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Journal of Cellular Biochemistry 93:579–587 (2004)

WDR26: A Novel Gb-Like , Suppresses MAPK Signaling Pathway

Ying Zhu,1 Yuequn Wang,1 Chunzhi Xia,2 Dali Li,1 Yongqing Li,1 Weiqi Zeng,1 Wuzhou Yuan,1 Hui Liu,1 Chuanbing Zhu,1 Xiushan Wu,1 and Mingyao Liu1,2* 1College of Life Sciences, Hunan Normal University, Changsha, 410081 Hunan, China 2Alkek Institute of Biosciences and Technology, and Department of Medical Biochemistry and Genetics, Texas A&M University System Health Science Center, Houston, Texas 77030

Abstract WD40 repeat play important roles in a variety of cellular functions, including cell growth, proliferation, , and intracellular signal transduction. Mitogen-activated protein kinases (MAPKs) are evolutionary conserved enzymes in cell signal transduction connecting cell-surface receptors to critical regulatory targets within cells and control cell survival, adaptation, and proliferation. Previous studies revealed that G-protein coupled receptors (GPCRs) play important roles in the signal transduction from extracellular stimuli to MAPKs and the WD40-containing Gb proteins as well as Gb-like proteins are involved in the stimulation and regulation of the MAPK signaling pathways. Here we report the identification and characterization of a novel human WD40 repeat protein, WD40 repeat protein 26 (WDR26). The cDNA of WDR26 is 3,729 bp, encoding a Gb-like protein of 514 amino acids in the cytoplasm. The protein is highly conserved in evolution across different species from yeast, Drosophila, mouse, to human. Northern blot analysis indicates that WDR26 is expressed in most of the examined human tissues, especially at a high level in skeletal muscle. Overexpression of WDR26 in the cell inhibits the transcriptional activities of ETS proteins, ELK-1 and c-fos serum response element (SRE), mediated by MEKK1. These results suggest that WDR26 may act as a negative regulator in MAPK signaling pathway and play an important role in cell signal transduction. J. Cell. Biochem. 93: 579–587, 2004. ß 2004 Wiley-Liss, Inc.

Key words: WD40 repeat protein; WD-40; Gb-like protein; WDR26; MAPK signaling pathway

Members of the WD-40 family of proteins, heterotrimeric GTP-binding proteins, play a including the well-known Gb subunits of variety of roles in intracellular signaling, RNA processing and degradation, expression, vesicular traffic and fusion, cytoskeletal assem- bly, and the as described in earlier reviews [Smith et al., 1999; Yu et al., 2000; Cabrera-Vera et al., 2003]. All proteins within Abbreviations used: MAPK, mitogen-activated protein the WD-40 family contain repeating sequences kinase; ERK, extracellular signal-related kinases; JNK, that are separated by approximately 40 amino Jun amino-terminal kinases; GPCRs, G-protein coupled acids. Each WD-40 repeat consists of two receptors; WDR26, WD40 repeat protein 26; MAPKK, signature sites: a poorly conserved site A MKK, or MEK, MAPK kinase; MAPKKK, MAPKK kinase; MEKK MEK kinase; EST, expressed sequence tags; WDR, containing a glycine and histidine (GH) pair, WD40 repeat protein; SRE, c-fos serum response element; and a well-conserved site B containing a SRF, serum response factor; BSA, bovine serum albumin; tryptophan and aspartate (WD) pair. Although DMEM, Dulbecco’s modified Eagle’s sodium. each protein contains four to eight WD-40 Ying Zhu, Yuequn Wang, and Xiushan Wu contributed repeating motifs, the separating distance and equally to the work. the actual sequence of individual repeats are Grant sponsor: Hunan Normal University (Special Profes- high variable. The functional diversity of WD- sorships); Grant sponsor: National Natural Science Foun- dation of China. 40 proteins suggests that WD-40 motifs may *Correspondence to: Mingyao Liu, PhD, College of Life not necessarily confer any particular function Sciences, Hunan Normal University, Changsha, Hunan while it may contributes to the formation of 410081, China. E-mail: [email protected] anti-parallel b-strands that stabilize the three- Received 26 March 2004; Accepted 29 April 2004 dimmensional b-propellers [Sondek et al., 1996; DOI 10.1002/jcb.20175 Smith et al., 1999]. ß 2004 Wiley-Liss, Inc. 转载 中国科技论文在线 http://www.paper.edu.cn

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Mitogen-activated protein kinases (MAPK) protein, WD40 repeat protein 26 (WDR26). are common components in signal transduction Northern blot analysis demonstrates that this connecting cell-surface receptors to critical gene is expressed in most of the human tissues, regulatory targets within cells and control a and especially at high levels in fetal brain and wide range of processes from the cell-cycle skeletal muscle of both fetal and adult stages. arrest and mating in yeast to cell proliferation This cytoplasmic protein contains five Gb-like and differentiation in metazoans (reviewed by WD40 repeats similar to hPIP1 and have Feng et al., 1998; Cobb, 1999; Kyriakis and similar sequence to WD40 repeat protein Avruch, 2001). The MAPK cascades are regu- 5 (WDR5), which can be induced by BMP-2 lated through three-tiered cascades composed of and dramatically accelerate the program of a MAPK, MAPK kinase (MAPKK, MKK, or osteoblastic differentiation [Gori et al., 2001]. MEK) and a MAPKK kinase or MEK kinase Transfection of WDR26 in mammalian cells can (MAPKKK or MEKK), which work in series and significantly suppress the transcriptional acti- comprise a module (reviewed by English et al., vation of MEKK-mediated SRE and ELK-1. 1999). In mammals there are at least four These results suggest that this new Gb-like distinct groups of MAPKs: extracellular sig- WD40 repeat protein may act as a repressor in nal-related kinases (ERK)-1/2, Jun amino- MAPK signaling pathway to mediate cellular terminal kinases (JNK1/2/3), p38 proteins functions. (p38 a/b/g), and ERK5, which are activated by specific MAPKKs and phosphorylate specific MATERIALS AND METHODS cellular targets. As reviewed by Chang and Cloning the Full-Lenghth cDNA and Karin [2001], one of the most explored targets of Bioinformatics Analysis MAPK signaling modules is transcription fac- tors, such as c-jun, Elk-1 which regulates tran- In order to identify novel Gb-like proteins in scription immediate early cell signal transduction, we used the consensus through binding to the serum response element sequence of WD40 region as the subject to (SRE) [Gille et al., 1995]. For example, most search human expressed sequence tags (EST) MAPKs phosphorylate ETS transcription fac- database with BLASTx algorithm (http:// tors that are involved in induction of fos www.ncbi.nlm.nih.gov/BLAST). Through com- , whose products heterodimerize with bined BLAST search and PCRs analysis as Jun proteins to form activation protein 1 (AP-1) previously described [Zeng et al., 2002], we complexes [Treisman, 1996]. Presumably each identified four overlapping ESTs—AW968761, MAPKKK confers responsiveness to distinct BG825203, BI757309, and BF971220, belong- stimuli, such as growth factors, hormones, ing to the same novel gene. A heart cDNA stress, and inflammation. library was constructed as PCR template with The multiplicity of both GPCR and MAPK the kits obtained from TAKARA1.50-RACE signaling pathways suggests the activities of PCR was performed with TAKARA 50-RACE both G-protein coupled receptors (GPCRs) and Core Kit to confirm the 50 terminus of WDR26. MAPKs is tightly regulated, depending on Jellyfish 1.4 was used to find the open reading cooperation of a number of activators and frame (ORF) and the deduced translated pro- suppressors. Although, we have known a lot duct. Then, the coding sequence was cloned about the activation process from receptors to from human heart library. All the primers and MAPKs, the knowledge on the mechanism of reaction conditions for these PCRs are listed in negative regulation is far from well developed. Table I and these PCR products were confirmed Recently, our laboratory identified a human by sequencing (Biotech1). For mammalian PAK1-interacting protein, hPIP1, which is a expression, the WDR26 ORF was subcloned negative regulator of p21-activated protein into the BamHI site of pCMV-tag2C (Strata- kinase (PAK). Overexpression of hPIP1 can gene1). The full-length sequence of WDR26 inhibit the Cdc42-stimulated PAK kinase activ- was submitted to GeneBank with an accession ity as well as PAK-mediated JNK and NF-kB number AY304473. signaling pathways through interaction with BLASTn (http://www.ncbi.nlm.nih.gov/BLAST) the N-terminal regulatory domains of PAK1 and Pfam 9.0 [Bateman et al., 2002] were used [Xia et al., 2001b]. Here we report the identifica- to analyze genomic structure and the pro- tion and characterization of a novel Gb-like tein domain, respectively. The homologues of 中国科技论文在线 http://www.paper.edu.cn

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TABLE I. Sets of Specific Oligonucleotide Primers and PCR Conditions

Primers Sequences Programs Cycles

E1 50-GCAAGAGTCAGGATGTCG-30 948C for 30 s; 508C for 30 s; 728C for 1 min 30 E2 50-CCAAAGCTCAGAGCAGTC-30 E3 50-GGAGGCACTTCAAGTTCTAC-30 948C for 30 s; 508C for 30 s; 728C for 1 min 30 E4 50-TCCATCACTCAAGCACCA-30 E5 50-ACTGGAGGTCAGCGTGGGC-30 948C for 30 s; 528C for 30 s; 728C for 1 min 30 E6 50-CGGGCTTCAGAAATGGTT-30 RT1 50-TCAGCGGCGTCAAT-30 308C for 10 min; 508C for 60 min; 808C for 2 min 1 S1 50-CGGAATTTGGTAGCAGAAGG-30 948C for 4 min; 568C for 1 min; 728C for 1 min 30 A1 50-CATGGAAGGAGACTGGGATAAG-30 S2 50-CAGTCTGGTTGAGCCCTAA-30 948C for 1 min; 588C for 1 min; 728C for 1 min 30 A2 50-GGCACTTCAAGTTCTACGC-30 Os 50-CAGGATCCCCGCCTCCTCTTCCT-30 948C for 30 s; 558C for 30 s; 728C for 2 min 30 Oa 50-GGATCCCAACTATCCATGCTACTGCAT-30

E1 to E6 are for expressed sequence tags (ESTs) analysis (E1 at the 3 terminus of AW968761 and E2 at 5 terminus of BG825203, E3 at the 3 terminus of BG825203 and E4 at the 5 terminus of BI757309, E5 at the 3 terminus of BI757309, and E6 at the 5 terminus of BF971220). RT1, S1, A1, S2, and A2 are for 50-RACE PCR. Os and Oa are for cloning the ORF of WD40 repeat protein 26 (WDR26) from heart cDNA library. The template of all the PCR are human heart cDNA library.

WDR26 were found with BLASTp (http://www. constructs for SRE-Luc was obtained from ncbi.nih.nlm.gov), and the most similar hits of Stratagene and reporter constructs for ELK-1- them were used to perform sequence alignment Luc were kindly provided from Dr. K.L. Guan at with ClustalW 1.8 [Jeanmougin et al., 1998] and the University of Michigan. Luciferase activity phylogenetic tree analysis with MegAlign pro- assays were performed according to the proto- gram (DNAstar, Inc., Madison, WI). cols of Stratagene. Each experiment was per- formed in triplicates and each assay was Cell Culture, Transient Transfection, repeated at least three times. The mean of the Immunocytochemistry, and Fluorescence Imaging data from three individual transfected wells are COS-7 was maintained in Dulbecco’s modi- presented. fied Eagle’s sodium (DMEM), supplemented Northern Analysis of WDR26 Expression with 10% fetal calf serum at 378C in a humi- in Human Fetal and Adult Tissues dified atmosphere of 5% CO2. Cells were transfected with pCMV-WDR26 with Lipofec- To study the expression pattern of WDR26 in tAMINE (Invitrogen, Carlsbad, CA) according different human tissues at fetal and adult to the method described before [Xia et al., stages, a RNA filter of purified adult human 2001a]. Forty-eight hours after transfection, mRNAs of multiple tissues (Clontech, Inc., cells were fixed with 4% paraformaldehyde for Palo Alto, CA) and a RNA filter of total RNA of 15 min at room temperature (RT), then blocked fetal (gestation 17-week) multiple tissues were with 0.2% bovine serum albumin (BSA) for hybridized with specific 32P-labeled cDNA as 30 min, followed by incubation with monoclonal described previously [Luo et al., 2002]. The antibody against Flag (M2 monoclonal, Sigma- therapeutically aborted fetuses (gestation 17 Aldrich, St. Louis, MO) for 1 h at RT, wash with week) were obtained under the approval of PBS (pH 7.4), and incubated with FITC-con- Health Center of Changsha Hospital of Women jugated phalloidin anti-mouse IgG (Molecular and Children, China, with the consent of the Probe, Inc., Eugene, OR). Actin filaments were patients and the regulation of university policy. labeled by rhodamine-conjugated phalloidin The RNA filter was prepared as described in (Sigma), and nuclear were stained with DAPI. previous studies [Luo et al., 2002]. Fluorescent images of cells were captured on a cooled charge-coupled device camera mounted RESULTS on an Olympus inverted research microscope 1 Molecular Cloning and Domain using Ultraview imaging software (Olympus , Structure of WDR 26 Inc., Melville, NY) [Guo et al., 2003]. During the search for novel Gb-like proteins Transient Expression Reporter Gene Assays in cell signaling and development, we identified COS-7 cells were transfected with LipofectA- a novel gene through scanning the human EST MINE (Invitrogen) according to the method database with WD40 consensus sequence. described before [Xia et al., 2001a]. The reporter Combined the results of the overlapping EST 中国科技论文在线 http://www.paper.edu.cn

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analysis, Northern blotting and 50-ACE the adult and fetal multiple tissue northern blot 3,729 bp full-length cDNA of the novel gene were performed using WDR26 cDNA as the was confirmed, which was named WDR26 as probe. A 3.7 kb transcript of WDR26 was approved by HUGO nomenclature committee. detected in most adult tissues with the highest The nucleotide sequence data reported here is expression detected in skeletal muscle and available in GenBank with the accession num- heart (Fig. 3A). In human fetal tissues, ber AY304473. WDR26 was detected mostly in skeletal muscle Alignment between the cDNA sequence and and brain with low level of expression in liver, indicates that WDR26 is iden- lung, and heart (Fig. 3B). The results indicate tical to the genomic sequence of PAC RP11- that WDR26 is expressed in most of human 397G23 on 1q42.13, spanning tissues and the expression level is much higher approximately 46 kb in reverse manner on the in skeletal muscle than in other tissues both genome and organized into 14 exons. All exon– during early developmental stages and in adult intron junctions contain the gt/ag consensus tissues. Furthermore, we noticed that the splice site (the data not shown here). The expression of WDR26 is similar to hPIP1,both translation start codon ATG is in the second of which are strongly expressed in skeletal exon and the TAA stop codon in the 14th exon. muscle. The complete cDNA of WDR26 consists of an WDR26 Protein Is a Cytoplasmic Protein ORF of 1,545 bp from 199 to 1,743, a 198 bp 50- Suppressing SRE- and ELK-1-Mediated untranslated terminus and a long 30-untrans- Transcriptional Activation lated terminus of 2,029 bp with a consensus polyadenylation signal (AATAAA) (Fig. 1A). To examine the subcellular location of The deduced WDR26 protein is 514 amino acids WDR26, the pCMV-WDR26 was transfected and its calculated relative molecular mass is into COS-7 cells, and 48 h after the transfection, 58,603 Da (59 kDa) (Fig. 1A). This protein the cells were visualized with epifluorence contains a WD40 region (199 amino acid to 494 microscope after labeled with FITC rhodamine amino acid), including five WD40 repeats in for actin and DAPI for nuclei. As shown in tandem arrays (Fig. 1B). Figure 4A, WDR26 protein distributes evenly in cytoplasm when overexpressed in the cells WDR26 Belongs to a Novel Protein Family (Fig. 4A, arrow). The combined image shows Conserved During Evolution that WDR26 protein with actin and nucleus in The amino acid sequence of WDR26 is highly the cell (Fig. 4D). Although we consider WDR26 identical to its homologues in other species, as a cytoplasmic protein, we could not rule out whose functions are unknown. Sequence align- the possibility that the protein also exists in the ment of these proteins demonstrates that it is nucleus as shown in the fluorescence staining of one of the most conserved proteins during the protein in the cell. evolution (Fig. 2A). The sequence identity Recent studies have revealed the importance between human and mouse homologues is even of multiple intracellular signaling pathways higher than that of Gb-subunit family of mediated by activation of GPCRs, through proteins. We tried to analyze the evolutionary which GPCRs transduce extracellular signals relationship between the 5-WD40-repeat Gb- into nucleus to regulate the activity of various like proteins and Gb proteins with phylogenetic transcript factors, such as c-fos serum response tree analysis (Fig. 2B). The data shows that de- element (SRE), in cellular processes. Although spite their similar domain structure, these Gb- WDR26 shares WD-40 repeat domains with Gb- like proteins such as hPIP1 and WDR26 belong subunits, the potential role of WDR26 is not to new subfamilies of proteins differing from clear. As a first step in our understanding of the Gb-subfamily of proteins. Furthermore, WDR26 in cell signal transduction, we exam- WDR26 and its homologues make up a new ined whether WDR26 is directly or indirectly conserved subfamily not known before (Fig. 2B). involved in the regulation of transcription factors, especially in the MAPK pathway. As WDR 26 Is Widely Expressed at Embryo an important nuclear effector of MAPK signal- and Adult Stages ing pathway, the c-fos SRE forms a ternary To characterize the transcript of WDR26 with complex together with serum response factor respect to its size and expression distribution, (SRF) and ETS proteins. To examine the effect 中国科技论文在线 http://www.paper.edu.cn

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Fig. 1. A: Nucleotide sequence and deduced protein sequence are numbered at the left side of each line, respectively. The of the human WD40 repeat protein 26 (WDR26) gene. WDR26 putative polyadenylation signal sequence AATAAA is under- encodes a polypeptide of 514 amino acids. The initiation ATG lined. B: The domain structure of WDR26. The boxes indicate the and termination TAA codons are boxed and shaded in gray. location of five WD40 repeats with WD-repeat 1 (aa 200–237), Amino acids are identified by their one-letter code. The five WD-repeat 2 (aa 246–283), WD-repeat 3 (291–328), WD- WD40 repeats are underlined. Both nucleotides and amino acids repeat 4 (413–453), and WD-repeat 5 (458–496). 中国科技论文在线 http://www.paper.edu.cn

Fig. 2. WDR26 is conserved during evolution. A: Comparison 56% to Drosophila melanogaster CG7611-PA, 38% to Arabi- of the amino acid sequences of WD40 region among WDR26 dopsis thaliana NP_196473, 61% to Anopheles gambiae and its homologues: H, human WDR26; M, Mus musculus EAA11448.1, 32% to Schizosaccharomyces pombe trp-asp BAC35923; R, Rattus LOC289325; D, Drosophila melanogaster repeat protein T38653. B: Un-rooted phylogenetic tree analysis CG7611-PA, A, Arabidopsis thaliana NP_196473; P, Anopheles of WD40 repeat proteins shows that WDR26 belong to a new gambiae EAA11448.1; Y, Schizosaccharomyces pombe trp-asp subfamily differing from Gb proteins and the PAK inhibitor hPIP1. repeat protein T38653. Identical residues fitting the WD40 repeat WDR26 and its homologues from other species shown in (A), five consensus have been boxed and are shaded in dark. Human human Gb proteins, and several Gb-like proteins were used in WDR26 protein is 99% identical to Mus musculus BAC35923, the unrooted phylogenetic tree analysis. 中国科技论文在线 http://www.paper.edu.cn

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Fig. 3. Expression of WDR26 in human adult (A) and 17-week-fetal tissues (B) analyzed using Northern blot. The RNA filters were hybridized with a 32P random-labeled cDNA probe, which contains coding sequence of WDR26. The same filters were also hybridized with b-actin to normalize for loading differences. A band at 3.7 kb was detected.

of WDR26 in this pathway, we performed across different species and organisms. Expres- reporter gene assays to measure the modulation sion of WDR26 was found in most of the tissues of SRE and ELK-1 by WDR26 in the cell. COS-7 tested with the highest in human adult skeletal cells were co-transfected with the expression muscle and heart, in fetal skeletal muscle and plasmids pCMV-WDR26, pFC-MEKK, pSRE- brain. Overexpression of WDR26 in the cell Luc, and pFA2-Elk-Luc, which encodes for negatively regulates MAPK signaling pathway luciferase controlled by SRE and Elk-1 respec- by significantly inhibiting the activities of SRE tively. As shown in Figure 5A, expression of and ELK-1, which are the targets of ERK, JNK WDR26 significantly reduced the endogenous and p38 [Treisman, 1996]. SRE-luciferase activity by 60%. Furthermore, Structural study on WD40 repeat proteins co-expression of WDR26 with MEKK1 strongly revealed that the propeller ring structure con- inhibited MEKK1-stimulated SRE-luciferase structed with WD40 repeats is conserved among activity, suggesting a potential role of this this superfamily, suggesting that WDR26 may protein in MEKK-mediated signal transduc- have a similar ternary structure like Gb- tion. We then tested the effect of WDR26 on the subfamily of proteins. Therefore, it is interest- transcriptional activity of Elk-1, a member of ing to demonstrate the opposite roles of Gb-like the ternary complex. As observed in the SRE- protein, WDR26, and the Gb proteins in MAPK luciferase assays, we found that expression of pathways. Besides WDR26, there are more Gb- WDR26 strongly inhibited the endogenous like proteins that act as regulatory factors in transcriptional activity of Elk-1 and the signaling pathway, such as hPIP1 and its MEKK-mediated Elk-1 transcriptional activity homologue in yeast skb15, the negative regula- (Fig. 5B). Taken together, our results suggest tors in Cdc42/Rac-PAK medicated JNK path- that WDR26 is a new WD40 repeat protein that way in mammalian [Xia et al., 2001b] and yeast potentially participates in the MAPK signaling cells [Kim et al., 2001]. All these Gb-like pathways in the cell. proteins share a WD region consisting five or six WD-40 repeats, which is different from the seven WD-40 repeats of Gb subunits. The DISCUSSION phylogenetic tree analysis also demonstrates WD-40 repeat proteins play important roles that Gb-like proteins, including WDR26 and its in a variety of cellular functions from cell pro- homologues, belong to a new WD40 repeat liferation, cell apoptosis, to different cell signal protein superfamily differing from Gb proteins transduction pathways. MAPKs are important (Fig. 3B). Together, these data suggest that the signal transducing enzymes that are involved in new subfamily of Gb-like proteins may coop- many facets of cellular regulation; therefore, erate with Gb proteins in the regulation of cell their activity should be tightly regulated. In this signaling pathways. report, we described the identification of a novel MAPK pathways are involved in multiple Gb-like protein, WDR26. The protein contains cellular living processes through phosphorylat- five WD-40 repeats and is highly conserved ing their specific endpoint targets, such as ELK- 中国科技论文在线 http://www.paper.edu.cn

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Fig. 4. WDR26 is a cytoplasmic protein. A: Cells expressing Flag-tagged WDR26 were stained with monoclonal antibody against Flag epitope of WDR26 and FITC-conjugated phalloidin. B: Cells in (A) stained with rhodamine-conjugated phalloidin. C: The nucleus of cells in (A) stained with DAPI. D: The combined image of (A–C). [Color figure can be viewed in the online issue, which is available at www.interscience. wiley.com.]

1 and SRE, which compose a ternary complex together with SRF to induce expression of c-fos and other early growth response genes that control the transition from quiescence to pro- liferation [Gille et al., 1995; Graf et al., 1997]. WDR26 is a conserved gene during evolution with high sequence identity among homologues from various species, suggesting a potential role in regulating some essential cellular processes, such as cell growth and proliferation, through the MAPK signaling pathway. The exact func- tion of WDR26 in the cell is not clear and is under active investigation.

ACKNOWLEDGMENTS We are grateful to all members of the laboratory of molecular developmental genetics the College of Life Sciences in Hunan Normal University for their excellent technical assis- tance and encouragement.

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