The Signaling Pathways of BCRABL

The transforming capacity of BCR-ABL is largely based on its ability to associate and interact with numerous proteins that connect it to various pathways of intracellular signaling. To understand the structural characteristics of BCR-ABL that facilitate these types of interactions, it is first necessary to dissect the functional sequences of the ABL and BCR proteins, respectively, and then to analyze their contribution to the transformed phe-notype in the chimeric protein.

ABL encodes a 145-kd protein (p145ABL) that functions as a nonreceptor tyrosine phosphokinase.45 ABL is primarily located in the nucleus but also found in the cytoplasm where it can also be associated with adhesion molecules. Its ubiquitous expression and the rigorous control of its tyrosine kinase activity hint at an important role in cellular biology.46 ABL knockout mice have a high incidence of neonatal death, have defects in Band T-cell development, are more susceptible to infections, and show numerous developmental abnormalities of various organ systems.47-49 The ABL protein appears to be involved in signal transduction and regulation of cell growth, as overexpression of

ABL results in growth arrest of cells during G1 possibly through direct interaction with the p53 and the retinoblastoma tumor suppressor products.46 It has also been suggested that ABL is activated in the stress response to DNA damaging agents regulating induction of the SEKl/stress-activated protein kinase pathway.50'51 The N-terminal segment of ABL has structural similarity to C-SRC. It contains three SRC homology (SH) domains, SH1 to SH3, the catalytic domain, and a myristoyla-tion sequence facilitating cytoplasmic localization. The C-ter-minal segment of ABL includes a DNA-binding domain, nuclear localization signals, and a binding site for actin (Figure 4-3). SH2 and SH3 regulate the tyrosine kinase function of ABL. SH2 attaches to tyrosine phosphorylated proteins that are targets for ABL kinase activity through three conserved arginine residues.52,53 Defects in the functional integrity of SH2 cause a decrease in phosphotyrosine binding and a decrease in the transforming capabilities of ABL. Conversely, exchanging the SH2 domain by SH2 domains from other proteins maintains the transforming activities of ABL, but changes the cellular target proteins of tyrosine phosphorylation. SH3 has been ascribed negative regulatory activity on the tyrosine kinase function of ABL and may possibly interact with cellular inhibitors.54,55 Mutational analysis of ABL has demonstrated that deletion of SH3 facilitates the transforming activity of ABL even in the absence of BCR or GAG sequences.56

An example of structural alterations of ABL that facilitate its transformation into a protein with leukemogenic potential comes from murine leukemias. The Abelson murine leukemia virus (A-MuLV) is a replication defective virus that in vivo induces primarily nonthymic pre-B-cell lymphomas in susceptible mice.21 A-MuLV is a fusion product of viral GAG sequences and a viral homologue of cellular ABL. GAG-ABL encodes a GAG-ABL protein of 160 kd (p160GAG-ABL) and, by internal in-frame deletion, a second smaller protein of 120 kd (p120GAG-ABL).57 In either case, GAG is attached to the N-ter-minus of ABL from which the SH3 domain has been lost (Figure 4-3). Furthermore, the GAG sequences enhance the stability of the GAG-ABL fusion protein thus facilitating their transformation.58 These events lead to localization of the fusion molecule to the cytoplasm and increased phosphotyrosine kinase activity, a mechanism that appears also at the core of BCR-ABL transforming activities.

The BCR gene codes for two proteins of 160 kd and 190 kd, respectively.59,60 Although the role of the BCR proteins is still largely unknown, several multifunctional domains of BCR have been identified. The N-terminal coiled-coil motif contains a 63 amino acid sequence, common to both p210BCR-ABL and p190BCR-ABL, that seems to be crucial for enhancing F-actin binding and tyrosine kinase activity of ABL.61 The N-terminal portion of BCR itself possesses serine/threonine kinase activity

Figure 4-3. The protein domains of ABL, BCR, the BCR-ABL fusion proteins p190BCR-ABL' p210BCR-ABL' p230BCR-ABL, and GAG-ABL from the Abelson murine leukemia virus (AMuLV). Note that the BCR-ABL proteins contain the SH3 domain of ABL that is missing in GAG-ABL.

and contains tyrosine residue 177 (Y177), which is phosphory-lated by the ABL tyrosine kinase and serves as a docking site for various adapter proteins (see below).62 The central portion of BCR is occupied by DBL protooncogene-like sequences. DBL catalyzes the exchange of guanine nucleotides on the RAS-related molecule Cdc42Hs and is also referred to as GDP-GTP exchange factor (GEF).63>64 The C-terminus of BCR contains sequences homologous to the catalytic domains of GTPase activating proteins (GAP) of the GTP protein binding family p21rho and p21 rac (Figure 4-3). Members of this family control the rate of GTP hydrolysis of active RAS proteins to their inactive GDP-bound form thus creating a connection to downstream intracellular signaling pathways.65

What determines the transforming activity of ABL in the BCR-ABL fusion? ABL can be activated by itself through internal deletions and point mutations, or by fusion with GAG. In the case of BCR-ABL, however, the SH3 domain is retained. It has been suggested that parts of BCR, by steric hindrance through binding of the first exon of BCR to the SH2 domain of ABL, may interfere with the adjacent SH3 regulatory domain thereby constitutively enhancing the ABL tyrosine phosphoki-nase activity. In addition, alterations of the N-terminus of ABL

that redistribute its location from the nucleus into the cytoplasm and enhance its F-actin binding capacity contribute to the transforming characteristics of BCR-ABL.66,67

The functional domains of BCR-ABL facilitate a multitude of interactions with other cytoplasmic proteins, referred to as adapter proteins, that link BCR-ABL to downstream intracellu-lar signaling pathways. These connections influence expression of gene complexes that generate the phenotypic manifestations of CML that are expressed in altered regulations of cell cycle, programmed cell death (apoptosis), and adhesion (Figure 4-4). An ever-increasing number of proteins is being identified that are complexed with BCR-ABL or represent substrates for tyro-sine phosphorylation by BCR-ABL. These include RAS,68 the phosphatidylinositol-3-kinase (PI3-kinase),69,70 proteins of the JAK/STAT pathway,71 NFkB,72 Src kinases (Hck and Lyn),73 JUN,74 MYC, and focal adhesion kinases (FAK).75

Involvement of RAS proteins has been determined as a major signaling pathway of BCR-ABL. RAS is a G-protein that occupies a key role in signal transduction, proliferation, and malignant transformation. In the active, GTP-bound state, RAS proteins link signals of activated growth factor receptors to downstream mitogenic effectors.76 The state of activity of RAS

Fak Src Pathway

Figure 4-4. Through its tyrosine kinase activity, BCR-ABL can attach to numerous adapter proteins. On phosphorylation, these in turn connect BCR-ABL to downstream protein kinases and enzymes that further transmit the "message" of BCR-ABL to the nucleus. The most important of these downstream effectors are RAS, the JAK/STAT system, and the PI-3K. The signaling network activates the transcription and expression of genes that impact proliferation and differentation of the cell, its regulation of apoptosis, and adhesion to the microenvironment.

Figure 4-4. Through its tyrosine kinase activity, BCR-ABL can attach to numerous adapter proteins. On phosphorylation, these in turn connect BCR-ABL to downstream protein kinases and enzymes that further transmit the "message" of BCR-ABL to the nucleus. The most important of these downstream effectors are RAS, the JAK/STAT system, and the PI-3K. The signaling network activates the transcription and expression of genes that impact proliferation and differentation of the cell, its regulation of apoptosis, and adhesion to the microenvironment.

is regulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). GAPs enhance the rate of GTP hydrolysis to GDP and negatively regulate RAS function, whereas GEFs induce exchange of GDP for GTP on RAS and act as activators.77 Evidence for a role of RAS in BCR-ABL signaling is derived from the observation in murine myeloid cells that activated RAS is constitutively elevated, and the fact that dominant negative RAS mutants in RAT-1 fibroblast cells completely block transformation by BCR-ABL.78'79 BCR-ABL is linked to RAS through several adapter proteins such as GRB2, SHC, CRKL, and CBL. GRB2 is linked with its SH2 domain to the conserved tyrosine residue Y177 within the first exon of BCR in the BCR-ABL molecule.80 The importance of this association for the activation of RAS is evident from the observation that mutant forms of GRB2 reverse BCR-ABL-induced transformation.81 Binding of GRB2 to BCR-ABL activates a further adapter protein, SOS ("son-of-sevenless"), which in turn recruits RAS. CRKL, an adapter protein that is tyrosine-phosphorylated only in Ph-positive cells, is the most prominent phosphoprotein in CML cells. It binds to proline-rich sequences of BCR-ABL and activates RAS through its ability to bind to SOS and another CRK SH3-binding guanine nucleotide-releasing protein termed C3G.82,83 Signaling downstream of RAS is less well characterized. One major path appears to involve association with RAF1 and subsequent activation of MEK kinase and extracellular signal-regulated kinase (ERK). A second pathway involves MEK kinases (MEKK) and the JUN kinase (JNK) pathway, also referred to as stress-activated protein kinase (SAPK) pathway.84

Alternatively, RAS can be activated by inhibiting its negative regulator RAS GAP. Recently, another BCR-ABL adapter protein has been identified called p62dok. Hematopoietic progenitor cells isolated from patients with CML in the chronic phase contain constitutively tyrosine-phosphorylated p62dok protein. Under these conditions, p62dok associates with RAS GAP thus inhibiting its activity.85 p62dok was found to form complexes with the SH2 domain-containing phosphatidylinositol polyphosphate 5-phosphatase SHIP1 in hematopoietic cells expressing BCR-ABL, suggesting more complex interactions of these proteins in BCR-ABL transformed hematopoietic progenitor cells.86 Other instances where negative regulatory pathways may be antagonized by the activity of p210BCR-ABL include protein-tyrosine phosphatase-lB (PTP-1B), which counteracts signaling of p210BCR-ABL and the ABL-interactor molecule (ABI-1).87,88

A second major pathway of BCR-ABL signaling involves PI3-kinase. PI3-kinase can be activated upon stimulation with multiple growth factors. It is composed of an 85-kd (p85) regulatory subunit that acts as an adapter sequence through its SH2 and SH3 domains, and a 110-kd (p110) catalytic sequence that is responsible for its enzymatic activity.89 PI3-kinase may be linked to BCR-ABL through CBL (casitas B-lineage lymphoma protein), a protooncogene product that is tyrosine-phosphorylated in BCR-ABL expressing cells. Through interaction of PI3-kinase protein kinase B (PKB) and AKT, BCR-ABL is directly linked to regulatory elements of apoptosis favoring expression of antiapoptotic effects mediated by BCL-XL and BCL-2.90 It has also been suggested that BCR-ABL positive cells are able to downregulate cyclin-dependent kinase inhibitor p27, thus interfering with the regulation of the cell cycle protein and promoting a proliferative and antiapoptotic phenotype of the CML cells.91-92

Constitutive activation of JAK/STAT proteins has been reported in cell lines and samples from patients expressing BCR-ABL.93-94 STAT proteins (signal transducers and activators of transcription) link cytokine receptor stimulation to gene transcription. Once tyrosine phosphorylated by JAK family kinases, STAT proteins dimerize, translocate to the nucleus, and bind to specific DNA elements.93,94 Constitutive tyrosine phosphorylation and activation of STAT1, 5, and 6 have been demonstrated in CML cells. Although the significance of STAT activation in CML is still unclear and differences in activation of specific STAT proteins seem to exist in pi90BCR-ABL and p210BCR-ABL-containing cells, it has been suggested that they may represent another link to induce antiapoptotic mechanisms and growth-factor independent growth.95-99

Another important target of p210BCR-ABL signaling in CML is MYC, a transcription factor involved in cell cycle regulation. Evidence for a role of MYC stems from experiments that demonstrated high levels of MYC RNA in p210BCR-ABL transformed cells, facilitation of transformation by overexpression of MYC, and blocking of transformation by expression of a mutant, dominant negative form of MYC.100

CRKL seems to establish a link between p210BCR-ABL and focal adhesion molecules such as FAK, actin, and paxillin.101,102 Recently, NFkB has been implicated in signaling pathways of p210BCR-ABL. NFkB is a transcription factor that translocates to the nucleus upon activation by growth-factor or cytokine stimulation. Transcription of NFkB has been identified as an inhibitor of apoptosis, but its regulation is complex and it may also be activated in cells undergoing apoptosis in response to serum starvation.103 It has been suggested that BCR-ABL activates NFKB-dependent transcription. Activation is dependent on tyrosine phosphorylation of NFkB and is partially RAS-dependent. Although NFkB does not appear to be necessary to enable BCR-ABL to protect cytokine-dependent 32D myeloid cells from apoptosis induced by cytokine deprivation or DNA damage, it is nevertheless required for BCR-ABL mediated tumorigenicity in nude mice and for transformation of primary bone marrow cells.104

The activity of p210BCR-ABL generates activation or suppression of a set of target genes that confer the malignant pheno-type on the Ph-positive cell. Some of the early genes identified have been FOS, JUN, and MYC. However, most of the data on altered gene expression through activation of p210BCR-ABL are derived from cell lines transformed by ectopic expression of p210BCR-ABL rather than acquiring the translocation naturally. Many target genes remain unidentified to date.105

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  • eemil
    How does the BCRAbl protein affect intracellular signaling pathways?
    4 months ago

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