In this study, we have taken two complementary approaches to examine the role of Jak-STAT activation in v-Abl function. First, we have defined a region in the carboxyl terminus of v-Abl that is required for Jak1 association both in vitro and in vivo. Deletion of this domain abrogates the ability of v-Abl to activate Jak1 and STATs in different cell lines. In addition, BAF/3 transfectants expressing the Jak1 binding mutant of v-Abl are unable to proliferate in the absence of IL-3 and are less tumorigenic in nude mice than are BAF/3 cells expressing the wild-type protein. Second, we have used a kinase-inactive mutant of Jak1 to show that Jak1 function is required for v-Abl to activate STATs and to stimulate proliferation of BAF/3 cells independent of cytokines. Together, these results indicate that Jak1 is important in several aspects of v-Abl function.
The Jak1 interaction domain of v-Abl maps to a novel region (aa 858 to 1080) in the carboxyl terminus of the protein, which was previously of unknown function. To our knowledge, no other signaling protein has been shown to interact with this domain. Although this region is required for Jak1 association, it is possible that other v-Abl domains or other Abl-interacting proteins contribute to Jak1 activation by v-Abl. Using in vitro binding assays, we can occasionally detect interaction of Jak1 with a GST fusion protein expressing the SH2 and the kinase domains of v-Abl. Our previous studies have demonstrated that the Abl kinase activity is required for v-Abl-dependent activation of the Jak-STAT pathway. Interestingly, association of Jak1 and v-Abl in vivo is diminished but not lost at nonpermissive temperature (9
). It is therefore possible that the binding of Jak1 to the C terminus of v-Abl serves primarily to bring Jak1 close to the v-Abl catalytic (kinase) domain and that the interaction between v-Abl SH2 domain and tyrosine-phosphorylated Jak1 further stabilizes this complex.
Cells that are naturally dependent on cytokines for growth can, in the presence of v-Abl, proliferate independently of cytokines. Our observations suggest that Jak1 is required for v-Abl-mediated proliferation of BAF/3 cells. This is especially interesting because several signaling molecules previously suggested to be involved in proliferative signals by v-Abl, including Shc, p62 Dok, and myc, do not show a reduced level of activation in cells expressing the Jak1 binding mutant form of v-Abl. Therefore, it appears that Jak1 may play an important role in mitogenic signals downstream of v-Abl. Although recent studies suggest that STATs may be required for maximal proliferation induced by cytokines (29
), it is not clear whether proliferation of v-Abl-expressing transfectants is dependent on STAT activation. In addition to STATs, Jaks may target other signaling pathways. For instance, a link between Jaks and Ras has been suggested (56
). Consistent with a possible role for Jak1 in Ras regulation, we found that the level of the GTP-bound form of Ras is slightly diminished downstream of the Jak1 binding mutant of v-Abl. The level of GTP Ras in BAF/3 cells expressing this mutant, however, is still higher than that seen in parental cells. This may be due to the recruitment of signaling proteins such as Grb-2, which have been shown to bind the proline-rich sequences 3′ of the Abl kinase domain (43
). Despite Ras activation, we and others did not detect a significant level of activated ERK1/ERK2 MAP kinases downstream of v-Abl (reference 36
and data not shown). It is possible that downstream of v-Abl, and in the context of BAF/3 cells, Ras is involved in regulation of other signaling proteins, such as PI3-kinase. Ras and PI3-kinase have been reported to regulate each other (12
). Consistent with this, and in accord with a recent report suggesting the requirement for Jak1 in activation of PI3-kinase (1
), we found a diminished level of p85 phosphorylation downstream of the Jak1 binding mutant of v-Abl (data not shown). Although the exact molecular mechanism underlying the proliferation defect in cells expressing the v-Abl mutant awaits further studies, our observations suggest that Jak1 activation downstream of v-Abl may serve to regulate multiple signaling pathways.
The ability of v-Abl to bind and activate Jak proteins led us to question whether this interaction is important in cellular transformation. Indeed, Jak proteins have been implicated in oncogenic processes (28
). A gain-of-function mutant allele of hopscotch
, a homologue of Drosophila
Jak, can also lead to transformation (19
). Our data clearly show that in BAF/3 cells, v-Abl–Jak1 association and Jak1 activation are important in cytokine-independent proliferation. However, factor-independent growth per se does not lead to transformation (7
). Signaling pathways activated by the abl
oncogene therefore might not solely mimic events triggered by receptor-cytokine interaction. Because we have been unable to obtain A-MuLV that contains the Jak1 binding mutant of v-Abl, we addressed the importance of Jak activation by v-Abl in nude-mouse assays with BAF/3 cells. We found that BAF/3 cells expressing the Jak1 binding mutant of v-Abl are somewhat impaired in generating tumors in nude mice. Since these assays were done with a long-term tissue culture cell line, they do not necessarily completely mimic the transformation process in vivo. Cellular transformation appears to require the activation of several different complementary pathways. Since secondary genetic alterations tend to accumulate in long-term tissue culture cell lines, some of these pathways may already be active in the BAF/3 cell line, making these cells more permissive for transformation by the Jak1 binding mutant of v-Abl. Alternatively, since some tumors do eventually appear in these mice, the Jak1 binding domain of v-Abl may be important for high-efficiency tumor formation but not for absolute transformation. Analysis of primary cells expressing the mutant v-Abl and examination of Jak1-deficient mice for efficiency of v-Abl-induced transformation are needed to further address the importance of Jak1 in transformation by v-Abl.
Although the human oncogenic form of Abl, Bcr-Abl, and the murine oncogenic form v-Abl have many common characteristics, several structural and functional differences between the two have been reported. It appears that the activation of Jak-STAT signaling by these two forms of Abl may be another example of biological differences between these oncoproteins. Unlike v-Abl, Bcr-Abl does not associate with Jak proteins (5
). It is possible that sequences within the Bcr region or the Abl SH3 domain which are present in Bcr-Abl, but not v-Abl, contribute to protein folding or protein-protein interactions rendering C-terminal sequences inaccessible for Jak1 binding. Alternatively, the C terminus of Bcr-Abl might not be able to bind Jak proteins. Comparison of Abl protein sequences reveals that although there is a high degree of homology (99%) between murine and human c-Abl in the SH2 and kinase domains (15
), the C-terminal region (corresponding to aa 858 to 1080 of v-Abl) are 68% identical. Therefore, it is possible that critical amino acids required for Jak1 binding within this region are not present in the human Bcr-Abl oncoprotein. Interestingly, constitutive activation of Jak proteins has not been consistently detected in all Bcr-Abl-expressing cells (4
). These data are consistent with the observation that kinase-inactive mutants of Jak2 do not block constitutive activation of Stat5 in Bcr-Abl-expressing BAF/3 cells (23
). Thus, activation of STATs by Bcr-Abl may be Jak independent. It is possible that Bcr-Abl directly activates STATs or that it targets its kinase activity to STATs through a different interacting protein.
One of the paradoxes of v-Abl biology is that although A-MuLV can bind to most cell types, the tumors that develop in mice infected with this virus are almost exclusively pre-B-cell leukemias. The etiology of this specificity remains unknown. Accumulating evidence suggests that the carboxyl terminus of v-Abl may play an important role in regulating this function of the oncoprotein. Experiments in which various regions of src
oncogenes were used to generate hybrid retroviral genomes have shown that the C-terminal domain of v-Abl, in addition to the 3′ end of the Abl kinase domain, is sufficient to confer a pre-B-cell transforming property to src
). Although all C-terminal truncation mutants of A-MuLV examined thus far can transform NIH 3T3 cells, they exhibit reduced efficiency of bone marrow transformation. The majority of these mutants are also somewhat impaired in their ability to generate tumors in mice (22
). The Abl oncoprotein has been shown to activate many signaling pathways, several of which may be required for transformation. It is possible that in some cells (e.g., NIH 3T3 cells), signaling pathways that require the intact carboxyl terminus of v-Abl, such as the cytokine signaling pathway, are not essential for transformation, perhaps due to the activation of other compensatory pathways. Given our data and the transformation phenotype of carboxyl-terminal truncation mutants of A-MuLV, it would be interesting to examine whether activation of the Jak-STAT pathway by the carboxyl terminus of v-Abl may play a unique role in the selectivity with which A-MuLV functions in its in vivo targets.