Signaling Pathways Activated by Oncogenic Forms of Abl Tyrosine Kinase*

v-Abl, encoded by the Abelson murine leukemia virus, is a nonreceptor tyrosine kinase with potent oncogenic activity in mice (for review, see Refs. 1 and 2). A similar human oncoprotein, BCR-ABL, is critical in the pathogenesis of 95% of chronic myelogenous leukemia (CML) and 10% of acute lymphocytic leukemia (for review, see Refs. 3 and 4). The c-Abl proto-oncoprotein was identified as the normal cellular homolog of v-Abl (for review, see Ref. 2). The increased tyrosine kinase activities of v-Abl and BCR-ABL, compared with the cellular c-Abl, correlate with their transforming activities. The molecular mechanisms by which these activated tyrosine kinases cause malignant transformation have remained obscure until the last few years when there have been reports that multiple signaling pathways are activated by v-Abl and/or BCRABL. The intent of this review is to: 1) synthesize the current understanding of v-Abl signaling, 2) identify those signaling pathways that are critical for transformation, and 3) compare v-Abl signaling to BCR-ABL signaling, which has been reviewed elsewhere recently (for review, see Refs. 5 and 6). There are several reasons for studying v-Abl. First, it is a potent transforming oncoprotein, and understanding its immediate substrates and final targets will help us understand the processes required for malignant transformation. Second, although in vitro v-Abl can transform many cell types, in vivo it only transforms pro or preB cells, the early B-lineage cells that have partially or completely rearranged their heavy chain genes (for review, see Ref. 12). The striking pro/preB cell tropism for transformation, in the absence of any evidence of pro/preB-specific viral infection, is likely to reveal regulatory paths that are unique to the early B-lymphocyte lineage. Finally, studying v-Abl in mice provides a convenient approach to identify activities that may be common to (or shared by) v-Abl and the human oncoprotein BCR-ABL. The v-abl oncogene in Abelson murine leukemia virus encodes a fusion protein in which a portion of retroviral Gag protein replaces the SH3 domain of c-Abl (Fig. 1) (for review, see Ref. 1) (7, 8). Removal of the SH3 domain constitutively activates the tyrosine kinase, and a myristoylation site in the Gag moiety confers localization to the inner plasma membrane; both modifications are important in the transforming activity of v-Abl (9). Infection of neonatal mice by Abelson murine leukemia virus results in rapid, 100% fatality because of pro/preB cell tumors. In vitro, v-Abl transforms pro/preB cells as well as myeloid cells and a subset of 3T3 fibroblasts (10) (for review, see Refs. 11 and 12). The human oncogene, BCR-ABL, is the result of a reciprocal chromosomal translocation in which the breakpoint cluster region (BCR) gene on chromosome 22 becomes fused to the c-ABL proto-oncogene on chromosome 9. It encodes a fusion protein in which part of the SH3 domain of c-ABL is replaced by portions of the BCR protein (for review, see Refs. 3 and 4). Different forms of BCR-ABL result when different portions of BCR are included; however, all BCR-ABL proteins have tyrosine kinase activities intermediate between the weaker c-ABL and the stronger v-Abl (13). In vitro BCR-ABL expression confers growth factor independence but is fully transforming only for certain cells (for review, see Ref. 14). v-Abl and BCR-ABL share a C terminus that is unique among non-receptor kinases. It contains a nuclear localization signal, a proline-rich region capable of associating with SH3-containing proteins, a sequence-independent DNA-binding domain and an actin-binding domain (for review, see Refs. 15 and 16).

substrate of the epidermal growth factor and platelet-derived growth factor receptors, that also associates with Shc (36) and inhibits ERK activation in response to epidermal growth factor signaling. 2 BCR-ABL also binds directly to Grb2 (37). Because this association involves the BCR portion of the protein, which is absent in v-Abl, this connection is unique to the human oncogene. However, BCR-ABL also connects to p21 ras and Grb2 via Shc (38), probably by virtue of Shc binding to the SH2 domain (23).
What p21 ras Effectors Are Important following v-Abl Activation?-Although p21 ras often activates the Raf serine kinase leading to activation of ERK, for v-Abl, there are branch points from Raf that exclude ERK. ERK is activated in v-Abl-transformed cells by a poorly understood Raf-independent path (39) and is not activated by BCR-ABL (40,41).
A Ras/Raf-dependent pathway is required for v-Abl-dependent induction of c-myc transcription, which appears to be ERK-independent (20). Expression of dominant negative Myc blocks transformation by either v-Abl or BCR-ABL (42), establishing the importance of this pathway. In the c-myc induction pathway Raf ultimately activates cyclin-dependent kinases, which phosphorylate Rb family proteins and thus activate E2F transcription factors (20). In addition to c-myc, v-Abl also induces mRNA encoding other E2F-dependent genes, which are required for cells to enter S phase including dihydrofolate reductase, ribonucleotide reductase, cyclin A, and cyclin E. 3 The connection from v-Abl/p21 ras /Raf to cyclindependent kinases may be through activation of the Cdc25A phosphatase (43), but there is presently no direct evidence for this. BCR-ABL also activates E2F proteins, leading to induction of c-myc transcription (44), presumably by a Ras-dependent pathway similar to that for v-Abl (20).
Recently, expression of the p19 ARF gene has been shown to be increased in a way that depends on p21 ras (45), E2F activators (46), and c-Myc (47). Up-regulation of p19 ARF leads to an increase in p53 levels (for review, see Ref. 48). Because v-Abl activates p21 ras and E2F proteins and induces c-Myc, it may be that v-Abl also causes an increase of p53 via p19 ARF . It is attractive to speculate that relative induction of p19 ARF versus c-myc and S phase genes might determine whether a cell becomes transformed or undergoes apoptosis in response to v-Abl. In support of this notion, there is evidence that a p53-dependent path inhibits transformation by v-Abl in vivo 4 and in vitro (49) and plays a role in BCR-ABL-dependent blast transformation in CML (50). Also, v-Abl can upregulate p19 ARF and cause p53-dependent apoptosis in Abelson virus-infected primary preB cells (51). Unlike preB transformants derived from normal mice, those from Ink4a/Arf Ϫ/Ϫ mice bypass the crisis that characterizes the transition from primary transformant to established, fully malignant cell line (51).

The Rac GTP-binding Protein Is Activated by v-Abl
Dominant negative Rac blocks v-Abl transformation, establishing a requirement for Rac activation in v-Abl transformation (52).
In this system, ERK and JNK activation was also inhibited by dominant negative Rac, suggesting that these effectors are downstream of v-Abl/Rac (52). Rac is not required for v-Abl-dependent induction of c-myc transcription, which depends on p21 ras and Raf (20), but Rac is necessary for activation transcription dependent on serum or 12-O-tetradecanoylphorbol-13-acetate response elements (52). Studies with dominant negative Rac also show that Rac is required for BCR-ABL-induced leukemogenesis (53). It is not currently clear whether activation of Rac by v-Abl or BCR-ABL proceeds directly or by activation of p21 ras or PI3K, as has been observed in other systems (for review, see Ref. 22).
JNK is activated by both v-Abl and BCR-ABL but by different pathways. v-Abl-dependent JNK activation requires Rac (52), whereas BCR-ABL-dependent JNK activation requires p21 ras (41). Phosphatases may provide another path to JNK activation because BCR-ABL associates with SHPT1, which regulates JNK activity (54). Dominant negative c-Jun inhibits BCR-ABL transformation, demonstrating the importance of JNK activation for BCR-ABL transformation (41).

PI3K Is Activated by v-Abl
v-Abl and BCR-ABL, but not c-Abl, associate with and activate PI3K (55). Interestingly, the accumulation of PI3K products phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4bisphosphate correlates better with v-Abl or BCR-ABL transformation than does association of Abl kinases with PI3K (55,56). v-Abl and BCR-ABL may activate PI3K by more than one pathway because activated p21 ras activates PI3K in other systems (57), and recent work suggests activation of Jak1 by v-Abl might contribute to PI3K activation (58). Inhibition of PI3K blocks proliferation of BCR-ABL-dependent cells, establishing the importance of PI3K for BCR-ABL activity (59). Akt kinase is an important effector of BCR-ABL-activated PI3K because a dominant negative mutant of Akt inhibits BCR-ABL-dependent transformation of murine bone marrow cells (60).

v-Abl Activates Protein Kinase C
IL-3-dependent mast cells transformed with a temperature-sensitive form of v-Abl revealed that v-Abl activates phospholipase C-mediated breakdown of phosphatidylcholine, generating diacylglycerol, which then activates PKC (61). The anti-apoptotic effect of v-Abl in this system was blocked by inhibiting PKC activity, indicating a functional role for PKC. Subsequent studies in the same cells show that the v-Abl/PKC pathway causes an increase in bcl-X L mRNA, which may be responsible for the anti-apoptotic effect (62).

v-Abl Activates Jak/STAT Pathways
The recent discovery that STAT1, -3, -5, and -6 are constitutively activated in v-Abl-transformed proB or preB cells (58,63) led to the attractive model that constitutive activation of STATs by v-Abl confers cytokine independence and is critical for transformation. In normal cells, nuclear translocation of STATs occurs only in response to cytokine binding to receptor and activation of receptorassociated Janus kinases (Jaks) (for review, see Ref. 64). Evidence is accumulating to support the constitutive STAT activation model. The domains of v-Abl that associate with Shc, Jak, and Abi were mapped by direct studies and are indicated by red bars. The region where Crk, Crkl, Nck, and Grb2 bind to v-Abl is inferred from studies on c-Abl and BCR-ABL and is indicated by a blue bar. The region of BCR-ABL that associates with Grb2 is also shown by a blue bar. Jak1 and Jak3 associate directly with v-Abl (58). Deletion of 200 amino acids in the DNA-binding portion of the v-Abl C terminus (Fig. 1) that are required for association with Jak1 results in a mutant v-Abl, which cannot provide cytokine-independent survival of BAF/3 pro-B lymphoblastoid cells. This provides evidence that Jak binding and STAT activation are important for v-Abl-dependent transformation (58). Furthermore, in murine tumors resulting from abl/myc retroviruses, constitutive activation of STAT3 makes the cells IL-6-independent (65). However, cytokine independence is not sufficient to cause transformation because a combination of IL-4 and IL-7 signaling cannot substitute for an active v-Abl kinase in transformed pre-B cells (66). STAT-independent paths from activated Jaks may also be important for v-Abl activity because cytokine-dependent suppression of apoptosis (67) and induction of the anti-apoptotic gene bcl-X L (68) result from a Jak-dependent, STAT-independent path in myeloid cells.
Contrary to v-Abl, BCR-ABL does not activate STATs by a Jakdependent pathway. JAK kinases are not consistently activated in BCR-ABL-positive cells (69,70), and activation of STAT5 by BCR-ABL is not blocked by dominant-negative JAK2 mutants (71). BCR-ABL does not associate with Jaks (71) even though its Cterminal region is identical to that of v-Abl (Fig. 1). Subcellular localization and tyrosine kinase activity differ significantly between the two proteins; the inner plasma membrane localization and/or high tyrosine kinase activity of v-Abl may be critical for Jak association and activation. Nevertheless, STAT1 and STAT5 are constitutively activated in BCR-ABL lines from CML patients (72), and primary peripheral blood cells from patients with CML have constitutive activation of STATs (70). Direct association of STAT SH2 domains with phosphorylated tyrosines on BCR-ABL could mediate Jak-independent activation, but no data are available to prove this (69,71).

Signaling Paths Responsible for Other Effects of v-Abl and BCR-ABL Are Partially Understood
Other downstream consequences are known to result from the action of either v-Abl or BCR-ABL, but the signaling paths leading to them are poorly understood. v-Abl has been reported to stabilize IB, thereby blocking activation of NF-B in preB cells (73). However, the role of NF-B appears to be different for BCR-ABL. Inhibition of NF-B by a non-degradable form of IB␣ showed that NF-B is required for BCR-ABL-mediated tumorigenicity in nude mice and transformation of primary bone marrow cells (74). Activation of NF-B in this system is Ras-dependent.
Both v-Abl and BCR-ABL activate proteasome-dependent degradation of specific proteins. In 3T3 fibroblasts, proteasome-dependent degradation of the cyclin-dependent kinase inhibitor p27 Kip occurs when mitogen-starved or density-arrested cells enter S following v-Abl activation. 4 In CML cells expressing BCR-ABL, proteasome-dependent degradation of the inhibitory protein Abi-2 occurs through a Ras-independent pathway (34). Degradation of p27 and Abi-2 may be induced by a common pathway, but this is not yet proven.
v-Abl and BCR-ABL affect the expression of genes that regulate apoptosis. v-Abl induces bcl-X L mRNA in pre-mast cells by a PKCdependent path (62) but causes up-regulation of Bax in myeloid progenitor cells (75). v-Abl induces both Bcl-2 and Bcl-X L in preB cells (66), but it remains to be shown that this is important for transformation. BCR-ABL induces Bcl-2 mRNA in a Ras-dependent pathway, and Bcl-2 has been shown to be essential for BCR-ABL-mediated transformation (76,77).

Perspectives
The numerous signaling pathways activated by v-Abl are summarized in Fig. 2. The ultimate effect, transformation or apoptosis, is likely to be determined by the relative strength of these signals in different cells. There is much left to learn. With the exception of E2F-dependent genes, few genes have been identified as functionally important final targets of the signaling pathways activated by v-Abl and BCR-ABL. Furthermore, no pathway has been completely characterized and few have been compared in different cell types. Thus, there are likely to be many more connections and many more examples of cross-talk and feedback than we currently understand. There may also be connections that vary in different types of cells. In addition, it is clear that we have little understanding of how a single protein, such as p21 ras or E2F-1, may signal multiple downstream effectors and how the relative strength of signaling to different effectors may be determined.
v-Abl and BCR-ABL activate a remarkably similar set of signaling pathways including p21 ras , Rac, and STATs and induction of c-myc mRNA. However, there are several significant differences. BCR-ABL does not activate ERK but v-Abl does, BCR-ABL does not associate with or activate Jaks but v-Abl does, BCR-ABL associates directly with Grb2 but v-Abl does not, and BCR-ABL activates JNK through Ras whereas v-Abl activates JNK through Rac.
It seems likely that the transforming versus apoptotic activities of v-Abl and BCR-ABL result from a delicate balance between many signaling pathways. Many of these, such as induction of c-myc and other E2F-dependent genes, degradation of p27, and activation of JNK, Rac, and PI3K, lead to cell cycle progression. Other signals such as induction of Bcl-X L and Bcl-2 provide antiapoptotic signals. Finally, it is possible that others, by inducing p19 ARF may lead to p53-dependent cell cycle arrest or apoptosis.

FIG. 2. Summary of signaling pathways activated by v-Abl tyrosine kinase.
Yellow rectangles indicate proteins that have been shown to be required for v-Abl-dependent transformation. Dashed lines indicate paths that are inferred for v-Abl from studies in which the signaling path was activated in response to a different oncoprotein or a mitogen.