formed between mutants and the N-region Raf-1 phosphopeptide were drastically reduced. Our results therefore suggest that the RKIP conserved pocket may constitute a novel phosphoamino-acid binding motif and is absolutely required for RKIP function.     

Raf kinase inhibitory protein regulates Raf-1 but not B-Raf kinase activation

Raf kinase inhibitory protein (RKIP; also known as phosphatidylethanolamine-binding protein or PEBP) is a modulator of the Raf/MAPK signaling cascade and a suppressor of metastatic cancer. Here, we show that RKIP inhibits MAPK by regulating Raf-1 activation; specifically, RKIP acts subsequent to Raf-1 membrane recruitment, prevents association of Raf-1 and p21-activated kinase (PAK), and blocks phosphorylation of the Raf-1 kinase domain by PAK and Src family kinases. Mutation of the PAK and Src phosphorylation sites on Raf-1 to aspartate, a phosphate mimic, prevented RKIP association with or inhibition of Raf-1 signaling. Interestingly, although RKIP can interact with B-Raf, RKIP depletion had no effect on activation of B-Raf. Because c-Raf-1 and B-Raf are both required for maximal MAPK stimulation by epidermal growth factor in neuronal and epithelial cell lines, we determined whether RKIP significantly affects MAPK signaling. In fact, RKIP depletion increased not only the amplitude but also the sensitivity of MAPK and DNA synthesis to epidermal growth factor stimulation by up to an order of magnitude. These results indicate that selective modulation of c-Raf-1 but not B-Raf activation by RKIP can limit the dynamic range of the MAPK signaling response to growth factors and may play a critical role in growth and development.     

RKIP downregulation induces the HBx-mediated Raf-1 mitochondrial translocation

The Raf-1 kinase inhibitory protein (RKIP) can regulate multiple key signaling pathways. Specifically, RKIP binds to Raf-1 kinase and inhibits the Ras-Raf-1-MEK1/2- ERK1/2 pathway. Additionally, Raf-1 has been shown to translocate to mitochondria and thereby protect cells from stress-mediated apoptosis. Recently, HBx was found to stimulate the mitochondrial translocation of Raf-1, contributing to the anti-apoptotic effect. We found that RKIP was downregulated during HBx-mediated hepatocarcinogenesis. In this study, we show that RKIP bound to Raf-1 and consequently inhibited the translocation of Raf-1 into mitochondria. This promoted the apoptosis of cells treated with apoptotic stimulus. Thus, the downregulation of RKIP increased the level of free Raf-1 and thereby elevated the mitochondrial translocation of Raf-1 during HBx-mediated hepatocarcinogenesis. The elevated Raf-1 mitochondrial translocation induced the increased anti-apoptotic effect and subsequently promoted HBx-mediated hepatocarcinogenesis.     

Raf Kinase Inhibitory Protein Function Is Regulated via a Flexible Pocket and Novel Phosphorylation-Dependent Mechanism

Raf kinase inhibitory protein (RKIP/PEBP1), a member of the phosphatidylethanolamine binding protein family that possesses a conserved ligand-binding pocket, negatively regulates the mammalian mitogen-activated protein kinase (MAPK) signaling cascade. Mutation of a conserved site (P74L) within the pocket leads to a loss or switch in the function of yeast or plant RKIP homologues. However, the mechanism by which the pocket influences RKIP function is unknown. Here we show that the pocket integrates two regulatory signals, phosphorylation and ligand binding, to control RKIP inhibition of Raf-1. RKIP association with Raf-1 is prevented by RKIP phosphorylation at S153. The P74L mutation increases kinase interaction and RKIP phosphorylation, enhancing Raf-1/MAPK signaling. Conversely, ligand binding to the RKIP pocket inhibits kinase interaction and RKIP phosphorylation by a noncompetitive mechanism. Additionally, ligand binding blocks RKIP association with Raf-1. Nuclear magnetic resonance studies reveal that the pocket is highly dynamic, rationalizing its capacity to interact with distinct partners and be involved in allosteric regulation. Our results show that RKIP uses a flexible pocket to integrate ligand binding- and phosphorylation-dependent interactions and to modulate the MAPK signaling pathway. This mechanism is an example of an emerging theme involving the regulation of signaling proteins and their interaction with effectors at the level of protein dynamics.Raf kinase inhibitory protein (RKIP/PEBP1) is a signaling modulator that regulates key signal transduction cascades in mammalian cells (reviewed in reference 16). A negative regulator of mitogen-activated protein kinase (MAPK) signaling (42), RKIP inhibits Raf kinase by binding directly to Raf-1, thereby preventing the phosphorylation and activation of Raf-1 (8, 38). RKIP functions as a regulator of the spindle checkpoint and promotes genomic stability by preventing MAPK from inhibiting Aurora B kinase (10). Consistent with this role, RKIP suppresses lung metastasis by prostate tumor cells in an orthotopic murine model (15). RKIP may be a general metastasis suppressor for solid tumors, since RKIP expression is low or undetectable in prostate and breast tumors, melanoma, hepatocellular carcinoma, and colorectal tumors (1, 2, 14, 15, 19, 34). Finally, RKIP suppresses NF-κB activation (43), inhibits G protein-coupled receptor (GPCR) kinase 2 (GRK2)-mediated downregulation of GPCRs (28), and potentiates the efficacy of chemotherapeutic agents (5). Thus, RKIP regulates three key mammalian signaling pathways involving MAPK, GPCR, and NF-κB signaling.RKIP is a member of the phosphatidylethanolamine binding protein (PEBP) family, which extends from bacteria to humans and consists of more than 400 proteins (16, 33). X-ray crystallographic studies have demonstrated that highly conserved sequences cluster around a pocket capable of binding anions, including o-phosphorylethanolamine (PE), acetate, and cacodylate (3, 35). This pocket is the only clearly identifiable feature for potential ligand binding within the RKIP architecture. Although the ligand-binding pocket shares homology with phospholipid binding domains, PEBP associates with phospholipid membranes primarily via peripheral, ionic interactions rather than more integrally inserting itself into the membrane (reference 39 and data not shown). The fact that RKIP interacts with protein targets such as Raf-1 and is phosphorylated by other protein kinases raises the possibility that the pocket mediates protein-protein interactions.The physiological role of the ligand-binding pocket is illustrated by studies of plant and yeast PEBPs. In the plant homologue of RKIP, mutation of the conserved DPDxP motif within the pocket (the equivalent of P74L) causes tomato plants to switch developmentally from shoot growth to flowering (32). The Saccharomyces cerevisiae RKIP/PEBP homologue, Tfs1p, functions as a negative regulator of RasGAP (Ira2), leading to upregulation of yeast Ras, activation of adenylyl cyclase, and increased cyclic AMP activation of protein kinase A (6). Yeast Ras signaling is inhibited by the corresponding P74L mutation in the pocket of Tfs1p, blocking Tfs1p interaction with Ira2. These results highlight the functional importance of the pocket among eukaryotic RKIP/PEBP family members. However, the molecular mechanism by which the pocket influences RKIP function and the significance of ligand binding to the pocket are unknown.Previous work has established the phosphorylation-mediated control of RKIP function. RKIP binds Raf-1, inhibiting Raf-1 activation and consequent signaling to MAPK (38, 42). When RKIP residue S153 is phosphorylated by protein kinase C (PKC), which occurs following cell stimulation with growth factors such as epidermal growth factor (EGF) or serum, RKIP can no longer bind to Raf-1, and thus it is inactivated as a Raf-1 inhibitor (8). Phosphorylation at S153 promotes the association of RKIP with, and inhibition of, GRK2, a kinase that phosphorylates and downregulates GPCRs such as the β-adrenergic receptor (28). Thus, S153 phosphorylation of RKIP is a key regulatory element of its association with and inhibition of different targets. The importance of the pocket and that of S153 phosphorylation have been independently established, but it is not clear whether these regulatory elements are functionally linked. Addressing this question is important for advancing our understanding of the molecular mechanism of RKIP function, which is likely to be pertinent to many RKIP/PEBP family members.In the present study, using cellular, biochemical, and structural approaches, we demonstrate that the highly conserved ligand-binding pocket integrates two regulatory signals, phosphorylation and ligand binding, to control RKIP function. Our results suggest that, in contrast to the mechanisms for other pocket-containing single-domain proteins, the structure and/or dynamics of the pocket influences RKIP interaction with and phosphorylation by kinases. This mechanism is likely conserved among RKIP homologues in eukaryotes.     

Activation of Raf-1 signaling by protein kinase C through a mechanism involving Raf kinase inhibitory protein

Protein kinase C (PKC) regulates activation of the Raf-1 signaling cascade by growth factors, but the mechanism by which this occurs has not been elucidated. Here we report that one mechanism involves dissociation of Raf kinase inhibitory protein (RKIP) from Raf-1. Classic and atypical but not novel PKC isoforms phosphorylate RKIP at serine 153 (Ser-153). RKIP Ser-153 phosphorylation by PKC either in vitro or in response to 12-O-tetradecanoylphorbol-13-acetate or epidermal growth factor causes release of RKIP from Raf-1, whereas mutant RKIP (S153V or S153E) remains bound. Increased expression of PKC can rescue inhibition of the mitogen-activated protein (MAP) kinase signaling cascade by wild-type but not mutant S153V RKIP. Taken together, these results constitute the first model showing how phosphorylation by PKC relieves a key inhibitor of the Raf/MAP kinase signaling cascade and may represent a general mechanism for the regulation of MAP kinase pathways.     

RKIP regulates MAP kinase signaling in cells with defective B-Raf activity

MAP kinase (MAPK) signaling results from activation of Raf kinases in response to external or internal stimuli. Here, we demonstrate that Raf kinase inhibitory protein (RKIP) regulates the activation of MAPK when B-Raf signaling is defective. We used multiple models including mouse embryonic fibroblasts (MEFs) and primary keratinocytes from RKIP- or Raf-deficient mice as well as allografts in mice to investigate the mechanism. Loss of B-Raf protein or activity significantly reduces MAPK activation in these cells. We show that RKIP depletion can rescue the compromised ERK activation and promote proliferation, and this rescue occurs through a Raf-1 dependent mechanism. These results provide formal evidence that RKIP is a bona fide regulator of Raf-1. We propose a new model in which RKIP plays a key role in regulating the ability of cells to signal through Raf-1 to ERK in B-Raf compromised cells.     

Raf kinase inhibitory protein regulates aurora B kinase and the spindle checkpoint

Raf kinase inhibitory protein (RKIP or PEBP) is an inhibitor of the Raf/MEK/MAP kinase signaling cascade and a suppressor of cancer metastasis. We now show that RKIP associates with centrosomes and kinetochores and regulates the spindle checkpoint in mammalian cells. RKIP depletion causes decreases in the mitotic index, the number of metaphase cells, and traversal times from nuclear envelope breakdown to anaphase, and an override of mitotic checkpoints induced by spindle poisons. Raf-1 depletion or MEK inhibition reverses the reduction in the mitotic index, whereas hyperactivation of Raf mimics the RKIP-depletion phenotype. Finally, RKIP depletion or Raf hyperactivation reduces kinetochore localization and kinase activity of Aurora B, a regulator of the spindle checkpoint. These results indicate that RKIP regulates Aurora B kinase and the spindle checkpoint via the Raf-1/MEK/ERK cascade and demonstrate that small changes in the MAP kinase (MAPK) pathway can profoundly impact the fidelity of the cell cycle.     

Raf Kinase Inhibitory Protein Protects Cells against Locostatin-Mediated Inhibition of Migration

Background

Raf Kinase Inhibitory Protein (RKIP, also PEBP1), a member of the Phosphatidylethanolamine Binding Protein family, negatively regulates growth factor signaling by the Raf/MAP kinase pathway. Since an organic compound, locostatin, was reported to bind RKIP and inhibit cell migration by a Raf-dependent mechanism, we addressed the role of RKIP in locostatin function.

Methods/Findings

We analyzed locostatin interaction with RKIP and examined the biological consequences of locostatin binding on RKIP function. NMR studies show that a locostatin precursor binds to the conserved phosphatidylethanolamine binding pocket of RKIP. However, drug binding to the pocket does not prevent RKIP association with its inhibitory target, Raf-1, nor affect RKIP phosphorylation by Protein Kinase C at a regulatory site. Similarly, exposure of wild type, RKIP-depleted HeLa cells or RKIP-deficient (RKIP^−/−) mouse embryonic fibroblasts (MEFs) to locostatin has no effect on MAP kinase activation. Locostatin treatment of wild type MEFs causes inhibition of cell migration following wounding. RKIP deficiency impairs migration further, indicating that RKIP protects cells against locostatin-mediated inhibition of migration. Locostatin treatment of depleted or RKIP^−/− MEFs reveals cytoskeletal disruption and microtubule abnormalities in the spindle.

Conclusions/Significance

These results suggest that locostatin''s effects on cytoskeletal structure and migration are caused through mechanisms independent of its binding to RKIP and Raf/MAP kinase signaling. The protective effect of RKIP against drug inhibition of migration suggests a new role for RKIP in potentially sequestering toxic compounds that may have deleterious effects on cells.     

Cilia movement regulates expression of the Raf-1 kinase inhibitor protein

Renal epithelial cell primary cilia act as mechanosensors in response to changes in luminal fluid flow. To determine the role of cilia bending in the mechanosensory function of cilia, we performed proteomic analysis of collecting duct cell lines with or without cilia that were kept stationary or rotated to stimulate cilia bending. Expression of the Raf-1 kinase inhibitor protein (RKIP), an inhibitor of the MAPK pathway, was significantly elevated in rotated cilia (+) cells. This was compared with RKIP levels in cilia (-) cells that were stationary or rotated as well as in cilia (+) cells that were stationary. This result was confirmed in cilia knockout adult mice that had lower renal RKIP levels compared with adult mice with cilia. Downstream of RKIP, expression of phosphorylated ERK was decreased only in cells that had cilia and were subjected to constant cilia bending. Furthermore, elevated RKIP levels were associated with reduced cell proliferation. Blockade of PKC abrogated ciliary bending-induced increases in RKIP. In summary, we found that ciliary movement may help control the expression of the Raf-1 kinase inhibitor protein and thus maintain cell differentiation. In terms of polycystic kidney disease, loss of cilia and therefore sensitivity to flow may lead to reduced RKIP levels, activation of the MAPK pathway, and contribute to the formation of cysts.     

Mechanical stress promotes matrix synthesis of mandibular condylar cartilage via the RKIP-ERK pathway

Mandibular hypoplasia is a common jaw deformity that affects breathing, occlusal function and facial aesthetics. Stimulating mandibular condylar growing with functional appliances is an ordinary but controversial treatment method in orthodontics. Therefore, it is vital to clarify how functional appliances affect condylar growing. Raf-1 kinase inhibitor protein (RKIP), as an endogenous inhibitory molecule of the ERK signaling, is postulated to involve in stress-induced response to articular cartilage. This study was to reveal the role of RKIP in regulating cartilage matrix synthesis with functional appliance treatment. Here, position rat mandibular forward simulating functional appliance effect to examine the stress-induced modification of mandibular condylar in vivo, meanwhile rat mandibular condylar chondrocytes (Mccs) were subjected to cyclic tensile stress (CTS, 16%, 1 HZ). The results showed that mandibular forward therapy enhanced condylar cartilage growth. The thicknesses of all layers of condylar cartilage were increased significantly. RKIP expression was also increased in the mature cartilage layer. In addition, CTS could enhance extracellular matrix formation and cartilage marker expression (aggrecan and collagen II), which shared a similar expression pattern with RKIP in Mccs. However, CTS induced up-regulation of collagen II and aggrecan was blocked by RKIP knockdown. Nuclear p-ERK, targeting downstream of RKIP, showed a decrease after CTS,which was disappeared in RKIP-knockdown Mccs. Taken together, physiological mechanical stimulation promotes cartilage growth modification by up-regulating RKIP through inhibiting ERK signaling pathway.     

Raf激酶抑制蛋白在信号传导通路及凋亡中的关键作用

细胞维持自身的完整性存在众多调控,避免它从一个生物学状态到另一个状态随意的转换。Raf激酶抑制蛋白（Rafkinase inhibitor protein,RKIP）,属于磷脂酰乙醇胺结合蛋白（phosphatidylethanolamine binding protein,PEBP）家族的成员,是新的一类信号级联传导的调节器来维持细胞自身平衡。RKIP可以抑制MAP激酶途径（Raf-1／MEK／ERK）,G蛋白偶联途径（G—protein coupled receptor,GPCR）,以及NF-κb途径。正因为RKIP在这些信号传导中的作用,使其在细胞凋亡过程中发挥了关键性的作用。更加深入的了解RKIP如何在肿瘤细胞调控表达,了解它如何对细胞凋亡、信号传导产生影响可以为人类治疗癌症起到极大的推动作用。     

1H, 13C, 15N backbone and side-chain resonance assignments of the human Raf-1 kinase inhibitor protein

Raf-1 kinase inhibitor protein (RKIP) plays a pivotal role in modulating multiple signaling networks. Here we report backbone and side chain resonance assignments of uniformly ¹⁵N, ¹³C labeled human RKIP.     

Crystal structures of YBHB and YBCL from Escherichia coli, two bacterial homologues to a Raf kinase inhibitor protein.

In rat and human cells, RKIP (previously known as PEBP) was characterized as an inhibitor of the MEK phosphorylation by Raf-1. In Escherichia coli, the genes ybhb and ybcl possibly encode two RKIP homologues while in the genomes of other bacteria and archaebacteria other homologous genes of RKIP have been found. The parallel between the cellular signaling mechanisms in eukaryotes and prokaryotes suggests that these bacterial proteins could be involved in the regulation of protein phosphorylation by kinases as well. We first showed that the proteins YBHB and YBCL were present in the cytoplasm and periplasm of E. coli, respectively, after which we determined their crystallographic structures. These structures verify that YBHB and YBCL belong to the same structural family as mammalian RKIP/PEBP proteins. The general fold and the anion binding site of these proteins are extremely well conserved between mammals and bacteria and suggest functional similarities. However, the bacterial proteins also exhibit some specific structural features, like a substrate binding pocket formed by the dimerization interface and the absence of cis peptide bonds. This structural variety should correspond to the recognition of multiple cellular partners.     

Regulation of Raf-1 by direct feedback phosphorylation

The Raf-1 kinase is an important signaling molecule, functioning in the Ras pathway to transmit mitogenic, differentiative, and oncogenic signals to the downstream kinases MEK and ERK. Because of its integral role in cell signaling, Raf-1 activity must be precisely controlled. Previous studies have shown that phosphorylation is required for Raf-1 activation, and here, we identify six phosphorylation sites that contribute to the downregulation of Raf-1 after mitogen stimulation. Five of the identified sites are proline-directed targets of activated ERK, and phosphorylation of all six sites requires MEK signaling, indicating a negative feedback mechanism. Hyperphosphorylation of these six sites inhibits the Ras/Raf-1 interaction and desensitizes Raf-1 to additional stimuli. The hyperphosphorylated/desensitized Raf-1 is subsequently dephosphorylated and returned to a signaling-competent state through interactions with the protein phosphatase PP2A and the prolyl isomerase Pin1. These findings elucidate a critical Raf-1 regulatory mechanism that contributes to the sensitive, temporal modulation of Ras signaling.     

Raf激酶抑制蛋白的研究进展

Raf激酶抑制蛋白(RKIP)是磷脂酰乙醇胺结合蛋白家族的成员。RKIP通过与Raf-1结合,抑制了Ras/Raf-1/MEK/ERK信号转导通路,并在NF-κB及G蛋白偶联受体(GPCR)信号转导通路中也起重要调节作用。RKIP参与细胞凋亡、肿瘤转移、神经发育以及精子发生等病理生理过程,通过研究RKIP能为治疗相关疾病提供新思路新靶点。本文主要介绍RKIP的生物功能,着重于其在神经系统、肿瘤和生殖系统中的研究进展。     

Reduction of Raf Kinase Inhibitor Protein Expression by Bcr-Abl Contributes to Chronic Myelogenous Leukemia Proliferation

Chronic myelogenous leukemia (CML) is characterized by a reciprocal chromosomal translocation (9;22) that generates the Bcr-Abl fusion gene. The Ras/Raf-1/MEK/ERK pathway is constitutively activated in Bcr-Abl-transformed cells, and Ras activity enhances the oncogenic ability of Bcr-Abl. However, the mechanism by which Bcr-Abl activates the Ras pathway is not completely understood. Raf kinase inhibitor protein (RKIP) inhibits activation of MEK by Raf-1 and its downstream signal transduction, resulting in blocking the MAP kinase pathway. In the present study, we found that RKIP was depleted in CML cells. We investigated the interaction between RKIP and Bcr-Abl in CML cell lines and Bcr-Abl⁺ progenitor cells from CML patients. The Abl kinase inhibitors and depletion of Bcr-Abl induced the expression of RKIP and reduced the pERK1/2 status, resulting in inhibited proliferation of CML cells. Moreover, RKIP up-regulated cell cycle regulator FoxM1 expression, resulting in G₁ arrest via p27^Kip1 and p21^Cip1 accumulation. In colony-forming unit granulocyte, erythroid, macrophage, megakaryocyte, colony-forming unit-granulocyte macrophage, and burst-forming unit erythroid, treatment with the Abl kinase inhibitors and depletion of Bcr-Abl induced RKIP and reduced FoxM1 expressions, and inhibited colony formation of Bcr-Abl⁺ progenitor cells, whereas depletion of RKIP weakened the inhibition of colony formation activity by the Abl kinase inhibitors in Bcr-Abl⁺ progenitor cells. Thus, Bcr-Abl represses the expression of RKIP, continuously activates pERK1/2, and suppresses FoxM1 expression, resulting in proliferation of CML cells.