Abl tyrosine kinase

EVOLUTIONARY HOMOLOGS part 3/3

Nuclear activities of Abl

The nuclear function of the c-Abl tyrosine kinase is not well understood. In order to identify nuclear substrates of Abl, a constitutively active and nuclear form of the protein was constructed. Active nuclear Abl efficiently phosphorylates c-Jun, a transcription factor not previously known to be tyrosine phosphorylated. After phosphorylation of c-Jun by Abl on Tyr170, c-Jun and Abl interact via the SH2 domain of Abl. Surprisingly, elevated levels of c-Jun activate nuclear Abl, resulting in activation of the JNK serine/threonine kinase. This phosphorylation circuit generates nuclear tyrosine phosphorylation and represents a reversal of previously known signaling models (Barila, 2000).

These data can be summarized in a model, according to which elevated c-Jun levels can activate Abl in the nucleus. Activation requires the central part of the c-Jun protein and the presence of Tyr170 in order to be efficient. In turn, and consistent with previous reports, activation of nuclear Abl results in an increase in JNK activity. This may occur through a mechanism that involves a positive effect on a JNK activator, a negative effect on a JNK repressor, or it could even be direct. Once activated, JNK may then phosphorylate c-Jun at the N-terminal sites and increase c-Jun stability, DNA binding and transcriptional activity. If JNK then dissociates from Jun after phosphorylation, as proposed, this would allow more JNK-free Jun to activate Abl and result in a positive feedback loop (Barila, 2000).

The mechanism by which c-Jun could achieve activation of nuclear Abl is not clear. One possibility is that c-Jun activates Abl allosterically, by direct binding to Abl. The data show that nuclear Abl is regulated by intramolecular interactions involving the SH2-catalytic domain linker, as occurs for the bulk cellular Abl protein and for Abl synthesized in reticulocyte lysate. It is assumed that c-Jun binding to Abl may interfere with the regulatory intramolecular interactions. Initially, tyrosine phosphorylation of c-Jun may not be required for this activation and could occur, for example, via interaction of the proline-rich sequences in c-Jun with the Abl SH3 domain. Alternatively, basal phosphorylation of c-Jun by Abl may suffice to initiate the positive feedback loop. Although Abl and/or Arg seem to be required for c-Jun activation of JNK, the possibility cannot be excluded that c-Jun, or other AP-1 family members, can activate JNK by a mechanism that does not involve Abl in other cell types or under different physiological circumstances. It will be interesting to test whether some of the severe defects in mice lacking c-Jun can be attributed to defects in activating JNK or related kinases (Barila, 2000 and references therein).

The ubiquitously expressed nonreceptor tyrosine kinase c-Abl contains three nuclear localization signals, however, it is found in both the nucleus and the cytoplasm of proliferating fibroblasts. A rapid and transient loss of c-Abl from the nucleus is observed upon the initial adhesion of fibroblasts onto a fibronectin matrix, suggesting the possibility of nuclear export. The C terminus of c-Abl does indeed contain a functional nuclear export signal (NES) with the characteristic leucine-rich motif. The c-Abl NES can functionally complement an NES-defective HIV Rev protein (RevDelta3NI) and can mediate the nuclear export of glutathione-S-transferase. The c-Abl NES function is sensitive to the nuclear export inhibitor leptomycin B. Mutation of a single leucine (L1064A) in the c-Abl NES abrogates export function. The NES-mutated c-Abl, termed c-Abl NES(-), is localized exclusively to the nucleus. Treatment of cells with leptomycin B also leads to the nuclear accumulation of wild-type c-Abl protein. The c-Abl NES(-) is not lost from the nucleus when detached fibroblasts are replated onto fibronectin matrix. Taken together, these results demonstrate that c-Abl shuttles continuously between the nucleus and the cytoplasm and that the rate of nuclear import and export can be modulated by the adherence status of fibroblastic cells (Taagepera, 1998).

The large carboxyl-terminal segment of c-Abl contains a DNA-binding domain that is necessary for the association of c-Abl with chromatin. The DNA-binding activity of c-Abl is lost during mitosis when the carboxyl-terminal segment becomes phosphorylated. In vitro phosphorylation of the DNA-binding domain by cdc2 kinase abolishes DNA binding. Homozygous mutant mice expressing a c-Abl tyrosine kinase without the DNA-binding domain have been reported to die of multiple defects at birth. Thus, binding of the c-Abl tyrosine kinase to DNA may be essential to its biological function (Kipreos, 1992).

The c-Abl tyrosine kinase protein is implicated in the signaling pathway as well as in transcription, DNA repair, apoptosis, and several other vital biological processes essential for cell proliferation or differentiation. The interaction of c-Abl with DNA is important for some of these functions, but the exact nature of this interaction is still a matter of controversy. The present study addresses the DNA-binding properties of the human c-Abl protein. Using CASTing experiments, the consensus binding site 5'-AA/CAACAAA/C was determined. The central highly conserved AAC triplet appears to constitute the crucial core element in the binding sequences of the c-Abl protein. The c-Abl DNA-binding domain recognizes specific sequences and interacts with deformed DNA structures such as four-way junctions and bubble DNA containing a large single-stranded loop, as determined by electromobility shift assay, melting temperature studies, and binding to specific oligonucleotides covalently linked to beads. Additional competition experiments suggest that the interaction mainly involves contacts within the minor groove of the double helix. The DNA-binding properties of c-Abl are reminiscent of those of high-mobility group (HMG)-like proteins such as LEF-1 and SRY. However, the circular permutation and ring closure assays and DNA unwinding experiments reveal that, unlike HMGs, c-Abl does not bend its target sequence. In addition, it is shown that the protein potentiates the DNA relaxation activity of topoisomerase I. These findings indicate that the interaction of c-Abl with DNA is both sequence-selective and structure-dependent (David-Cordonnier, 1998).

The c-abl proto-oncogene encodes a nuclear tyrosine kinase that can phosphorylate the mammalian RNA polymerase II (RNAP II) on its C-terminal repeated domain (CTD). Phosphorylation of the CTD has previously been shown to require the tyrosine kinase and the SH2 domain of Abl. A CTD-interacting domain (CTD-ID) at the C-terminal region of c-Abl is also required. Deletion of the CTD-ID causes a two-fold increase in the Km of binding to polymerase. Direct binding of the CTD-ID to CTD and to RNAP II can be demonstrated in vitro. Phosphorylation of the CTD by c-Abl is observed in cotransfected COS cells. Mutant Abl proteins lacking the CTD-ID, while capable of autophosphorylation, neither phosphorylate nor associate with the CTD in vivo. Transient overexpression of c-Abl also leads to a four- to fivefold increase in the phosphotyrosine content of the RNAP II large subunit. Moreover, the large subunit of RNAP II can be coprecipitated with c-Abl. Tyrosine phosphorylation of the coprecipitated RNAP II is dependent on the presence of the CTD-ID in Abl. The ability of c-Abl to phosphorylate and associate with RNAP II can be correlated with the enhancement of transcription by c-Abl. Taken together, these observations demonstrate that c-Abl can function as a CTD kinase in vitro as well as in vivo (Baskaran, 1996).

The carboxyl-terminal repeated domain (CTD) of the mammalian RNA polymerase II is a substrate for the Abl. This specificity is conferred in part by the SH2 domain. The Abl SH2 domain binds the tyrosine-phosphorylated (Tyr[P]) CTD and is required for the processive and stoichiometric phosphorylation of the 52 tyrosines in the CTD. Mutation of the Abl SH2, or exchanging it with that of Src (which does not bind the Tyr[P]-CTD) abolishes processivity and reduces the CTD kinase activity without any effect on autophosphorylation or the phosphorylation of nonspecific substrates. These results demonstrate that the SH2 domain of the Abl tyrosine kinase plays an active role in catalysis and suggests that SH2 domain and the tyrosine kinase domain may act in concert to confer substrate specificity (Duyster, 1995).

The E2F family of transcription factors regulates cell cycle progression, and deregulated expression of E2F-1 can lead to neoplastic transformation. In myeloid cells, introduction and expression of the Abelson leukemia virus causes growth factor independence. The p120 v-Abl protein activates E2F-1-mediated transcription through a physical interaction with the E2F-1 transcription factor. BCR-Abl and c-Abl also stimulate E2F-1-mediated transcription. These results suggest a new mechanism by which v-Abl leads to factor-independent myeloid cell proliferation: the activation of E2F-1-mediated transcription (Birchenall-Roberts, 1997).

The decision to enter the cell division cycle is governed by the interplay between growth activators and growth inhibitors. The retinoblastoma protein (see Drosophila Rb ) is an example of a growth inhibitor whose main function appears to be the binding and inactivation of key cell cycle activators. One target of RB is a proto-oncoprotein, the c-Abl tyrosine kinase. RB binds to the ATP-binding lobe in the kinase domain and inhibits the nuclear pool of c-Abl in quiescent and G1 cells. Phosphorylation of RB at G1/S releases c-Abl, leading to the activation of this nuclear tyrosine kinase. An Abl mutant was constructed, replacing the ATP-binding lobe of c-Abl with that of c-Src. The mutant protein AS2 is active as a tyrosine kinase and can phosphorylate Abl substrates, such as the C-terminal repeated domain of RNA polymerase II. AS2, however, does not bind to RB, and its activity is not inhibited by RB. As a result, the nuclear pool of AS2 is no longer cell cycle regulated. Excess AS2, but not its kinase-defective counterpart, can overcome RB-induced growth arrest. Interestingly, wild-type c-Abl, in both its kinase-active and -inactive forms, can also overcome RB. Furthermore, overexpression of a kinase-defective c-Abl in rodent fibroblasts accelerates the transition from quiescence to S phase and cooperates with c-Myc to induce transformation. These effects, however, do not occur with the kinase-defective form of AS2. Thus, the growth-stimulating function of the kinase-defective c-Abl is dependent on the binding and the abrogation of RB function. RB function can be abolished by the overproduction of one of its binding proteins; this is consistent with the hypothesis that RB induces cell cycle arrest by acting as a "molecular matchmaker" to assemble protein complexes. Exclusive engagement of RB by one of its many targets is incompatible with the biological function of this growth suppressor protein (Welch, 1995).

The v-abl oncogene of Abelson murine leukemia virus encodes a deregulated form of the cellular nonreceptor tyrosine kinase. v-Abl activates c-myc transcription, and c-Myc is an essential downstream component in the v-Abl transformation program. To explore the mechanism by which v-Abl activates c-myc transcription, a cotransfection assay was developed. Transactivation of a c-myc promoter by v-Abl requires the SH1 (tyrosine kinase) and SH2 domains of v-Abl; the C-terminal domains are not required for transactivation. The assay also identifies the E2F site in the c-myc promoter as a v-Abl-responsive element. In addition, multimerized E2F sites were shown to be sufficient to confer v-Abl-dependent activation on a minimal promoter. This is the first identification of a v-Abl response element for transcriptional activation. v-Abl tyrosine kinase-dependent changes in proteins binding the c-myc E2F site have also been demonstrated, including induction of a complex containing DP1, p107, cyclin A, and cdk2. Identification of v-Abl-dependent changes in E2F-binding proteins provides an important link between v-Abl, transcription, cell cycle regulation, and control of cellular growth (Wong, 1995).

The retinoblastoma protein (pRb) acts to constrain the G1-S transition in mammalian cells. Phosphorylation of pRb in G1 inactivates its growth-inhibitory function, allowing for cell cycle progression. Phosphorylation of S780 results in a lose of Rb's ability to bind to E2F. Phosphorylation of S807 and/or S811 is required to abolish Rb binding to c-Abl, while modification of threonine 821 and or T826 is required to abolish Rb binding to LXCXE-containing proteins such as simian virus 40 large T antigen. Although several cyclins and associated cyclin-dependent kinases (cdks) have been implicated in pRb phosphorylation, the precise mechanism by which pRb is phosphorylated in vivo remains unclear. By selectively inhibiting either cdk4/6 or cdk2, it has been shown that endogenous D-type cyclins, acting with cdk4/6, are able to phosphorylate pRb only partially, a process that is likely to be completed by cyclin E-cdk2 complexes. Cyclin E-cdk2 is unable to phosphorylate pRb in the absence of prior phosphorylation by cyclin D-cdk4/6 complexes. Complete phosphorylation of pRb, inactivation of E2F binding, and activation of E2F transcription occur only after the sequential action of at least two distinct G1 cyclin kinase complexes (Lundberg, 1998).

The growth suppression function of RB is dependent on its protein binding activity. RB contains at least three distinct protein binding functions: (1) the A/B pocket, which binds proteins with the LXCXE motif; (2) the C pocket, which binds the c-Abl tyrosine kinase; and (3) the large A/B pocket, which binds the E2F family of transcription factors. Phosphorylation of RB, which is catalyzed by cyclin-dependent protein kinases, inhibits all three protein binding activities. LXCXE binding is inactivated by the phosphorylation of two threonines (Thr821 and Thr826), while the C pocket is inhibited by the phosphorylation of two serines (Ser807 and Ser811). The E2F binding activity of RB is inhibited by two sets of phosphorylation sites acting through distinct mechanisms. Phosphorylation at several of the seven C-terminal sites can inhibit E2F binding. Phosphorylation of two serine sites in the insert domain can inhibit E2F binding, but this inhibition requires the presence of the RB N-terminal region. RB mutant proteins lacking all seven C-terminal sites and two insert domain serines can block Rat-1 cells in G1. These RB mutants can bind LXCXE proteins, c-Abl, and E2F even after they become phosphorylated at the remaining nonmutated sites. Thus, multiple phosphorylation sites regulate the protein binding activities of RB through different mechanisms, and a constitutive growth suppressor can be generated through the combined mutation of the relevant phosphorylation sites in RB (Knudsen, 1997).

c-Ab1 is a non-receptor protein-tyrosine kinase lacking a clear physiological role. A clue to its normal function is suggested by overexpression of Ab1 in fibroblasts, which leads to inhibition of cell growth. This effect requires tyrosine kinase activity and the Ab1 C-terminus. c-Ab1 is localized to the cell nucleus, where it can bind DNA, and interacts with the retinoblastoma protein, a potential mediator of the growth-inhibitory effect. Nuclear localization of Ab1 can be directed by a pentalysine nuclear localization signal in the Ab1 C-terminus. There are two additional basic motifs in the Ab1 C-terminus, either of which may function independently of the pentalysine signal to localize Ab1 to the nucleus. Both c-Ab1 and transforming Ab1 proteins inhibit entry into S phase and this effect is absolutely dependent on nuclear localization. The Ab1 cytostatic effect requires both the Rb and p53 tumor suppressor gene products. These results indicate that Ab1 inhibits cell proliferation by interacting with central elements of the cell cycle control apparatus in the nucleus, and suggests a direct connection between p53 and Rb in this growth-inhibitory pathway (Wen, 1996).

Transformation of B-lineage precursors by the Abelson murine leukemia virus appears to arrest development at the pre-B stage. Abelson-transformed pre-B cell lines generally retain transcriptionally inactive, unrearranged immunoglobulin kappa alleles. Nontransformed pre-B cells expanded from mouse bone marrow efficiently transcribe unrearranged kappa alleles. In addition, they contain activated complexes of the NF-kappa B/Rel transcription factor family, in contrast to their Abelson-transformed counterparts. Using conditionally transformed pre-B cell lines, it has been shown that the v-abl viral transforming protein, a tyrosine kinase, blocks germ-line kappa gene transcription and negatively regulates NF-kappa B/Rel (Drosophila homologs Dorsal and Dif) activity. An active v-abl kinase specifically inhibits the NF-kappa B/Rel-dependent kappa intron enhancer, which is implicated in promoting both transcription and rearrangement of the kappa locus. v-abl inhibits the activated state of NF-kappa B/Rel complexes in a pre-B cell via a post-translational mechanism that results in increased stability of the inhibitory subunit I kappa B alpha (Drosophila homolog: Cactus). This analysis suggests a molecular pathway by which v-abl inhibits kappa locus transcription and rearrangement (Klug, 1994).

Deregulated expression of v-abl and BCR/abl genes has been associated with myeloproliferative syndromes and myelodysplasia, both of which can progress to acute leukemia. These studies identify the localization of the oncogenic form of the abl gene product encoded by the Abelson murine leukemia virus in the nuclei of myeloid cells and the association of the v-Abl protein with the transcriptional regulator cyclic AMP response element-binding protein (See Drosophila CrebB-17A). The specific domains within each of the proteins responsible for this interaction have been mapped. Complex formation is a prerequisite for transcriptional potentiation of CREB. Transient overexpression of the homologous cellular protein c-Abl also results in the activation of promoters containing an intact CRE. These observations identify a novel function for v-Abl, that of a transcriptional activator that physically interacts with a transcription factor (Birchenall-Roberts. 1995).

The p53 tumor suppressor is inhibited and destabilized by Mdm2. However, under stress conditions, this downregulation is relieved, allowing the accumulation of biologically active p53. c-Abl is important for p53 activation under stress conditions. In response to DNA damage, c-Abl protects p53 by neutralizing the inhibitory effects of Mdm2. Does this neutralization involves a direct interplay between c-Abl and Mdm2, and what is the contribution of the c-Abl kinase activity? The kinase activity of c-Abl is required for maintaining the basal levels of p53 expression and for achieving maximal accumulation of p53 in response to DNA damage. Importantly, c-Abl binds and phosphorylates Mdm2 in vivo and in vitro. Hdm2 (human Mdm2) phosphorylation at Tyr394 was characterized. Substitution of Tyr394 by Phe394 enhances the ability of Mdm2 to promote p53 degradation and to inhibit its transcriptional and apoptotic activities. The results suggest that phosphorylation of Mdm2 by c-Abl impairs the inhibition of p53 by Mdm2, hence defining a novel mechanism by which c-Abl activates p53 (Goldberg, 2002).

Abl response to DNA damage

The gene mutated in the autosomal recessive disorder ataxia telangiectasia (AT), designated ATM (for "AT mutated"), is a member of a family of phosphatidylinositol-3-kinase-like enzymes involved in cell-cycle control, meiotic recombination, telomere length monitoring and DNA-damage response. Previous studies have demonstrated that AT cells are hypersensitive to ionizing radiation and are defective at the G1/S checkpoint after radiation damage. Because cells lacking the protein tyrosine kinase c-Abl are also defective in radiation-induced G1 arrest, an investigation was made into the possibility that ATM might interact with c-Abl in response to radiation damage. The results show that ATM binds c-Abl constitutively in control cells but not in AT cells. These results demonstrate that the SH3 domain of c-Abl interacts with a DPAPNPPHFP motif (residues 1,373-1,382) of ATM. The results also reveal that radiation-induction of c-Abl tyrosine kinase activity is diminished in AT cells. These findings indicate that ATM is involved in the activation of c-Abl by DNA damage and this interaction may in part mediate radiation-induced G1 arrest. Other studies show that exposure of cells to ionizing radiation also induces c-Abl/p53 complexes and causes a p53-dependent downregulation of Cdk2 and subsequent G1 arrest (Shafman, 1997).

The c-Abl protein tyrosine kinase is activated by ionizing radiation and certain other DNA-damaging agents, whereas the DNA-dependent protein kinase (DNA-PK), consisting of a serine/threonine kinase and Ku DNA-binding subunits, requires DNA double-strand breaks or other DNA lesions for activation. c-Abl interacts constitutively with DNA-PK. Ionizing radiation stimulates binding of c-Abl to DNA-PK and induces an association of c-Abl with Ku antigen. DNA-PK phosphorylates and activates c-Abl in vitro. Cells deficient in DNA-PK are defective in c-Abl activation induced by ionizing radiation. In a potential feedback mechanism, c-Abl phosphorylates DNA-PK, but not Ku, in vitro. Phosphorylation of DNA-PK by c-Abl inhibits the ability of DNA-PK to form a complex with DNA. Treatment of cells with ionizing radiation results in phosphorylation of DNA-PK that is dependent on c-Abl (Kharbanda, 1997).

The DNA-dependent protein kinase (DNA-PK) controls the repair of double-stranded DNA breaks in mammalian cells. The protein kinase subunit of DNA-PK (DNA-PKcs) is targeted to DNA breaks by association with the Ku DNA-binding heterodimer. A Ku association site is present at the carboxyl terminus of DNA-PKcs (amino acids 3002-3850) near the protein kinase domain. Correspondingly, the nuclear c-Abl tyrosine kinase that associates with DNA-PK also binds to the kinase homology domain. The c-Abl SH3 domain binds to amino acids 3414-3850 of DNA-PKcs. c-Abl phosphorylates C-terminal fragments of DNA-PKcs, particularly amino acids 3414-3850. c-Abl phosphorylation of DNA-PKcs disassociates the DNA-PKcs.Ku complex. Thus, Ku and c-Abl provide opposing functions with regard to DNA-PK activity (Jin, 1997).

Activation of the c-Abl protein tyrosine kinase by certain DNA-damaging agents contributes to downregulation of Cdk2 and G1 arrest by a p53-dependent mechanism. This study investigates the potential role of c-Abl in apoptosis induced by DNA damage. Transient transfection studies with wild-type, but not kinase-inactive, c-Abl demonstrate induction of apoptosis. Cells that stably express inactive c-Abl exhibit resistance to ionizing radiation-induced loss of clonogenic survival and apoptosis. Cells null for c-abl are also impaired in the apoptotic response to ionizing radiation. Cells deficient in p53 undergo apoptosis in response to expression of c-Abl and exhibit decreases in radiation-induced apoptosis when expressing inactive c-Abl. These findings suggest that c-Abl kinase regulates DNA damage-induced apoptosis (Yuan, 1997a).

Cancer chemotherapeutic agents such as cisplatin exert their cytotoxic effect by inducing DNA damage and activating programmed cell death (apoptosis). The tumor-suppressor protein p53 is an important activator of apoptosis. Although p53-deficient cancer cells are less responsive to chemotherapy, their resistance is not complete, which suggests that other apoptotic pathways may exist. A p53-related gene, p73, which encodes several proteins as a result of alternative splicing, can also induce apoptosis. The amount of p73 protein in the cell is increased by cisplatin. This induction of p73 is not seen in cells unable to carry out mismatch repair and in which the nuclear enzyme c-Abl tyrosine kinase is not activated by cisplatin. The half-life of p73 is prolonged by cisplatin and by co-expression with c-Abl tyrosine kinase; the apoptosis-inducing function of p73 is also enhanced by the c-Abl kinase. Mouse embryo fibroblasts deficient in mismatch repair or in c-Abl do not upregulate p73 and are more resistant to killing by cisplatin. These results indicate that c-Abl and p73 are components of a mismatch-repair-dependent apoptosis pathway that contributes to cisplatin-induced cytotoxicity (Gong, 1999).

c-Abl, a non-receptor tyrosine kinase, is activated by agents that damage DNA. This activation results in either arrest of the cell cycle in phase G1 or apoptotic cell death, both of which are dependent on the kinase activity of c-Abl. p73, a member of the p53 family of tumor-suppressor proteins, can also induce apoptosis. The apoptotic activity of p73alpha requires the presence of functional, kinase-competent c-Abl. Furthermore, p73 and c-Abl can associate with each other, and this binding is mediated by a PxxP motif in p73 and the SH3 domain of c-Abl. p73 is a substrate of the c-Abl kinase and the ability of c-Abl to phosphorylate p73 is markedly increased by gamma-irradiation. Moreover, p73 is phosphorylated in vivo in response to ionizing radiation. These findings define a pro-apoptotic signaling pathway involving p73 and c-Abl (Agami, 1999).

c-Abl and Atm in oxidative stress response are mediated by protein kinase Cdelta

c-Abl and Atm have been implicated in cell responses to DNA damage and oxidative stress. However, the molecular mechanisms by which they regulate oxidative stress response remain unclear. In this report, deficiency of c-Abl and deficiency of ATM are shown to differentially alter cell responses to oxidative stress; these signaling proteins function by induction of antioxidant protein peroxiredoxin I (Prx I) via Nrf2 and cell death, both of which require protein kinase C (PKC) delta activation and are mediated by reactive oxygen species. c-abl^-/- osteoblasts display enhanced Prx I induction, elevated Nrf2 levels, and hypersusceptibility to arsenate, which are reinstated by reconstitution of c-Abl; Atm^-/- osteoblasts show the opposite. These phenotypes correlate with increased PKC delta expression in c-abl^-/- osteoblasts and decreased PKC delta expression in Atm^-/- cells, respectively. The enhanced responses of c-abl^-/- osteoblasts can be mimicked by overexpression of PKC delta in normal cells and impeded by inhibition of PKC delta, and diminished responses of Atm^-/- cells can be rescued by PKC delta overexpression, indicating that PKC delta mediates the effects of c-Abl and ATM in oxidative stress response. Hence, these results unveiled a previously unrecognized mechanism by which c-Abl and Atm participate in oxidative stress response (Li, 2004).

How does c-Abl or Atm regulate the protein level of PKC delta? c-Abl or Atm may affect the transcription of PKC delta gene, the stability of PKC delta mRNA or protein, or the translation efficiency of PKC delta mRNA. RT-PCR assays did not reveal any significant difference in the levels of PKC delta mRNA among wild-type and c-Abl-deficient osteoblasts, suggesting that c-Abl regulates PKC delta expression posttranscriptionally. It was found that c-Abl deficiency inhibits activation-induced degradation of PKC delta, but the molecular mechanism behind this warrants further investigation. Studies have indicated that in cells expressing activated Src (Y527F), PKC delta was down-regulated. This down-regulation is a result of phosphorylation-mediated degradation. It is speculated that c-Abl, a member of the Src family, may have a similar function in regulating the level of PKC delta. It has also been shown that c-Abl interacts with PKC delta in response to oxidative stress. c-Abl is able to phosphorylate PKC delta in fibroblasts. Unfortunately, PKC delta immunoprecipitated from c-Abl-deficient and control osteoblasts did not show significant difference in phosphorylation at tyrosine residues. One possible explanation is that PKC delta might have multiple sites for tyrosine phosphorylation that are carried out by several kinases. Hence, c-Abl deficiency would not make a detectable difference. The role for Atm in the regulation of PKC delta expression is even less clear. RT-PCR analysis revealed no significant difference in the levels of PKC delta mRNA, suggesting that the regulation, like that of c-Abl, occurs at posttranscriptional levels. Surprisingly, degradation of PKC delta was similar in Atm^-/- osteoblasts and wild-type cells. One likely explanation is that the portion of degraded PKC delta molecules in Atm^-/- osteoblasts may have a shortened lifespan, whereas the rest have a normal lifespan. Treatment of Atm^-/- osteoblasts with MG132, a proteosome inhibitor, appeared to increase the PKC delta levels to that of control osteoblasts. The molecular mechanisms by which Atm regulates PKC delta protein levels need further investigation. Because Atm interacts with c-Abl and can activate it, it is possible that there exists a tertiary complex composed of PKC delta, c-Abl, and Atm in the cells, and that c-Abl may mediate the function of Atm in controlling PKC delta expression (Li, 2004).

Another layer of complexity is that Prx I/PAG is also a c-Abl interacting protein. c-Abl, a nonreceptor tyrosine kinase, plays a negative role in Prx I induction. Without c-Abl, osteoblasts show an enhanced induction of Prx I. On the basis of these facts, it is proposed that in normal osteoblasts, the induction of Prx I is suppressed, facilitating the activation of c-Abl. When c-Abl is deficient, the suppression is lifted and more Prx I is expressed. Therefore, a feedback circuit may exist that controls the activity of c-Abl in response to stress. Alternatively, interaction between c-Abl and Prx I may be involved in regulating the antioxidant activity of Prx I, for example, phosphorylation of Prx I by c-Abl. One such example is that Prx I could be phosphorylated by cdc2 and this phosphorylation reduces the activity of Prx I (Li, 2004).

Drug resistance of BCR-ABl variants

The Abl tyrosine kinase inhibitor STI-571 is effective therapy for stable phase chronic myeloid leukemia (CML) patients, but the majority of CML blast-crisis patients that respond to STI-571 relapse because of reactivation of Bcr-Abl signaling. Mutations of Thr-315 in the Abl kinase domain to Ile (T315I) have been described in STI-571-resistant patients and likely cause resistance from steric interference with drug binding. Mutations of Tyr-253 in the nucleotide-binding (P) loop of the Abl kinase domain to Phe or His have been identified in patients with advanced CML and acquired STI-571 resistance. Bcr-Abl Y253F demonstrates intermediate resistance to STI-571 in vitro and in vivo when compared with Bcr-Abl T315I. The response of Abl proteins to STI-571 is influenced by the regulatory state of the kinase and by tyrosine phosphorylation. The sensitivity of purified c-Abl to STI-571 is increased by a dysregulating mutation (P112L) in the Src homology 3 domain of Abl but decreased by phosphorylation at the regulatory Tyr-393. In contrast, the Y253F mutation dysregulates c-Abl and confers intrinsic but not absolute resistance to STI-571 that is independent of Tyr-393 phosphorylation. The Abl P-loop is a second target for mutations that confer resistance to STI-571 in advanced CML, and the Y253F mutation may impair the induced-fit interaction of STI-571 with the Abl catalytic domain rather than sterically blocking binding of the drug. Because clinical resistance induced by the Y253F mutation might be overcome by dose escalation of STI-571, molecular genotyping of STI-571-resistant patients may provide information useful for rational therapeutic management (Roumiantsev, 2002).

The inadvertent fusion of the bcr gene with the abl gene results in a constitutively active tyrosine kinase (Bcr-Abl) that transforms cells and causes chronic myelogenous leukemia. Small molecule inhibitors of Bcr-Abl that bind to the kinase domain can be used to treat chronic myelogenous leukemia. Crystal structures of the kinase domain of Abl in complex with two such inhibitors, imatinib (also known as STI-571 and Gleevec) and PD173955 (Parke-Davis), is reported. Both compounds bind to the canonical ATP-binding site of the kinase domain, but they do so in different ways. As shown in a crystal structure of Abl bound to a smaller variant of STI-571, STI-571 captures a specific inactive conformation of the activation loop of Abl in which the loop mimics bound peptide substrate. In contrast, PD173955 binds to a conformation of Abl in which the activation loop resembles that of an active kinase. The structure suggests that PD173955 would be insensitive to whether the conformation of the activation loop corresponds to active kinases or to that seen in the STI-571 complex. In vitro kinase assays confirm that this is the case and indicate that PD173955 is at least 10-fold more inhibitory than STI-571. The structures suggest that PD173955 achieves its greater potency over STI-571 by being able to target multiple forms of Abl (active or inactive), whereas STI-571 requires a specific inactive conformation of Abl (Nagar, 2002).

The Bcr-Abl fusion protein kinase causes chronic myeloid leukemia and is targeted by the signal transduction inhibitor STI-571/Gleevec/imatinib (STI-571). Sequencing of the BCR-ABL gene in patients who have relapsed after STI-571 chemotherapy has revealed a limited set of kinase domain mutations that mediate drug resistance. To obtain a more comprehensive survey of the amino acid substitutions that confer STI-571 resistance, an in vitro screen of randomly mutagenized BCR-ABL was performed and all of the major mutations previously identified in patients were recovered along with numerous others that illuminate novel mechanisms of acquired drug resistance. Structural modeling implies that a novel class of variants acts allosterically to destabilize the autoinhibited conformation of the ABL kinase to which STI-571 preferentially binds. This screening strategy is a paradigm applicable to a growing list of target-directed anti-cancer agents and provides a means of anticipating the drug-resistant amino acid substitutions that are likely to be clinically problematic (Azam, 2003).

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