raf kinase inhibitory protein (rkip): a physiological regulator and future therapeutic target

Review

10.1517/14728220802383940 © 2008 Informa UK Ltd ISSN 1472-8222 1275All rights reserved: reproduction in whole or in part not permitted

General

Raf kinase inhibitory protein (RKIP): A physiological regulator and future therapeutic target Lingchun Zeng , Akira Imamoto & Marsha Rich Rosner † † The University of Chicago, Ben May Department for Cancer Research, IL, USA

Background : Raf kinase inhibitory protein (RKIP) belongs to the phosphatidylethanolamine binding protein (PEBP) family that is expressed in both prokaryotic and euakaryotic organisms. Objective : In this review, we discuss the role of RKIP as a modulator of signal transduction, the relationship of RKIP to other members of the PEBP family, and the role of RKIP in human health and disease. Results/conclusion : In mammals, RKIP regulates activation of MAPK, NF- κ B and G protein coupled receptors (GPCRs). As a modulator of key signaling pathways, RKIP affects various cellular processes including cell differentiation, the cell cycle, apoptosis and cell migration. Emerging evidence suggests that RKIP is implicated in several human diseases or disorders, among them metastatic tumorigenesis and Alzheimer’s disease.

Keywords: Alzheimer’s disease , cancer , cancer metastasis , GPCR , MAPK , NF- κ B , PEBP , RKIP

Expert Opin. Ther. Targets (2008) 12(10):1275-1287

1. Introduction

Raf kinase inhibitory protein (RKIP), also called phosphatidylethanolamine binding protein 1 (PEBP1), is the prototype member of the PEBP family that has an evolutionarily conserved ligand binding domain [1] . Initially, a soluble cytosolic protein of 23 kDa was purified from bovine brain [2] , then characterized as a phosphatidylethanolamine binding protein (PEBP) [3] . Rat and human cDNA encoding this protein were identified later [4-8] . Upon further functional characteriza tion of this protein, PEBP1 was named RKIP because of its ability to bind and inhibit Raf kinase [9] . RKIP is widely expressed in most tissues at various developmental stages, and it is most abundant in brain, testis, epididymis, liver and kidney [10,11] .

Although the precise cellular function of RKIP and the molecular mechanisms by which it acts have not been elucidated completely, emerging experimental evidence indicates that RKIP is implicated in several fundamental signal transduction pathways that control cellular growth, differentiation, apoptosis and migration. Pathophysiologically, RKIP has been discovered to be associated with various human diseases, including cancer. In this review, the modulation of several fundamental signal cascades by RKIP, the relationship of RKIP to other PEBP family members and the roles of RKIP in human diseases are discussed. For additional information, readers may refer to other reviews published recently [12-17] .

2. The biochemical and structural nature of RKIP

The mammalian RKIP gene encodes a 187 amino acid protein with a molecular weight of ∼ 21 kDa. RKIP, like other members of the PEBP family, is characterized by an evolutionarily conserved binding pocket. Although RKIP can bind hydrophobic

1. Introduction

2. The biochemical and structural

nature of RKIP

3. RKIP ortholog genes defi ne

an unique subfamily in the

PEBP superfamily

4. RKIP modulates Raf, GPCR

and NF-κB signaling pathways

5. RKIP in oncogenesis and

cancer metastasis

6. RKIP in cell division and

genomic stability

7. RKIP and apoptosis

8. RKIP and cell migration

9. RKIP in neural system and

neurodegenerative diseases

10. The PEBP family and cell

differentiation

11. Insights from RKIP

knockout mice

12. Expert opinion

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Raf kinase inhibitory protein (RKIP): A physiological regulator and future therapeutic target

1276 Expert Opin. Ther. Targets (2008) 12(10)

ligands including phosphatidylethanolamine and locostatin in vitro , the physiological relevance of these interactions has not been established unambiguously [3] . Most experimental findings indicate that RKIP modulates different signal cascades by direct or indirect interactions with other proteins. RKIP is able to bind various proteins to modulate their signaling pathways including Raf-1, B-Raf, MAPK/extracellular signal regulated kinase kinase (MEK), G protein-coupled receptor kinase 2 (GRK2), and NF- κ B inducing kinase (NIK)/TGF- β activated kinase 1 (TAK1)/I-kappa-B kinase (IKK) [9,18-23] . Furthermore, the interaction between RKIP and other proteins such as Raf-1 can be regulated by phosphorylation depending on the cellular context [18] . Some of the key phosphorylation sites such as Ser153 are critical for regulating the association between RKIP and Raf-1 [18] . Lastly, the N-terminal residues 2 – 12 of RKIP form a natural cleavage peptide, hippocampal cholinergic neurostimulating peptide (HCNP), that is involved in neuronal development and differentiation [24-26] .

The three-dimensional structure of human and rat RKIP has been determined by X-ray crystallography [27,28] . The structure consists of a large central β -sheet flanked by a smaller β -sheet on one side, and an α β α extension on the other ( Figure 1 ). Sequence alignments for members of the PEBP protein family reveal two conserved central regions, CR 1 (residues 64 – 91) and CR 2 (residues 111 – 123), that

represent a consensus signature for the PEBP family and form part of the ligand-binding site [27,28] .

3. RKIP ortholog genes defi ne an unique subfamily in the PEBP superfamily

To place RKIP in an evolutionary context, we compared 32 non-redundant PEBP protein sequences obtained from the NCBI Gene database for the best characterized species from Mammalia, plants and fungi. A more comprehensive phylogenetic analysis of the PEBP family will be reported elsewhere. The accession numbers and relevant information for the 32 PEBP members are listed in Table 1 .

The resulting unrooted neighbor-joining phylogenetic tree based on the 32 full-length PEBP protein sequences is presented in Figure 2 . This phylogenetic tree indicates that the PEBP proteins segregate into several evolutionarily distinct subfamilies, including RKIP, PEBP4, mitochondrial ribosomal protein L38 (MRPL38) and some other undefined groups. Interestingly, based on the available experimental data, the different PEBP subfamilies exhibit both similar and distinct biological functions. For example, some RKIP-related subfamilies (including RKIP, PEBP2 and PEBP4) all inhibit the MAPK signaling pathway [9,18] . MRPL38 is one of the protein components of the large 39S subunit of the mitochondrial ribosome [29] . By contrast,

S153

Phospholipidbinding pocket

Figure 1 . Three-dimensional structure of Raf kinase inhibitory protein (RKIP). The structure of RKIP has been resolved by X-ray crystallography. RKIP is dominated by the ligand binding domain (residues 17 – 172). Hippocampal cholinergic neurostimulating peptide (HCNP) is a 11 amino acid peptide (residues 2 – 12) cleaved from RKIP, and the P74 residue is the residue corresponding to P76 of Tomato gene Self-Pruning (SP) that is mutated to L leading to developmental abnormalities. The S153 phosphorylation site is indicated.

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Zeng, Imamoto & Rosner

Expert Opin. Ther. Targets (2008) 12(10) 1277

Table 1 . The PEBP members used in phylogenetic analysis.

Gene Accession No Species UniGene Chromosome AA

Hs-RKIP NP_002558 Homo sapiens Hs.433863 12q24.23 187

Hs-PEBP4 NP_659399 Homo sapiens Hs.491242 8p21.3 223

Hs-MRPL38 NP_115867 Homo sapiens Hs.442609 17q25.3 346

Mm-PEBP1 NP_061346 Mus musculus Mm.195898 563.0 cM 187

Mm-PEBP2 NP_083871 Mus musculus Mm.293018 6 G1 187

Mm-PEBP4 NP_082836 Mus musculus Mm.23509 14 D2 242

Mm-MRPL38 NP_077139 Mus musculus Mm.29974 11 E2 380

Rn-RKIP NP_058932 Rattus norvegicus Rn.29745 12q16 187

Rn-PEBP2 XP_575702 Rattus norvegicus Rn.198710 4q43 187

Rn-PEBP4 XP_001080758 Rattus norvegicus Rn.215546 15p11 296

Rn-MRPL38 NP_001009369 Rattus norvegicus Rn.34217 10q32.3 346

Bt-PEBP1 NP_001028795 Bos taurus Bt.59089 17 187

Bt-PEBP4 XP_001250505 Bos taurus Bt.87688 8 101

Bt-MRPL38 NP_001030566 Bos taurus Bt.49524 19 346

Dm-CG17919 NP_649644 Drosophila melanogaster Dm.20147 3R, 83E4-83E4 202



Dm-CG18594 NP_651051 Drosophila melanogaster Dm.20618 3R, 94A16-94A16 176


Dm-CG6180 NP_609588 Drosophila melanogaster Dm.2238 2L, 33F3-33F3 257

Dm-MRPL38 NP_511152 Drosophila melanogaster Dm.2916 X, 12E5-12E5 416


Dm-A5 NP_476998 Drosophila melanogaster Dm.2837 2L, 22A1-22A1 210

At-TFL1 NP_196004 Arabidopsis thaliana At.1041 5 177

At-TSF NP_193770 Arabidopsis thaliana At.470 4 175

At-BFT NP_201010 Arabidopsis thaliana At.55676 5 177

At-E12A11 NP_173250 Arabidopsis thaliana At.48205 1 173

At-FT NP_176726 Arabidopsis thaliana At.469 1 175

At-AT5G01300 NP_195750 Arabidopsis thaliana At.48967 5 162

Sc-MRPL35 NP_010608 Saccharomyces cerevisiae IV 367

Sc-TFS1 NP_013279 Saccharomyces cerevisiae XII 219

Sc-YLR179C NP_013280 Saccharomyces cerevisiae XII 201

The redundant sequences (alternately spliced transcripts and short truncated sequences) were eliminated following two criteria as described previously [109] . This data set includes 32 PEBP members and the members are named using the abbreviation of the genus and species names followed by the genes.

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RKIP/PEBP1 expression promotes apoptosis [30] and antimetastasis functions [31] whereas PEBP4 has been reported to inhibit apoptosis [32] . Thus, the distinct biological features of PEBP members are consistent with the classification of the PEBP family ( Figure 2 ) and further suggest that RKIP homologs define a unique subfamily with similar but also distinct biological functions.

4. RKIP modulates Raf, GPCR and NF- k B signaling pathways

Only after the discovery that RKIP inhibits Raf-1 signaling [9] , was its function studied extensively by several groups. Emerging results have implicated RKIP as a modulator of fundamental signal cascades, including mediators of MAPK, G protein coupled receptor (GPCR) and NF- κ B signal transduction cascades ( Figure 3 ).

The Raf-MEK-MAPK signaling pathway is essential for cellular proliferation, differentiation, apoptosis, survival and

migration [33,34] , and it’s deregulation contributes to many human diseases including cancer [35,36] and developmental disorders [37] . The regulation of the Raf-MEK-MAPK cascade is complex and not completely understood but involves scaffold protein association as well as phosphorylation or de-phosphorylation of the signaling components (reviewed in [38] ). In an effort to isolate regulatory proteins for Raf-1, Yeung et al. discovered that RKIP can bind to Raf-1 and disrupt downstream MAPK signaling ( Figure 3 ) [9] , and overexpressed RKIP can act as a competitive inhibitor for MEK, the downstream target of Raf-1 [22] . However, studies based upon RKIP knockdown have demonstrated that RKIP inhibits activation of Raf-1 and has no effect on constitutively activated Raf signaling [21] . Furthermore, RKIP is a phosphoprotein, and PKC-mediated phosphorylation of RKIP (Ser153) dramatically decreases its association with Raf-1 [18] . Substitution of Ser153 by Glu does not impair the affinity of RKIP to Raf-1, suggesting that steric hindrance by the phosphate group rather than negative

RKIP

MRPL38

PEBP4

At-AT5G01300Dm-CG18594Dm-CG10298Dm-A5Dm-CG17917Rn-PEBP4Mm-PEBP4Bt-PEBP4Hs-PEBP4Dm-CG30060At-TSFAt-FT

At-BFTAt-E12A11

At-TFL1

Sc-YLR179CSc-TFS1Sc-MRPL35Dm-MRPL38Hs-MRPL38Mm-MRPL38Bt-MRPL38Rn-MRPL38Dm-CG17919Dm-CG7054Dm-CG6180Bt-PEBP1Rn-PEBP2Mm-PEBP2Rn-RKIPMm-PEBP1Hs-RKIP

52

83

6596

80

77

7155

83

69

99

9293

88

Figure 2 . Phylogenetic classifi cation of the most studied phosphatidylethanolamine binding protein (PEBP) family genes. The phylogenetic tree, based on 32 full-length PEBP protein sequences, was constructed as described previously [109] . Briefl y, multiple alignments of protein sequences were created by ClustalW [110] , and the alignments were used for phylogenetic tree construction by the MEGA4 program [111] . The phylogenetic tree was constructed by maximum parsimony method. To assess the confi dence of individual nodes, a bootstrap analysis with 1000 replications was performed using the same program package. The Raf kinase inhibitory protein (RKIP), PEBP4 and mitochondrial ribosomal protein L38 (MRPL38) subfamilies are indicated on the right of the genes.

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charge is required to inhibit the association of RKIP with Raf-1 ( Figure 3 ) [18] . RKIP binding to Raf-1 directly inhibits Raf-1 activation via blocking two key activation sites on Raf-1, Ser338 and Tyr341, which are phosphorylated by p21-associated kinase (PAK) and Src, respectively [21] . Interestingly, PKC can also regulate Raf directly, by a mechanism distinct from that of receptor tyrosine kinases [39] . These findings suggest that the mechanism by which RKIP regulates Raf is more complex than anticipated previously.

RKIP can also modulate another important signaling pathway initiated by G protein-coupled receptors (GPCRs). With more than 800 members, GPCRs comprise the largest family of membrane receptors. These receptors control essential physiological processes, including neurotransmission, hormone and enzyme release, inflammation and blood pressure regulation [40] . After agonist binding, GPCR promotes dissociation of G α -GTP from G β γ subunits, and both G α -GTP and G β γ stimulate downstream effectors [41] . This pathway is controlled by feedback inhibition, and GRK2 is one such inhibitor that phosphorylates activated GPCRs and initiates their internalization, ultimately shutting down GPCR signaling ( Figure 3 ) [42,43] . Interestingly, GRK2 is a

target of RKIP. RKIP phosphorylated at Ser153 by PKC is released from Raf-1 and binds to GRK2, blocking its activity and ultimately enhancing GPCR signaling ( Figure 3 ). The physiological relevance of this regulation was validated in cardiomyoctes where knockdown of RKIP lead to decreased β -adrenergic receptor-stimulated cAMP levels and contractile activity [19] .

Additionally, RKIP was reported to regulate activation of NF- κ B. The nuclear NF- κ B family of transcription factors has a critical role in coordinating the expression of a large number of genes that control immune and stress responses [44,45] . RKIP can antagonize NF- κ B in response to stimulation with TNF- α and IL-1 β , and may act upstream of NF- κ B to inhibit one or more kinases including NIK, TAK1 or IKK α / β ( Figure 3 ) [23] . Since these studies were primarily based on RKIP overexpression using in vitro systems, RKIP knockout cells and in vivo models are required to provide a better understanding of the role of RKIP in NF- κ B signaling.

Taken together, although the molecular mechanisms by which RKIP influences Raf-MEK-MAPK, GPCR, NF- κ B signaling as well as crosstalk between these pathways are not delineated completely, RKIP serves as an important

Nucleus

RKIP

GRK2

PRKIP

PKA

GPCR

PKC

Ras

MEK

Raf

ERK P

RTK

IKK complex

IκBP

Cytokine R

NF-κB

NF-κB

Degradation

Figure 3 . Role of Raf kinase inhibitory protein (RKIP) in Raf, G-protein-coupled receptor (GPCR), and NF- k B signal pathways. In quiescent cells, RKIP binds Raf-1 and inhibits downstream MAPK signaling. Upon stimulation by mitogenic signals, RKIP is phosphorylated and released from Raf-1. Phosphorylated RKIP binds to and inhibits G protein-coupled receptor kinase 2 (GRK2), thereby enhancing GPCR signaling. Additionally, RKIP may antagonize NF- κ B signaling in the context of TNF- α and IL-1 β stimulation by inhibition of upstream kinases NIK, TAK1 and/or IKK α / β . ERK: Extracellular signal-regulated kinase; MEK: MAPK/extracellular signal regulated kinase kinase; RTK: Receptor tyrosine kinase.

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modulator of physiological responses to various extra-cellular stimuli including mitogenic, inflammation and stress stimulus ( Figure 3 ).

5. RKIP in oncogenesis and cancer metastasis

The link between RKIP and cancer was first established using a prostate model. In search for genes that regulate metastasis process in prostate cancer, Keller and co-workers discovered that RKIP is downregulated by more than 70% in metastatic C4-2B cells compared with non-metastatic LNCaP cells [46] . Restoration of RKIP expression prevented invasion and metastasis in an orthotopic prostate cancer mouse model but not growth of the primary tumors [31] . RKIP expression in clinical samples of prostate cancer is reduced in tumor cells, and almost completely abrogated in metastatic cells. These results indicate that RKIP expression correlates inversely with prostate tumor progression and metastasis [31,47] . Subsequently, many reports revealed a reduction or loss of RKIP expression in various cancer cell lines or tumors/metastases including melanoma [20,48] , insulinomas [49] , breast cancer [50] , hepatocellular carcinoma [51,52] , anaplastic thyroid cancer [53] and colorectal cancer [54-56] . Furthermore, decreased expression of RKIP serves as a prognostic marker for prostate [47] and colorectal [54,55] cancer but whether RKIP has prognostic value for other cancers remains to be determined. Collectively, these data

RKIP

MEK

Raf

ERK

Aurora B kinase

Spindle checkpoint

Mitogenic signals

Figure 4 . Model of Raf kinase inhibitory protein (RKIP) regulation of Aurora B kinase and the spindle checkpoint. Depletion of RKIP leads to hyperactivation of Raf-MAPK/extracellular signal regulated kinase kinase (MEK)-MAPK signaling, thereby inhibiting Aurora B kinase which is essential for spindle checkpoint regulation. ERK: Extracellular signal-regulated kinase.

suggest RKIP may act as a tumor and/or metastasis suppressor for solid tumors.

Despite this compelling evidence, there are several puzzling observations that need to be resolved. First, a recent report demonstrated that RKIP is expressed homogeneously in both primary and metastatic melanoma samples from a large clinical cohort, and the RKIP staining intensity did not correlate with ERK phosphorylation or the clinical course of the disease in patients [57] . This surprising finding argues against a critical role for RKIP in blocking MAPK signaling in melanoma as suggested previously [48] . Second, no significant inverse correlation between RKIP expression and phospho-ERK staining was observed for node-postive breast cancer or for matched lymph node metastases, although RKIP expression was indeed downregulated in the lymph node metastases [50] . Third, efficient knockdown of RKIP in Merkel cell carcinoma cell line UISO did not elevate the level of phospho-ERK even following growth factor stimulation, suggesting that RKIP is not critical for silencing MAPK signaling in this system [58] . These observa tions suggest that the relationship between RKIP and MAPK signaling is more subtle and complex than suggested previously. It is possible that differences in the ratio between RKIP and phospho-RKIP explain some of the discrepancies between these different reports. Elucidation of the mechanism(s) by which RKIP regulates tumor progression and metastasis in a variety of tumor types as well as analyses of larger clinical sample sizes should provide a better understanding of the biological role of RKIP as a tumor suppressor.

6. RKIP in cell division and genomic stability

The cell-division cycle is regulated by the coordinated activities of both positive and negative growth-regulatory signals [59] , and deregulation of the cell cycle leads to genomic instability and cancer progression [60] . One of the key steps in cell cycle progression occurs during mitosis when proper kinetochore attachment to chromosomes enables equal and ordered separation of chromosomal DNA to daughter cells. Many aspects of this process are controlled by Aurora kinases [61] . The MAPK pathway also play a critical role in the regulation of the cell cycle [62] . Since RIKP can modulate MAPK signaling as discussed above, it is likely that RKIP plays one or more roles in cell cycle control. Recently, we demonstrated that RKIP associates with centrosomes and kinetochores and regulates the spindle checkpoint in cultured mammalian cells [16,63] . Specifically, RKIP depletion results in hyperactivation of the Raf-1/MEK/ERK signaling cascade, leading to Aurora B kinase inhibition, bypass of the spindle checkpoint and the generation of chromosomal abnormalities ( Figure 4 ) [63] . A recent report that loss of RKIP correlates positively with chromosomal losses supports an important role for RKIP in the maintenance of genomic stability [64] .

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7. RKIP and apoptosis

Apoptosis, or programmed cell death (PCD), is a distinct genetic and biochemical pathway essential for embryonic development and normal tissue homeostasis of metazoans [65-67] . Deregulation of apoptosis leads to a variety of human disorders including cancer [68] . Apoptosis contributes to the cytotoxic action of most chemotherapeutic drugs, and disruption of apoptotic pathways leads to drug resistance [69] .

Manipulation of RKIP expression appears to alter the sensitivity of drug-resistant cancer cells to apoptosis. In prostate cancer cells resistant to 9-nitrocamptothecin (9NC), overexpression of RKIP sensitizes the cells to 9NC-induced apoptosis, and, conversely, knockdown of RKIP abrogates 9NC-induced apoptosis in 9NC-senstive cells [30] . 9NC induces RKIP expression in 9NC-senstive cells but not in 9NC-resistant cells, and elevated RKIP inhibits the two key survival pathways mediated by Raf-MEK-MAPK and NF- κ B ( Figure 5 ) [30] . In non-Hodgkin’s lymphoma B cells, the chemotherapy drug Rituximab-mediated upregulation of RKIP expression suggesting that inhibition of Raf-MEK-MAPK is one important mechanism underlying Rituximab-induced apoptosis ( Figure 5 ) [70] . RKIP overexpression in prostate and melanoma cells was shown to sensitize TNF-related apoptosis inducing ligand (TRAIL)-induced apoptosis via inhibition of NF- κ B and upregulation of the TRAIL receptor death receptor 5 (DR5) [71] . Very recently, oncogenic B-Raf

IKK complex

Degradation

NF-κBIκB MEK

Raf

ERKNF-κB

RKIP

Apoptosis

Chemotherapy drugs

Figure 5 . Model of Raf kinase inhibitory protein (RKIP) promoting drug-induced cell apoptosis. Chemotherapy drugs induce RKIP expression, and the elevated RKIP proteins block either Raf-MAPK/extracellular signal regulated kinase kinase (MEK)-MAPK, NF- κ B or both of these two survival pathways. ERK: Extracellular signal-regulated kinase.

V600E was shown to induce RKIP expression as part of the mechanism by which overactive B-Raf induces senescence and apoptosis in nontumorigenic cells [72] . These findings suggest that RKIP may be a general mediator of cell apoptosis induced by aberrant signaling or chemotherapeutic agents through a mechanism involving inhibition of Raf-MEK-ERK and/or NF- κ B depending upon the particular stimulus and cellular environment.

Interestingly, PEBP4 was shown to have an antiapoptotic role in several cancer cell lines. Overexpression of PEBP4 inhibits TNF- α or TRAIL-induced apoptosis of prostate and breast cancer cells via activation of the AKT pathway; conversely, knockdown of PEBP4 promotes apoptosis and cell cycle arrest by downregulation of cyclin A and cyclin E [32,73,74] . Thus, RKIP and PEBP4, members of different PEBP subfamilies ( Figure 3 ), may have distinct and even opposite biological effects on apoptosis depending upon the particular cellular context.

8. RKIP and cell migration

Cell migration is a critical process in embryonic development and in many physiological and disease states [75] . For example, cell migration is one of the key steps required for cancer metastasis, although the underlying mechanisms has not been fully elucidated [76] . Only a few reports address the link between RKIP and cell migration, and the results are controversial. One study reported that RKIP plays a positive role in cell migration as evidenced by decreased cell migration in RKIP-depleted cells [77] . Overexpression of RKIP was shown to inhibit cell–cell adhesion by downregulation of E-cadherin and promote cell-subsratum adhesion and cell migration by upregulation of β 1 integrin [78] . However, several studies have reported the opposite effect of RKIP on cell migration; namely, that overexpression of RKIP inhibits cell migration or invasion [48,52] . These discrepancies regarding the role of RKIP in cell migration may be due to differences in cell systems and/or artifacts of overexpression and will probably require RKIP knockout systems as well as more detailed mechanistic studies to resolve.

9. RKIP in neural system and neurodegenerative diseases

RKIP is highly expressed in neural tissue [11] and, given the importance of MAPK, GPCR and NF- κ B signaling in the nervous system, is likely to play multiple roles. Surprisingly, the most important function of RKIP in the brain that has been elucidated to date relates to its role as a precursor for a neural peptide. HCNP, a novel undecapeptide cleaved from the N-terminal of RKIP, promotes differentiation of septo-hippocampal cholinergic neurons by stimulating production of choline acetyltransferase (ChAT) [24] . HCNP can act independently and also cooperatively with nerve growth factor (NGF) to enhance development of the cholinergic

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phenotype in the medial septal nucleus [25,79] . However, whether full-length RKIP functions similarly to HCNP remains to be determined.

Recently, RKIP and HCNP have been implicated in neurodegenerative diseases including Alzheimer’s disease (AD), the most common form of dementia in ageing human populations [80] . RKIP is significantly downregulated in the hippocampal CA1 field of late-onset AD patients [81] . Additionally, in the AD transgenic mouse model Tg2576, hippocampal RKIP is decreased compared with RKIP hippo-campal expression in non-transgenic littermates, and the decreased RKIP correlates with accumulation of amyloid- β (A β ) plaques, a hallmark of AD formation [82] . Since cholinergic dysfunction is implicated in age-related memory disturbances characteristic of AD [83,84] , these observations suggest that reduction of RKIP or its derivative HCNP may decrease differentiation of hippocampus cholinergic neurons and thereby contribute to neuronal dysfunction and pathogenesis of AD. However, considering the small sample size used for these studies, more work is required to establish a clear link between RKIP and/or HCNP and AD pathogenesis.

Several important questions relating to HCNP function remain to be addressed. For example, what is the mechanism by which HCNP induces production of choline ChAT, what is the specific receptor mediating this effect, what is the enzyme responsible for the cleavage of HCNP from RKIP and how this cleavage is regulated? Answers to these questions should advance our understanding of the role of HCNP and/or RKIP in neural development and disease.

10. The PEBP family and cell differentiation

The first link between PEBP genes and cell differentiation came from studying the role of the plant PEBP homolog, centroradialis (CEN), in inflorescence architecture. A natural loss-of-function mutation of CEN, a PEBP family gene in Antirrhinum , causes the normally indeterminate inflorescence to terminate in a flower [85] . CEN orthologs from other plants have been shown to retain the same conserved function of inhibiting flowering while maintaining both apical and inflorescence meristem indeterminacy. These genes include Arabidopsis Terminal Flower 1 ( TFL1 ) [86-88] , Tomato Self-Pruning ( SP ) [89] and Pea Determinate ( DET ) and Late Flowering [90] . Interestingly, a paralog of CEN termed Flowering Locus T ( FT ) that has an opposite function was also identified in Arabidopsis ; FT promotes flowering, and loss of FT delays flowering [91,92] . These findings suggest that flowering in Arabidopsis is controlled strictly by positive and negative signaling pathways regulated by FT and TFL1, respectively. Similarly, mutation of the SP Tomato gene at a conserved site within the pocket switches the plant from shoot growth to flowering [93] . These studies highlight the important role of PEBP family members in regulating plant cell differentiation and physiology.

There is also limited evidence suggesting that RKIP can regulate mammalian cell differentiation. RKIP is expressed in the differentiated spinous and granular layers of normal skin but not in the basal layer of the epidermis and undifferentiated carcinoma [94] . RKIP expression is associated with skin differentiation, and overexpression of RKIP enhances human keratinocyte differentiation [94] . RKIP expression also correlates with macrophage and dendritic cell differentiation, and overexpression of RKIP induces the expression of macrophage-specific mature marker CD11c and CD36 [95] .

Taken together, these studies indicate that RKIP and its homologs can have a profound effect on cell differentiation and development. Further experiments involving animal or other vertebrate models will be required to test the role of RKIP and/or other PEBP family members in developmental processes rigorously.

11. Insights from RKIP knockout mice

To better understand the function of RKIP in vivo , RKIP knockout mice have been generated using a gene trap embryonic stem (ES) cell line. Unexpectedly, the homozygous RKIP knockout mice are viable and fertile, suggesting that RKIP is not essential for embryonic development [96] . The mice may have some minor defects in olfactory functions, and reduced reproduction rates for male mice [96,97] . However, we have generated RKIP -/- mice by a similar approach and were able to obtain viable pups at expected Mendelian rates (unpublished data). This discrepancy may be explained by the different genetic backgrounds of the mice used.

One important issue that remains to be determined is whether the visibly normal phenotype of RKIP -/- mice results from genetic compensation by related genes PEBP2 or PEBP4. As noted above, PEBP2 and PEBP4 are reported to block MAPK signaling [32,98] . Furthermore, we observe expression of PEBP2 in both RKIP +/+ and RKIP -/- mouse embryonic fibroblasts (MEFs) (unpublished data). The available RKIP mouse model and MEFs are valuable tools for testing or validating the findings obtained from cell lines, and double knockouts of RKIP and either PEBP2 or PEBP4 may help address the role of RKIP in development, cancer or other disease processes.

12. Expert opinion

The ligand binding domains within the PEBP family of proteins are conserved in both prokaryotes and eukaryotes [1,99] , suggesting an ancient common origin for this basic functional unit. Diversity within the flexible loops of the PEBP structure has undoubtedly contributed to functional divergence within the PEBP subfamilies throughout evolution. RKIP functions in a variety of biological processes, including cell differentiation, migration, apoptosis and the

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cell cycle. Furthermore, growing evidence suggests that RKIP dysfunction is implicated in the progression to several human diseases, including cancer and AD.

To date, therapeutic agents that target RKIP specifically have not been developed. Since inactivation of B-Raf by mutation [100,101] or inhibition [102] can lead to a dependence of MAPK signaling on Raf-1, RKIP expression may be an effective approach for treatment. RKIP is decreased in certain tumor and metastatic tissues; thus, one strategy for treatment of these diseases is to restore RKIP expression by gene therapy. However, expressing RKIP in every cancer cell is still a great challenge, and gene therapy suffers from other potential problems such as specificity and safety. Other strategies do exist, such as using small compounds to induce RKIP expression or modulation of other pathways that directly regulated by RKIP (e.g., MAPK, NF- κ B and GPCR pathways). Alternatively, drugs could be developed to potentiate RKIP interaction with and inhibition of its targets. Finally, it may be possible to leverage the spindle checkpoint defect in RKIP-deficient cells by treating them with chemotherapeutic agents such as Taxol or Aurora kinase inhibitors to induce mitotic catastrophe.

Although RKIP biology has been partially delineated, many important issues remain to be addressed. A few of these questions are listed below: i) How is the function of RKIP regulated? Phosphorylation of particular sites (such as Ser153) may be one of the important regulatory mechanisms; ii) In addition to the MAPK, NF- κ B and GPCR pathways, does RKIP modulate other critical signal pathways? A systematic identification of RKIP binding partners through proteomic strategies may help to address this question; iii) Many studies localize RKIP in the cytosol but a few reports show that RKIP can be localized to the membrane or secreted to extracellular fluid [103-105] . Does RKIP have discrete functions related to its intracellular or extracellular location? iv) The function of RKIP’s homolog genes PEBP2 and PEBP4 has not been not extensively addressed. Is there any interplay or crosstalk among these genes? v) How is RKIP expression regulated on both a transcriptional and post-transcriptional level? Recently, RKIP transcription was

reported to inhibited by Snail [106] but the significance of this regulation is not yet clear. DNA methylation is an important epigenetic mechanism for gene silencing, and this is also an interesting topic for RKIP. Several reports show that hypermethylation is not the cause of RKIP down-regulation in melanoma cell lines [48] , prostate cancer cell lines [106] , and 28 human colorectal cancers [55] . However, a recent paper reports that methylation of the RKIP promoter is a major mechanism by which RKIP expression is silenced in colorectal cancer [64] . Therefore, whether promoter hypermethylation regulates RKIP expression remains to be determined, and larger sample sizes may help to address this issue; vi) How does regulation of the MAPK cascade by RKIP differ from that of other inhibitors such as Sprouty [107] ; and finally vii) RKIP was reported to be an inhibitor of serine proteases [103] , and a yeast PEBP family gene TFS1 was shown to inhibit carboxypeptidase Y [108] , suggesting that inhibition of proteases may be another important function of RKIP. What is the physiological relevance of these findings?

Answers to these questions will advance our under -standing of the role of RKIP in human physiology and pathology. With the current availability of RKIP mouse models and derived cells, we are now well positioned to rigorously anlalyze the role of RKIP in these various biological processes.

Acknowledgements

We thank Alexey E Granovsky for assistance with the RKIP structure figure.

Declaration of interest

Work from the author’s laboratory was supported by grants from the National Institutes of Health (NS33858 and CA112310 to M Rosner), the University of Chicago Cancer Research Center (to M Rosner) and a gift from the Cornelius Crane Trust for Eczema Research (to M Rosner). The authors declare no conflicts of interest.

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Affi liation Lingchun Zeng , Akira Imamoto & Marsha Rich Rosner † † Author for correspondence The University of Chicago, Ben May Department for Cancer Research, 929 East 57th Street, Chicago, IL 60637, USA Tel: +1 312 702 0380 ; Fax: +1 312 702 4476 ; E-mail: [email protected]

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