Malignant activation of several DH domain-encoding oncogenes including dbl
has been shown to result from molecular alterations that result in N- or C-terminal truncations, although the DH and PH domains remain intact (8
). Here we describe the molecular alteration responsible for formation of the lbc
oncogene. Our results demonstrate both at the genomic and cDNA levels that the lbc
oncogene transcriptional unit is derived by C-terminal truncation of the lbc
proto-oncogene located on chromosome 15 and subsequent fusion with unrelated sequence derived from chromosome 7. This conclusion is based on the finding that the onco-lbc
genomic clone TL encodes lbc
transcribed sequence which maps to chromosomes 15 and 7. Analysis of onco-lbc
cDNA clones shows that the truncation site corresponds to the end of the proto-lbc
PH domain. We further find that the ensuing chromosome 7-derived sequence in the onco-lbc
cDNA supplies a short in-frame 3′ sequence followed by a termination codon and 3′ untranslated sequence. This chromosome 7-derived sequence does not hybridize to any of the human tissue mRNAs that we have tested by Northern blotting (results not shown). Taken together, these results indicate that the fused chromosome 7-derived sequence in the onco-lbc
C terminus provides an in-frame stop codon for the truncated proto-lbc
The original LBC leukemia DNA whose transfection into NIH 3T3 cells yielded the lbc
oncogene was classified as a lymphoid blast crisis phase of a Ph+ (Philadelphia chromosome) chronic myeloid leukemia sample, and we have previously shown that lbc
is expressed in human leukemic lymphoblastic cell lines (49
). Here we report that Southern blot analysis reveals no difference in the gross lbc
gene structures of normal human and LBC sample DNAs. This strongly suggests (but does not prove) that the original leukemia cells did not contain the structurally altered onco-lbc
gene, and hence it is likely that the genetic alteration which gave rise to onco-lbc
occurred serendipitously during the course of the transfection process. This is a common event responsible for generating several transfection-derived cellular oncogenes (6
). However, primary human leukemia samples can exhibit considerable cellular heterogeneity, and the conversion of chronic-phase chronic myeloid leukemia to blast phase is reported to be accompanied by acquisition of multiple poorly defined chromosomal alterations (44
). Therefore, it may also be that only a small fraction of the original LBC cells contained a rearranged lbc
gene not detectable by Southern blot analysis of LBC DNA. Precedent for this is provided by several reported cases in which activated ras
oncogenes occur in only a fraction of the neoplastic cells (46
); furthermore, the in vivo assay used to detect the lbc
oncogene is known to be sufficiently sensitive to detect oncogenes present in low levels in a sample (50
). While PCR analysis could resolve this issue, no additional LBC sample is available for further study. In either case, for pathobiological relevance, lbc
oncogene activation would be expected to occur in more than a single cancer sample, and this possibility is currently being investigated.
Isolation of lbc
proto-oncogene cDNAs demonstrates that onco- and proto-lbc
encode identical N termini coding for an EF hand motif and DH and PH domains. After base 1972, however, the proto-lbc
open reading frame extends for an additional novel 1,434 bp not present in onco-lbc
. Whether isolated from skeletal muscle tissue or from hematopoietic cells (unpublished results), we have found this proto-oncogenic C terminus to be invariant in sequence. Therefore, the oncogenic form of lbc
essentially represents the N-terminal half of the proto-oncogene. Comparison of the translated sequence of the proto-specific C terminus to those of known proteins reveals an ~110-amino-acid region (residues 651 to 763) with similarity to an extensive list of proteins, many of which are cyto-matrix associated, such as trichohyalin, plectin, caldesmon, INCENP, and myosin. In all of these proteins and in Lbc, this region is rich in the residues E/Q/R, and this likely provides the basis for the observed homology. While this region in Lbc is predicted to form an α-helical structure, it is shorter than in the homologous proteins (110 residues versus at least 300 residues). The role of this region in the known proteins appears to fall into two categories. First, this region is predicted to form a rod-like α-helical domain crucial for dimerization and higher-order assembly, such as for caldesmon (21
) and myosin (4
). Second, this homology region is strongly implicated in affecting protein-protein association, such as INCENP association with microtubules (31
) and the association of the plectin rod domain with vimentin or lamin B (11
). Therefore, this region in Lbc may play such a role. Intriguingly, a predicted α-helical region is also present in the dbl
proto-oncogene product and, analogous to Lbc, is missing in the Dbl oncoprotein (42
), strongly suggesting that structural and/or functional features of such domains in this family of oncogenes can normally suppress transforming activity. While in the case of Dbl this region encodes a heptad repeat motif characteristic of a coiled-coil structure (41
), this does not seem to be the case for Lbc. The α-helical region of Lbc also contains a putative leucine zipper that may confer additional protein-protein association, although this motif is found in many proteins of different categories and it is far from being a specific pattern. Following the α-helical region is a proline-rich sequence (residues 782 to 790). This sequence contains a minimal PXXP core motif (P, proline; X, any amino acid) shown to provide SH3 domain binding sites (38
). Therefore, this region in Lbc may be a potential SH3 binding site, and its precise role is under investigation.
Expression of the proto-lbc
cDNA in NIH 3T3 and COS cells yields a protein product corresponding to a predicted size of 102 kDa that does not appear to be heavily posttranslationally modified in mammalian cells. We report here that both the onco- and proto-Lbc protein products promote formation of GTP-bound Rho in COS cells, demonstrating their GEF activity in vivo. In the absence of exogenous exchange activity, a higher percentage of exogenously expressed RhoA was found in the GTP-bound form (48%) compared to the level observed for other Ras-related small GTP binding proteins such as Ras and Ral (10 to 20%) (10
). This finding has been observed by others with COS-7 cells (12
), but the reason is not clear. Expression of proto-Lbc increases the percentage of GTP-bound Rho by ~30%, and expression of onco-Lbc results in an increase of 44%.
When the transforming activities of proto- versus onco-Lbc cDNAs were compared by NIH 3T3 focus formation assays, proto-Lbc was found to have ≤10% of the level of activity of onco-Lbc, even though the cDNAs were expressed at comparable levels under the control of the same promoter. While proto-Lbc-induced foci are much reduced in number, they nevertheless display characteristic Lbc morphology, indicating that relatively high-level expression of proto-Lbc by the strong promoter of the pSRαNeo vector can lead to weak transforming activity. However, loss of the proto-Lbc C terminus clearly has a major effect on amplifying transforming activity. An analogous situation is observed for Dbl where high-level expression of proto-Dbl results in weak transformation (41
), although only the structurally altered oncoprotein is potently transforming (41
). While a considerable difference in the transforming activities of onco- and proto-Lbc is observed, the difference in GEF activities between these two forms in vivo is modest. This suggests that GEF activity levels alone may not be sufficient to account for the biological difference between onco- and proto-Lbc and that loss of C-terminal function synergizes with GEF activity to elicit potent transformation. However, detection of a potentially greater difference in GEF activities between the two forms upon measurement of endogenous GTP-Rho cannot be ruled out.
Analysis of the role of the proto-Lbc C terminus in transformation reveals that deletion of the proline-rich motif results in an ~twofold increase in transforming activity compared to that of full-length proto-Lbc, suggesting that the proline-rich sequence may make a modest contribution to controlling transforming activity. Further truncation of the α-helical region results in a significant increase in transforming activity, resulting in ~50% of the activity of onco-Lbc. While this demonstrates that the α-HEL 15 construct is significantly transforming, its activity is not equal to that of ONC 4A. This indicates that the remaining 235-amino-acid sequence between the PH domain and the α-helical region, which does not encode any known domains or motifs, appears to exert a significant inhibitory effect on transformation. Since an onco-Lbc cDNA mutant that contains a deletion of the chromosome 7-derived C terminus (TR4) still retains a high level of transforming activity, these findings demonstrate that it is loss of the proto-Lbc C terminus, rather than gain of unrelated sequence by the truncated proto-oncogene, that confers potent oncogenicity.
All DH domains are closely followed by a PH domain, indicating some coordinate function (5
). Mutational analysis of the onco-Lbc DH domain demonstrates that an intact DH domain is necessary for Lbc transforming ability. This result is in agreement with that found for many other DH domain-encoding oncoproteins (8
) and confirms our earlier findings that activation of the Rho signaling pathway by Lbc is an integral part of Lbc transformation (45
). In addition, we find that deletion of the entire onco-Lbc PH domain, or mutation of the conserved tryptophan at position 404 in the PH domain, significantly inhibits Lbc transformation. This illustrates the critical role of the PH domain in Lbc transformation, and the importance of the conserved W residue to PH domain function, and is consistent with the findings for other DH domain-encoding oncoproteins (8
). Although we have not directly tested the GEF activities of the Lbc PH domain mutants in vitro, almost certainly they still retain exchange activity, since our earlier in vivo results obtained by using microinjection show that these mutants are still fully capable of inducing Rho-dependent actin cytoskeletal changes in fibroblasts, in contrast to onco-Lbc DH domain mutants, which retain no cytoskeletal activity (37
). Taken together, these results indicate that the PH domain does not directly determine GEF activity but may regulate it in some way in vivo that is required during cellular transformation.
Within the past few years, the importance of correct intracellular targeting for oncoprotein activity has been brought to light (28
). Therefore, we analyzed the subcellular localization of Lbc in order to gain more insight into its mechanism of transformation. High-speed cell fractionation analysis revealed a substantial difference between onco- and proto-Lbc localization. The proto-Lbc product was observed to localize predominantly to the particulate, membrane-associated fraction. Interestingly, similar findings have been reported for the Ras exchange factor, Ras-GRF (7
), and for Tiam-1, a Rac exchange factor (47
), indicating that membrane localization may be a common feature of GTP exchange factors for Ras superfamily small GTP proteins involved in cell growth control. In contrast to the proto-Lbc localization, >50% of onco-Lbc is in the soluble, cytosolic fraction. These results indicate that Lbc transforming activity may correlate with release from a membrane-associated location.
Analysis of Lbc PH domain mutants indicate that the PH domain does not play a major role in determining proto-Lbc membrane localization, although previous data indicate that it may influence onco-Lbc localization to the cytoskeleton (37
). Other exchange factors such as Ras-GRF and Tiam-1 each encode an N-terminal PH domain in addition to a second PH domain that is in tandem to the DH domain in these proteins (7
). In both cases, the N-terminal PH domain is shown to be required for the particulate localization of these proteins (7
). Lbc does not contain an additional PH domain, and it may be that isolated PH domains such as those present in Ras-GRF and Tiam-1 play a different role than those of PH domains found in tandem with DH domains. Furthermore, in the case of Ras-GRF, replacement of the N-terminal PH domain with a heterologous PH domain still targets the protein to the particulate fraction but is not sufficient for Ras-GRF activation, indicating that the PH domain has an additional, critical function other than membrane localization (7
). This result is in agreement with our observations reported here that the Lbc PH domain has some currently unknown critical function required for cell transformation other than membrane localization. Understanding of the considerable diversity of known PH domain ligands (17
) is increased by the report that the PH domain of Dbl confers cytoskeletal, rather than membrane, localization (56
). Additional reports on analogous PH domains of the Ras exchangers Sos (35
) and Vav (16
) indicate a role for phospholipid binding and signal transduction via phosphatidylinositol 3-kinase and present the possibility that the Lbc PH domain could serve a similar function.
Interestingly, the proto-Lbc C terminus alone was observed to localize predominantly to the particulate fraction. While the weakly transforming proto-Lbc PP43 mutant also strongly localizes to the particulate fraction, the more active α-HEL mutant shows some relocalization to the cytosolic fraction. This indicates that a cytosolic localization correlates with a gain in transforming activity and that the greatest cytosolic concentration occurs with the fully transforming onco-Lbc that lacks the C terminus. These results indicate that at least one function of the Lbc C terminus is to confer correct location of the proto-Lbc product required for controlling oncogenic activity. Part of this requirement may be to mediate interaction with currently unknown elements that also localize to the membrane and serve to inhibit transformation. In addition, membrane localization of proto-Lbc may serve to limit access to the physiological substrate of Lbc GEF activity, Rho. Rho localizes predominantly to the cytosol, although a small fraction is thought to cycle to and from the membrane (1
), and this fraction is presumed to be the biologically active fraction. Thus, abnormal cytosolic localization of onco-Lbc could result in sustained Rho activation and consequent promotion of oncogenicity. Based on the results reported here, future experiments will address the precise identification of cellular components that interact with the C terminus and regulate proto-Lbc subcellular targeting and cell transformation.