©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of AF-6 and Canoe as Putative Targets for Ras (*)

(Received for publication, October 10, 1995; and in revised form, November 7, 1995)

Masamitsu Kuriyama (1) Naozumi Harada (1) Shinya Kuroda (1) Takaharu Yamamoto (1) Masato Nakafuku (1) Akihiro Iwamatsu (2) Daisuke Yamamoto (3) Raj Prasad (4) Carlo Croce (4) Eli Canaani (5) Kozo Kaibuchi (1)(§)

From the  (1)Division of Signal Transduction, Nara Institute of Science and Technology, Ikoma 630-01, Japan, (2)Central Laboratories for Key Technology, Kirin Brewery Co. Ltd., Yokohama 236, Japan, (3)Mitsubishi Kasei Institute of Life Sciences and ERATO Yamamoto Behavior Genes Project, Machida 194, Japan, (4)Thomas Jefferson University, Philadelphia, Pennsylvania 19107, and (5)Weizmann Institute of Science, Rehovot 76100, Israel

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Ras (Ha-Ras, Ki-Ras, N-Ras) is implicated in the regulation of various cell functions such as gene expression and cell proliferation downstream from specific extracellular signals. Here, we partially purified a Ras-interacting protein with molecular mass of about 180 kDa (p180) from bovine brain membrane extract by glutathione S-transferase (GST)-Ha-Ras affinity column chromatography. This protein bound to the GTPS (guanosine 5`-(3-O-thio)triphosphate, a nonhydrolyzable GTP analog)bulletGST-Ha-Ras affinity column but not to those containing GDPbulletGST-Ha-Ras or GTPSbulletGST-Ha-Ras with a mutation in the effector domain (Ha-Ras). The amino acid sequences of the peptides derived from p180 were almost identical to those of human AF-6 that is identified as the fusion partner of the ALL-1 protein. The ALL-1/AF-6 chimeric protein is the critical product of the t (6:11) abnormality associated with some human leukemia. AF-6 has a GLGF/Dlg homology repeat (DHR) motif and shows a high degree of sequence similarity with Drosophila Canoe, which is assumed to function downstream from Notch in a common developmental pathway. The recombinant N-terminal domain of AF-6 and Canoe specifically interacted with GTPSbulletGST-Ha-Ras. The known Ras target c-Raf-1 inhibited the interaction of AF-6 with GTPSbulletGST-Ha-Ras. These results indicate that AF-6 and Canoe are putative targets for Ras.


INTRODUCTION

Ras (Ha-Ras, Ki-Ras, N-Ras) is a signal-transducing guanine nucleotide-binding protein for tyrosine kinase-type receptors such as epidermal growth factor receptors and the Src family, leading to a mitogenic response and differentiation (for reviews, see Refs. 1 and 2). Ras has GDP-bound inactive and GTP-bound active forms, the latter of which makes physical contact with targets. Intensive investigations revealed that the Raf kinase family, consisting of c-Raf-1 (for reviews, see (3) and (4) ), A-Raf(5) , and B-Raf(6, 7, 8, 9) , is one of the direct targets for Ras. The activated Raf phosphorylates MAP (^1)kinase kinase and activates it. Consequently the activated MAP kinase kinase activates MAP kinase, leading to the expression of certain genes such as c-fos (for reviews, see (10) and (11) ). Several molecules interacting with activated Ras in addition to Raf have been identified in mammals. These include phosphatidylinositol-3-OH kinase(12) , Ral GDS(13, 14) , and Rin1(15) . On the basis of these observations, a variety of Ras targets may account for the pleiotropic functions of Ras. To understand the molecular mechanism of pleiotropic functions of Ras, it is essential to identify novel targets for Ras.

In the present study, we discovered and partially purified another putative target for Ras with a molecular mass of about 180 kDa (p180) by use of GST-Ha-Ras affinity column chromatography and identified it as AF-6(16) , whose structure resembles that of Drosophila Canoe, which is involved in the Notch signaling pathway(17) .


EXPERIMENTAL PROCEDURES

Materials and Chemicals

All materials used in the nucleic acid study were purchased from Takara Shuzo Co. Ltd. (Kyoto, Japan). Expression plasmids, pGEX, pMal-c2, and pRSET were obtained from Pharmacia Biotech (Tokyo, Japan), New England Biolabs Inc. (Beverly, MA), and Invitrogen Corp. (San Diego, CA), respectively. Other materials and chemicals were obtained from commercial sources. [S]GTPS and [S]methionine were purchased from DuPont NEN. A rabbit polyclonal antibody against a 16-mer peptide corresponding to 561-576 aa of human AF-6 (RVEQQPDYRRQESRTQ) was generated and purified.

Plasmid Construction

Plasmids, pGEX-Ha-Ras, pGEX-R-Ras, pGEX-RalA, and pGEX-RhoA were constructed as described previously(18) . To obtain the in vitro translated N-terminal domain of AF-6 and Canoe, pRSET-AF-6 (36-848 aa), pRSET-AF-6 (36-206 aa), and pRSET-Canoe (1-217 aa) were constructed as follows. The 2.4-kilobase cDNA fragment encoding AF-6 (36-848 aa) was amplified by polymerase chain reaction from human brain Quick clone cDNA (Clontech Laboratories Inc., Palo Alto, CA). For the shorter N-terminal domain of AF-6, the 0.51-kilobase cDNA fragment encoding AF-6 (36-206 aa) and the 0.65-kilobase cDNA fragment encoding Canoe (1-217 aa) were amplified by polymerase chain reaction from the AF-6 cDNA clone K12 (16) and Canoe cDNA clone in pBluescript-SK (17) , respectively. These cDNA fragments, having an artificial termination codon and also artificial KpnI sites at both terminals, were cloned into the KpnI site of pRSET. To produce the shorter N-terminal domain of AF-6 and N-terminal domain of c-Raf-1 as MBP fusion proteins, the cDNA fragments encoding AF-6 (36-206 aa) and c-Raf-1 (1-149 aa) were subcloned into the KpnI site of pMal-c2-KpnI in which an additional KpnI site was introduced adjacent to the BamHI site.

Preparation of Bovine Brain Membrane Extract

The homogenate of bovine brain gray matter, 190 g, was prepared and centrifuged at 20,000 times g for 30 min at 4 °C as described(19) . The precipitate was suspended into 360 ml of homogenizing buffer (25 mM Tris/HCl at pH 7.5, 5 mM EGTA, 1 mM dithiothreitol, 10 mM MgCl(2), 10 µM (p-amidinophenyl)-methanesulfonyl fluoride, 1 mg/liter leupeptin, 10% sucrose) to prepare the crude membrane fraction(19) . The proteins in this fraction were extracted by addition of an equal volume of homogenizing buffer containing 4 M NaCl. After shaking for 1 h at 4 °C, the membrane fraction was centrifuged at 20,000 times g for 1 h at 4 °C. The supernatant was dialyzed against buffer A (20 mM Tris/HCl at pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, 5 mM MgCl(2)) three times. Solid ammonium sulfate was then added to a final concentration of 40% saturation. The 0-40% precipitate was dissolved into 16 ml of buffer A, dialyzed against buffer A three times, and used as the membrane extract.

GST-Ha-Ras Affinity Column Chromatography

The membrane extract (16 ml) was eluted through 2.5 ml of a glutathione-Sepharose 4B column (Pharmacia Biotech Inc.) to remove endogenous GST. One-tenth of the pass-through fraction was loaded on 0.25 ml of glutathione-Sepharose 4B columns containing respective GST-small G proteins loaded with guanine nucleotides as described(19) . After washing the columns with 0.825 ml of buffer A three times, the bound proteins were coeluted with respective GST-small G proteins by addition of 0.825 ml of buffer A containing 10 mM glutathione and 0.2 M NaCl three times. To prepare affinity-purified p180 for peptide sequencing, the pass-through fraction (16 ml) was loaded onto a 1-ml glutathione-Sepharose column containing 24 nmol of GTPSbulletGST-Ha-Ras. The proteins were eluted by addition of 10 ml of buffer A containing 10 mM glutathione and 0.2 M NaCl, and fractions of 1 ml each were collected. The p180 protein appeared in fractions 2-10. The same procedures were repeated 12 times.

Peptide Sequence Analysis of p180

The affinity-purified p180 was dialyzed three times against distilled water and concentrated by freeze-drying. The concentrated samples were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes(20) . The immobilized p180 was digested, fractionated, and subjected to amino acid sequencing as described(20) .

Interaction of in Vitro Translated AF-6, Canoe, and Bacterial AF-6 with GST-Small G Proteins

In vitro translation of pRSET-AF-6 (36-848 aa), pRSET-AF-6 (36-206 aa), and pRSET-Canoe (1-217 aa) were performed as described(19) . In vitro translated products labeled with [S]methionine were mixed with glutathione-Sepharose 4B beads containing the respective GST-small G proteins loaded with guanine nucleotides(19) . The bound proteins were then coeluted with GST-small G proteins by addition of glutathione. The eluates were resolved by SDS-PAGE and vacuum-dried followed by autoradiography. The shorter N-terminal domain of AF-6 (36-206 aa) was expressed as an MBP fusion protein (MBP-AF-6) and purified using amylose resin (New England Biolabs). MBP-AF-6 (0.15 nmol) was subjected to the GST-small G protein affinity column chromatography as described above. For competition assay with c-Raf-1, 1.5 nmol of MBP-c-Raf-1 was simultaneously added to the incubation mixture.

Other Procedures

SDS-PAGE proceeded as described previously (21) . The BLAST program was used for protein homology search(22) . Immunoblot analysis of p180 was carried out as described(23) .


RESULTS AND DISCUSSION

To detect molecules interacting with Ha-Ras, the bovine brain membrane extract was loaded onto GST-Ha-Ras affinity columns. The proteins bound to the affinity columns were coeluted with GST-Ha-Ras by addition of glutathione. Proteins with a molecular mass of about 180 kDa (p180) and 195 kDa (p195) were detected in the glutathione eluate from the GTPSbulletGST-Ha-Ras affinity column but not from those containing GST or GDPbulletGST-Ha-Ras (Fig. 1). Neither p180 nor p195 was detected in the eluate of the affinity column for GTPSbulletGST-Ha-Ras, which has a mutation in the effector-interacting domain(1, 2) . We further confirmed the specificity of the interaction by affinity column chromatography using GST-R-Ras, GST-RalA, and GST-RhoA. Less p180 and p195 were eluted from the GTPSbulletGST-R-Ras affinity column but not from the GDPbulletGST-R-Ras affinity column (data not shown). Neither p180 nor p195 was eluted from the GST-RalA or GST-RhoA affinity column (data not shown).


Figure 1: Purification of Ha-Ras-interacting proteins. The membrane extract was loaded onto a glutathione-Sepharose 4B column containing either GST, GDPbulletGST-Ha-Ras, GTPSbulletGST-Ha-Ras, or GTPSbulletGST-Ha-Ras. Bound proteins were coeluted with the respective GST-fusion proteins by addition of glutathione. Aliquots (40 µl each) from the glutathione eluates were resolved by SDS-PAGE followed by silver staining. Lane 1, GST; lane 2, GDPbulletGST-Ha-Ras; lane 3, GTPSbulletGST-Ha-Ras; lane 4, GTPSbulletGST-Ha-Ras. The arrow and arrowhead denote the positions of p180 and p195, respectively. The results are representative of three independent experiments.



To identify the GTPSbulletHa-Ras-interacting molecule, p180 was subjected to amino acid sequencing as described under ``Experimental Procedures.'' Six peptide sequences derived from p180 were determined. These were: 1) STATTQDVLE; 2) DMPETSFTR; 3) LPYLVELSPDG; 4) PGIVQETTFDLG; 5) YAPDDIPNINS; and 6) LLLEWQFQK. All six peptide sequences were almost identical to the deduced amino acid sequence of human AF-6, which is the fusion partner of the ALL-1 protein(16) . The ALL-1/AF-6 chimeric protein is the critical product of the t (6:11) abnormality associated with some human leukemia. Furthermore, p180 was recognized by the antibody raised against human AF-6 (Fig. 2). The calculated molecular mass of human AF-6 is 181,777 Da, which is close to the apparent molecular mass of p180 estimated by SDS-PAGE. We therefore concluded that p180 is the bovine counterpart of human AF-6 and hereafter referred to it as AF-6. Since this antibody cross-reacted with p195 weakly (data not shown), p195 may be an isoform or an alternatively spliced form of AF-6.


Figure 2: Immunoblot analysis of p180. Protein p180 was immunoblotted against the anti-AF-6 antibody. Lane 1, with preimmune serum; lane 2, with the anti-AF-6 antibody. The arrow denotes the position of p180. The results are representative of three independent experiments.



To address whether or not recombinant AF-6 interacts with GTPSbulletHa-Ras, GST-small G proteins immobilized on beads were mixed with the in vitro translated N-terminal domain of AF-6 (36-848 aa), and interacting proteins were coeluted with GST-small G proteins by the addition of glutathione. The in vitro translated AF-6 (36-848 aa) was coeluted with GTPSbulletGST-Ha-Ras but weakly with GDPbulletGST-Ha-Ras, GTPSbulletGST-Ha-Ras, GST-R-Ras, GST-RalA, and GST-RhoA (Fig. 3a). The band with GTPSbulletGST-Ha-Ras was very faint (lane 5), and the bands with GDPbulletGST-RalA and GTPSbulletGST-RalA (lanes 8 and 9) were a little bit stronger than those with GDPbulletGST-R-Ras and GTPSbulletGST-R-Ras (lanes 6 and 7). The weak bands detected in the eluates other than that from GTPSbulletGST-Ha-Ras may result from the weak interaction of AF-6 with the respective small G proteins. Although some AF-6 in the membrane extract was slightly retained on the GTPSbulletGST-R-Ras affinity column (data not shown), the in vitro translated AF-6 was not. This may be due to the lower affinity of AF-6 for GTPSbulletGST-R-Ras than that for GTPSbulletGST-Ha-Ras. To determine the Ras-interacting domain of AF-6 more accurately, a similar experiment was performed using the shorter N-terminal domain of AF-6 (36-206 aa). A similar retention was observed when the shorter N-terminal domain of AF-6 (36-206 aa) was employed (Fig. 3b).


Figure 3: Interaction of AF-6 with activated Ha-Ras. a, the in vitro translated AF-6 (36-848 aa) was mixed with GST-small G proteins immobilized to glutathione-Sepharose 4B beads. The interacting proteins were coeluted with GST-small G proteins by addition of glutathione. Aliquots (40 µl) of the eluates were subjected to SDS-PAGE and vacuum-dried followed by autoradiography. Lane 1, in vitro translated AF-6; lane 2, GST; lane 3, GDPbulletGST-Ha-Ras; lane 4, GTPSbulletGST-Ha-Ras; lane 5, GTPSbulletGST-Ha-Ras; lane 6, GDPbulletGST-R-Ras; lane 7, GTPSbulletGST-R-Ras; lane 8, GDPbulletGST-RalA; lane 9, GTPSbulletGST-RalA; lane 10, GDPbulletGST-RhoA; lane 11, GTPSbulletGST-RhoA. The arrow denotes the position of AF-6. b, the in vitro translated AF-6 (36-206 aa) or Canoe (1-217 aa) was mixed with glutathione-Sepharose 4B beads containing GST-Ha-Ras. Lane 1, in vitro translated AF-6; lane 2, GST; lane 3, GDPbulletGST-Ha-Ras; lane 4, GTPSbulletGST-Ha-Ras; lane 5, GTPSbulletGST-Ha-Ras; lane 6, in vitro translated Canoe; lane 7, GST; lane 8, GDPbulletGST-Ha-Ras; lane 9, GTPSbulletGST-Ha-Ras; lane 10, GTPSbulletGST-Ha-Ras. The arrow and arrowhead denote the positions of AF-6 and Canoe, respectively. c, MBP-AF-6 was mixed with GST-Ha-Ras affinity beads. Lane 1, GST; lane 2, GDPbulletGST-Ha-Ras; lane 3, GTPSbulletGST-Ha-Ras; lane 4, GTPSbulletGST-Ha-Ras; lane 5, GTPSbulletGST-Ha-Ras with MBP-c-Raf-1. The results are representative of three independent experiments.



A homology search of the GenBank protein data base revealed a high degree of sequence similarity of AF-6 with Drosophila Canoe (Fig. 4), which is assumed to function downstream from Notch in a common developmental pathway(17) . Since Canoe was presumed to interact with Ras in the same manner as AF-6, the interaction of Canoe was investigated. The in vitro translated N-terminal domain of Canoe (1-217 aa) was also coeluted with GTPSbulletGST-Ha-Ras and scarcely with GST, GDPbulletGST-Ha-Ras, and GTPSbulletGST-Ha-Ras (Fig. 3b).


Figure 4: Schematic representation of AF-6 and Canoe structures. The numbers indicate the amino acid sequence identities in each domain. DHR, Dlg homology repeat.



We examined whether or not AF-6 directly interacts with GTPSbulletHa-Ras. The shorter N-terminal domain of AF-6 (36-206 aa) was expressed as an MBP fusion protein (MBP-AF-6) and mixed with immobilized GST-Ha-Ras. Interacting proteins were coeluted with GST-Ha-Ras by the addition of glutathione. MBP-AF-6 was coeluted with GTPSbulletGST-Ha-Ras but not with GST, GDPbulletGST-Ha-Ras, or GTPSbulletGST-Ha-Ras (Fig. 3c). The band corresponding to molecular mass of about 55 kDa may be a degraded product of MBP-AF-6.

The apparent K(d) values for MBP-AF-6 and MBP-c-Raf-1 were estimated to be about 250 and 200 nM, respectively, under the conditions (data not shown). Since c-Raf-1 interacts with activated Ras via the effector domain(1, 2) , we examined whether or not c-Raf-1 competes with AF-6 for interaction with activated Ha-Ras. An excess amount of MBP-c-Raf-1 inhibited the interaction of the MBP-AF-6 with GTPSbulletGST-Ha-Ras (Fig. 3c).

In this study, we purified a Ras-interacting protein (p180) from a bovine brain membrane extract. We identified it as AF-6, which has a GLGF/DHR motif and shows a high degree of sequence similarity with Drosophila Canoe(16, 17) . The recombinant AF-6 and Canoe specifically interacted with activated Ha-Ras. Furthermore, c-Raf-1 inhibited the interaction of AF-6 with activated Ha-Ras. These results indicate that AF-6 and Canoe serve as putative targets for Ras.

We showed that activated Ras interacted with the N-terminal domains of AF-6 and Canoe. These domains show a high degree of sequence similarity to each other, indicating that this unique domain confers specificity for the GTPbulletRas complex. The direct interaction of c-Raf-1, A-Raf, B-Raf, phosphatidylinositol-3-OH kinase, Ral GDS, and Rin1 with activated Ras has been demonstrated(3, 4, 5, 6, 7, 8, 9, 12, 13, 14, 15) . The Ras-interacting interfaces of these proteins have been determined. There is no obvious homology among Ras-interacting interfaces of c-Raf-1, phosphatidylinositol-3-OH kinase, Ral GDS, Rin1, and AF-6/Canoe, indicating that activated Ras can recognize a variety of target interfaces. This diversity of Ras-interacting interfaces may allow a range of downstream pathways from Ras to induce appropriate cellular responses to extracellular signals.

AF-6 and Canoe are homologous to each other and share a common domain organization (Fig. 4)(16, 17) . The most highly conserved region among them is a GLGF/DHR motif, which is found in a number of other proteins including Drosophila discs-large tumor suppressor gene product (Dlg)(24) , dishevelled gene product (25, 26) , an intracellular protein-tyrosine phosphatase (PTP-meg)(27) , postsynaptic density protein 95 (PSD-95)(28) , and a tight junction-associated protein ZO-1(29, 30) . The GLGF/DHR motif is thought to function to localize them at the specialized sites of cell-cell contact by forming a complex with specific proteins such as protein 4.1 homologues(31) . The structural feature of AF-6 and Canoe suggests that they locate at the junction of plasma membrane and cytoskeleton, where they may regulate signal transduction and cytoskeleton.

The N terminus of AF-6 flanked by the GLGF/DHR motif also shares a high homology with that of Canoe, to both of which activated Ras binds specifically. Canoe has been postulated to function downstream from Notch and to mediate interactions between the Notch cascade and other signaling pathways(17) . Although AF-6 function remains obscure, the similar structural feature and property of AF-6 and Canoe suggest that the AF-6/Canoe family may serve as an intracellular signaling component controlled by two distinct signaling pathways such as Ras and Notch. Our preliminary experiments suggest that Canoe is genetically linked to Ras1 in Drosophila eye development. Further studies are required to understand the roles of AF-6/Canoe family in signal transduction.


FOOTNOTES

*
This study was supported by grants-in-aid for Scientific Research and for Cancer Research from the Ministry of Education, Science, and Culture, Japan(1995) and by a grant from the Yamanouchi Foundation for Research on Metabolic Disease(1995). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-7437-2-5440; Fax: 81-7437-2-5449; kaibuchi@bs.aist-nara.ac.jp.

(^1)
The abbreviations used are: MAP, mitogen-activated protein; Ral GDS, Ral guanine nucleotide dissociation stimulator; GST, glutathione S-transferase; AF-6, ALL-1 fusion partner from chromosome 6; ALL, acute lymphoblastic leukemia; GTPS, guanosine 5`-(3-O-thio)triphosphate; aa, amino acids; MBP, maltose-binding protein; G proteins, GTP-binding proteins; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Dr. Takahisa Hachiya and Tosiaki Miyazaki (MBL, Japan) for preparing the polyclonal antibody against human AF-6. We are also grateful to Masako Nishimura for secretarial assistance.


REFERENCES

  1. Satoh, T., Nakafuku, M., and Kaziro, Y. (1992) J. Biol. Chem. 267, 24149-24152 [Free Full Text]
  2. McCormick, F. (1994) Curr. Opin. Genet. Dev. 4, 71-76 [Medline] [Order article via Infotrieve]
  3. Blenis, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5889-5892 [Abstract]
  4. Daum, G., Eisenmann-Tappe, I., Fries, H.-W., Troppmair, J., and Rapp, U. R. (1994) Trends Biochem. Sci. 19, 474-480 [CrossRef][Medline] [Order article via Infotrieve]
  5. Vojtek, A. B., Hollenberg, S. M., and Cooper, J. A. (1993) Cell 74, 205-214 [Medline] [Order article via Infotrieve]
  6. Moodie, S. A., Paris, M. J., Kolch, W., and Wolfman, A. (1994) Mol. Cell. Biol. 14, 7153-7162 [Abstract]
  7. Jaiswal, R. K., Moodie, S. A., Wolfman, A., and Landreth, G. E. (1994) Mol. Cell. Biol. 14, 6944-6953 [Abstract]
  8. Catling, A. D., Reuter, C. W. M., Cox, M. E., Parsons, S. J., and Weber, M. J. (1994) J. Biol. Chem. 269, 30014-30021 [Abstract/Free Full Text]
  9. Yamamori, B., Kuroda, S., Shimizu, K., Fukui, K., Ohtsuka, T., and Takai, Y. (1995) J. Biol. Chem. 270, 11723-11726 [Abstract/Free Full Text]
  10. Cano, E., and Mahadevan, L. C. (1995) Trends Biochem. Sci. 20, 117-122 [CrossRef][Medline] [Order article via Infotrieve]
  11. Marshall, C. J. (1995) Cell 80, 179-185 [Medline] [Order article via Infotrieve]
  12. Rodriguez-Viciana, P., Warne, P. H., Dhand, R., Vanhaesebroeck, B., Gout, I., Fry, M. J., Waterfield, M. D., and Downward, J. (1994) Nature 370, 527-532 [CrossRef][Medline] [Order article via Infotrieve]
  13. Kikuchi, A., Demo, S. D., Ye, Z. H., Chen, Y. W., and Williams, L. T. (1994) Mol. Cell. Biol. 14, 7483-7491 [Abstract]
  14. Spaargaren, M., and Bischoff, J. R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 12609-12613 [Abstract/Free Full Text]
  15. Han, L., and Colicelli, J. (1995) Mol. Cell. Biol. 15, 1318-1323 [Abstract]
  16. Prasad, R., Gu, Y., Alder, H., Nakamura, T., Canaani, O., Saito, H., Huebner, K., Gale, R. P., Nowell, P. C., Kuriyama, K., Miyazaki, Y., Croce, C. M., and Canaani, E. (1993) Cancer Res. 53, 5624-5628 [Abstract]
  17. Miyamoto, H., Nihonmatsu, I., Kondo, S., Ueda, R., Togashi, S., Hirata, K., Ikegami, Y., and Yamamoto, D. (1995) Genes & Dev. 9, 612-625
  18. Shimizu, K., Kuroda, S., Yamamori, B., Matsuda, S., Kaibuchi, K., Yamauchi, T., Isobe, T., Irie, K., Matsumoto, K., and Takai, Y. (1994) J. Biol. Chem. 269, 22917-22920 [Abstract/Free Full Text]
  19. Yamamoto, T., Matsui, T., Nakafuku, M., Iwamatsu, A., and Kaibuchi, K. (1995) J. Biol. Chem. 270, 30557-30561 [Abstract/Free Full Text]
  20. Iwamatsu, A. (1992) Electrophoresis 13, 142-147 [Medline] [Order article via Infotrieve]
  21. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  22. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403-410 [CrossRef][Medline] [Order article via Infotrieve]
  23. Harlow, E., and Lame, D. (1988) Antibodies: A Laboratory Manual , Cold Spring Harbor laboratory, Cold Spring Harbor, NY
  24. Woods, D. F., and Bryant, P. J. (1991) Cell 66, 451-464 [Medline] [Order article via Infotrieve]
  25. Theisen, H., Purcell, J., Bennett, M., Kansagara, D., Syed, A., and Marsh, J. L. (1994) Development 120, 347-360 [Abstract/Free Full Text]
  26. Klingensmith, J., Nusse, R., and Perrimon, N. (1994) Genes & Dev. 8, 118-130
  27. Gu, M. X., York, J. D., Warshawsky, I., and Majerus, P. W. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5867-5871 [Abstract]
  28. Cho, K. O., Hunt, C. A., and Kennedy, M. B. (1992) Neuron 9, 929-942 [Medline] [Order article via Infotrieve]
  29. Itoh, M., Nagafuchi, A., Yonemura, S., Kitani-Yasuda, T., Tsukita, S., and Tsukita, S. (1993) J. Cell Biol. 121, 491-502 [Abstract]
  30. Willott, E., Balda, M. S., Fanning, A. S., Jameson, B., Van Itallie, C., and Anderson, J. M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7834-7838 [Abstract/Free Full Text]
  31. Lue, R. A., Marfatia, S. M., Branton, D., and Chishti, A. H. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9818-9822 [Abstract/Free Full Text]

©1996 by The American Society for Biochemistry and Molecular Biology, Inc.