Inhibition of v-Abl transformation in 3T3 cells overexpressing ... - Nature

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Oncogene (2001) 20, 4926 ± 4934 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc

Inhibition of v-Abl transformation in 3T3 cells overexpressing dierent forms of the Abelson interactor protein Abi-1 Alexandra Ikeguchi1, Hen-Ying Yang2, Guangxia Gao2 and Stephen P Go*,2 1

Department of Medicine, Columbia University, College of Physicians and Surgeons, New York, NY 10032, USA; 2Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University, College of Physicians and Surgeons, New York, NY 10032, USA

The abi-1 gene encodes a protein that binds and is phosphorylated by the Abelson protein tyrosine kinase. Constructs expressing a full-length abi-1 cDNA, and a smaller cDNA arising from an alternatively spliced form, were generated and tested for their eect on transformation of NIH3T3 cells by the Abelson murine leukemia virus. Overexpression of both forms of the protein strongly inhibited transformation by the wild-type P160 strain of the virus, but not by the non-interacting mutant P90A strain. The inhibition required the SH3 domain of Abi-1, suggesting that a direct interaction was required for the eect. Rare breakthrough P160 transformants of the Abi-1 overexpressing lines were found to have downregulated Abi-1 protein levels by a post-transcriptional mechanism. Oncogene (2001) 20, 4926 ± 4934. Keywords: Abelson murine leukemia virus; interacting protein

Introduction The Abelson gene (c-abl) encodes a tyrosine kinase involved in mitogenic signaling from growth factor receptors and in the response to DNA damage (see Van Etten, 1999 for recent review). Activated forms of the gene can lead to leukemias and sarcomas in several settings. The gene was ®rst identi®ed as the oncogene of the Abelson murine leukemia virus, an acute transforming retrovirus capable of inducing a rapid pre-B cell lymphoma in infected mice (Abelson and Rabstein, 1970). A second retrovirus transducing the abl gene, the Hardy-Zuckerman sarcoma virus, causes sarcomas in cats (Bergold et al., 1987). In addition, nearly all cases of chronic myelogenous leukemia (CML) in humans are initiated by chromosomal translocation that fuses the BCR and ABL genes to produce an activated BCR-ABL kinase (de Klein et al., 1982). The (9 : 22) translocation creates a key cytological marker for CML, the Philadelphia chromosome. These activated forms of Abl can initiate many

*Correspondence: SP Go Received 17 October 2000; revised 27 March 2001; accepted 2 April 2001

mitogenic and anti-apoptotic signals that are important in tumor expansion and survival. The activation of the ras pathway is probably one of the most important of these signals (Sawyers et al., 1995). The kinase activity of c-Abl for its critical substrates is normally thought to be closely regulated, perhaps by intracellular location, by phosphorylation, and by noncovalent association with other proteins, such as Shc (Rael et al., 1996), p62Dok (Carpino et al., 1997; Yamanashi and Baltimore, 1997), Crk (Nichols et al., 1994; Ren et al., 1994), PAG/msp23 (Wen and Van Etten, 1997), and Abi-1 (Shi et al., 1995) and Abi-2 (Dai and Pendergast, 1995). The oncogenic forms of the kinase are able to overcome the potential regulatory eects of these proteins, either by overexpression, relocalization to distinct intracellular locations, or acquisition of higher constitutive kinase activity or loss of normal inhibitory interactions. The Abi-1 gene product was originally identi®ed in a yeast two-hybrid screen for proteins interacting with the Abelson tyrosine kinase (Shi et al., 1995). A second highly related protein, termed Abi-2, was identi®ed in a similar screen (Dai and Pendergast, 1995). The Abi proteins are bound and phosphorylated by the Abl kinase in vivo. Abi-1 contains sequence similarity to homeobox proteins, several prominent proline-rich sequences that serve as binding sites for other proteins, and a src homology 3 (SH3) domain at its very carboxyterminus. The Abi-1 protein interacts with cAbl in two distinct ways: the SH3 domain of Abi-1 interacts with PxxP motifs in the carboxyterminal portion of c-Abl; and, reciprocally, the SH3 domain of c-Abl interacts with the proline-rich regions of Abi-1. The normal form of c-Abl can thus interact through both domains. The activated v-Abl protein encoded by the wild-type Abelson MuLV genome, P160v-abl, however, has lost its SH3 domain during recombination with the parental helper virus and thus can only interact with Abi-1 in its carboxyterminus. A deletion mutant of Abelson virus lacking the carboxyterminus of v-Abl, the P90Av-abl strain, has lost the carboxyterminal PxxP motifs (Go et al., 1981; Murtagh et al., 1986) and thus can no longer bind to Abi-1 (Shi et al., 1995). The role of Abi-1 in the normal physiology of the cell remains unclear. The Drosophila homologue of the mammalian genes is thought to serve as a positive

Inhibition of v-Abl transformation by Abi-1 A Ikeguchi et al

signaling molecule in enhancing the ability of Abl to phosphorylate at least one of its substrates, Ena (Juang and Homann, 1999). Mammalian Abi-1 was independently identi®ed (Biesova et al., 1997) as a protein interacting with Eps8, a prominent substrate of the EGF and PDGF receptors and thought to be involved in mitogenic signaling (Fazioli et al., 1993). Thus, Abi1 might function in the intracellular response to growth factor exposure. Abi-1 may also interact with the cytoskeletal components such as dynamin and synaptojanin (So et al., 2000). More recently, Abi-1 has also been shown to directly associate with Sos, the guanosine exchange factor important for Ras activation (Fan and Go, 2000; Scita et al., 1999). Whether this association normally is associated with a positive or negative signal is not known. However, the highlevel overexpression of Abi-1 can have signi®cant negative regulatory eects on EGF-mediated signaling (Fan and Go, 2000; Shi et al., 1995). Thus, overexpression of Abi-1 can reduce and delay the activation of the extracellular response kinases (Erks) that normally follows EGF exposure. The initial report describing the identi®cation of Abi-1 showed that the high-level expression of the protein in NIH3T3 cells caused a strong inhibition of transformation by the P160v-abl protein, while there was no eect on transformation by the noninteracting P90A strain or by v-Src (Shi et al., 1995). Subsequent analysis of the structure of the Abi-1 mRNA and gene revealed that the construct used in these studies was truncated at the 5' end. Thus, the inhibitory activity of the construct could have been attributable to its potential interference with the normal function of the endogenous Abi-1 protein in a dominant negative manner. The intact protein might not, therefore, be predicted to exhibit similar inhibitory activity. In addition, alternatively spliced forms of Abi-1 mRNA have been reported (Biesova et al., 1997; Taki et al., 1998), and these forms may not all show equivalent biological activities. To address these issues, we have now performed further tests of full-length Abi-1 constructs.

RACE procedures, and the DNA product was cloned and sequenced. The longest products contained 310 additional base pairs of new sequence (Figure 1). The added sequence contained a new ATG codon in frame with the original ORF, and conceptual translation from this start predicted the presence of 85 residues of new sequence at the aminoterminus of the protein. The new ATG was present in the context of a good consensus sequence for translation initiation. Since no stop codon was present upstream of this ATG, it is formally possible that additional upstream sequences could encode still longer proteins. However, the newly predicted aminoterminus is a near-perfect match to the aminoterminal sequence of the putative human homologue, e3B1 (Biesova et al., 1997), suggesting that this is the correct initiation codon. Subsequent analysis of mRNAs isolated from these cells by Northern blot revealed that in addition to the major abi-1 mRNA species, at least one additional species of smaller size could be detected (data not shown). To identify this form, PCR was performed with primers speci®c for the central portion of the abi-1 gene on a template of cDNA prepared from WEHI-3B mRNA. Two products diering in size by approximately 90 bp were indeed identi®ed. These products were cloned and the nucleotide sequences were determined. The longer product (of size 534 bp) corresponded to the original cDNA. The shorter product (of size 447) was identical but lacked 87 bp of the original sequence from the central portion of the coding region. The shorter mRNA could be translated through the missing sequences in frame to form a protein lacking 29 residues of internal sequences (Figure 1). The sequence analysis of the new clones revealed several dierences from the published sequence. To resolve these discrepancies, the original clones and the new clones were resequenced. Several errors in the original sequence, including four short stretches with changes in reading frame, were identi®ed and corrected (Figure 1). The new sequence brings the predicted mouse Abi-1 protein into closer similarity to human ABI-1 and ABI-2 sequences. The long form corre-

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Results cDNAs clones of the Abi-1 mRNA: Full-length clones and an alternatively spliced form The Abi-1 gene was ®rst recovered from a cDNA library of mRNAs isolated from the murine WEHI-3B cell line (Shi et al., 1995). The original report of the Abi-1 gene presented the nucleotide sequence of a cDNA clone containing 2755 bp, predicted to encode a protein of 394 residues (Shi et al., 1995). Careful analyses of Abi-1 mRNA by Northern blot revealed that the major species present in most tissues was longer than the longest clones isolated originally, even after accounting for the presence of poly(A) sequences. To characterize the full-length mRNA, a cDNA library from mouse liver mRNA was used as template for 5'

Figure 1 Amino acid sequences of the Abi-1 proteins encoded by the cDNAs used in this study. The long form includes 85 extra residues at the amino terminus more than previously reported (shaded; (Shi et al., 1995)); other changes from the published sequences are underlined. The 29 residues missing in the short form of the protein arising from alternative mRNA splicing are boxed Oncogene


4928

sponds to the full-length e3B1 sequence (Biesova et al., 1997), and the short form to the B48 cDNA reported from normal peripheral blood (Taki et al., 1998). Inhibition of transformation by overexpression of various forms of Abi-1 The initial study of Abi-1 function demonstrated that overexpression of the protein in NIH3T3 cells could strongly inhibit transformation by the Abelson murine leukemia virus (Shi et al., 1995). Cell lines expressing high levels of Abi-1 were resistant to transformation by viruses encoding the P160v-Abl protein, which binds to Abi-1, but not by those encoding the shorter P90Av-Abl protein, which lacks Abi-1 binding sites. The fact that these studies were carried out with constructs expressing a truncated form of Abi-1 raised the possibility that the inhibition could be due to a dominant interfering activity: that is, to the ability of the shorter form to compete with the endogenous full-length form for binding to v-Abl. Thus, the full-length form might not inhibit v-Abl like the short form, and might even promote v-Abl transformation. Alternatively, it could be that the binding of any form of Abi-1 to v-Abl would inhibit the transforming activity, perhaps by blocking access to critical substrates of the kinase. In this case the overexpression of any form of Abi-1 would render cells resistant to transformation. The isolation of an alternatively spliced form of Abi-1 (Figure 1) raised the additional question of whether both forms could inhibit transformation.

To test for inhibition of transformation, cell lines that overexpressed either of the two alternatively spliced, full-length forms of Abi-1 were generated. The coding regions were inserted into the expression plasmid pSRa to direct the synthesis of the proteins with a myc epitope tag at their carboxyterminal end. Each DNA was mixed with the pGKpuro plasmid DNA encoding resistance to puromycin, and the mixture was used to transform NIH3T3 cells by the calcium phosphate method. Several puromycin-resistant colonies arising from each transformation were picked and expanded, lysates were prepared, and the levels of Abi-1 expression were determined by Western blot using a monoclonal antibody speci®c for the myc epitope (Figure 2). Ten out of 23 clones expressed high levels of the longer form, while 13 out of 22 clones expressed the alternatively spliced shorter form. This represents a frequency of cotransformation very similar to that seen for other genes, and suggests no particular toxicity or cytostatic eect of high-level Abi-1 expression. Crude estimates of the level of overexpression on Western blots with anti-Abi-1 sera suggest that 10 ± 100-fold times the low endogenous levels of Abi-1 were achieved in these lines. The Abi-1-expressing clones showed no signi®cant changes in cell morphology, growth rate, or culture density at con¯uence (data not shown). Serial 10-fold dilutions of Abelson virus stocks of the strains encoding P160 and P90A proteins were prepared, and used to infect Abi-1-expressing and control NIH3T3 cells at approximately 30% con¯u-

a

b

Figure 2 Overexpression of Abi-1 in NIH3T3 cells. NIH3T3 cells were cotransfected with plasmids containing Abi-1 cDNA with a 3' myc tag and the gene for puromycin resistance. Puromycin resistant clones were picked and expanded, and whole cell lysates were prepared. Western blots of cellular extracts were performed using the mouse anti-myc monoclonal antibody Oncogene


ence. After growth to con¯uence, the cells were maintained in low serum for 2 weeks and transformed colonies were counted (Table 1). Control cells not expressing Abi-1 were sensitive to both viruses. Cell lines overexpressing either of the two forms of Abi-1 were strongly resistant to transformation by the P160 virus, like those lines described previously that overexpressed the 5' truncated forms (Shi et al., 1995). The apparent titer of P160 virus on these lines was reduced 100 ± 1000-fold as compared to nonexpressors. One of the lines (12S) was an exception, and was sensitive to virus transformation; reexamination of this line revealed that Abi-1 expression had been lost during the cell passages after infection (Figure 3; Table 1). In all cases, the cells showed no resistance to transformation by the P90A strain that lacked the binding site for Abi-1; the lines were as sensitive as the parental or the control lines. Thus, there was no resistance to infection per se, such as would be caused by loss of the virus receptor. These results suggest that the overexpression of either of the two full-length Abi-1 forms inhibited P160 A-MuLV transformation, and that the inhibition required the interaction region in the C-terminus of vAbl. Abi-1 expression is lost in rare P160 transformants derived from Abi-expressing lines Although the Abi-1 expressing cell lines were dramatically resistant to transformation by the P160 strain of

virus, they were not totally resistant, and rare transformed colonies were detected upon infection. To examine the mechanism of breakthrough transformation in these colonies, foci of morphologically transformed cells were picked, expanded, and passaged without the use of trypsin in low serum concentrations to maintain selection for the transformed phenotype. Lysates of these cultures were prepared, and the levels of Abi-1 protein was then assessed by Western blot, using anti-myc antibodies to detect the epitope tag. (Figure 4). In every case, the transformed lines showed no detectable levels of Abi-1 protein. The loss of Abi-1 protein was seen in lines expressing either of the forms derived from the alternatively spliced mRNAs. This result strongly suggests that overexpression of Abi-1 was incompatible with morphological transformation by P160v-Abl, and conversely that loss of Abi-1 protein expression was required for transformation by the virus. In many situations, genes originally introduced into cells by transformation are unstable and are lost at high frequency, either by DNA rearrangements or by chromosome segregation; in other situations transcription of the introduced gene is suppressed. To determine whether the loss of Abi-1 protein in these rare transformed lines was due to loss of the DNA, loss of transcription, or post-transcriptional regulation, we examined the levels of Abi-1 mRNA. RNA was extracted from both the parental Abi-1 overexpressing cell lines and the rare P160 transformants. A labeled

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Table 1 Inhibition of A-MuLV transformation by overexpression of Abi-1 Cell line 8S 9S 12S 1L 9L 22L

Abi-1 expression after mocka

FFUb (P160)

Abi-1 expression after P160a

FFUb (P90A)

Abi-1 expression after P90Aa

None + ++ None ++ +

4.86103 5.06101 5.96103 3.56103 516101 516101

None + None None ++ +

1.46105 1.36105 4.76105 4.56105 1.16105 1.56105

None ++ ++ None ++ +

a

Levels of Abi-1 expression assessed by Western blot. None, no detectable expression. +, intermediate level. ++, high levels of expression. Transformation susceptibility of cell lines. Apparent titer of A-MuLV preparations as focus-forming units (FFU) per ml

b

Figure 3 Abi-1 protein expression in NIH3T3 cells used in the A-MuLV focus forming assay as assessed by Western blot using antibody to myc. In each group of three, the ®rst lane represents cells that were mock infected, the second cells that were infected with P160, and the third cells that were infected with P90A Oncogene


4930

a

b

Figure 4 Abi-1 protein expression in breakthrough transformants. Individual transformed foci from the A-MuLV focus forming assay were picked and expanded, and whole cell lysates were prepared. (a) Western blot using anti-Abl rabbit monoclonal antibody K12. (b) Western blot detecting Abi-1 after probing with anti-myc antibody. In each panel, lysates are from the parental NIH3T3 cell line (lane 1), mock infected Abi-1 overexpressing cell lines (short form, lane 2 and long form, lane 10), P160 infected Abi-1 negative cell lines (short form, lane 3 and long form, lane 11), and P160 infected Abi-1 overexpressing clones (short form, lanes 4 ± 9 and long form, lanes 12 ± 17)

Figure 5 Abi-1 messenger RNA in breakthrough transformants. Whole cell RNA was extracted from P160 transformed clones derived from Abi-1 overexpressing cell lines, uninfected Abi-1 overexpressing cell lines, and parental NIH3T3 cells. The RNAs were used to protect a labeled antisense probe, and the products were resolved by polyacrylamide gel electrophoresis. Yeast RNA with RNase (lane 1), yeast RNA without RNase (lane 2), NIH3T3 (lane 3), uninfected cell lines overexpressing Abi-1 short form (lanes 4 and 5) and long form (lanes 6 and 7), and P160 transformed clones derived from cell lines overexpressing Abi-1 short form (lanes 8 ± 11) and long form (lanes 12 ± 15)

antisense RNA probe was prepared by in vitro transcription from a template DNA containing the 3' portion of the Abi-1 cDNA and also including the region encoding the myc epitope tag. This probe could Oncogene

distinguish between the endogenous Abi-1 mRNA and the RNAs derived from the transformed expression construct. The probe was annealed to the RNA preparations and digested, and the protected fragments


were examined by gel electrophoresis and autoradiography (Figure 5). Endogenous Abi-1 transcripts were not detected in any of the cell lines, consistent with the very low expression of the gene in NIH3T3 cells (Shi et al., 1995). The myc-tagged Abi-1 mRNAs were readily detected in the parental lines, with a wide range of levels in dierent clones. Surprisingly, equal or even higher levels of the Abi-1 mRNAs were detected in the transformed cell lines. Thus, the loss of Abi-1 protein expression in these cells was not caused by loss of the Abi-1 DNA or RNA but rather must have been achieved by regulation of translation or at the level of protein stability. The Abi-1 expressing lines were not resistant to transformation by the P90A strain of v-Abl, and thus no strong selective pressure (beyond that seen in NIH3T3 cells) was associated with transformation by this virus. To determine whether the P90A transformants showed changes in the expression of Abi-1, foci of transformed cells were picked randomly from infected cultures and expanded as before. Cell lysates were prepared, and analysed by Western blot to determine the levels of epitope-tagged Abi-1 protein. Most of the transformants still exhibited detectable levels of Abi-1 protein (Figure 6). However, several of the lines had lost expression. Furthermore, several of

the lines initially expressing the short form now expressed both the original protein and a larger protein migrating slightly more slowly than the long form, suggesting a rearrangement in the transfected gene. Thus, there may be some modest selective pressure against Abi-1 expression associated with P90A transformation. The result suggests that the loss of Abi-1 protein can be induced during transformation even with a v-Abl kinase lacking a direct interaction site, although at lower frequency than with the P160 interaction-competent form.

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The SH3 domain of Abi-1 is required for inhibition of transformation The observation that Abi-1 overexpression caused an inhibition of transformation by only the P160 strain of A-MuLV and not the P90A strain suggests that a direct interaction between Abi-1 and the v-Abl kinase was required for the eect. To test this notion directly, cell lines were engineered to overexpress a mutant of Abi-1 lacking the C-terminal SH3 domain (AbiD394 ± 475; (Fan and Go, 2000)). This mutant, here termed Abi-1DSH3, fails to interact with P160 v-Abl in vitro and in vivo, and fails to inhibit Erk2 activation by EGF (Fan and Go, 2000; Shi et al., 1995). The coding

a

b

Figure 6 Abi-1 protein expression in P90A transformed clones. NIH3T3 cell lines overexpressing Abi-1 were infected with P90A virus, and transformed foci were picked and expanded. Whole cell lysates were prepared and subjected to Western blot analysis. (a) Immunodetection with K12 anti-Abl antibodies. (b) Immunodetection with 9E10 anti-myc antibodies. In each panel, lysates are from the parental NIH3T3 cell line (lane 1), mock infected Abi-1 overexpressing cell lines (short form, lane 2 and long form, lane 10), P90A infected Abi-1 negative cell lines (short form, lane 3 and long form, lane 11), and P90A infected Abi-1 overexpressing clones (short form, lanes 4 ± 9 and long form, lanes 12 ± 17) Oncogene


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region for Abi-1DSH3 was inserted into the pMT21 plasmid to direct the expression of the protein with a myc epitope tag at the C-terminus. NIH 3T3 cells were cotransformed with a mixture of the DNA expressing Abi-1DSH3 and the plasmid DNA pBabepuro, encoding puromycin resistance, and the recipient cells were selected by growth in 2 mg/ml puromycin. Several puromycin-resistant colonies were picked and expanded, and the level of Abi-1DSH3 expression was assessed by Western blot using the 9E10 monoclonal antibody as before. Clones expressing high (C11, C14) and intermediate (C3, C5) levels of the protein, and clones cotransformed only with the empty pMT21 vector DNA (f2), were chosen for further analysis (Figure 7). Serial 10-fold dilutions of the P160 strain of A-MuLV were used to infect these lines, and transformed foci were counted after 2 weeks as before (Table 2). All the lines were highly sensitive to transformation by the virus, and even the lines expressing very high levels of Abi-1DSH3 were as sensitive as the controls. These results indicate that the SH3 domain of Abi-1 is required for its inhibition of transformation, and suggest that a direct interaction of the protein with v-Abl is required for its activity. Discussion The results presented above demonstrate that the highlevel overexpression of the full-length Abi-1 protein, like the N-terminally truncated Abi-1D1 ± 85 version used previously (Shi et al., 1995), can also inhibit v-Abl transformation of ®broblast cells. The magnitude of the inhibition of transformation was similar for the two forms of the protein. In addition, an alternatively spliced form that lacks an internal block of 29 residues

Figure 7 Overexpression of Abi-1DSH3 in NIH3T3 cells. NIH3T3 cells were cotransfected with plasmids expressing Abi1DSH3 with a myc epitope tag and a gene for puromycin resistance. Puromycin resistant clones were picked and expanded, and whole cell lysates were prepared. Western blots of cellular extracts were performed using monoclonal anti-myc antibody. Lanes C, individual lines expressing Abi-1DSH3; Lane f2, a line transformed by the empty vector DNA without insert. Lane M, molecular weight size markers Oncogene

Table 2 Lack of inhibition of A-MuLV transformation by deletion mutant Abi-1DSH3 Cell line f2 C3 C5 C11 C14

Abi-1DSH3 expressiona

FFUb (P160)

Nonec + + ++ ++

46105 2.26104 66105 36105 46105

a

Levels of Abi-1DSH3 expression assessed by Western blot. None, no detectable expression. +, intermediate level. ++, high levels of expression. bTransformation susceptibility of cell lines. Apparent titer of A-MuLV preparations as focus-forming units (FFU) per ml. cLine arising after transformation with empty vector lacking insert

was similarly able to inhibit transformation. This form of the protein lacked one of its putative SH3 binding sites, suggesting that this particular site was not crucial to the activity of the protein. The inhibition required high-level expression, and was lost whenever Abi-1 expression fell to low levels. We note that although both full-length and the Nterminally truncated forms of the protein show similar inhibitory activity, their mechanisms of action may not be identical. In transient transfection assays, the fulllength Abi-1 protein potently blocked v-Abl induced activation of Erk2, a signaling kinase downstream of the Ras eector molecule (Fan and Go, 2000). This inhibition of Ras signaling is a plausible means by which the full-length protein blocks v-Abl transformation. Signi®cantly, the inhibition occurred with no changes in the levels of v-Abl protein. While the truncated protein Abi-1D1 ± 85 also blocked Erk2 activation by v-Abl, it caused a strong downregulation of the v-Abl protein levels as detected by Western blots (Fan and Go, 2000). One possibility is that the truncated Abi-1 can under some circumstances act to target both the protein itself and associated proteins for proteosomal degradation through the ubiquitin pathway. This change in v-Abl levels may re¯ect a distinct mechanism of inhibition for the truncated version of Abi-1. The inhibition of P160 transformation by high level expression of Abi-1 was not absolute and could be overcome at low frequency to give rise to rare transformed colonies. The transformed clones expressed normal levels of v-Abl, but had without exception lost detectable expression of the Abi-1 protein, including both long and short forms (Figure 4b). The loss of protein expression occurred without a signi®cant loss of Abi-1 mRNA levels, suggesting posttranscriptional downregulation. These results are consistent with the possibility that Abi-1 is being targeted for rapid degradation upon transformation, and may be analogous to the loss of Abi-2 protein by ubiquitin-mediated proteosomal degradation that was detected upon transformation by BCR ± ABL in a dierent setting (Dai et al., 1998). The loss would then allow for the transforming activity of the P160v-abl kinase to act on essential downstream targets.


In contrast to the eects on P160, there was very little inhibition of P90A transformation by the overexpression of Abi-1. This result suggests that the strongest inhibition requires an interaction between the two proteins, and that the P90A protein escapes the eect through the loss of the carboxyterminal binding site for Abi-1. However, it should be noted that is also possible that the P90A kinase may evade the eects for other reasons. For example, P90A lacks a large portion of the wild-type protein, and is known to fail to interact with many other partners, including the Jak1 kinase (Danial et al., 1998) and actin (McWhirter and Wang, 1993; Van Etten et al., 1994), that may modulate P160 function. These alterations may allow the protein to indirectly avoid the Abi-1 eects. Further, even in this case, there were some eects on Abi-1 levels and structure in the P90A transformed clones; expression was sometimes lost, and altered proteins were expressed in some clones. Thus, P90A may still interact indirectly with some function of Abi1. However, additional experiments suggest that a direct interaction of Abi-1 and v-Abl is required for inhibition of transformation. Overexpression of a mutant form of Abi-1 lacking the C-terminal SH3 domain needed to bind to v-Abl, even at very high protein levels, did not cause detectable inhibition of transformation. Thus, the inhibition by Abi-1 may well involve a direct binding to v-Abl. The overexpression of Abi-1 is likely to bind up proteins critical for v-Abl transformation and block their normal function. These proteins may well include Eps8 or Sos and components of the Ras pathway, but other partners may also be involved. However Abi-1 may act, the loss of Abi-1 protein upon transformation in mouse ®broblasts provides a useful marker of transformation susceptibility and therefore of transformation itself. If a similar loss of expression occurs consistently in CML and not in other leukemias, it may serve as a useful marker in clinical settings as well.

Materials and methods DNA manipulations A mouse liver cDNA library was used to isolate additional 5' Abi-1 sequences with a 5' RACE kit (Clontech). Two oligonucleotides (5'-CATCCTAATACGACTCACTATAGGGC-3', provided with the kit, and 5'-ACTATACTGAACTACTGCAGCTTCCTC-3', derived from the Abi-1 coding sequence), were used as DNA polymerase chain reaction (PCR) primers. Touchdown PCR conditions described by the manufacturer were utilized in a Perkin Elmer thermal cycler. The PCR products were resolved by agarose gel electrophoresis and cloned using standard methods (Maniatis et al., 1982). Sequencing was performed on plasmid templates using an automated BioSequencer (Applied Biosystems). The fulllength cDNA of each alternatively spliced form of Abi-1 was inserted into the pSRa expression vector, which expresses ORFs with an appended 3' myc epitope tag (gift of Audrey Minden, Columbia University).

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Preparation of cell lines and analysis of proteins NIH3T3 cells were cotransfected with plasmids pSRa-Abi-1 or pMT21-Abi-1DSH3, and with pGKpuro or pBabe-puro, to provide a selectable puromycin resistance marker, by the calcium phosphate precipitation method. The cells were grown in Dulbecco's modi®ed Eagle's medium (DMEM) supplemented with 10% bovine calf serum (BCS) and 2 ± 10 mg/ml of puromycin. Puromycin resistant clones were expanded and analysed for expression of Abi-1. Cells were lysed in RIPA buer (150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0, 1 mM PMSF, and 10 mg/ml each of aprotinin and leupeptin), and whole-cell lysates were subjected to Western blot analysis with 9E10 a-myc antibodies (Santa Cruz). For subsequent experiments, Abi-1 overexpressing cell lines were maintained in DMEM supplemented with 10% BCS and 2.5 mg/ml puromycin. v-Abl transformed cell lines were prepared by picking morphologically transformed foci and expanding in DMEM supplemented with 5% BCS and 2.5 mg/ml puromycin. Expression of P160 or P90Av-Abl protein was determined by Western blot analysis of whole cell lysates with rabbit monoclonal antibody K12 (Santa Cruz). A-MuLV focus-forming assays P160 viral stocks were prepared by transfection of ecotropic phoenix cells (Kinsella and Nolan, 1996) with pGDN-v-Abl DNA containing the A-MuLV provirus; supernatant virus was harvested after 48 h. In some experiments, P160 stocks were also prepared by cotransfection of NIH3T3 cells with the A-MuLV proviral DNA plus pNCA M-MuLV helper DNA. P90A stocks were prepared by rescuing virus from transformed nonproducer NIH3T3 cells (Rosenberg and Witte, 1980) with M-MuLV helper virus. To determine the virus concentration, infections were performed by inoculation of cells with 10-fold serial dilutions of viral stocks. Upon reaching con¯uence, the cells were maintained in DMEM supplemented with 2% BCS and 2.5 mg/ml puromycin for 2 weeks. Transformed foci were then counted. RNA analysis by RNase protection Whole cell RNA was prepared from parental NIH3T3 cells and Abi-1 overexpressing lines by the RNAzol B RNA isolation method (TEL-TEST). An antisense RNA probe was prepared by insertion of a 375-bp cDNA fragment encompassing the myc tag and 3' segment of Abi-1 (nucleotides 1178 ± 1428) into pBluescript SK (Stratagene). The resulting clone was linearized and used as template for in vitro transcription with T3 RNA polymerase according to the Maxiscript protocol (Ambion). Ten micrograms of each RNA sample was hybridized to the probe, and the products were digested with RNase A plus T1 using the RPA II ribonuclease protection assay kit (Ambion). The digests were resolved by electrophoresis on a 5% polyacrylamide/8 M urea gel and detected by autoradiography according to the speci®cations of the manufacturer.

Acknowledgments This work was supported by Public Health Service grant P01 CA 23767 from the National Cancer Institute. H-Y Yang and G Gao are Associates, and SP Go is an Investigator of the Howard Hughes Medical Institute. Oncogene


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