Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 as a Putative Effector of Rap2 to Activate the c-Jun N-terminal Kinase

Abstract

Little is known about the specific signaling roles of Rap2, a Ras family small GTP-binding protein. In a search for novel Rap2-interacting proteins by the yeast two-hybrid system, we isolated isoform 3 of the human mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), a previously described but uncharacterized isoform. Other isoforms of MAP4K4 in humans and mice are known as hematopoietic progenitor kinase (HPK)/germinal center kinase (GCK)-like kinase and Nck-interacting kinase, respectively. MAP4K4 belongs to the STE20 group of protein kinases and regulates c-Jun N-terminal kinase (JNK). MAP4K4 interacted with Rap2 through its C-terminal citron homology domain but did not interact with Rap1 or Ras. Interaction with Rap2 required the intact effector region of Rap2. MAP4K4 interacted preferentially with GTP-bound Rap2 over GDP-bound Rap2 in vitro. In cultured cells, MAP4K4 colocalized with Rap2, while a mutant MAP4K4 lacking the citron homology domain failed to do so. Furthermore, Rap2 enhanced MAP4K4-induced activation of JNK. These results suggest that MAP4K4 is a putative effector of Rap2 mediating the activation of JNK by Rap2. Little is known about the specific signaling roles of Rap2, a Ras family small GTP-binding protein. In a search for novel Rap2-interacting proteins by the yeast two-hybrid system, we isolated isoform 3 of the human mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), a previously described but uncharacterized isoform. Other isoforms of MAP4K4 in humans and mice are known as hematopoietic progenitor kinase (HPK)/germinal center kinase (GCK)-like kinase and Nck-interacting kinase, respectively. MAP4K4 belongs to the STE20 group of protein kinases and regulates c-Jun N-terminal kinase (JNK). MAP4K4 interacted with Rap2 through its C-terminal citron homology domain but did not interact with Rap1 or Ras. Interaction with Rap2 required the intact effector region of Rap2. MAP4K4 interacted preferentially with GTP-bound Rap2 over GDP-bound Rap2 in vitro. In cultured cells, MAP4K4 colocalized with Rap2, while a mutant MAP4K4 lacking the citron homology domain failed to do so. Furthermore, Rap2 enhanced MAP4K4-induced activation of JNK. These results suggest that MAP4K4 is a putative effector of Rap2 mediating the activation of JNK by Rap2. Rap2 belongs to the Ras family of small GTP-binding proteins. The mammalian Ras family consists of Ras proteins (Ha-Ras, Ki-Ras, and N-Ras), Rap proteins (Rap1A, Rap1B, Rap2A, and Rap2B), R-Ras, R-Ras2/TC21, R-Ras3/M-Ras, Ral proteins (RalA and RalB), Rheb, Rin, and Rit (for a review, see Ref. 1Campbell S.L. Khosravi-Far R. Rossman K.L. Clark G.J. Der C.J. Oncogene. 1998; 17: 1395-1413Crossref PubMed Scopus (925) Google Scholar). Orthologs of both Rap1 and Rap2 are present in the nematode Caenorhabditis elegans, suggesting that Rap1 and Rap2 play important and distinct roles. However, little is known about the specific roles of Rap2 in cell signaling. The Ras family proteins regulate many cellular processes including proliferation, differentiation, and apoptosis (1Campbell S.L. Khosravi-Far R. Rossman K.L. Clark G.J. Der C.J. Oncogene. 1998; 17: 1395-1413Crossref PubMed Scopus (925) Google Scholar). In regulating these processes, they serve as molecular switches by cycling between GTP-bound active and GDP-bound inactive forms. The GTP-bound forms interact physically with downstream effectors. Interactions of Ras family proteins with effectors are mediated by effector regions (in the case of Ras, amino acids 32–40). The effector regions of the Ras family proteins also play critical roles in differential recognition of effectors. An effector region identical to that of Ras is present in Rap1. Rap1 interacts with most of the effectors of Ras, including Raf-1 and B-raf, both of which are upstream of the “classical” mitogen-activated protein kinase (MAPK), 1The abbreviations used are: MAPK, mitogen-activated protein kinase; MAP4K, MAP kinase kinase kinase kinase; HPK, hematopoietic progenitor kinase; GCK, germinal center kinase; HGK, HPK/GCK-like kinase; SH, Src homology; NIK, Nck-interacting kinase; CNH, citron homology; JNK, c-Jun N-terminal kinase; GST, glutathione S-transferase; HA, hemagglutinin; GTPγS, guanosine 5′-O-(thiotriphosphate). the extracellular signal-regulated kinase. Although Rap1 interacts with both Raf-1 and B-raf, it activates only B-raf (1Campbell S.L. Khosravi-Far R. Rossman K.L. Clark G.J. Der C.J. Oncogene. 1998; 17: 1395-1413Crossref PubMed Scopus (925) Google Scholar). Rap1 does not have its specific effector, like the other Ras family members sharing the identical effector region. The effector region of Rap2 differs from those of Ras and Rap1 by a single amino acid: amino acid 39 in Rap2 is Phe, while in Ras and Rap1 it is Ser. This difference could confer on Rap2 an ability to interact with its specific effectors and play signaling roles distinct from those of Rap1. Here, we report the identification of a novel Rap2-interacting protein, human MAP kinase kinase kinase kinase (MAP4K) 4 isoform 3, a previously described but uncharacterized isoform of MAP4K4 (2Yao Z. Zhou G. Wang X.S. Brown A. Diener K. Gan H. Tan T.-H. J. Biol. Chem. 1999; 274: 2118-2125Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 3Su Y.-C. Han J. Xu S. Cobb M. Skolnik E.Y. EMBO J. 1997; 16: 1279-1290Crossref PubMed Scopus (221) Google Scholar, 4Wright J.H. Wang X. Manning G. LaMere B.J. Le P. Zhu S. Khatry D. Flanagan P.M. Buckley S.D. Whyte D.B. Howlett A.R. Bischoff J.R. Lipson K.E. Jallal B. Mol. Cell. Biol. 2003; 23: 2068-2082Crossref PubMed Scopus (100) Google Scholar). MAP4K4 exhibits structural homology to hematopoietic progenitor kinase (HPK) and germinal center kinase (GCK) (for a review, see Ref. 5Kyriakis J.M. J. Biol. Chem. 1999; 274: 5259-5262Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Thus, human MAP4K4 was designated HPK/GCK-like kinase (HGK) (2Yao Z. Zhou G. Wang X.S. Brown A. Diener K. Gan H. Tan T.-H. J. Biol. Chem. 1999; 274: 2118-2125Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Mouse MAP4K4 was discovered as a kinase that interacts with Nck, an adaptor protein composed of one Src homology (SH) 2 and three SH3 domains, and hence designated Nck-interacting kinase (NIK) (3Su Y.-C. Han J. Xu S. Cobb M. Skolnik E.Y. EMBO J. 1997; 16: 1279-1290Crossref PubMed Scopus (221) Google Scholar) (Fig. 1A). MAP4K4 belongs to a large group of kinases related to Saccharomyces cerevisiae MAP4K, STE20. The STE20 group is divided into two families (for a review, see Ref. 6Dan I. Watanabe N.M. Kusumi A. Trends Cell Biol. 2001; 11: 220-230Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar). One is the p21-activated kinase family, whose members are activated by the Rho family small GTP-binding proteins Cdc42 and Rac. The other is the GCK family, which consists of eight subfamilies. GCK and HPK, also known as MAP4K2 and MAP4K1, respectively, belong to the GCK-I subfamily; MAP4K4 belongs to the GCK-IV subfamily. Members of both GCK-I and -IV subfamilies are upstream of c-Jun N-terminal kinase (JNK), a “stress-activated” MAPK. In the present study, we show that Rap2 enhances activation of JNK by MAP4K4. We postulate that Rap2 plays signaling roles distinct from those of Rap1 by regulating JNK. Two-hybrid Screening—A cDNA fragment encoding amino acids 1–168 of human Rap2A was subcloned into plasmid pBTM116 to yield pBTM116-Rap2A. pBTM116-Rap2A and the human fetal brain cDNA library cloned in pACT2 (Clontech, Palo Alto, CA) were co-transformed into the S. cerevisiae L40 reporter strain. Approximately 4.7 × 106 co-transformants were examined for HIS3 and β-galactosidase expression as described previously (7Kohno H. Tanaka K. Mino A. Umikawa M. Imamura H. Fujiwara T. Fujita Y. Hotta K. Qadota H. Watanabe T. Ohya Y. Takai Y. EMBO J. 1996; 15: 6060-6068Crossref PubMed Scopus (241) Google Scholar). In Vitro Binding Assay—Full-length Rap2A (amino acids 1–183) and Ha-Ras were expressed in Sf9 insect cells as fusion proteins with glutathione S-transferase (GST) using the baculovirus expression system as described previously (7Kohno H. Tanaka K. Mino A. Umikawa M. Imamura H. Fujiwara T. Fujita Y. Hotta K. Qadota H. Watanabe T. Ohya Y. Takai Y. EMBO J. 1996; 15: 6060-6068Crossref PubMed Scopus (241) Google Scholar). GST-Rap2A or GST-Ha-Ras was immobilized on glutathione-Sepharose resin (Amersham Biosciences). The coding sequence for full-length MAP4K4 isoform 3 (amino acids 1–1212) (Fig. 1A) was generated by using a PCR-amplified cDNA sequence encoding amino acids 1–208 (common to all isoforms) and a cDNA clone encoding amino acids 38–1212 of this isoform (KIAA0687), provided by Dr. Takahiro Nagase (Kazusa DNA Research Institute, Chiba, Japan). HEK293T cells were transfected with pCIneo-HA-MAP4K4 or pCIneo-HA-MAP4K4ΔCNH expressing HA-tagged full-length MAP4K4 or its deletion mutant lacking the CNH domain using Polyfect reagent (Qiagen). Cells were harvested, homogenized in buffer A (20 mm Tris/HCl, pH 7.6, 100 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, 5 mm MgCl2, 0.1% Nonidet P-40) containing protease inhibitors (Roche Applied Science), and centrifuged at 100,000 × g for 1 h. The supernatant (the soluble fraction) was incubated with the glutathione-Sepharose resin carrying immobilized GST-Rap2A or GST-Ha-Ras preloaded with GTPγS or GDP as described previously (7Kohno H. Tanaka K. Mino A. Umikawa M. Imamura H. Fujiwara T. Fujita Y. Hotta K. Qadota H. Watanabe T. Ohya Y. Takai Y. EMBO J. 1996; 15: 6060-6068Crossref PubMed Scopus (241) Google Scholar). Fluorescence Microscopy—NIH3T3 cells were fixed with formaldehyde and processed for immunofluorescence confocal microscopy (LSM 5 PASCAL, Carl Zeiss) with monoclonal anti-Myc antibody (9E10; Cell Signaling Technology, Beverly, MA) and polyclonal anti-HA antibody (sc-805; Santa Cruz Biotechnology, Santa Cruz, CA) as described previously (8Umikawa M. Obaishi H. Nakanishi H. Sato-Horikawa K. Takahashi K. Hotta I. Matsuura Y. Takai Y. J. Biol. Chem. 1999; 274: 25197-25200Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). JNK Activation Assay—pFLAG-CMV2-JNK2 and pGEX2T-c-Jun(1–79) were kindly provided by Dr. Katsuji Yoshioka (Kanazawa University, Ishikawa, Japan) (9Ito M. Yoshioka K. Akechi M. Yamashita S. Takamatsu N. Sugiyama K. Hibi M. Nakabeppu Y. Shiba T. Yamamoto K.I. Mol. Cell. Biol. 1999; 19: 7539-7548Crossref PubMed Scopus (231) Google Scholar). 293T cells were homogenized in buffer A containing protease inhibitors, and homogenates were subjected to Western immunoblotting with polyclonal anti-phospho-JNK (Thr-183/Tyr-185) antibody (number 9251; Cell Signaling Technology). The homogenates were also subjected to an immunocomplex kinase assay as described previously (8Umikawa M. Obaishi H. Nakanishi H. Sato-Horikawa K. Takahashi K. Hotta I. Matsuura Y. Takai Y. J. Biol. Chem. 1999; 274: 25197-25200Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) except that FLAG-JNK2 was immunoprecipitated with anti-FLAG M2 resin (Sigma), and the levels of phosphorylation of GST-c-Jun(1–79) were assessed by Western immunoblotting with polyclonal anti-phospho-c-Jun (Ser-63) antibody (number 9246; Cell Signaling Technology). To identify a specific effector of Rap2, we carried out a yeast two-hybrid screening for Rap2-interacting proteins using a human fetal brain cDNA library. From this library, three independent positive clones, pACT2-H-1, -2, and -3, were isolated which encoded the C-terminal portions of MAP4K4 (Fig. 1A). MAP4K4 contains the N-terminal kinase domain bearing homology to STE20, the intervening region that includes a coiled-coil domain, and the C-terminal regulatory domain termed the CNH domain. The CNH domain was encoded by all the three clones, suggesting that it interacts with Rap2. Several Ras-interacting proteins contain regions of about 100 amino acids that mediate interactions with Ras; the Ras-binding domain and the Ras-associating domain (1Campbell S.L. Khosravi-Far R. Rossman K.L. Clark G.J. Der C.J. Oncogene. 1998; 17: 1395-1413Crossref PubMed Scopus (925) Google Scholar). However, these domains were not found within the CNH domain of MAP4K4. When the CNH domain was divided into N- and C-terminal portions, each portion did not interact with Rap2 (Fig. 1A). This suggests that the entire CNH domain is required for interaction with Rap2. Alternatively, a region mediating the interaction may overlap the boundary between the two portions. Next, we examined the interaction of MAP4K4 with Rap2 mutants and other small GTP-binding proteins by the two-hybrid assay (Table I). MAP4K4 strongly interacted with Rap2 and its activated mutant, Rap2(G12V), while it interacted weakly with the dominant negative mutant, Rap2(S17N). MAP4K4 barely interacted with the effector region mutant, Rap2(F39S). MAP4K4 failed to interact with Rap1, its activated mutant, Rap1(G12V), or Ras. In comparison, Raf-1 failed to interact with only the dominant negative mutant, Rap2(S17N). Both MAP4K4 and Raf-1 failed to interact with RhoA (data not shown). These results indicate that, unlike Raf-1, MAP4K4 interacts with only Rap2. They also indicate that the interaction requires the GTP-bound form of Rap2 and the intact effector region of Rap2.Table ISpecific interaction of MAP4K4 with Rap2 in the yeast two-hybrid assaypACT2-H-1pGAD-Raf-1pACT2HIS3aCo-transformants were examined for HIS3 expression: ++, strong expression; ±, weak expression; -, no expression.β-GalbCo-transformants were also examined for β-galactosidase (β-Gal) expression by the o-nitrophenyl-β-d-galactopyranosidase assay (7).HIS3β-GalHIS3β-GalunitsunitsunitspBTM116-Rap2A++53.7++78.9-0.8pBTM116-Rap2A(G12V)++54.9++83.4-0.9pBTM116-Rap2A(S17N)±18.5-4.3-2.3pBTM116-Rap2A(F39S)±7.1++95.6-1.7pBTM116-Rap1A-1.6++53.3-1.2pBTM116-Rap1A(G12V)-3.6++55.7-2.1pBTM116-Ha-Ras-2.8++144.8-2.7a Co-transformants were examined for HIS3 expression: ++, strong expression; ±, weak expression; -, no expression.b Co-transformants were also examined for β-galactosidase (β-Gal) expression by the o-nitrophenyl-β-d-galactopyranosidase assay (7Kohno H. Tanaka K. Mino A. Umikawa M. Imamura H. Fujiwara T. Fujita Y. Hotta K. Qadota H. Watanabe T. Ohya Y. Takai Y. EMBO J. 1996; 15: 6060-6068Crossref PubMed Scopus (241) Google Scholar). Open table in a new tab We further examined this interaction in vitro (Fig. 1, B and C). For this purpose, HA-tagged full-length MAP4K4 (HA-MAP4K4) or its C-terminal deletion mutant (HA-MAP4K4Δ CNH, amino acids 1–909) expressed in 293T cells was examined for interaction with immobilized GST fusion protein of Rap2 or Ras bound with GTPγS (Fig. 1B). HA-MAP4K4 interacted with GST-Rap2A (lane 5) but not with GST-Ha-Ras (lane 3). HA-MAP4K4ΔCNH, lacking the CNH domain, did not interact with GST-Rap2A (lane 6) or GST-Ha-Ras (lane 4). Next, HA-MAP4K4 was examined for interaction with GTPγS-bound, GDP-bound, or nucleotide free forms of GST-Rap2A (Fig. 1C). HA-MAP4K4 interacted with the GTPγS-bound form of GST-Rap2A (lane 2) preferentially over the GDP-bound form of GST-Rap2A (lane 3). HA-MAP4K4 did not interact with the nucleotide free form of GST-Rap2A (lane 4). These results again indicate that MAP4K4 interacts with Rap2 specifically through the CNH domain and that the interaction is GTP-dependent. To assess whether Rap2 interacts with MAP4K4 in cells, we examined their subcellular localizations by indirect immunofluorescence microscopy (Fig. 2). When expressed alone, Myc-tagged Rap2 (Myc-Rap2A) showed a vesicular staining pattern that was particularly intense in the perinuclear region of the cell (panel a). Unlike Myc-Rap2A, HA-MAP4K4 showed a diffuse staining pattern in the cytoplasm when expressed alone (panel b). Strikingly, when co-expressed with Myc-Rap2A (panels c–e), HA-MAP4K4 co-localized almost completely with Myc-Rap2A. In contrast to full-length HA-MAP4K4, HA-MAP4K4ΔCNH failed to co-localize with Myc-Rap2A (panels f–h); Myc-Rap2A again showed a vesicular perinuclear staining pattern (panel f), while HA-MAP4K4ΔCNH showed a diffuse cytoplasmic staining pattern (panel g). The staining pattern of HA-MAP4K4ΔCNH in cells co-expressing Myc-Rap2A was the same as that in cells expressing HA-MAP4K4ΔCNH alone (data not shown). The absence of co-localization was also observed when HA-MAP4K4 was co-expressed with Myc-Rap2A(S17N) (panels i–k) or Myc-Ha-Ras (panels l–n). HA-MAP4K4 showed a diffuse cytoplasmic staining pattern (panels j and m), while Myc-Rap2A(S17N) showed a fine vesicular cytoplasmic staining pattern (panel i), and Myc-Ha-Ras showed a perinuclear and plasma membrane-associated staining pattern (panel l). The staining pattern of Myc-Rap2A(S17N) or Myc-Ha-Ras in cells co-expressing HA-MAP4K4 was the same as that in cells expressing Myc-Rap2A(S17N) or Myc-Ha-Ras alone (data not shown). MAP4K4 activates JNK (2Yao Z. Zhou G. Wang X.S. Brown A. Diener K. Gan H. Tan T.-H. J. Biol. Chem. 1999; 274: 2118-2125Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 3Su Y.-C. Han J. Xu S. Cobb M. Skolnik E.Y. EMBO J. 1997; 16: 1279-1290Crossref PubMed Scopus (221) Google Scholar). We therefore tested whether Rap2 affects this process. FLAG-tagged JNK (FLAG-JNK2) was expressed alone, with HA-MAP4K4, with Myc-Rap2A, or with both of them in 293T cells (Fig. 3). Activation of JNK was examined by assessing the extent of phosphorylation of JNK within cell homogenates by Western immunoblotting with anti-phospho-JNK antibody that detects activated JNK (Fig. 3A). Expression of FLAG-JNK2 alone resulted in no detectable phosphorylation of JNK (lane 1). Co-expression of HA-MAP4K4 with FLAG-JNK2 resulted in phosphorylation of JNK to some extent (lane 2), while co-expression of Myc-Rap2A with FLAG-JNK2 resulted in less phosphorylation (lane 3). In contrast, co-expression of both HA-MAP4K4 and Myc-Rap2A with FLAG-JNK2 resulted in marked phosphorylation (lane 4). This phosphorylation of JNK most likely reflects phosphorylation of FLAG-JNK2, since co-expression of both HA-MAP4K4 and Myc-Rap2A without FLAG-JNK2 resulted in no detectable phosphorylation (data not shown). Activation of JNK was also examined by an immunocomplex kinase assay. FLAG-JNK2 was precipitated from cell homogenates with anti-FLAG M2 resin and examined for its kinase activity toward a GST-fusion protein of the c-Jun N terminus, GST-c-Jun(1–79) (Fig. 3B). Compared with the basal kinase activity of FLAG-JNK2 (lane 1), the kinase activity of FLAG-JNK2 co-expressed with HA-MAP4K4 was elevated to some extent (lane 2). The kinase activity of FLAG-JNK2 co-expressed with Myc-Rap2A was similar to the basal kinase activity (lane 3). In contrast, the kinase activity of FLAG-JNK2 co-expressed with both HA-MAP4K4 and Myc-Rap2A was markedly elevated (lane 4). Thus, Rap2 enhances MAP4K4-induced activation of JNK. The functional difference between Rap2 and its close relative Rap1, sharing 60% amino acid identity, has been unclear. In other words, it has not been clear why Rap2 in addition to Rap1 exists. One possible answer is that Rap1 and Rap2 interact with different effectors and regulate different signaling pathways. Consistent with this, we have identified in the present study MAP4K4 as a putative effector of Rap2 that does not interact with Rap1. The effector region of Rap2 is different from that of Rap1 by a single amino acid, Phe-39. The importance of this amino acid in specific interaction with MAP4K4 was demonstrated by the substitution of Phe with Ser, present in the corresponding position in Rap1, which resulted in severe attenuation of interaction. The interaction may be conserved among multicellular organisms, as the C. elegans ortholog of MAP4K4 (MIG-15) interacts with the C. elegans ortholog of Rap2 (C25D7.7 protein) but not with orthologs of Rap1 (C27B7.8 protein) or Ras (LET-60) 2M. Oshiro and K. Kariya, unpublished observation. . Rap2 interacts with the CNH domain of MAP4K4. The CNH domain was originally found in citron that interacts with Rho and Rac (11Madaule P. Furuyashiki T. Reid T. Ishizaki T. Watanabe G. Morii N. Narumiya S. FEBS Lett. 1995; 377: 243-248Crossref PubMed Scopus (150) Google Scholar). Subsequently, it was found in Rom2p, a GDP/GTP exchange factor for S. cerevisiae Rho protein (Rho1p), and the myotonic dystrophy kinase-related Cdc42-binding kinase (MRCKα) (12Ozaki K. Tanaka K. Imamura H. Hihata T. Kameyama T. Nonaka H. Hirano H. Matsuura Y. Takai Y. EMBO J. 1996; 15: 2196-2207Crossref PubMed Scopus (185) Google Scholar, 13Chen X.-Q. Tan I. Leung T. Lim L. J. Biol. Chem. 1999; 274: 19901-19905Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Although all three proteins interact with Rho family small GTP-binding proteins, their CNH domains do not mediate the interactions directly. The CNH domain is also present in the C-terminal portions of GCK-I and -IV subfamilies of the STE20 group kinases (5Kyriakis J.M. J. Biol. Chem. 1999; 274: 5259-5262Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 6Dan I. Watanabe N.M. Kusumi A. Trends Cell Biol. 2001; 11: 220-230Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar). GCK/MAP4K2, which belongs to the GCK-I subfamily, interacts with Rab8 small GTP-binding protein through its C-terminal portion containing the CNH domain (14Ren M. Zeng J. De Lemos-Chiarandini C. Rosenfeld M. Adesnik M. Sabatini D.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5151-5155Crossref PubMed Scopus (89) Google Scholar). GCK/MAP4K2 interacts with the GTP-bound form of Rab8 preferentially over the GDP-bound form, and the interaction requires the intact effector region of Rab8. This suggests that GCK/MAP4K2 might act as an effector of Rab8 (14Ren M. Zeng J. De Lemos-Chiarandini C. Rosenfeld M. Adesnik M. Sabatini D.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5151-5155Crossref PubMed Scopus (89) Google Scholar). Since our present study indicated that the CNH domain in MAP4K4 interacts with Rap2, it is interesting to hypothesize that some of the CNH domains in the STE20 group kinases may share a region that mediates interactions with small GTP-binding proteins. We thank Dr. Katsuji Yoshioka and Dr. Takahiro Nagase for plasmids. We also thank Kyoko Kise for help in preparation of the manuscript, Asako Umikawa for technical assistance, and Makiko Gushi for discussion.