Association of Krev-1/Rap1a with Krit1, a Novel Ankyrin Repeat-Containing Protein Encoded by a Gene Mapping to 7Q21-22

Association of Krev-1/rap1a with Krit1, a novel ankyrin repeat-containing protein encoded by a gene mapping to 7q21-22

Ilya Serebriiskii1, Joanne Estojak1, Gonosuke Sonoda2, Joseph R Testa2 and Erica A Golemis1

1Division of Basic Sciences and 2Medical Sciences, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, Pennsylvania 19111, USA

Krev-1/rap1A is an evolutionarily conserved Ras-family Wojtaszek et al., 1993) have failed to con®rm more GTPase whose cellular function remains unclear, but than a very weak anti-oncogenic activity, initially which has been proposed to function as a tumor leaving its role as a regulator of Ras in question. suppressor gene, and may act as a Ras antagonist. To Adding an additional dimension to the understand- elucidate Krev-1 activity, we have used LexA-Krev-1 in a ing of Krev-1 function, it has also been shown to be a two-hybrid screen of a HeLa cell cDNA library. Of the component of a large complex with cytochrome b of two cDNA classes isolated, one contained a single isolate human neutrophils (Quinn et al., 1989, 1992). encoding the known Krev-1 interactor Raf, while the Cytochrome b558 is a component of the NADPH second contained multiple isolates coding for a previously oxidase which is responsible for producing a undescribed protein which we have designated Krit1 (for ``respiratory burst'' of free radicals that degrade Krev Interaction Trapped 1). The full length Krit1 foreign objects: other components of the oxidase cDNA encodes a protein of 529 amino acids, with an include p47phox and p67phox, and the small GTP- amino-terminal ankyrin repeat domain and a novel binding protein Rac (reviewed in Bokoch, 1995; carboxy-terminal domain required for association with Bokoch and Knaus, 1994). Studies indicating Krev-1 Krit1. Krit1 interacted strongly with Krev-1 but only helps activate NADPH oxidase (Eklund et al., 1991; weakly with Ras, suggesting it might speci®cally regulate Maly et al., 1994) are particularly provocative given Krev-1 activities. Krit1 mRNA and protein are expressed recent demonstration that Ras signaling proceeds in endogenously at low levels, with tissue speci®c variation. part through inducing superoxide production by Intriguingly, the Krit cDNA has been mapped by FISH activation of the NADPH oxidase (Irani et al., 1997), to chromosome 7q21-22, a region known to be frequently as control of cellular superoxide levels may provide a deleted or ampli®ed in multiple forms of cancer. novel mechanism by which Krev-1 regulates cell transformation by Ras. Keywords: Krit1; Krev-1/Rap1a; 7q21-22; ankyrin Further insights into potential Krev-1 activities repeat; interaction derive from studies of homologs of the protein in lower eukaryotes. Dominant gain-of-function mutants of the Drosophila Ras3 gene (Pizon et al., 1988), a gene 88% identical to human Krev-1, result in a Introduction development defect of altered cell fate in differentiation of the ommatidia composing the eye (Hariharan et al., The human gene encoding the small GTP-binding 1991). The yeast gene RSR1/BUD1 (57% identity to protein Krev-1/rap1a was isolated by Pizon et al.in KRev-1) (Ruggieri et al., 1992) was ®rst identi®ed in 1988 based on extensive sequence conservation with the S. cerevisiae as a multicopy suppressor of a cdc24 Drosophila Ras gene (Pizon et al., 1988) and by mutation which causes lethality due to inability to bud Kitayama et al. in 1989 based on its ability to revert (Bender and Pringle, 1989). A series of genetic studies cells transformed by the oncoprotein Ras to a normal initally implicated both RSR1/BUD1 and Krev-1 as cell morphology (Kitayama et al., 1989). Because of the controlling agents for cell polarity (Chant et al., 1991; sequence conservation and the reversion phenotype, Chant and Herskowitz, 1991; Ruggieri et al., 1992). In Krev-1 was initially hypothesized to function anti- subsequent work, a number of studies have addressed oncogenically as a negative eector competing with the interaction of RSR1 with signal transduction Ras for downstream targets important for control of pathways relevant to cytoskeletal controls. In yeast, cell signaling and cell morphology. This hypothesis was these studies have begun to establish connections supported by the discovery that Krev-1 could bind between RSR1 with speci®c GEF and GAP proteins tightly to a number of Ras eectors, including Ras (Bender, 1993; Park et al., 1993; Powers et al., 1991); GTPase-activating protein (GAP) (Frech et al., 1990), and with CDC24 and Rho-class GTPases including the oncogene Ras (Nassar et al., 1995; Zhang et al., Cdc42 (Zheng et al., 1995), implying cross-signaling 1993) and Ral-GEF (Herrmann et al., 1996), as it to MAP kinase cascades involved in dierentiation seemed likely that Krev-1 might act to sequester these control (Simon et al., 1995; Zhao et al., 1995) and proteins from Ras. However, a number of subsequent regulation of actin-based processes in mammals (Coso studies performed to characterize the antioncogenic et al., 1995; Nobes and Hall, 1995). Finally, the function of Krev-1 (Buss et al., 1991; Jelinek and tuberous sclerosis-2 (TSC2) product, tuberin, has been Hassell, 1992; Sakoda et al., 1992; Sato et al., 1994; shown to possess GAP activity for Krev-1, linking Krev-1 to the growth derangement accompanying Correspondence: EA Golemis development of tuberous sclerosis (Wienecke et al., Received 25 February 1997; revised 13 May 1997; accepted 1995). Together, this wide array of studies suggest a 13 May 1997 very complex Krev-1 function related to cell morphol- Ankyrin-repeat protein Krit1 binds Krev-1/rap1A I Serebriiskii et al 1044 ogy and cell polarity, while emphasizing that much and demonstrated the presence of an in-frame stop remains to be clari®ed. codon 251 bp upstream of the assigned ATG for We have been utilizing the S. cerevisiae budding initiation of protein translation, with no intervening pathway in a variety of screening strategies to elucidate splice sites. Use of the program TSSW to scan the cell growth controls in mammals (Khazak et al., 1995; 1.2 kb of sequence ¯anking the cDNA start site Law et al., 1996). As part of this approach, and indicated a number of potential promoter elements, because of the intrinsic interest of Krev-1 biology, we including a preferred TATAAAA site for initiation of used this gene as the bait in a two-hybrid screen to transcription 731 bp upstream of the endpoint of the identify partner proteins which might yield clues as to cDNA (757 bp upstream of the ATG) and additional its function. As a result of this screen, we here report clustered binding sites for transcription factors. An the isolation and preliminary characterization of additional potential TATAAAA is identi®ed at 228 bp KRIT1, a novel gene whose protein product interacts upstream of the cDNA endpoint (254 upstream of the strongly and speci®cally with Krev-1, and which maps ATG). Sequence for this genomic clone has similarly to human chromosome 7q21-22, a region proposed to been deposited to Genbank (accession number encompass an antioncogene. U90269).

Krit1 protein structural features Results and discussion Inspection of the Krit1 predicted protein sequence revealed several features of note. The 529 amino acid Cloning of Krit1 sequence has a predicted molecular weight of 60.8 kD We used LexA-fused Krev-1 in an interaction trap/two (re¯ecting the presence of a large percentage of hybrid approach (Golemis et al., 1996; Gyuris et al., aromatic residues, particularly tyrosines), and a 1993) to screen a HeLa cDNA library to identify novel calculated pl of 7.9. The Krit1 protein sequence partner proteins. From a screen of >500 000 possesses a number of regions predicted to be transformants, we isolated 8 clones which were positive hydrophobic, particularly at the carboxy-terminal end after puri®cation of plasmid and retransformation into of the protein; a single transmembrane domain is naive yeast containing LexA-Krev-1. Of the eight, one predicted at aa 339 ± 359 (Hofmann and Stoel, 1993). interacted with intermediate anity as assayed by two- Krit1 is not strongly homologous to any previously hybrid, and proved to be a partial cDNA for the described protein. However, comparison of the Krit1 oncoprotein Raf, initiating at amino acid 56, at the protein sequence of Genbank by BLAST (Altschul et approximate beginning of the Ras binding domain or al., 1990) indicated a potential relation to two groups of RBD (Nassar et al., 1995). Since Raf has been described proteins. The carboxy-terminal region of Krit 1 (aa as interacting with Krev-1, isolation of this cDNA *280 ± 433), originally isolated as interacting with indicated that the LexA-Krev1 `bait' was folded Krev-1, may be weakly related to a family of appropriately to allow physiological interactions. cytoskeletal and membrane-associated proteins de®ned The other seven clones emerging from the screen by erythrocyte protein 4.1 (Conboy et al., 1986), appeared to interact very strongly with Krev-1, and including among other moesin, azrin, and merlin when sequenced proved to be multiple independent (Trofatter et al., 1993). This assignment is not certain, isolates of the cDNA encoding a novel protein which since the use of additional homology prediction we have designated Krit1 (for Krev-1 Interaction algorithms that incorporate structural information Trapped 1). This cDNA lacked a suitable candidate (including Prosite, Blocksearch, and 3-D threading methionine for initiation of translation, and was (Moult, 1996)) indicates that particular residues therefore presumed to be a partial cDNA. 5'-RACE thought to be characterisitic of the Band 4.1 protein was used with human kidney cDNA to extend the family are not well conserved in Krit1, despite the Krit1 clone, resulting in the addition of 398 base pairs general homology of the region (Figure 2a). In contrast, of novel sequence. The assembled cDNA comprised the amino-terminal end of Krit 1 (aa *83 ± 215) contain 1986 base pairs (bp) with an additional 18 bp poly-A 4 ankyrin repeats with unambiguous match to tail. A methionine codon located 26 bp from the 5' end previously de®ned consensus sequence (Bork, 1993) of the assembled sequence initiates the translation of a (Figure 2b). Ankyrin motifs have been shown to be 529 amino acid open reading frame (ORF), followed interactive domains, and these may mediate association by * 370 bp of 3' untranslated sequence containing an of Krit-1 with additional cellular partners. While Alu repeat family member (Figure 1a). Repeated multiple classes of proteins possess ankyrin repeats, eorts at 5' RACE did not result in the isolation of they are extremely abundant in cytoskeletal and additional upstream sequence; and of multiple ex- membrane attached proteins. In conjunction with the pressed sequence tags (EST) corresponding to Krit1 in hydrophobic character and predicted transmembrane the dbEST or TIGR databases, all commence domain for Krit1 and the known localization of Krev-1 comparably to or 3' of the assembled Krit1 clone. to endosomal compartments (Beranger et al., 1991; The Krit1 cDNA sequence has been deposited in Pizon et al., 1994), the presence of multiple ankyrin Genbank (accession number U90268). repeats suggests that Krit1 might similarly be associated The Krit1 cDNA was used as a probe to screen a with intracellular membranes. human genomic gt11 library. This screen resulted in the isolation of a 9.1 kb insert encompassing the 5' end of Speci®city of Krit1 interaction with Krev-1 the Krit1 cDNA (Figure 1b). Analysis of the sequence of this clone led to the identi®cation of three exons of All proteins de®ned to date as interacting with Krev-1 the Krit1 cDNA (bp 1 ± 136, 137 ± 250 and 251 ± 396), also interact to some degree with Ras. To investigate the Ankyrin-repeat protein Krit1 binds Krev-1/rap1A I Serebriiskii et al 1045

Figure 1 (a) Krit1 cDNA and protein coding sequence. Numbers in italics, nucleotides; numbers in plain text, amino acid coding sequence. Bold letters represent ankyrin repeat region (aa 83 ± 215) and possible Band 4.1 family homology region (aa 280 ± 433.) Nucleotide sequence in italics (bp 1700 ± 1986, Alu repeat element. ? and bold g indicate start of cDNA isolated by two-hybrid screen (bp 399). *, stop codon. (b) Partial KRIT1 genomic structure. Top line, Krit1 cDNA; bottom line, KRIT1 genomic clone. White boxes represent assigned exonic sequence. Cross-hatched boxes represent Alu repeat sequences. Black vertical line with ¯anking dashed line on genomic sequence, location preferred TATAAA box and predicted mRNA initiation site. Large arrow on cDNA represents Krit1 protein coding sequence Ankyrin-repeat protein Krit1 binds Krev-1/rap1A I Serebriiskii et al 1046 Table 1 Interaction of Krit1 with Krev-1 and Ras a LexA-KRev-1 LexA-Ras LexA-Bicoid JG4-5/Raf 5 (++) 638 (++++) 7(7) JG4-5/Krit11 ± 529 232 (++++) 2 (+) 2(7) JG4-5/Krit11 ± 483 3.3 (7) nd nd JG4-5/Krit11 ± 303 51(7) nd nd JG4-5/Krit11 ± 529** 91 (++++) nd nd JG4-5/Krit1126 ± 529 197 (++++) nd nd JG4-5 51(7) 2(7) 3(7) A two-hybrid assay was used to map the domain of Krit1 conferring association with Krev-1 and compare interaction of Krit1 and Raf with Krev-1 versus Ras and a negative control, Bicoid. The JG4-5 vector expresses truncated forms of Krit1 or Raf as translational fusions to a transcriptional activation domain. pJK103 was used as a LacZ reporter (2lexAop-LacZ), while EGY48 was used as a LEU2 reporter (6lexAop-LEU2), (Estojak et al., 1995). Numbers shown re¯ect beta-galactosidase values: ++++, ++, +, and 7 represent relative growth on media lacking leucine. nd, not determined

b a series of truncated derivatives of Krit1 to interact with Krev-1 (Figure 3, Table 1). The isolated amino-

terminal domain of the Krit1 protein (Krit11 ± 303), containing the intact ankyrin repeat motif, completely failed to interact with Krev-1. As noted above,

Krit1126-530 associated with Krev-1 as well as full length Krit1, again implying that interaction is speci®ed in the Figure 2 Krit1 protein structure and homologies. (a) Alignment with Band 4.1 family proteins. The sequences for human carboxy-terminal region of the protein. Finally, a short erythrocyte band 4.1 protein (Acc. M14993), human ezrin (Acc. deletion of the carboxy-terminal end of the Krit1 X51521), mouse moesin (Acc. P26038) and mouse merlin (Acc. protein (aa 483 ± 529) resulted in near-total loss of L27105) are aligned with the carboxy-terminal region of Krit1. protein interaction, while substitution of two missense Amino acid positions are noted at right. Capital letters represent mutations at aa 368 and 393 caused some diminish- identity of conservative amino acid substitution amount at least 4 sequences. Bold letters represent Prosite signature patterns to ment of interaction (91 versus 232 2 b-galactosidase). establish identity of band 4.1 family members; +/7 beneath All Krit1 derived proteins were con®rmed to be alignments indicates Krit1 match to Prosite pattern. (b) expressed to similar levels by Western analysis (not Alignment of ankyrin repeats. Shown, four tandem ankyrin shown) and neither Krit1 nor Raf interacted with the repeats derived from Krit1. Ankyrin consensus (Bork, 1993), shown at bottom. C, hydrophobic residue. Amino acid position LexA-bicoid negative control. of Krit1 is noted at right. Note, the Krit1 protein sequence Cumulatively, these results indicate that Krit1 identi®es a large number of ankyrin-repeat containing proteins interacts preferentially with Krev-1, while Raf inter- from Genbank; of these, the closest relative is a C. elegans protein acts preferentially with Ras. The carboxy-terminal whose function is currently unknown (gene ZK265.1, Acc. region of Krit1 is necessary and sucient to mediate Z81143; 55% similarity, 30% identity over ankyrin repeats and ¯anking region), followed by the Drosophila protein large Forked interaction with Krev-1, while the amino-terminal (Acc. D21203) and the murine SH3P2 (Acc. U58888) ankyrin repeat containing region may be utilized for association with additional cellular proteins.

speci®city of Krit1 interaction with Krev-1, we ®rst used Krit1 expression properties the carboxy-terminal domain of Krit1 gene originally

isolated by two-hybrid screen (Krit1126 ± 530)inan To assess the expression pattern of the Krit1 transcript, interaction mating approach (Finley and Brent, 1994) 32P-labeled cDNA spanning the Krit1 coding region in which we tested for association with 25 distinct but excluding the carboxy-terminal Alu sequence was proteins similarly fused to LexA (see Materials and used to probe a multiple tissue Northern blot (MTN1, methods). When so tested, Krit1 interacted strongly Clontech). Weak cross-hybridizing signals were ob- with Krev-1, very weakly with Ras, and not at all with served in heart, muscle, and brain, but not in other any non-speci®c proteins, supporting the idea that this tissues including lung, liver, kidney and placenta. A protein is a speci®c partner for Krev-1. We then single hybridizing species of *3.5 kb was observed in quantitated the interaction of full length Krit1 brain, which was the most intense individual signal

(Krit11 ± 529) with Krev-1 and Ras (Table 1). Krit11 ± 529 obtained. Multiple cross-hybridizing species in the associated strongly with Krev-1: approximately 232 b- 3.0 ± 4.5 kb size range, including at least three distinct galactosidase units and very strong growth on medium bands and a diuse background hybridization, were lacking leucine, as compared to 2 b-galactosidase units observed in heart and muscle. The summed intensity of

and weak growth on leucine-minus medium for Krit11-529 the heart and muscle hybridizing bands was approxi- with Ras. In contrast, similarly expressed Raf, used as a mately equivalent to the single band observed in brain. control, interacted weakly with Krev-1 (5 b-galactosi- Repeated eorts to raise antisera to Krit1, utilizing dase units and moderate growth on leucine-minus both peptide and full-length protein based approaches medium) but very strongly with Ras (638 b-galactosi- were unsuccessful, limiting assay of endogenous protein dase units and very strong growth on leucine-medium). expressing patterns. However, it appear likely that the To more exactly de®ne the domain of Krit1 required endogenous Krit1 protein is expressed at low levels in for interaction with Krev-1, we compared the ability of mammalian cells, based on the relatively low Ankyrin-repeat protein Krit1 binds Krev-1/rap1A I Serebriiskii et al 1047

Krit11–529

Krit11–483

Krit11–303

Krit11–529**

Krit1126–529

Figure 3 Derivatives of Krit1. Thick line, coding sequence; thin line 5' and 3' noncoding sequence. Dark boxed region, ankyrin repeat region. Light boxed region, putative Band 4.1 homology region abundance of the Krit1 mRNA, and the fact that its coding sequence contains a high proportion of disfavored codons for amino acid translation (IS and EG, unpublished results). Further, we have experimen- tally demonstrated that transfection of a plasmid designed to express epitope-tagged Krit1 from the strong CMV promoter into Cos cells, or infection of insect cells with baculovirus expressing Krit1, does not result in detectable levels of protein synthesis even though the Krit 1 mRNA is eciently transcribed, and other similarly transfected controls are well expressed. In contrast, Krit1 protein is expressed well in both yeast (which demonstrate a pattern of codon preference not greatly removed from mammals) and bacteria. At Figure 4 Chromosomal assignment of KRIT1. Localization of this time we are uncertain as to the source of the the KRIT1 probe to human chromosome 7 by FISH. Figure limited expression of the Krit1 protein in higher depicts copies of chromosome 7 from several dierent metaphase eukaryotes. Finally, in support of the idea that the spreads, demonstrating speci®c hybridization signals at chromosome 7q21-22. Photograph represents computer-enhanced, merged Krit1 protein is expressed and functional, rather than a image of ¯uorescein signals (bright dots) and DAPI stained disfunctional mRNA, inspection of the dbEST chromosomes database identi®ed several murine transcripts homologous to Krit1; one typical, AA175084, was 89% identical on the nucleotide level over 450 base pairs, Sargent et al., 1994; Weiss et al., 1987; Yamada et al., encoding a peptide with 92% identity, 95% similarity 1990). While a role for Krev-1/rap1A has not been to aa 94 ± 243 of Krit 1. This relatively strong investigated in these classes of malignancies, a char- conservation pattern suggests the Krit1 protein acteristic feature of chromosome 7 monosomy and 7q22 maintains a cellular function across species. deletions is their occurrence as secondary events following hyperactivation of Ras or mutation of the Ras-GAP protein neuro®bromatosis 1 (NF-1) gene Chromosomal assignment of Krit1 to 7q21-22 (Ballester et al., 1990; Martin et al., 1990; Xu et al., Because of the prior description of Krev-1 as a 1990; reviewed in Luna-Fineman et al., 1995). Insofar as regulator of oncogenesis, it was of interest to Krev-1 possesses a function as a regulator of Ras determine whether KRIT1 chromosomal localization activity, or a competitive inhibitor of Ras binding to suggested a role in cancer. Fluorescence in situ eectors, deletion of Krit1, a potential positive regulator hybridization (FISH) was used for chromosomal of Krev-1 activity, may provide an indirect mechanism assignment of the KRIT1 gene. FISH using a labeled to enhance Ras-dependent tumor progression. KRIT1 probe spanning the protein coding region In summary, we have de®ned a novel partner protein revealed speci®c labeling on human chromosome 7 for Krev-1, whose speci®city of interaction, sequence (Figure 4). Hybridization was detected on chromosome characteristics, and chromosomal location make it an 7 in 33 of 39 metaphase spreads examined. Seventy-®ve intriguing candidate for further study relative to of 204 (37%) ¯uorescence signals hybridized to the oncogenesis. Based on its interaction pro®le, it seems long arm of chromosome 7; all ¯uorescent signals on likely that this protein may prove to have a function chromosome 7 were located at 7q21-22 often at or near speci®cally related to Krev-1 rather than Ras activity, the boundary between these two bands. although this is not yet proven; for example, while Localization of KRIT1 to 7q21-22 is particularly both Ras and Krev-1 have been previously shown to interesting, in that 7q22 has been frequently implicated bind Ral-GDS, only Ras is able to activate this protein as a site of chromosomal alterations in a variety of (Urano et al., 1996). Our current results do not address cancers, including myeloid disorders and uterine the cellular function of Krit1. One intriguing leiomyomata (Kere et al., 1987; Luna-Fineman et al., possibility, given the frequent association of ankyrin 1995; Ogata et al., 1993; Pedersen-Bjergaard et al., 1982; repeat-containing proteins with the cytoskeleton, is that Ankyrin-repeat protein Krit1 binds Krev-1/rap1A I Serebriiskii et al 1048 Krit1 may act in localizing the Krev-1 to an http://dot.imgen.bcm.tmc.edu:9331/gene-®nder/gf.html, was appropriate cellular compartment for function. It is used to identify promoter elements. To obtain the Krit1 hoped that the further characterization of the KRIT1 genomic clone, a human genomic library derived from gene and protein may lead to insights into the ultimate human placental DNA, cloned in the gt11 bacteriophage 32 role of Krev-1 in tumor progression and cellular vector was screened using a P-labeled 1.4 kb fragment of the Krit1 cDNA. Positive clones were detected and growth control. puri®ed, subcloned to vector, and sequenced utilizing standard techniques (Ausubel et al., 1987 ± 1996). The

two point mutations in Krit11 ± 529** arose spontaneously Materials and methods during PCR subcloning of the full-length cDNA.

Two-hybrid screen and interaction analysis Fluorescence in situ hybridization Two hybrid screening (Golemis et al., 1996), interaction Normal human metaphase spreads were prepared accord- mating (Finley and Brent, 1994), and interaction anity ing to the method of (Fan et al., 1990). Human determination (Estojak et al., 1995) were performed as lymphocytes were cultured for 72 h at 378CinRPMI previously described. For library screening, a HeLa library 1640 medium containing phytohemagglutinin and 10% (Gyuris et al., 1993) was probed using LexA-fused to full fetal bovine serum. Cultures were synchronized by length Krev-1 as bait, and EGY48 and pJK103 as treatment with 5-bromodeoxyuridine (0.18 mg/ml, Sigma) reporters. For interaction mating, the panel of LexA- for 16 h, followed by release from the block by incubation fused proteins screened included LexA-Max, c-Myc, Cdc2, in fresh medium containing thymidine (2.5 mg/ml) for 6 h. SSN6, bicoid, daughterless, hairy, CLN3, goosecoid, Cdi1, A KRIT1 cDNA clone was labeled with biotin-16-dUTP by Cyclin D, Rb, Cdk3, LAR, TR, p53 and others. For nick translation. Fluorescence in situ hybridization (FISH) analysis of Krit1 truncations, betagalactosidase values were and detection of immuno¯uorescence were performed determined for 2 ± 4 independent colonies, with standard essentially as described previously (Bell et al., 1995). The deviation from Table 1 values less than 20%; growth on chromosome preparations were stained with diamidino-2- leucine- medium was scored essentially as described in phenylindole (DAPI) and observed with a Zeiss Axiophot (Estojak et al., 1995), ranging from ++++ (rapid growth ¯uorescence microscope. Digitized images were captured in 1 ± 2 days) to 77 (no detectable growth in 4 or more with a cooled CCD camera connected to a computer work days). station. Images of DAPI staining and ¯uorescein signals were merged using Oncor Image software, version 1.6. DNA manipulations Extension of Krit1 cDNA was performed using a 5'RACE- Ready cDNA kit from Clontech. The Krit1 cDNA sequence was used to screen expressed sequence tag Acknowledgements (EST) databases maintained by the Institute of Genome We are grateful to G Rall for comments on the manuscript. Research (TIGR) (Consortium, 1995) and Genbank using We thank A Makris for the gift of the LexA-Ras fusion the BLAST algorithm (Altschul et al., 1990). Sequence construct, J Cherno for the gift of the human placental alignments utilized the package of programs available library and R Finley and R Brent for use of the LexA- through UWGCG (Devereux et al., 1984), as well as fusion panel. These studies were supported by funds from additional programs as noted in text. The program TSSW, the National Institues of Health, R29 CA63366 (to EG) created by VV Solovyev and AA Salamov and located at and core grant CA-06927.

References

Altschul SF, Gish W, Miller W, Myers WE and Lipman DJ. Chant J, Corrado K, Pringle JR and Herskowitz I. (1991). (1990). J. Mol. Biol., 215, 403 ± 410. Cell, 65, 1213 ± 1224. Ausubel FM, Brent R, Kingston R, Moore D, Seidman J, Chant J and Herskowitz I. (1991). Cell, 65, 1203 ± 1212. Smith JA and Struhl K. (1987 ± 1996). Current Protocols in Conboy J, Kan YW, Shohet SB and Mohandas N. (1986). Molecular Biology. New York: John Wiley & Sons. Proc.Natl.Acad.Sci.USA,83, 9512 ± 9516. Ballester R, Marchuk D, Boguski M, Saulino A, Letcher R, Consortium Institute for Genomic Research. (1995). Nature, Wigler M and Collins R. (1990). Cell, 63, 851 ± 859. 377, 3 ± 174. Bell DW, Taguchi T, Jenkins NA, Gilbert DH, Copeland Coso OA, Chiariello M, Yu J-C, Teramoto HCP, Xu N, Miki NG, Gilks CB, Zweidler-McKay P, Grimes HL, Tsichlis T and Gutkind JS. (1995). Cell, 81, 1137 ± 1146. PN and Testa JR. (1995). Cytogenet. Cell Genet., 70, 263 ± Devereux J, Haeberlie P and Smithies O. (1984). Nuc. Acids 267. Res., 12, 387 ± 397. Bender A. (1993). Proc. Nat. Acad. Sci. USA, 90, 9926 ± Eklund EA, Marshall M, Gibbs JB, Crean CD and Gabig 9929. TG. (1991). J. Biol. Chem., 266, 13964 ± 13970. Bender A and Pringle JR. (1989). Proc. Nat. Acad. Sci. USA, Estojak J, Brent R and Golemis EA. (1995). Mol. Cell. Biol., 86, 9976 ± 9980. 15, 5820 ± 5829. Beranger F, Goud B, Tavitian A and de Gunzburg J. (1991). Fan Y-S, Davis LM and Shows TB. (1990). Proc. Natl. Acad. Proc. Nat. Acad. Sci. USA, 88, 1606 ± 1610. Sci. USA, 87, 6223 ± 6227. Bokoch GM. (1995). Trends Cell Biol., 5, 109 ± 113. Finley R and Brent R. (1994). Proc. Natl. Acad. Sci. USA, Bokoch GM and Knaus UG. (1994). Curr. Op. Immun., 6, 91, 12980 ± 12984. 98 ± 105. Frech M, John J, Pizon V, Chardin P, Tavitian A, Clark R, Bork P. (1993). Proteins, 17, 363 ± 374. McCormick F and Wittinghofer A. (1990). Science, 249, Buss JE, Quilliam LA, Kato K, Casey PJ, Solski PA, Wong 169 ± 171. G, Clark R, McCormick F, Bokoch GM and Der CJ. (1991). Mol. Cell. Biol., 11, 1523 ± 1530. Ankyrin-repeat protein Krit1 binds Krev-1/rap1A I Serebriiskii et al 1049 Golemis EA, Gyuris J and Brent R. (1996). In Current Powers S, Gonzales E, Christensen T, Cubert J and Broek D. Protocols in Molecular Biology, (eds.) Ausubel FMR, (1991). Cell, 65, 1225 ± 1231. Brent R, Kingston D, Moore J, Seidman SJ and K Struhl. Quinn M, Mullen M, Jesaitis A and Linner J. (1992). Blood, John Wiley and Sons: New York, pp. 20.1.1 ± 20.1.28. 79, 1563 ± 1573. Gyuris J, Golemis EA, Chertkov H and Brent R. (1993). Cell, Quinn MT, Parkos CA, Walker L, Orkin SH, Dinauer MC 75, 791 ± 803. and Jesaitis AJ. (1989). Nature, 342, 198 ± 200. Hariharan IK, Carthew RW and Rubin GM. (1991). Cell, 67, Ruggieri R, Bender A, Matsui Y, Powers S, Takai Y, Pringle 717 ± 722. JR and Matsumoto K. (1992). Mol. Cell Biol., 12, 758 ± Herrmann C, Horn G, Spaargaren M and Wittinghofer A. 766. (1996). J. Biol. Chem., 271, 6794 ± 6800. Sakoda T, Kaibuchi K, Kishi K Kishida S, Doi K, Hoshino Hofmann K and Stoel W. (1993). Biol. Chem., 347, 166. M, Hattori S and Takai Y. (1992). Oncogene, 7, 1705 ± Irani K, Xia Y, Zweier JL, Sollott SJ, Der CJ, Fearon ER, 1711. Sundaresan M, Finkel T and Goldschmidt-Clermont PJ. Sargent MS, Weremowicz S, Rein MS and Morton CC. (1997). Science, 275, 1649 ± 1652. (1994). Canc. Gen.s & Cytogen., 77, 65 ± 68. Jelinek MA and Hassell JA. (1992). Oncogene, 7, 1687 ± 1698. Sato KY, Polakis PG, Haubruck H, Fasching CL, Kere J, Ruutu T, Lahtinen R and de la Chapelle A. (1987). McCormick F and Stanbridge EJ. (1994). Cancer Res., Blood, 70, 1349 ± 1353. 54, 552 ± 559. Khazak V, Woychik N, Sadhale P, Brent R and Golemis EA. Simon M-N, De Virgillio C, Souza B, Pringle JR, Abo A and (1995). Mol. Biol. Cell., 6, 759 ± 775. Reed SI. (1995). Nature, 376, 702 ± 705. Kitayama H, Sugimoto Y, Matsuzaki T, Ikawa Y and Noda Trofatter JA, MacCollin MM, Rutter JL, Murrell JR, Duyao M. (1989). Cell, 56, 77 ± 84. MP,ParryDM,EldridgeR,KleyN,MenonAG,Pulaski Law SF, Estojak J, Wang B, Mysliwiec T, Kruh GD and K, Haase VH, Ambrose CM, Munroe D, Bove C, Haines Golemis EA. (1996). Mol. Cell. Biol., 16, 3327 ± 3337. JL, Martuza RL, MacDonald ME, Seizinger BR, Short Luna-Fineman S, Shannon KM and Lange BJ. (1995). MP, Buckler AJ and Gusella JF. (1993). Cell, 72, 791 ± Blood, 85, 1985 ± 1999. 800. Maly F-E, Quilliam LA, Dorseuil O, Der CJ and Bokoch Urano T, Emkey R and Feig LA. (1996). EMBO J., 15, 810 ± GM. (1994). J. Biol. Chem., 269, 18743 ± 18746. 816. Martin GA, Viskochil D, Bollag G, McCabe PC, Crosier WJ, Weiss K, Williams KD, Dahl GV, Wang W, Johnson FL, Haubruck H, Conroy L, Clark R, O'Connell P, Cawthon Murphy SB and Dow LW. (1987). Leukemia, 1, 97 ± 104. RM, Innis MI and McCormick F. (1990). Cell, 63, 843 ± Wienecke R, Konig A and DeClue JE. (1995). J. Biol. Chem., 849. 270, 16409 ± 16414. Moult J. (1996). Curr. Opin. Biotech., 7, 422 ± 427. Wojtaszek PA, Stumpo DJ, Blackshear PJ and Macara IG. Nassar N, Horn G, Herrmann C, Scherer A, McCormick F (1993). Oncogene, 8, 755 ± 760. and Wittinghofer A. (1995). Nature, 375, 554 ± 560. XuG,LinB,TanakaK,DunnD,WoodD,GestelandR, Nobes CD and Hall A. (1995). Cell, 81, 53 ± 62. White R, Weiss R and Tamano F. (1990). Cell, 63, 835 ± Ogata T, Ayosawa D, Namba M, Takahashi E, Oshimura M 841. and Oishi M. (1993). Mol. Cell. Biol., 13, 6036 ± 6043. Yamada T, Shippey CA, Martineau M and Secker-Walker Park H-O, Chant J and Herskowitz I. (1993). Nature, 365, LM. (1990). Genes Chrom. Cancer, 2, 88 ± 93. 269 ± 274. Zhang X-f, Settleman J, Kyriakis JM, Takeuchi-Suzuki E, Pedersen-Bjergaard J, Vindelov L, Philip P, Ruutu P, Elledge SJ, Marshall MS, Bruder JT, Rapp UR and Elmgreen J, Repo H, Christensen IJ, Killmann S-A and Avruch J. (1993). Nature, 364, 308 ± 313. Jensen G. (1982). Blood, 60, 172 ± 179. Zhao Z-S, Leung T, Manser E and Lim L. (1995). Mol. Cell. Pizon V, Chardin P, Lerosey I, Olofsson B and Tavitian A. Biol., 15, 5246 ± 5257. (1988). Oncogene, 3, 201 ± 204. Zheng Y, Bender A and Cerione RA. (1995). J. Biol. Chem., Pizon V, Desjardins M, Bucci C, Parton RG and Zeriat M. 270, 626 ± 630. (1994). J. Cell Sci., 107, 1661 ± 1670.