Protein kinase PKN1 associates with TRAF2 and is involved in TRAF2-NF-κB signaling pathway

BBRC Biochemical and Biophysical Research Communications 314 (2004) 688–694 www.elsevier.com/locate/ybbrc

Protein kinase PKN1 associates with TRAF2 and is involved in TRAF2-NF-jB signaling pathwayq,qq Yusuke Gotoh,a,1 Kumiko Oishi,a,1,2 Hideki Shibata,a,3 Akiko Yamagiwa,a,2 Takayuki Isagawa,a Tamako Nishimura,a Emiko Goyama,a Mikiko Takahashi,b Hideyuki Mukai,a,b and Yoshitaka Onoa,b,* a

Graduate School of Science and Technology, Kobe University, Kobe 657-8501, Japan b Biosignal Research Center, Kobe University, Kobe 657-8501, Japan Received 18 December 2003

Abstract PKN1 is a fatty acid and Rho-activated serine/threonine protein kinase whose catalytic domain is highly homologous to protein kinase C (PKC) family. In yeast two-hybrid screening for PKN1 binding proteins, we identified tumor necrosis factor a (TNFa) receptor-associated factor 2 (TRAF2). TRAF2 is one of the major mediators of TNF receptor superfamily transducing TNF signal to various functional targets, including activation of NF-jB, JNK, and apoptosis. FLAG-tagged PKN1 was co-immunoprecipitated with endogenous TRAF2 from HEK293 cell lysate, and in vitro binding assay using the deletion mutants of TRAF2 showed that PKN1 directly binds to the TRAF domain of TRAF2. PKN1 has the TRAF2-binding consensus sequences PXQX (S/T) at amino acid residues 580–584 (PIQES), and P580AQ582A mutant was not co-immunoprecipitated with TRAF2. Furthermore, the reduced expression of PKN1 by RNA interference (RNAi) down-regulated TRAF2-induced NF-jB activation in HEK293T cells. These results suggest that PKN1 is involved in TRAF2-NF-jB signaling pathway. Ó 2003 Elsevier Inc. All rights reserved. Keywords: PKN; PRK; PKC; TRAF; NF-jB; RNA interference; Luciferase; Kinase; Rho; TNF

q This work was supported in part by research grants from Japan Society for the Promotion of Science. qq Abbreviations: aa, amino acid(s); GST, glutathione S-transferase; IKK, IjB kinase; JNK, c-Jun N-terminal kinase; LMP1, latentinfection membrane protein 1; MAPK, mitogen-activated protein kinase; MAPKKK, MAPK kinase; MEKK, MAPK/ERK kinase; NF-jB, nuclear factor-jB; NIK, NF-jB-inducing kinase; PKC, protein kinase C; RNAi, RNA interference; SDS–PAGE, SDS–polyacrylamide gel electrophoresis; siRNA, small interfering RNA; TAK1, TGF-bactivated kinase; TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor; TRAF, TNF receptor-associated factor; WT, wild type. * Corresponding author. Fax: +81-78-803-5782. E-mail address: [email protected] (Y. Ono). 1 Y.G. and K.O. contributed equally to this work. 2 Present address: Center for Developmental Biology, RIKEN, Kobe 650-0047, Japan. 3 Present address: Laboratory of Molecular and Cellular Regulation, Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.

0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.12.148

PKN is a fatty acid-activated serine/threonine protein kinase that has a catalytic domain highly homologous to those of protein kinase C (PKC) family members in the carboxyl-terminal part and a unique regulatory region in the amino-terminal part [1–8]. PKN makes a family that comprises at least three gene products, including PKN14 (PKNa/PRK1), PKN2 (PKNc/PRK2), and PKN3 (PKNb) [5,8,9]. The amino-terminal region of the PKN1 contains three repeats of the anti-parallel coiled-coil (ACC) domain and C2-like region, and is assumed to restrict the protein kinase activity of the catalytic domain in the absence of activators [4,5,8,10, 11]. ACC domains were reported to function as binding interfaces to various associated proteins as follows: small GTPase RhoA [12,13]; intermediate filament

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The nomenclature employed here are PKN1, PKN2, and PKN3 for PKNa/PRK1, PKNc/PRK2, and PKNb, respectively, throughout the manuscript to avoid confusion.

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proteins [14,15]; a-actinin [16]; a paraneoplastic cerebellar degeneration-associated antigen PCD17/CDR62, which is a potential transcription factor [17]; a neuron-specific basic helix–loop–helix transcription factor NDRF/NeuroD2 [18]; and a centrosome- and Golgi-localized PKNassociated protein (CG-NAP) [19]. The carboxyl-terminal part of C2-like region functions as an essential autoinhibitory domain [11], which is sensitive to arachidonic acid, one of the activators of PKN1 [2]. ACC domain, C2-like region, and the catalytic domain are well conserved among PKN family members [5,8,9,20], although each “linker region” located between C2-like region and the catalytic domain is relatively diverse. Previously we identified Graf and Graf2, which are GTPase activating proteins for RhoA and Cdc42Hs, as the isoformspecific binding partners of PKN3 by using the linker region of PKN3 as bait by yeast two-hybrid screening [21]. In the present study, we screened HeLa cDNA library for isoform-specific binding partners of PKN1 by twohybrid system using the linker region of PKN1 as bait, and identified TNFa receptor (TNFR)-associated factor 2 (TRAF2) as the binding partner of PKN1. One of the roles of TRAF2 is modulating an early step in TNF receptor superfamily-induced activation of the NF-jB transcription factors, which play an evolutionarily conserved and critical role in regulating developmental events and immune responses through the expression of many of cytokines, chemokines, and adhesion molecules [22–26]. PKN1 bound to the carboxyl-terminal region of TRAF2 through the amino acid residues (aa) 580–584 (PIQES) of PKN1 both in vitro and in vivo, and the reduced expression of PKN1 by RNAi down-regulated TRAF2-induced NF-jB activation in HEK293T cells. These findings suggest that PKN1 is involved in TRAF2-NF-jB signaling pathway.

Materials and methods Expression constructs. The pGBKT7/PKN1 (511–633) and the GAD10/PKN1 (511–633) plasmids for two-hybrid screening were constructed by subcloning the fragment encoding aa 511–633 of PKN1 into EcoRI site of pGBKT7 or pGAD10. The pRc/CMV/hPKN1/ FLAG plasmid for FLAG-tagged PKN1 was described previously in [9] as pRc/CMV/hPKNa/FLAG plasmid. The pRc/CMV/hPKN1 (K644E)/FLAG, pRc/CMV/hPKN1 (T774A)/FLAG, pRc/CMV/ hPKN1 (P580A)/FLAG, and pRc/CMV/hPKN1 (P580AQ582A)/ FLAG were constructed by conversion of Lys644, Thr774, Pro580, and Gln582 to Ala by in vitro site-directed mutagenesis using QuikChange (Stratagene) according to the manufacturer’s instructions. The pBlueBacHis/GST/hPKN1 for GST-tagged PKN1 was described previously in [11] as pBlueBacHis/GST/PKNf. To make the pGBKT7/ TRAF2, the full-length TRAF2 was subcloned into pGBKT7. The pTB701/HA/TRAF2 for full-length TRAF2, pTB701/HA/TRAF2N for aa 1–236, and pTB701/HA/TRAF2C for aa 267–501 were made by subcloning the appropriate regions of TRAF2 into pTB701/HA. The pRSETA/TRAF2C for (His)6 -tagged aa 267–501 of TRAF2 was constructed by inserting an SacI fragment of pTB701/HA/TRAF2 into pRSETA. The pCR2/FLAG/TRAF2 plasmid and pCR2 empty vector

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were generous gifts from H. Nakano (Juntendo University, Japan). The pTAL-Luc and pNF-jB-Luc vectors for luciferase reporter gene assays were obtained from Clontech Laboratories. The pEF1a-b-gal vector was a generous gift from M. Tsuda and his colleagues (Toyama Medical and Pharmaceutical University, Japan). Yeast two-hybrid screening. pGBKT7/PKN1 (511–633) was used as bait in a two-hybrid screening of HeLa cDNA library according to the Matchmaker Two-hybrid System Protocol (Clontech). Primary positive clones were recovered and transfected into original yeast host strain. Appeared colonies were streaked onto synthetic complete plate lacking leucine, tryptophan, histidine, and adenine, but supplemented with 5 mM 3-amino-1,2,4-triazole and 20 lg/ml X-a-gal, and examined for growth and blue color development. Cell culture and transfection to cells. HEK293 cells and HEK293T cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 50 U/ml each of penicillin, and 5 lg/ml streptomycin in humidified 5% CO2 at 37 °C. Cells were transfected by lipofection using TRANS-IT (LT-1) (Mirus) according to manufacturer’s protocol. RNAi constructs. The pSUPER-PKN1 RNAi vector was constructed by ligation of the following oligonucleotide pair to pSUPER vector, which, obtained from Oligoengine [27]: 50 -gatccccGGAGCTGAAGCTGAAGGAGttcaagagaCTCCTTC AGCTTCAGCTCCtttttggaaa-30 and 50 -agc ttttccaaaaaGGAGCTG AAGCTGAAGGAGtctcttgaaC TCCTTCAGCTTCAGCTCCg gg-30 . As a negative control, pSUPER-PKN1 (nc) RNAi vector was also constructed using 50 -gatccccGGAGCTGATCCTGAAGGAGttcaaca caCTCCTTCAGGATCAGCTCCtttttg gaaa-30 and 50 -agcttttccaaaaa GGAGCTGATCCTGAAGGAGtctcttgaaCTCCTTCAGGATCAGC TCCggg-30 for producing siRNA duplex containing underlined 2 base pair change from that of pSUPER-PKN1. Antibodies. Antibodies used were as follows: Mouse monoclonal anti-FLAG (M2), anti-GST, anti-aTubulin (Sigma), anti-6His (Covance Research Products), anti-PRK1 (Transduction Laboratories), rat monoclonal anti-HA (3F10) (Roche Molecular Biochemicals), and rabbit polyclonal anti-TRAF2 (Sigma). Immunoprecipitation. HEK293 cells and HEK293T cells were plated at 2
106 /dish in 100 mm dishes 24 h before transfection with the indicated vectors. Twenty-four hours after transfection, cells were washed with PBS and lysed in 1 ml lysis buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2 , 1 mM EDTA, 1 mM DTT, 10 lg/ml leupeptin, and 1% NP40). Cell debris was removed by centrifugation at 15,000 rpm at 4 °C for 10 min. The cell extracts were incubated with the indicated antibodies at 4 °C for 2 h and with protein A–Sepharose for 2 h. The resulting immunoprecipitates were subjected to SDS–PAGE and immunoblot analysis with the indicated antibodies. Glutathione–Sepharose-pull down assay. GST-tagged PKN1 and (His)6 -tagged PKN1 were affinity purified from Sf9 cells, and GST only and (His)6 -tagged TRAF2C were affinity purified from Escherichia coli. Two micrograms of the each GST-tagged protein was incubated at 4 °C for 2 h with 2 lg of the indicated (His)6 -tagged protein and then precipitated with glutathione–Sepharose 4B beads (Amersham Biosciences). Co-precipitated (His)6 -tagged protein was detected by immunoblot analysis with an anti-6His antibody. Protein kinase assay. For autophosphorylation reaction, the beads-immobilized kinase was incubated in 25 ll reaction mixture (20 mM Tris–HCl, pH 7.5, 4 mM MgCl2 , 20 lM ATP, and 18.5 KBq [c-32 P]ATP) at 30 °C. After 5 min, the reaction was stopped by addition of SDS sample buffer and boiled for 5 min. A 25 ll aliquot of reaction mixture was subjected to SDS–PAGE. The gel was fixed, dried, and subjected to autoradiography. Reporter gene assay. For NF-jB reporter gene assay, HEK293T cells were plated at 2
105 /well in 6-well dishes 24 h before transfection with 3 lg of each pSUPER vector. Twenty-four hours after the first transfection, cells were additionally transfected with a total of 1 lg of vectors (including 0.2 lg pNF-jB-Luc or pTAL-Luc vector, 0.1 lg pEF1a-b-gal vector, and 0.7 lg pCR2/FLAG/TRAF2 vector or pCR2

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empty vector). The cells were harvested 48 h after the second transfection and luciferase activity was monitored via the Dual Luciferase Reporter assay system (Promega). Relative transfection efficiency was determined using the Galacto-Star Mammalian Reporter Gene Assay System (Applied Biosystems) according to manufacturer’s protocol.

Results Identification of TRAF2 as a PKN1-associating protein in the yeast two-hybrid system The aa 511–633 of PKN1, which corresponds to the linker region of the enzyme (Fig. 1A), was used as bait in a two-hybrid screening of HeLa cDNA library. From approximately 8.5 million transformants, five positive clones were obtained (data not shown). Three of these clones (clones #2, #4, and #5) encoded TRAF2, a member of the TRAF family [28]. The clone #2 encoded aa 225-end of TRAF2, and both clone #4 and clone #5 encoded aa 173-end of TRAF2. As shown in Fig. 1B, the TRAF2-fusion construct resulted in high a-galactosidase levels upon cotransfection with the PKN1 bait construct. In contrast, neither the combination of PKN1 and large T, p53, and TRAF2, nor large T and TRAF2 could exhibit the a-galactosidase activity. These results confirm the specificity of the association between PKN1 and TRAF2 in the two-hybrid system. PKN1 interacts with TRAF domain of TRAF2, and the TRAF2-binding motif, PIQES, of PKN1 is responsible for interaction between PKN1 and TRAF2 TRAF2 was originally identified by biochemical characterization of the intracellular factor that associated with the cytoplasmic domain of TNFR2 [28]. As TRAF2 does not contain intrinsic catalytic activity, protein–protein interactions are essential for TRAF2-mediated acti-

Fig. 1. TRAF2 is identified as a PKN1-associated protein. (A) Diagram of PKN1 construct and bait used for two-hybrid screening. The TRAF2-binding consensus sequences are indicated by bold. (B) Interaction between PKN1 and TRAF2 in the yeast two-hybrid system. Yeast host cells were co-transfected with expression plasmids encoding various proteins fused GAL4bd (left) and those fused to GAL4ad (right) as indicated in the left panel. Right panel shows developments of blue color 3 days after initiating assays. Murine tumor suppressor p53 and SV40 largeT antigen were used as controls.

vation of downstream signals [29]. To further examine the interaction of PKN1 with TRAF2, FLAG-tagged PKN1 was expressed in HEK293T cells and immunoprecipitated from cell extract using an anti-FLAG antibody. As shown in Fig. 2A, endogenous TRAF2 was clearly detected in the immunoprecipitate, suggesting that PKN1 binds to TRAF2 in mammalian cell. To identify the interaction site of TRAF2 with PKN1, deletion mutants of TRAF2 were constructed as shown in Fig. 2B. FLAG-tagged PKN1 was co-expressed with HA-tagged TRAF2, HAtagged TRAF2N (aa 1–236), or HA-tagged TRAF2C (aa 267–501) in HEK293 cells. TRAF2C (aa 267–501) corresponds to TRAF domain, which is known to mediate the interactions between TRAF proteins and the cytoplasmic domain of distinct members of the TNF receptor superfamily, including the TNFR2, CD40, and CD30 [28,30–34]. As shown in Fig. 2C, HA-tagged TRAF2 and HA-tagged TRAF2C, but not TRAF2N, were co-precipitated with FLAG-tagged PKN1 from cell lysates, suggesting that the TRAF domain of TRAF2 is responsible for the interaction of TRAF2 with PKN1. Next, to test whether PKN1 can directly interact with TRAF2, GST-tagged PKN1 and (His)6 -tagged TRAF2C (aa 267– 501) were affinity purified from Sf9 cells and E. coli, respectively. As shown in Fig. 2D, (His)6 -tagged TRAF2C was pulled down with GST-tagged PKN1, suggesting that PKN1 directly binds to TRAF domain of TRAF2. Some of TRAF2 associated proteins, including CD40, CD30, and LMP1, contain a PXQX (T/S) motif that has been demonstrated as the major consensus motif mediating the interactions with TRAF proteins [31,32,35–38]. We found a putative TRAF2-binding motif, PIQES (aa 580–584), in PKN1 (Fig. 1A). To evaluate the importance of this region for TRAF2binding, the effect of point mutations of this motif of PKN1 on TRAF2 association was assessed by immunoprecipitation experiments. Pro-580 and both Pro-580 and Gln-582 could be mutated to Ala without compromising the kinase activity of PKN1 in vitro (Fig. 2E), however, P580A mutant of PKN1 showed reduced binding affinity to TRAF2, and P580AQ582A mutant hardly bound to TRAF2 (Fig. 2F). In contrast, other mutations, which completely abolished catalytic activity of PKN1, Lys-644 in the ATP binding site to Glu (K644E mutant), and Thr-774 in the activation loop to Ala (T774A mutant), did not affect binding affinity to TRAF2 (Fig. 2F). These results indicate that the PIQES (aa 580–584) of PKN1 is critical for binding of PKN1 to TRAF2 in a kinase activity independent manner. RNAi of PKN1 down-regulated the TRAF2-induced NFjB activation TRAF2 plays a key role in transducing signals from the TNF receptor superfamily to NF-jB. It is also known that overexpression of TRAF2 leads to activation of

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Fig. 2. PKN1 interacts with TRAF2 both in vivo and in vitro. (A) Interaction of FLAG-tagged PKN1 with endogenous TRAF2 in HEK293T cells. HEK293T cells were transfected with pRc/CMV/PKN1 /FLAG. Cell extracts were immunoprecipitated with anti-FLAG antibody (aFLAG) or normal mouse IgG (normal). (B) Diagrams of TRAF2 deletion constructs. (C) Interaction of PKN1 with deletion mutants of TRAF2. HEK293 cells were co-transfected with pRc/CMV/PKN1/FLAG and each of TRAF2 expression plasmid. Cell extracts were immunoprecipitated with anti-FLAG antibody (aFLAG) or normal mouse IgG (normal). (D) GST-pull down assay. (His)6 -tagged TRAF2C was incubated with GST-tagged PKN1. (His)6 -tagged TRAF2C co-precipitated with GST-tagged PKN1 was detected by immunoblot analysis with anti-6His antibody (aHis). (E) In vitro autophosphorylation assay of various mutants of PKN1. In vitro autophosphorylation reactions were performed by using anti-FLAG immunoprecipitates prepared from HEK293 cells transfected with each plasmid. The lower panel shows the amount of immunoprecipitated PKN1 proteins. (F) Interaction of various mutants of PKN1 with TRAF2. HEK293 cells were co-transfected with pTB701/HA/TRAF2 and each of the PKN1 expression plasmids. Cell extracts were immunoprecipitated with anti-FLAG antibody (aFLAG) or normal mouse IgG (normal). WT, pRc/CMV/ PKN1/FLAG; K644E, pRc/CMV/PKN1 (K644E)/FLAG; T774A, pRc/CMV/PKN1 (T774A)/FLAG; P580A, pRc/CMV/PKN1 (P580A)/FLAG; and P580AQ582A, pRc/CMV/PKN1 (P580AQ582A)/FLAG.

NF-jB [34,39,40]. To investigate a possible role for PKN1 in TRAF2-induced NF-jB activation, we examined whether reduction of the expression level of PKN1 affects

NF-jB-dependent reporter gene (pNF-jB-Luc) expression. As shown in Fig. 3A, the amount of PKN1 was significantly reduced by transfection of pSUPER-PKN1

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Fig. 3. RNAi of PKN1 down-regulates the TRAF2-induced NF-jB activation. (A) Western blot analysis of PKN1, FLAG-tagged TRAF2, and aTubulin in HEK293T cells. HEK293T cells were co-transfected with FLAG-tagged TRAF2 expression vector and pSUPER-PKN1 (nc) (left) or pSUPER-PKN1 (right). Cells were harvested 72 h post-transfection and blot was probed with anti-PRK1 antibody (upper), antiFLAG antibody (middle), and anti-atubulin antibody (lower) as a loading control. (B,C) Effect of the reduced expression of PKN1 on NFjB-dependent transcription. HEK293T cells were transfected with pSUPER-PKN1 or pSUPER-PKN1 (nc) 24 h before pEF1a-b-gal vector, pNF-jB-Luc vector (B) or pTAL-Luc vector (C), and pCR2/FLAG/ TRAF2 vector or pCR2 empty vector transfection. Reporter luciferase values were normalized for transfection efficiency as determined by bgalactosidase expression. Results are expressed as percentage of the luciferase activity obtained with pSUPER-PKN1 (nc). The averages and standard deviations from four independent experiments are shown.

vector without any effect on the FLAG-TRAF2 expression level in HEK293T cells, whereas pSUPER-PKN1 (nc), which produced siRNA duplex 2 base pairs different from that produced by pSUPER-PKN1, did not show any effect on the expression level of both PKN1 and TRAF2. When FLAG-TRAF2 was expressed in HEK293T cells, 4- to 5-fold activation of NF-jB-dependent reporter compared to vector control was observed (data not shown). As shown in Fig. 3B, the reduced expression of PKN1 by RNAi down-regulated TRAF2-induced NF-jB activation in HEK293T cells, whereas RNAi of PKN1 did not affect thymidine kinase promoter activation (Fig. 3C). These results suggest that PKN1 is involved in TRAF2-induced NF-jB activation.

Discussion In this paper we suggested that PKN1 binds to TRAF2 in vitro and in vivo. In the TNFR signaling

pathway, for example, TRAF-associated proteins are recruited by TRAF proteins into the ligand-bound receptor complex at the plasma membrane [29,32,33,40– 43]. Thus, PKN1 also may be recruited by TRAF2 to the receptor complexes and phosphorylate downstream component(s). NF-jB is a dimer consisting of the various combinations of different Rel proteins, and most NF-jB dimers are bound to IjBs and retained in the cytoplasm in resting cells [22]. In response to various stimuli, IjBs are phosphorylated by IjB kinase (IKK) complex including IKKa, IKKb, and IKKc resulting in their polyubiquitination and degradation. The degradation of IjBs allows NF-jB to translocate into the nucleus and activates its target genes [44]. aPKC and atypical PKCs interact with IKKb in vitro and in vivo, and the catalytic domains are required for this interaction [45]. The catalytic domain of PKN1 is highly homologous to those of PKC family members, and the substrate specificity of these enzymes is basically similar to each other in vitro [2–4,8,46]. Thus, it is plausible that PKN1 recognizes IKK as a substrate and regulates IKK activity. Sanz et al. [47] reported that TRAF2-induced NF-jB activation is little or not affected by the k=iPKC dominant negative mutant, although it was inhibited by reduction of expression level of PKN1 by RNAi in the present study. Then the upstream signal leading to PKN1 seems to be different from those of PKCs even if PKN1 targets IKK. Recently we reported that PKN1 efficiently phosphorylates and activates MLTKa (MLK-like mitogenactivated protein triple kinase), which was recently identified as a MAPKKK for the p38 MAPK cascade [48]. Sun et al. [49] reported that co-incubation of PKN2 and MEKK2 leads to an increased phosphorylation level of MEKK2, suggesting that PKN2 phosphorylates or increases the activity of MEKK2. In mammalian cells, a number of MAPKKK family members, MEKK1, MEKK2, MEKK3, TAK1, COT/TPL-2, and NIK, are known to mediate the TNF receptor superfamily-induced NF-jB activation [50,51]. PKN might interact with and activate some of these members of the MAPKKK family as a MAPKKK kinase and affect the NF-jB activity. PKN1 is a downstream target of small GTPase RhoA [1,7,8]. RhoA activates the translocation of NF-jB complexes by inducing the phosphorylation of the IjBa isoform in Ser-32 and Ser-36 residues [52]. It was recently reported that RhoA can trigger an IKK-independent pathway leading to NF-jB activation [52,53]. Since NFjB activation by RhoA was reported to require IjB phosphorylation [52], a kinase other than IKKa and IKKb may phosphorylate IjB and thereby activate NFjB in response to RhoA. PKN1 might function as the IjB kinase responsible for this NF-jB activation. Six TRAF proteins including TRAF2 have been reported, each containing a highly conserved TRAF

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domain at the carboxyl terminus, and with the exception of TRAF1, a lesser conserved amino-terminal RING finger and zinc finger motif [28,31,33,54,55]. It has been shown that TRAF proteins bind to overlapping subsets of receptors, apparently by composition for the same binding sites [32,35,37,56,57]. Indeed, PKN1 is co-immunoprecipitated with other TRAF proteins including TRAF1, TRAF3, TRAF5, and TRAF6 from HEK293T cells (data not shown). On the other hand, PKN1 has at least two isoforms, PKN2 and PKN3 [5,8,9]. PKN2 has TRAF2-binding motif, PGQDS (aa 599–603), and was also co-immunoprecipitated with TRAF2 (data not shown). Not only TRAF2 but also some other TRAF proteins might transduce each signal through PKN1, and PKN2 as well as PKN1 might be involved in TRAF signaling pathway. Elucidation of the relevant combination might lead to some critical function of the PKN family member in the downstream of TNF receptor superfamily signaling.

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Acknowledgments We thank Dr. Y. Nishizuka for encouragement. We also thank Dr. T. Kato for critical reading and discussion on the manuscript.

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