Abl1 | GeneID:11350 | Mus musculus
[ ] NCBI Entrez Gene
|Gene ID||11350||Official Symbol||Abl1|
|Synonyms||AI325092; Abl; E430008G22Rik; MGC117749; c-Abl|
|Full Name||c-abl oncogene 1, receptor tyrosine kinase|
|Description||c-abl oncogene 1, receptor tyrosine kinase|
|Chromosome||2 B|2 21.0 cM|
|Also Known As||Abelson murine leukemia oncogene; OTTMUSP00000013005; v-abl Abelson murine leukemia oncogene 1|
Orthologs and Paralogs
|GeneID:491292||ABL1||XP_548413.2||Canis lupus familiaris|
[ ] Monoclonal and Polyclonal Antibodies
|1||abcam||ab63530||c Abl antibody (ab63530); Rabbit polyclonal to c Abl|
|2||abcam||ab62189||c Abl (phospho Y245) antibody (ab62189); Rabbit polyclonal to c Abl (phospho Y245)|
|3||abcam||ab61757||c Abl (phospho Y204) antibody (ab61757); Rabbit polyclonal to c Abl (phospho Y204)|
|4||abcam||ab55284||c Abl (phospho Y412) antibody (ab55284); Rabbit polyclonal to c Abl (phospho Y412)|
|5||abcam||ab54394||c Abl antibody [SPM328], prediluted (ab54394); Mouse monoclonal [SPM328] to c Abl, prediluted|
|6||abcam||ab47410||c Abl antibody (ab47410); Rabbit polyclonal to c Abl|
|7||abcam||ab47315||c Abl (phospho Y412) antibody (ab47315); Rabbit polyclonal to c Abl (phospho Y412)|
|8||abcam||ab4479||c Abl (phospho Y245) antibody (ab4479); Rabbit polyclonal to c Abl (phospho Y245)|
|9||abcam||ab4717||c Abl (phospho Y412) antibody (ab4717); Rabbit polyclonal to c Abl (phospho Y412)|
|10||abcam||ab16903||c Abl antibody [19-110] (ab16903); Mouse monoclonal [19-110] to c Abl|
|11||abcam||ab16905||c Abl antibody [24-21] (ab16905); Mouse monoclonal [24-21] to c Abl|
|12||abcam||ab3227||c Abl antibody [8E9] (ab3227); Mouse monoclonal [8E9] to c Abl|
|13||abcam||ab15130||c Abl antibody (ab15130); Rabbit polyclonal to c Abl|
|14||abcam||ab10528||c Abl antibody [ABL-148] (ab10528); Mouse monoclonal [ABL-148] to c Abl|
|15||abcam||ab15133||c Abl antibody, prediluted (ab15133); Rabbit polyclonal to c Abl, prediluted|
|16||sigma||C5365||Anti-phospho-c-Abl (pTyr245) antibody produced in rabbit ;|
|17||sigma||C5240||Anti-phospho-c-Abl (pTyr412) antibody produced in rabbit ;|
|18||sigma||A5844||Monoclonal Anti-c-Abl antibody produced in mouse ;|
|GO:0000287||Function||magnesium ion binding|
|GO:0030145||Function||manganese ion binding|
|GO:0046872||Function||metal ion binding|
|GO:0004715||Function||non-membrane spanning protein tyrosine kinase activity|
|GO:0019904||Function||protein domain specific binding|
|GO:0004672||Function||protein kinase activity|
|GO:0004713||Function||protein tyrosine kinase activity|
|GO:0030036||Process||actin cytoskeleton organization|
|GO:0006468||Process||protein amino acid phosphorylation|
|GO:0051726||Process||regulation of cell cycle|
MicroRNA and Targets
[ ] MicroRNA Sequences and Transcript Targets from miRBase at Sanger
|RNA Target||miRNA #||mat miRNA||Mature miRNA Sequence|
|Grap2||Grap2 / Abl1||Affinity Capture-Western||Liu SK (1998)|
|Pxn||Abl1 / Pxn||Affinity Capture-Western||Lewis JM (1998)|
|Pxn||Pxn / Abl1||Affinity Capture-Western||Lewis JM (1998)|
|Pxn||Abl1 / Pxn||Biochemical Activity||Lewis JM (1998)|
|Rin1||Abl1 / Rin1||Biochemical Activity||Han L (1997)|
|Rin1||Abl1 / Rin1||Reconstituted Complex||Han L (1997)|
- [ ] Liberatore RA, et al. (2009) "c-Abl-deficient mice exhibit reduced numbers of peritoneal B-1 cells and defects in BCR-induced B cell activation." Int Immunol. 21(4):403-414. PMID:19228876
- [ ] Vazquez MC, et al. (2009) "c-Abl modulates AICD dependent cellular responses: transcriptional induction and apoptosis." J Cell Physiol. 220(1):136-143. PMID:19306298
- [ ] Brightbill H, et al. (2009) "The effects of c-Abl mutation on developing B cell differentiation and survival." Int Immunol. 21(5):575-585. PMID:19299624
- [ ] Huang CC, et al. (2008) "An enhanced association of RACK1 with Abl in cells transfected with oncogenic ras." Int J Biochem Cell Biol. 40(3):423-431. PMID:17881279
- [ ] Huang Y, et al. (2008) "The c-Abl tyrosine kinase regulates actin remodeling at the immune synapse." Blood. 112(1):111-119. PMID:18305217
- [ ] Jia DY, et al. (2008) "c-Abl is involved in high glucose-induced apoptosis in embryonic E12.5 cortical neural progenitor cells from the mouse brain." J Neurochem. 106(4):1720-1730. PMID:18624912
- [ ] Bueno MJ, et al. (2008) "Genetic and epigenetic silencing of microRNA-203 enhances ABL1 and BCR-ABL1 oncogene expression." Cancer Cell. 13(6):496-506. PMID:18538733
- [ ] Kharas MG, et al. (2008) "Ablation of PI3K blocks BCR-ABL leukemogenesis in mice, and a dual PI3K/mTOR inhibitor prevents expansion of human BCR-ABL+ leukemia cells." J Clin Invest. 118(9):3038-3050. PMID:18704194
- [ ] Ganapathipillai SS, et al. (2008) "Coupling of mutated Met variants to DNA repair via Abl and Rad51." Cancer Res. 68(14):5769-5777. PMID:18632630
- [ ] Silberman I, et al. (2008) "T cell survival and function requires the c-Abl tyrosine kinase." Cell Cycle. 7(24):3847-3857. PMID:19098427
- [ ] Fanta S, et al. (2008) "Pharmacological inhibition of c-Abl compromises genetic stability and DNA repair in Bcr-Abl-negative cells." Oncogene. 27(31):4380-4384. PMID:18362889
- [ ] Oh AS, et al. (2008) "Tyrosine phosphorylation of the nuclear receptor coactivator AIB1/SRC-3 is enhanced by Abl kinase and is required for its activity in cancer cells." Mol Cell Biol. 28(21):6580-6593. PMID:18765637
- [ ] Lam QL, et al. (2007) "Impaired V(D)J recombination and increased apoptosis among B cell precursors in the bone marrow of c-Abl-deficient mice." Int Immunol. 19(3):267-276. PMID:17229817
- [ ] Grimmler M, et al. (2007) "Cdk-inhibitory activity and stability of p27Kip1 are directly regulated by oncogenic tyrosine kinases." Cell. 128(2):269-280. PMID:17254966
- [ ] Frasca F, et al. (2007) "Role of c-Abl in directing metabolic versus mitogenic effects in insulin receptor signaling." J Biol Chem. 282(36):26077-26088. PMID:17620332
- [ ] Backer S, et al. (2007) "Trio controls the mature organization of neuronal clusters in the hindbrain." J Neurosci. 27(39):10323-10332. PMID:17898204
- [ ] Preyer M, et al. (2007) "Delayed activation of Bax by DNA damage in embryonic stem cells with knock-in mutations of the Abl nuclear localization signals." Cell Death Differ. 14(6):1139-1148. PMID:17363963
- [ ] Borges HL, et al. (2007) "Reduction of apoptosis in Rb-deficient embryos via Abl knockout." Oncogene. 26(26):3868-3877. PMID:17173068
- [ ] Jin H, et al. (2007) "Abl tyrosine kinase promotes dorsal ruffles but restrains lamellipodia extension during cell spreading on fibronectin." Mol Biol Cell. 18(10):4143-4154. PMID:17686996
- [ ] Sfakianos MK, et al. (2007) "Inhibition of Rho via Arg and p190RhoGAP in the postnatal mouse hippocampus regulates dendritic spine maturation, synapse and dendrite stability, and behavior." J Neurosci. 27(41):10982-10992. PMID:17928439
- [ ] Zhou T, et al. (2007) "Crystal structure of the T315I mutant of AbI kinase." Chem Biol Drug Des. 70(3):171-181. PMID:17718712
- [ ] Krause DS, et al. (2006) "Requirement for CD44 in homing and engraftment of BCR-ABL-expressing leukemic stem cells." Nat Med. 12(10):1175-1180. PMID:16998483
- [ ] Hirao N, et al. (2006) "NESH (Abi-3) is present in the Abi/WAVE complex but does not promote c-Abl-mediated phosphorylation." FEBS Lett. 580(27):6464-6470. PMID:17101133
- [ ] Slupianek A, et al. (2006) "BCR/ABL modifies the kinetics and fidelity of DNA double-strand breaks repair in hematopoietic cells." DNA Repair (Amst). 5(2):243-250. PMID:16297667
- [ ] Wilkes MC, et al. (2006) "Transforming growth factor beta activation of c-Abl is independent of receptor internalization and regulated by phosphatidylinositol 3-kinase and PAK2 in mesenchymal cultures." J Biol Chem. 281(38):27846-27854. PMID:16867995
- [ ] Gao B, et al. (2006) "The tyrosine kinase c-Abl protects c-Jun from ubiquitination-mediated degradation in T cells." J Biol Chem. 281(40):29711-29718. PMID:16901904
- [ ] Stuart JR, et al. (2006) "c-Abl interacts with the WAVE2 signaling complex to induce membrane ruffling and cell spreading." J Biol Chem. 281(42):31290-31297. PMID:16899465
- [ ] Wang X, et al. (2006) "p53 functions as a negative regulator of osteoblastogenesis, osteoblast-dependent osteoclastogenesis, and bone remodeling." J Cell Biol. 172(1):115-125. PMID:16380437
- [ ] Tokonzaba E, et al. (2006) "Phosphoinositide, phosphopeptide and pyridone interactions of the Abl SH2 domain." Chem Biol Drug Des. 67(3):230-237. PMID:16611216
- [ ] Letterio J, et al. (2006) "Transforming growth factor-beta1 sensitivity is altered in Abl-Myc- and Raf-Myc-induced mouse pre-B-cell tumors." Stem Cells. 24(12):2611-2617. PMID:16945999
- [ ] Chen X, et al. (2006) "A kinase-independent function of c-Abl in promoting proteolytic destruction of damaged DNA binding proteins." Mol Cell. 22(4):489-499. PMID:16713579
- [ ] Moresco EM, et al. (2005) "Integrin-mediated dendrite branch maintenance requires Abelson (Abl) family kinases." J Neurosci. 25(26):6105-6118. PMID:15987940
- [ ] Devireddy LR, et al. (2005) "A cell-surface receptor for lipocalin 24p3 selectively mediates apoptosis and iron uptake." Cell. 123(7):1293-1305. PMID:16377569
- [ ] Boureux A, et al. (2005) "Abl tyrosine kinase regulates a Rac/JNK and a Rac/Nox pathway for DNA synthesis and Myc expression induced by growth factors." J Cell Sci. 118(Pt 16):3717-3726. PMID:16076903
- [ ] Lin H, et al. (2005) "Bcr-Abl-mediated suppression of normal hematopoiesis in leukemia." Oncogene. 24(20):3246-3256. PMID:15735695
- [ ] Zhao R, et al. (2005) "GATA6 is essential for embryonic development of the liver but dispensable for early heart formation." Mol Cell Biol. 25(7):2622-2631. PMID:15767668
- [ ] Carninci P, et al. (2005) "The transcriptional landscape of the mammalian genome." Science. 309(5740):1559-1563. PMID:16141072
- [ ] Woodring PJ, et al. (2005) "Mitotic phosphorylation rescues Abl from F-actin-mediated inhibition." J Biol Chem. 280(11):10318-10325. PMID:15632178
- [ ] Katayama S, et al. (2005) "Antisense transcription in the mammalian transcriptome." Science. 309(5740):1564-1566. PMID:16141073
- [ ] Tzeng SJ, et al. (2005) "The B cell inhibitory Fc receptor triggers apoptosis by a novel c-Abl family kinase-dependent pathway." J Biol Chem. 280(42):35247-35254. PMID:16115887
- [ ] Zeng L, et al. (2005) "Identification of TopBP1 as a c-Abl-interacting protein and a repressor for c-Abl expression." J Biol Chem. 280(32):29374-29380. PMID:15961388
- [ ] Stuart JR, et al. (2005) "c-Abl regulates early growth response protein (EGR1) in response to oxidative stress." Oncogene. 24(55):8085-8092. PMID:16091742
- [ ] Toyofuku T, et al. (2004) "Guidance of myocardial patterning in cardiac development by Sema6D reverse signalling." Nat Cell Biol. 6(12):1204-1211. PMID:15543137
- [ ] Li B, et al. (2004) "Distinct roles of c-Abl and Atm in oxidative stress response are mediated by protein kinase C delta." Genes Dev. 18(15):1824-1837. PMID:15289456
- [ ] Suzuki J, et al. (2004) "Loss of c-abl facilitates anchorage-independent growth of p53- and RB- deficient primary mouse embryonic fibroblasts." Oncogene. 23(52):8527-8534. PMID:15378021
- [ ] Watahiki A, et al. (2004) "Libraries enriched for alternatively spliced exons reveal splicing patterns in melanocytes and melanomas." Nat Methods. 1(3):233-239. PMID:15782199
- [ ] Gerhard DS, et al. (2004) "The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC)." Genome Res. 14(10B):2121-2127. PMID:15489334
- [ ] Woodring PJ, et al. (2004) "c-Abl phosphorylates Dok1 to promote filopodia during cell spreading." J Cell Biol. 165(4):493-503. PMID:15148308
- [ ] Kuribara R, et al. (2004) "Roles of Bim in apoptosis of normal and Bcr-Abl-expressing hematopoietic progenitors." Mol Cell Biol. 24(14):6172-6183. PMID:15226421
- [ ] Blackshaw S, et al. (2004) "Genomic analysis of mouse retinal development." PLoS Biol. 2(9):E247. PMID:15226823
A role for c-Abl in B cell development and signaling has been suggested by previous work showing that c-Abl-deficient mice have defects in bone marrow B cell development and that c-Abl-deficient B cells are hypoproliferative in response to antigen receptor stimulation. Here we show that in addition to defects in early B cell development, c-Abl-deficient mice have defects in peripheral B cell development, including reduced percentages of peritoneal B-1 cells as well as transitional and marginal zone B cells in the spleen. It has been shown that c-Abl kinase activity increases upon B cell receptor (BCR) stimulation and that one of the targets of tyrosine phosphorylation by c-Abl is CD19. However, the consequences of c-Abl activity on B cell activation and CD19 signaling remain unknown. Here, we show that c-Abl-deficient splenic B cells exhibit reduced calcium flux in response to CD19 cross-linking, consistent with a role for c-Abl in CD19-dependent signaling. Additionally, we show that c-Abl-deficient B cells are defective in their ability to be activated in response to antigen receptor engagement, suggesting a functional role for c-Abl in BCR-dependent activation signaling pathways.
APP intracellular domain (AICD) has been proposed as a transcriptional inductor that moves to the nucleus with the adaptor protein Fe65 and regulates transcription. The two proteins, APP and Fe65, can be phosphorylated by c-Abl kinase. Neprilysin has been proposed as a target gene for AICD. We found that AICD expression is decreased by treatment with STI-571, a c-Abl inhibitor, suggesting a modulation of AICD transcription by c-Abl kinase. We observed interaction between c-Abl kinase, the AICD fragment and the Fe65 adaptor protein. In addition, STI-571 reduces apoptosis in APPSw, and the apoptotic response induced by Fe65 over-expression was inhibited by with the expression of a kinase dead (KD) c-Abl and enhanced by over-expression of WT-c-Abl. However, in the APPSw cells, the ability of the KD-c-Abl to protect against Fe65 was reduced. Finally, in APPSw clone, we detected higher trans-activation of the pro-apoptotic p73 isoform, TAp73 promoter. Our results show that c-Abl modulates AICD dependent cellular responses, transcriptional induction as well as the apoptotic response, which could participate in the onset and progression of the neurodegenerative pathology, observed in Alzheimer's disease (AD).
c-Abl is a widely expressed Src family protein tyrosine kinase that is activated by chromosomal translocation in certain human leukemias. While shown in various experimental systems to regulate cell division and stress responses, its biological functions remain poorly understood. Although expressed at similar levels throughout B cell development, we found that the fraction of phosphorylated, active c-Abl peaks at the pro-B stage. We went on to perform a detailed analysis of B cell development in c-Abl-deficient mice. We confirmed a striking but variable decrease in pro- and pre-B cell numbers, a decrease in pre-B cell growth and an increase in pre-B cell apoptosis. This phenotype was not rescued by transgenic expression of a functional IgHC transgene and only partially rescued by the anti-apoptosis gene Bcl-x. Unlike their wild-type counterparts, c-Abl-deficient pre-B cells show a defect in Ca(2+) flux upon cross-linking of CD19, a co-receptor known to be involved in pre-B cell receptor signaling and failed to express CD25 on the cell surface. Despite these pre-B cell-signaling defects, selection for in-frame heavy-chain rearrangements was intact in the mutant mice. Remarkably, we were able to rescue the proliferative defect by culturing cells in vitro with large amounts of rIL-7. We conclude that c-Abl is required for normal B cell differentiation and survival.
The cellular RACK1 was shown in association with Abl in BALB/3T3 cells transfected with S-ras(Q(61)K) by immunoprecipitation. An identical finding was demonstrated with cells transfected with the embryonic E-ras, but not in cells without transformation. The Abl-RACK1 of transformed cells as resolvable with Triton X-114 was found with little affinity for FAK, PY(397)-FAK and integrin. Of interests, PY(397)-FAK in the membrane skeleton of transformed cells was shown in significant quantities on the Western blot. However the PY(397)-FAK of transformed cells was not functionally able to react with RACK1 and recruit cytokeratin-1, a substrate of Src, indicating that PY(397)-FAK is not operative to transmit integrin signals. In other words, the Abl-RACK1 of transformed cells cannot replace the Src-RACK1 of cells without transformation to bridge PY(397)-FAK and cytokeratin-1 for integrin signals, and the formation of Abl-RACK1 in transformed cells may block the association of PY(397)-FAK-RACK1. We characterized Abl and RACK1 from transformed cells by chromatography on a HiTrap-PEP(Taxol) affinity column, constructed from a beta-tubulin peptide specific for Taxol binding (PEP(Taxol)). However, the Triton X-100 cannot achieve the same resolution of Abl-RACK1 from plasma membrane as is shown with Triton X-114. A significant fraction of Abl was deposited at the membrane skeleton and was therefore not accessible with Triton X-100. Half of Abl resolved with Triton X-100 was demonstrated to have catalytic activity as shown with positive phosphotyrosine staining on the Western blot and competitive elution with a specific phosphate, such as sodium beta-glycerophosphate, from HiTrap-PEP(Taxol), but this was not associated with RACK1. No significant difference of RACK1 was found in Triton X-100 resolvable membrane preparations from cells with and without transformations. Future studies are planned to differentiate the mechanism operative for RACK1 associated and RACK1 freed Abl in cells transformed with oncogenic ras.
Actin dynamics during T-cell activation are controlled by the coordinate action of multiple actin regulatory proteins, functioning downstream of a complex network of kinases and other signaling molecules. The c-Abl nonreceptor tyrosine kinase regulates actin responses in nonhematopoietic cells, but its function in T cells is poorly understood. Using kinase inhibitors, RNAi, and conditional knockout mice, we investigated the role of c-Abl in controlling the T-cell actin response. We find that c-Abl is required for normal actin polymerization and lamellipodial spreading at the immune synapse, and for downstream events leading to efficient interleukin-2 production. c-Abl also plays a key role in signaling chemokine-induced T-cell migration. c-Abl is required for the appropriate function of 2 proteins known to be important for controlling actin responses to T-cell receptor (TCR) engagement, the actin-stabilizing adapter protein HS1, and the Rac1-dependent actin polymerizing protein WAVE2. c-Abl binds to phospho-HS1 via its SH2 domains and is required for full tyrosine phosphorylation of HS1 during T-cell activation. In addition, c-Abl is required for normal localization of WAVE2 to the immune synapse (IS). These studies identify c-Abl as a key player in the signaling cascade, leading to actin reorganization during T-cell activation.
Hyperglycemia causes direct apoptosis of neural progenitor cells (NPCs) in diabetic-induced neural tube defects in embryos. However, the underlying mechanisms are poorly understood. The present study is aimed to investigate the specific cellular proteins that may be involved in NPCs apoptosis as well as mechanisms by which the proteins regulate the oxidative stress-induced NPCs apoptosis. Our present results have shown that the expression of c-Abl was up-regulated in NPCs exposed to high glucose in vitro. The increased c-Abl was localized mainly in the nucleus. High glucose also induced an increase in nuclear p53 protein levels and the p53-c-Abl complex in NPCs. Administration of reactive oxygen species scavengers decreased the protein level of c-Abl, p53 and NPCs apoptosis. Inhibition of c-Abl reduced NPCs apoptosis and the nuclear protein level of p53 in response to high glucose. These results demonstrate that c-Abl is involved in the reactive oxygen species-activated apoptotic pathways in NPCs apoptosis. Inhibition of c-Abl may protect NPCs against insults induced by high glucose via the modulation of NPCs apoptotic machinery.
The mammalian genome contains several hundred microRNAs that regulate gene expression through modulation of target mRNAs. Here, we report a fragile chromosomal region lost in specific hematopoietic malignancies. This 7 Mb region encodes about 12% of all genomic microRNAs, including miR-203. This microRNA is additionally hypermethylated in several hematopoietic tumors, including chronic myelogenous leukemias and some acute lymphoblastic leukemias. A putative miR-203 target, ABL1, is specifically activated in these hematopoietic malignancies in some cases as a BCR-ABL1 fusion protein (Philadelphia chromosome). Re-expression of miR-203 reduces ABL1 and BCR-ABL1 fusion protein levels and inhibits tumor cell proliferation in an ABL1-dependent manner. Thus, miR-203 functions as a tumor suppressor, and re-expression of this microRNA might have therapeutic benefits in specific hematopoietic malignancies.
Some cases of pre-B cell acute lymphoblastic leukemia (pre-B-ALL) are caused by the Philadelphia (Ph) chromosome-encoded BCR-ABL oncogene, and these tend to have a poor prognosis. Inhibitors of the PI3K/AKT pathway reduce BCR-ABL-mediated transformation in vitro; however, the specific PI3K isoforms involved are poorly defined. Using a murine model of Ph+ pre-B-ALL, we found that deletion of both Pik3r1 and Pik3r2, genes encoding class IA PI3K regulatory isoforms, severely impaired transformation. BCR-ABL-dependent pre/pro-B cell lines could be established at low frequency from progenitors that lacked these genes, but the cells were smaller, proliferated more slowly, and failed to cause leukemia in vivo. These cell lines displayed nearly undetectable PI3K signaling function and were resistant to the PI3K inhibitor wortmannin. However, they maintained activation of mammalian target of rapamycin (mTOR) and were more sensitive to rapamycin. Treatment with rapamycin caused feedback activation of AKT in WT cell lines but not PI3K-deficient lines. A dual inhibitor of PI3K and mTOR, PI-103, was more effective than rapamycin at suppressing proliferation of mouse pre-B-ALL and human CD19+CD34+)Ph+ ALL leukemia cells treated with the ABL kinase inhibitor imatinib. Our findings provide mechanistic insights into PI3K dependency in oncogenic networks and provide a rationale for targeting class IA PI3K, alone or together with mTOR, in the treatment of Ph+ ALL.
Abnormal activation of DNA repair pathways by deregulated signaling of receptor tyrosine kinase systems is a compelling likelihood with significant implications in both cancer biology and treatment. Here, we show that due to a potential substrate switch, mutated variants of the receptor for hepatocyte growth factor Met, but not the wild-type form of the receptor, directly couple to the Abl tyrosine kinase and the Rad51 recombinase, two key signaling elements of homologous recombination-based DNA repair. Treatment of cells that express the mutated receptor variants with the Met inhibitor SU11274 leads, in a mutant-dependent manner, to a reduction of tyrosine phosphorylated levels of Abl and Rad51, impairs radiation-induced nuclear translocation of Rad51, and acts as a radiosensitizer together with the p53 inhibitor pifithrin-alpha by increasing cellular double-strand DNA break levels following exposure to ionizing radiation. Finally, we propose that in order to overcome a mutation-dependent resistance to SU11274, this aberrant molecular axis may alternatively be targeted with the Abl inhibitor, nilotinib.
C-Abl (Abl) regulates multiple cellular processes, including proliferation, survival, shape determination and motility, and participates in cellular responses to genotoxic and oxidative stress stimuli. Mice lacking Abl exhibit retarded growth, osteoporosis and defects in the immune system resulting in lymphopoenia and susceptibility to infections, leading to early death. To define the role of Abl in the regulation of adult T cells we ablated Abl exclusively in T cells by generating mice with floxed abl alleles and expressing an Lck-Cre transgene (Abl-T(-/-)). These mice exhibited thymic atrophy and abnormally reduced T cell numbers in the periphery. The thymic atrophy was caused by increased susceptibility of thymocytes to cell death. Importantly, Abl deficient T cells displayed abnormally reduced response to mitogenic stimulation in vitro. Consequently, Abl-T(-/-) mice exhibited impaired ability to reject syngeneic tumor, to induce T-mediated tumor cell killing, and to generate anti-tumor antibodies. These results demonstrate a cell-autonomous role for Abl in T cell function and survival.
Imatinib inhibits the kinase activity of Bcr-Abl and is currently the most effective drug for treatment of chronic myeloid leukemia (CML). Imatinib also blocks c-Abl, a physiological tyrosine kinase activated by a variety of stress signals including damaged DNA. We investigated the effect of pharmacological inhibition of c-Abl on the processing of irradiation-induced DNA damage in Bcr-Abl-negative cells. Cell lines and peripheral blood mononuclear cells (PBMCs) from healthy volunteers were treated with imatinib or dasatinib before gamma-irradiation. Inhibition of c-Abl caused an enhanced irradiation-induced mutation frequency and slowdown of DNA repair, whereas imatinib was ineffective in cells expressing a T315I variant of c-Abl. Mutation frequency and repair kinetics were also studied in c-Abl-/- murine embryonic fibroblasts (MEFs) retransfected with wild-type c-Abl (wt-Abl) or a kinase-defect variant of Abl (KD-Abl). Enhanced mutation frequency as well as delayed DNA repair was observed in cells expressing KD-Abl. These data indicate that pharmacological inhibition of c-Abl compromises DNA-damage response.
Overexpression and activation of the steroid receptor coactivator amplified in breast cancer 1 (AIB1)/steroid receptor coactivator-3 (SRC-3) have been shown to have a critical role in oncogenesis and are required for both steroid and growth factor signaling in epithelial tumors. Here, we report a new mechanism for activation of SRC coactivators. We demonstrate regulated tyrosine phosphorylation of AIB1/SRC-3 at a C-terminal tyrosine residue (Y1357) that is phosphorylated after insulin-like growth factor 1, epidermal growth factor, or estrogen treatment of breast cancer cells. Phosphorylated Y1357 is increased in HER2/neu (v-erb-b2 erythroblastic leukemia viral oncogene homolog 2) mammary tumor epithelia and is required to modulate AIB1/SRC-3 coactivation of estrogen receptor alpha (ERalpha), progesterone receptor B, NF-kappaB, and AP-1-dependent promoters. c-Abl (v-Abl Abelson murine leukemia viral oncogene homolog 1) tyrosine kinase directly phosphorylates AIB1/SRC-3 at Y1357 and modulates the association of AIB1 with c-Abl, ERalpha, the transcriptional cofactor p300, and the methyltransferase coactivator-associated arginine methyltransferase 1, CARM1. AIB1/SRC-3-dependent transcription and phenotypic changes, such as cell growth and focus formation, can be reversed by an Abl kinase inhibitor, imatinib. Thus, the phosphorylation state of Y1357 can function as a molecular on/off switch and facilitates the cross talk between hormone, growth factor, and intracellular kinase signaling pathways in cancer.
Previous studies on c-Abl-deficient mice have shown high post-natal mortality and lymphopenia. However, the mechanisms by which c-Abl may influence B lymphopoiesis remain obscure. In this study, we analyzed B cell sub-populations at various differentiation stages in the bone marrow (BM) of c-Abl-deficient mice. Phenotypic analyses revealed that c-Abl(-/-) pro-B cells were reduced to half of normal incidence and absolute number, while pre-B cells showed an even greater reduction. Both c-Abl(-/-) pro-B and pre-B cell populations showed considerably elevated apoptosis ex vivo and in short-term culture but their cell cycle progression was not impaired. In contrast, apoptosis of immature IgM(+)IgD(-) B lymphocytes remained at normal control levels. Inhibition of c-Abl activity by STI571 in normal BM cultures significantly increased apoptosis in B cell precursors while the survival of immature B cells was not affected. To determine whether c-Abl deficiency affects Ig heavy-chain rearrangement, we found that the frequency of V(D)J recombination was markedly reduced by 15-fold in c-Abl(-/-) pro-B cells compared with the control values. However, no perturbation in the levels of signal-end recombination intermediates was found. Taken together, we propose that c-Abl mediates a stage-specific anti-apoptotic response in precursor B cells and is required for efficient V(D)J recombination during B cell development.
p27Kip1 controls cell proliferation by binding to and regulating the activity of cyclin-dependent kinases (Cdks). Here we show that Cdk inhibition and p27 stability are regulated through direct phosphorylation by tyrosine kinases. A conserved tyrosine residue (Y88) in the Cdk-binding domain of p27 can be phosphorylated by the Src-family kinase Lyn and the oncogene product BCR-ABL. Y88 phosphorylation does not prevent p27 binding to cyclin A/Cdk2. Instead, it causes phosphorylated Y88 and the entire inhibitory 3(10)-helix of p27 to be ejected from the Cdk2 active site, thus restoring partial Cdk activity. Importantly, this allows Y88-phosphorylated p27 to be efficiently phosphorylated on threonine 187 by Cdk2 which in turn promotes its SCF-Skp2-dependent degradation. This direct link between transforming tyrosine kinases and p27 may provide an explanation for Cdk kinase activities observed in p27 complexes and for premature p27 elimination in cells that have been transformed by activated tyrosine kinases.
c-Abl is a cytoplasmic tyrosine kinase involved in several signal transduction pathways. Here we report that c-Abl is involved also in insulin receptor signaling. Indeed, c-Abl tyrosine kinase is activated upon insulin stimulation. Inhibition of c-Abl tyrosine kinase by STI571 attenuates the effect of insulin on Akt/GSK-3beta phosphorylation and glycogen synthesis, and at the same time, it enhances the effect of insulin on ERK activation, cell proliferation, and migration. This effect of STI571 is specific to c-Abl inhibition, because it does not occur in Abl-null cells and is restored in c-Abl-reconstituted cells. Numerous evidences suggest that focal adhesion kinase (FAK) is involved in mediating this c-Abl effect. First, anti-phosphotyrosine blots indicate that c-Abl tyrosine kinase activation is concomitant with FAK dephosphorylation in response to insulin, whereas c-Abl inhibition is accompanied by FAK phosphorylation in response to insulin, a response similar to that observed with IGF-I. Second, the c-Abl effects on insulin signaling are not observed in cells devoid of FAK (FAK(-/-) cells). Taken together these results suggest that c-Abl activation by insulin, via a modification of FAK response, may play an important role in directing mitogenic versus metabolic insulin receptor signaling.
During the embryonic development of the hindbrain, movements of neuronal clusters allow the formation of mature "pools", in particular for inferior olivary (ION) and facial motor (fMN) nuclei. The cellular mechanisms of neuron clustering remain uncharacterized. We report that the absence of the Rho-guanine exchange factor Trio, which can activate both RhoG and Rac1 in vivo, prevents the proper formation of ION and fMN subnuclei. Rac1, but not RhoG, appears to be a downstream actor in Trio-induced lamellation. In addition, we report that Cadherin-11 is expressed by a subset of neurons through the overall period of ION and fMN parcellations, and defects observed in trio mutant mice are located specifically in Cadherin-11-expressing regions. Moreover, endogenous Cadherin-11 is found in a complex with Trio when lamellation occurs. Altogether, those results establish a link between Trio activity, the subsequent Rac1 activation, and neuronal clusters organization, as well as a possible recruitment of the Cadherin-11 adhesive receptor to form a complex with Trio.
The non-receptor tyrosine kinase Abl contains nuclear localization (NLS) and nuclear export signals that drive its nucleo-cytoplasmic shuttling. The nuclear Abl tyrosine kinase is activated by DNA damage through ataxia telangiectasia mutated (ATM). Previous studies have suggested nuclear Abl to have proapoptotic activity. To determine the requirement for Abl nuclear import in DNA damage-induced apoptosis, we took a genetic approach by mutating the three NLS (muNLS) of abl1 in mouse embryonic stem (ES) cells through homologous recombination. Exposure of ES cells to genotoxins caused an ATM-dependent nuclear accumulation of Abl but not Abl muNLS. ES cells expressing Abl muNLS exhibited delayed Bax activation, reduced cytochrome c release and decreased caspase-9 activity in response to DNA damage. These results provide a genetic proof that Abl nuclear entry contributes to DNA damage-induced activation of the intrinsic apoptotic pathway.
The retinoblastoma protein RB regulates cell proliferation, differentiation and apoptosis. Homozygous knockout of Rb in mice causes embryonic lethality owing to placental defects that result in excessive apoptosis. RB binds to a number of cellular proteins including the nuclear Abl protein and inhibits its tyrosine kinase activity. Ex vivo experiments have shown that genotoxic or inflammatory stress can activate Abl kinase to stimulate apoptosis. Employing the Rb-null embryos as an in vivo model of apoptosis, we have shown that the genetic ablation of Abl can reduce apoptosis in the developing central nervous system and the embryonic liver. These results are consistent with the inhibitory interaction between RB and Abl, and provide in vivo evidence for the proapoptotic function of Abl.
The nonreceptor Abl tyrosine kinase stimulates F-actin microspikes and membrane ruffles in response to adhesion and growth factor signals. We show here that induced dimerization of Abl-FKBP, but not the kinase-defective AblKD-FKBP, inhibits cell spreading on fibronectin. Conversely, knockdown of cellular Abl by shRNA stimulates cell spreading. The Abl kinase inhibitor, imatinib, also stimulates cell spreading and its effect is overridden by the imatinib-resistant AblT315I. Expression of Abl but not AbkKD in Abl/Arg-deficient cells again inhibits spreading. Furthermore, Abl inhibits spreading of cells that express the activated Rac, RacV12, correlating with RacV12 localization to dorsal membrane protrusions. Ectopic expression of CrkII, a Rac activator that is inactivated by Abl-mediated tyrosine phosphorylation, antagonizes Abl-mediated dorsal membrane localization of RacV12. Ectopic expression of a dynamin-2 mutant, previously shown to induce Rac-GTP localization to the dorsal membrane, abolishes the stimulatory effect of imatinib on cell spreading. These results suggest that Abl tyrosine kinase, through CrkII phosphorylation and in collaboration with dynamin-2 can regulate the partitioning of Rac-GTP to favor dorsal ruffles during cell spreading. The Abl-dependent dorsal membrane localization of activated Rac explains its positive role in ruffling and negative role in cell spreading and migration.
The RhoA (Rho) GTPase is a master regulator of dendrite morphogenesis. Rho activation in developing neurons slows dendrite branch dynamics, yielding smaller, less branched dendrite arbors. Constitutive activation of Rho in mature neurons causes dendritic spine loss and dendritic regression, indicating that Rho can affect dendritic structure and function even after dendrites have developed. However, it is unclear whether and how endogenous Rho modulates dendrite and synapse morphology after dendrite arbor development has occurred. We demonstrate that a Rho inhibitory pathway involving the Arg tyrosine kinase and p190RhoGAP is essential for synapse and dendrite stability during late postnatal development. Hippocampal CA1 pyramidal dendrites develop normally in arg-/- mice, reaching their mature size by postnatal day 21 (P21). However, dendritic spines do not undergo the normal morphological maturation in these mice, leading to a loss of hippocampal synapses and dendritic branches by P42. Coincident with this synapse and dendrite loss, arg-/- mice exhibit progressive deficits in a hippocampus-dependent object recognition behavioral task. p190RhoGAP localizes to dendritic spines, and its activity is reduced in arg-/- hippocampus, leading to increased Rho activity. Although mutations in p190rhogap enhance dendritic regression resulting from decreased Arg levels, reducing gene dosage of the Rho effector ROCKII can suppress the dendritic regression observed in arg-/- mice. Together, these data indicate that signaling through Arg and p190RhoGAP acts late during synaptic refinement to promote dendritic spine maturation and synapse/dendrite stability by attenuating synaptic Rho activity.
Imatinib (Gleevec) is currently the frontline therapy for chronic myeloid leukemia (CML), a disease characterized by the presence of a constitutively activated chimeric tyrosine kinase protein Bcr-AbI. However, drug resistance often occurs at later stages of the disease, principally because of the occurrence of mutations in the kinase domain. Second generation Bcr-AbI inhibitors, such as dasatinib and nilotinib are capable of inhibiting many imatinib-resistant forms of the kinase but not the form in which threonine is mutated to isoleucine at the gatekeeper position (T315I). In this study, we present the crystal structure of the kinase domain of the c-AbI T315I mutant, as well as the wild-type form, in complex with a pyrrolopyridine inhibitor, PPY-A. The side chain of Ile315 is accommodated in the AbI T315I mutant structure without large conformational changes proximal to the site of mutation. In contrast to other inhibitors, such as imatinib and dasatinib, PPY-A does not occupy the hydrophobic pocket behind the gatekeeper residue. This binding mode, coupled with augmented contacts with the glycine-rich loop, appears to be critical for its ability to override the T315I mutation. The data presented here may provide structural guidance for the design of clinically useful inhibitors of Bcr-AbI T315I.
In individuals with chronic myeloid leukemia (CML) treated by autologous hematopoietic stem cell (HSC) transplantation, malignant progenitors in the graft contribute to leukemic relapse, but the mechanisms of homing and engraftment of leukemic CML stem cells are unknown. Here we show that CD44 expression is increased on mouse stem-progenitor cells expressing BCR-ABL and that CD44 contributes functional E-selectin ligands. In a mouse retroviral transplantation model of CML, BCR-ABL1-transduced progenitors from CD44-mutant donors are defective in homing to recipient marrow, resulting in decreased engraftment and impaired induction of CML-like myeloproliferative disease. By contrast, CD44-deficient stem cells transduced with empty retrovirus engraft as efficiently as do wild-type HSCs. CD44 is dispensable for induction of acute B-lymphoblastic leukemia by BCR-ABL, indicating that CD44 is specifically required on leukemic cells that initiate CML. The requirement for donor CD44 is bypassed by direct intrafemoral injection of BCR-ABL1-transduced CD44-deficient stem cells or by coexpression of human CD44. Antibody to CD44 attenuates induction of CML-like leukemia in recipients. These results show that BCR-ABL-expressing leukemic stem cells depend to a greater extent on CD44 for homing and engraftment than do normal HSCs, and argue that CD44 blockade may be beneficial in autologous transplantation in CML.
Abl interactor (Abi) was identified as an Abl tyrosine kinase-binding protein and subsequently shown to be a component of the macromolecular Abi/WAVE complex, which is a key regulator of Rac-dependent actin polymerization. Previous studies showed that Abi-1 promotes c-Abl-mediated phosphorylation of Mammalian Enabled (Mena) and WAVE2. In addition to Abi-1, mammals possess Abi-2 and NESH (Abi-3). In this study, we compared the three Abi proteins in terms of the promotion of c-Abl-mediated phosphorylation and the formation of Abi/WAVE complex. Although Abi-2, like Abi-1, promoted the c-Abl-mediated phosphorylation of Mena and WAVE2, NESH (Abi-3) had no such effect. This difference was likely due to their binding abilities as to c-Abl. Immunoprecipitation revealed that NESH (Abi-3) is present in the Abi/WAVE complex. Our results suggest that NESH (Abi-3), like Abi-1 and Abi-2, is a component of the Abi/WAVE complex, but likely plays a different role in the regulation of c-Abl.
The oncogenic BCR/ABL tyrosine kinase facilitates the repair of DNA double-strand breaks (DSBs). We find that after gamma-irradiation BCR/ABL-positive leukemia cells accumulate more DSBs in comparison to normal cells. These lesions are efficiently repaired in a time-dependent fashion by BCR/ABL-stimulated non-homologous end-joining (NHEJ) followed by homologous recombination repair (HRR) mechanisms. However, mutations and large deletions were detected in HRR and NHEJ products, respectively, in BCR/ABL-positive leukemia cells. We propose that unfaithful repair of DSBs may contribute to genomic instability in the Philadelphia chromosome-positive leukemias.
Transforming growth factor beta (TGF-beta) modulates a number of cellular phenotypes as divergent as growth stimulation and growth inhibition. Although the Smad pathway is critical for many of these responses, recent evidence indicates that Smad-independent pathways may also have a critical role. One such protein previously shown to regulate TGF-beta action independent of the Smad proteins is the c-Abl nonreceptor tyrosine kinase. In the current study we determined that TGF-beta receptor signaling activates c-Abl kinase activity in a subset of fibroblast but not epithelial cultures. This cell type-specific response occurs in a membrane-proximal locale independent of receptor internalization and upstream of dynamin action. Although c-Abl activation by TGF-beta is independent of Smad2 or Smad3, it is prevented by inhibitors of phosphatidylinositol 3-kinase or PAK2. Thus, c-Abl represents a target downstream of phosphatidylinositol 3-kinase-activated PAK2, which differentiates TGF-beta signaling in fibroblasts and epithelial cell lines and integrates serine/threonine receptor kinases with tyrosine kinase pathways.
The cross-talk of ubiquitination with other types of posttranscriptional modifications, such as phosphorylation, regulates the stability of many proteins. We have previously demonstrated that c-Jun is a substrate of Itch, a HECT-type E3 ubiquitin ligase. c-Jun is also a substrate of the tyrosine kinase c-Abl. Here we report that genetic ablation of c-Abl accelerated c-Jun degradation. Phosphorylation of the tyrosine within the PPXY motif by c-Abl inhibited c-Jun ubiquitination and its binding by Itch. The nuclear localization of c-Abl, triggered by T-cell activation signals, was essential for its activity in regulating c-Jun transcription activity. These findings define a potential molecular mechanism for the immunodeficiency in mice lacking the c-abl gene.
The Wiskott-Aldrich syndrome-related protein WAVE2 promotes Arp2/3-dependent actin polymerization downstream of Rho-GTPase activation. The Abelson-interacting protein-1 (Abi-1) forms the core of the WAVE2 complex and is necessary for proper stimulation of WAVE2 activity. Here we have shown that the Abl-tyrosine kinase interacts with the WAVE2 complex and that Abl kinase activity facilitates interaction between Abl and WAVE2 complex members. We have characterized various interactions between Abl and members of the WAVE2 complex and revealed that Abi-1 promotes interaction between Abl and WAVE2 members. We have demonstrated that Abl-dependent phosphorylation of WAVE2 is necessary for its activation in vivo, which is highlighted by the findings that RNA interference of WAVE2 expression in Abl/Arg-/- cells has no additive effect on the amount of membrane ruffling. Furthermore, Abl phosphorylates WAVE2 on tyrosine 150, and WAVE2-deficient cells rescued with a Y150F mutant fail to regain their ability to ruffle and form microspikes, unlike cells rescued with wild-type WAVE2. Together, these data show that c-Abl activates WAVE2 via tyrosine phosphorylation to promote actin remodeling in vivo and that Abi-1 forms the crucial link between these two factors.
p53 is a well known tumor suppressor. We show that p53 also regulates osteoblast differentiation, bone formation, and osteoblast-dependent osteoclast differentiation. Indeed, p53(-/-) mice display a high bone mass phenotype, and p53(-/-) osteoblasts show accelerated differentiation, secondary to an increase in expression of the osteoblast differentiation factor osterix, as a result. Reporter assays indicate that p53 represses osterix transcription by the minimal promoter in a DNA-binding-independent manner. In addition, p53(-/-) osteoblasts have an enhanced ability to favor osteoclast differentiation, in association with an increase in expression of macrophage-colony stimulating factor, which is under the control of osterix. Furthermore, inactivating p53 is sufficient to rescue the osteoblast differentiation defects observed in mice lacking c-Abl, a p53-interacting protein. Thus, these results identify p53 as a novel regulator of osteoblast differentiation, osteoblast-dependent osteoclastogenesis, and bone remodeling.
Signaling proteins are localized and regulated by Src homology 2 domains which recognize phosphotyrosine-containing sequences. Recently, noncanonical ligands have been proposed for Src homology 2 domains including that of Abl and its breakpoint cluster region fusion, which causes chronic myelogenous leukemia. Here, the Abl Src homology 2 domain's binding sites and affinities for phosphotyrosine- and phosphoserine-containing motifs, phosphoinositides as well as a pyridone-based peptidomimetic inhibitor were determined using nuclear magnetic resonance spectroscopy in order to define their roles. The cognate Crk peptide ligand was bound with an affinity of 69 microM and, like the higher affinity peptidomimetic, engages the phosphotyrosine and +3 hydrophobic pockets while putative phosphoserine-containing breakpoint cluster region ligands are ruled out. Surprisingly, phosphatidylinositol 4, 5 bisphosphate interacts with an overlapping site through an electrostatic mechanism that does not appear to involve hydrophobic insertion into micelles. The conserved Arg36 residue in the FLVRES motif is required for both phosphotyrosine binding and for localization to phosphatidylinositol 4, 5 bisphosphate-containing liposomes, while Arg59 in the betaD strand is necessary for the phosphoinositide interaction. Thus the Src homology 2 domain of Abl, a myristoylated and membrane-localized protein, is able to interact directly with phosphoinositides through a multifunctional basic site that overlaps the phosphotyrosine pocket.
Understanding the mechanisms leading to transformation of early B-lineage precursors is an important step leading to rational design of new treatments for precursor (pre)-B-cell leukemia. We used normal mouse pre-B cells to determine if and how transforming growth factor (TGF)-beta1 affects these precursors to the B-cell lineage and whether transformed pre-B cells respond to TGF-beta1. We found that normal pre-B cells proliferating in the presence of interleukin (IL)-7 enter cell-cycle arrest after exposure to TGF-beta1. However, clonally related IL-7-independent tumors induced by oncogenes abl + myc or raf + myc have reduced sensitivity to TGF-beta1. In contrast, tumor cells induced by myc alone remain sensitive to TGF-beta1 growth suppression. These results suggest that lesions in different molecular signaling pathways can lead to loss of TGF-beta1 sensitivity in a single cell type. The approach of using normal pre-B-cell lines and transformation by overexpression of different oncogenes provides a system to compare and contrast molecular pathways that lead to full malignancy.
Damaged DNA binding proteins (DDBs) play a critical role in the initial recognition of UV-damaged DNA and mediate recruitment of nucleotide excision repair factors. Previous studies identified DDB2 as a target of the CUL-4A ubiquitin ligase. However, the biochemical mechanism governing DDB proteolysis and its underlying physiological function in the removal of UV-induced DNA damage are largely unknown. Here, we report that the c-Abl nonreceptor tyrosine kinase negatively regulates the repair of UV-induced photolesions on genomic DNA. Biochemical studies revealed that c-Abl promotes CUL-4A-mediated DDB ubiquitination and degradation in a manner that does not require its tyrosine kinase activity both under normal growth conditions and following UV irradiation. Moreover, c-Abl activates DDB degradation in part by alleviating the inhibitory effect of CAND1/TIP120A on CUL-4A. These results revealed a kinase-independent function of c-Abl in a ubiquitin-proteolytic pathway that regulates the damage recognition step of nucleotide excision repair.
Dendrite arbor structure is a critical determinant of nervous system function that must be actively maintained throughout life, but the signaling pathways that regulate dendrite maintenance are essentially unknown. We report that the Abelson (Abl) and Abl-related gene (Arg) nonreceptor tyrosine kinases are required for maintenance of cortical dendrites in the mouse brain. arg-/- cortical dendrites initially develop normally and are indistinguishable from wild-type dendrites at postnatal day 21. Dendrite branches are not efficiently maintained in arg-/- neurons, leading to a reduction in dendrite arbor size by early adulthood. More severe dendrite loss is observed in abl-/-arg-/- neurons. Elevation of Arg kinase activity in primary cortical neurons promotes axon and dendrite branching. Activation of integrin receptors by adhesion to laminin-1 or Semaphorin 7A also promotes neurite branching in cortical neurons, but this response is absent in arg-/- neurons because of the reduced dynamic behavior of mutant neurite branches. These data suggest that integrin signaling through Abl and Arg support cortical dendrite branch maintenance by promoting dendrite branch dynamics in response to adhesive cues.
The lipocalin mouse 24p3 has been implicated in diverse physiological processes, including apoptosis due to interleukin-3 (IL-3) deprivation and iron transport. Here we report cloning of the 24p3 cell-surface receptor (24p3R). Ectopic 24p3R expression confers on cells the ability to undergo either iron uptake or apoptosis, dependent upon the iron content of the ligand: Iron-loaded 24p3 increases intracellular iron concentration without promoting apoptosis; iron-lacking 24p3 decreases intracellular iron levels, which induces expression of the proapoptotic protein Bim, resulting in apoptosis. Intracellular iron delivery blocks Bim induction and suppresses apoptosis due to 24p3 addition or IL-3 deprivation. We find, unexpectedly, that the BCR-ABL oncoprotein activates expression of 24p3 and represses 24p3R expression, rendering BCR-ABL(+) cells refractory to secreted 24p3. By inhibiting BCR-ABL, imatinib induces 24p3R expression and, consequently, apoptosis. Our results reveal an unanticipated role for intracellular iron regulation in an apoptotic pathway relevant to BCR-ABL-induced myeloproliferative disease and its treatment.
The cytoplasmic tyrosine kinase Abl is a Src substrate required for platelet-derived growth factor (PDGF) receptor signaling leading to Myc expression and DNA synthesis. Abl targets are, however, ill defined. Here we report that the small GTPase Rac is an important effector of its mitogenic function. PDGF-induced Rac activation was impaired in cells with inactive Abl and active Rac overcame the mitogenic defects found in these cells. Rac function required both a Jun N-terminal kinase (JNK) and a NADPH oxidase (Nox) pathway. Furthermore, co-activation of JNK and Nox were sufficient to mimic the Rac mitogenic rescue. Abl also regulated PDGF-induced JNK and Nox activation. Finally, we found that Myc is an important target of this signaling cascade: Myc induction was sensitive to small inhibitors of JNK and Nox activities and forced expression of Myc overcame the G1 block induced by dominant interfering mutants of mitogen-activated protein kinase kinase 4 (MKK4) and Nox2 activating subunit. We concluded that cytoplasmic Abl operates on a Rac/JNK and a Rac/Nox pathway for PDGF-induced Myc induction and DNA synthesis.
A variety of experimental evidence including findings in various mouse models indicates that the BCR-ABL oncogene is the cause of chronic myeloid leukemia (CML). Since normal hematopoietic cells in marrow and spleen are replaced with proliferating leukemic blasts, we determined whether this is an active process mediated by the leukemia cells. The lipocalin 24p3 was reported to be secreted by mouse hematopoietic cells deprived of IL-3, resulting in apoptosis induction in a variety of hematopoietic cells including bone marrow cells. Here, we show that BCR-ABL+ mouse hematopoietic cells induced persistent expression and secretion of 24p3. Importantly, BCR-ABL+ hematopoietic cells were resistant to the apoptotic effects of 24p3. The expression of the Bcr-Abl oncoprotein and its tyrosine kinase were required for induction of 24p3 expression. Co-culture studies showed that BCR-ABL+ cells induced apoptosis in BCR-ABL negative cells. Antisense 24p3/siRNA expression reduced the level of 24p3 protein in both BCR-ABL+ cells and in conditioned medium (CM) obtained from these cells. CM from BCR-ABL+ cells expressing antisense 24p3/siRNA had reduced apoptotic activity for target cells; 24p3 antibody also reduced the apoptotic activity of the CM. Leukemic mice induced by BCR-ABL+ cells expressing either antisense 24p3 or 24p3 siRNA had increased levels of normal hematopoiesis and reduced invasion of leukemia cells in marrow and spleen tissues. These findings indicate that suppression of normal hematopoiesis in BCR-ABL-induced leukemia is an active process involving secretion of the cell death-inducing factor 24p3 by mouse leukemia cells, raising the possibility that similar factors are involved in BCR-ABL+ CML.
Several lines of evidence suggest that GATA6 has an integral role in controlling development of the mammalian liver. Unfortunately, this proposal has been impossible to address directly because mouse embryos lacking GATA6 die during gastrulation. Here we show that the early embryonic deficiency associated with GATA6-knockout mice can be overcome by providing GATA6-null embryos with a wild-type extraembryonic endoderm with the use of tetraploid embryo complementation. Analysis of rescued Gata6-/- embryos revealed that, although hepatic specification occurs normally, the specified cells fail to differentiate and the liver bud does not expand. Although GATA6 is expressed in multiple tissues that impact development of the liver, including the heart, septum transversum mesenchyme, and vasculature, all are relatively unaffected by loss of GATA6, which is consistent with a cell-autonomous requirement for GATA6 during hepatogenesis. We also demonstrate that a closely related GATA factor, GATA4, is expressed transiently in the prehepatic endoderm during hepatic specification and then lost during expansion of the hepatic primordium. Our data support the proposal that GATA4 and GATA6 are functionally redundant during hepatic specification but that GATA6 alone is available for liver bud growth and commitment of the endoderm to a hepatic cell fate.
This study describes comprehensive polling of transcription start and termination sites and analysis of previously unidentified full-length complementary DNAs derived from the mouse genome. We identify the 5' and 3' boundaries of 181,047 transcripts with extensive variation in transcripts arising from alternative promoter usage, splicing, and polyadenylation. There are 16,247 new mouse protein-coding transcripts, including 5154 encoding previously unidentified proteins. Genomic mapping of the transcriptome reveals transcriptional forests, with overlapping transcription on both strands, separated by deserts in which few transcripts are observed. The data provide a comprehensive platform for the comparative analysis of mammalian transcriptional regulation in differentiation and development.
We have previously shown that F-actin exerts a negative effect on Abl tyrosine kinase activity. This inhibition results from a direct association of F-actin with the C terminus of Abl and accounts, in part, for the loss of Abl activity in detached fibroblasts. We report here that Abl from mitotic cells or cells treated with the protein phosphatase inhibitor okadaic acid remains active when detached from the extracellular matrix. Aspartic acid substitution of Thr(566), which is phosphorylated in mitotic or okadaic acid-treated cells, is sufficient to abolish F-actin-mediated inhibition and to maintain Abl activity despite cell detachment. A recent crystal structure of the Abl N-terminal region has revealed autoinhibitory interactions among the Src homology 3 (SH3), SH2, and kinase domains. We found that deletion of the SH2 domain also abolished the negative effect of F-actin on kinase activity. Immediately following the kinase domain in Abl is a proline-rich linker (PRL) that binds to several SH3 adaptor proteins. Interestingly, binding of the Crk N-terminal SH3 domain to the PRL also disrupted F-actin-mediated inhibition of Abl kinase. These results suggest that F-actin may reinforce the autoinhibitory interactions to regulate Abl kinase and that inhibition can be relieved through phosphorylation and/or protein interactions with the Abl PRL region.
Antisense transcription (transcription from the opposite strand to a protein-coding or sense strand) has been ascribed roles in gene regulation involving degradation of the corresponding sense transcripts (RNA interference), as well as gene silencing at the chromatin level. Global transcriptome analysis provides evidence that a large proportion of the genome can produce transcripts from both strands, and that antisense transcripts commonly link neighboring "genes" in complex loci into chains of linked transcriptional units. Expression profiling reveals frequent concordant regulation of sense/antisense pairs. We present experimental evidence that perturbation of an antisense RNA can alter the expression of sense messenger RNAs, suggesting that antisense transcription contributes to control of transcriptional outputs in mammals.
The inhibitory Fc receptors function to regulate the antigen-driven activation and expansion of lymphocytes. In B cells, the Fc gammaRIIB1 is a potent inhibitor of B cell antigen receptor (BCR) signaling when coligated to the BCR by engagement of antigen-containing immune complexes. Inhibition is mediated by the recruitment of the inositol phosphatase, SHIP, to the Fc gammaRIIB1 phosphorylated tyrosine-based inhibitory motif (ITIM). Here we show that BCR-independent aggregation of the Fc gammaRIIB1 transduces an ITIM- and SHIP-independent proapoptotic signal that is dependent on members of the c-Abl tyrosine kinase family. These results define a novel Abl family kinase-dependent Fc gammaRIIB1 signaling pathway that functions independently of the BCR in controlling antigen-driven B cell responses.
Expression of BCR-ABL is the leading cause of chronic myelogenous leukemia. In chronic myelogenous leukemia cells, c-Abl expression is silenced by promoter methylation. In addition, the level of c-Abl needs to be tightly and constantly regulated due to its cytotoxicity and its rapid degradation after activation. Yet the regulation of c-Abl expression remains unclear. In an effort to gain better understanding of c-Abl function, we performed a glutathione S-transferase-Abl pull-down screen and identified TopBP1, a topoisomerase IIbeta-binding protein that contains Brca1 C-terminal motifs and has been implicated in DNA damage response. Their physical interaction was verified by in vitro and in vivo assays with TopBP1 found as a substrate of Abl proteins. TopBP1 could repress the expression of c-Abl at both mRNA and protein levels. Reporter assays indicate that TopBP1 directly repressed the promoter activity of c-Abl. Furthermore, TopBP1 repressed expression of c-Abl through a novel mechanism that involved histone deacetylation and DNA methylation. This transcriptional repression was inhibited by c-Abl in a kinase-dependent manner. The dual antagonistic interplay between c-Abl and TopBP1 may also provide a mechanism for fine-tuning of c-Abl levels.
c-Abl is a tyrosine kinase that can act as a regulator of cell growth and apoptosis in response to stress. Using cell lines expressing c-Abl in an inducible manner, we identified genes whose expression was regulated by c-Abl kinase activity. Microarray analysis indicated that Early Growth Response-1 (EGR1) gene expression is induced by c-Abl kinase activity, which was confirmed at the message and protein levels. Promoter mapping experiments revealed that c-Abl utilizes three distal serum response elements (SREs) in the EGR1 promoter, which are transactivated by mitogen/extracellular receptor kinase (MEK/ERK) signaling. PD 95089, a specific inhibitor of MEK/ERK signaling, attenuated c-Abl-mediated upregulation of EGR1 expression in a dose-dependent manner. Similar results were obtained by using a dominant-negative mutant of mitogen/extracellular kinase. Significantly, hydrogen peroxide-induced EGR1 expression appears to be mediated by c-Abl, as cells expressing dominant negative c-Abl, and c-Abl-/- murine embryonic fibroblasts, are completely defective in hydrogen peroxide-induced EGR1 expression. In addition, c-Abl-induced apoptosis is partially mitigated by EGR1 activity, as cells devoid of EGR1 expression undergo reduced rates of c-Abl-induced apoptosis. Together, these results indicate that c-Abl promotes the induction of EGR1 through the MEK/ERK pathway in regulating apoptotic response to oxidative stress.
Cardiac chamber formation involves dynamic changes in myocardial organization, including trabeculation and expansion of the compact layer. The positional cues that regulate myocardial patterning, however, remain unclear. Through ligation of the Plexin-A1 receptor, the transmembrane-type semaphorin Sema6D regulates endocardial cell migration. Here, we demonstrate that knockdown of either Sema6D or Plexin-A1 leads to the generation of a small, thin ventricular compact layer and to defective trabeculation. In the heart, expression of the Plexin-A1 extracellular domain alone can rescue the defective trabeculation induced by suppression of Plexin-A1, but not that resulting from defective Sema6D expression. This indicates that reverse signalling by Sema6D occurs within the myocardium. In a ligand-dependent manner, Abl kinase is recruited to the cytoplasmic tail of Sema6D and activated, resulting in phosphorylation of Enabled and dissociation from Sema6D. Constitutive activation of Sema6D signalling enhances the migration of myocardial cells into the trabeculae, whereas inhibition arrests cells within the compact layer. Thus, Sema6D coordinates both compact-layer expansion and trabeculation, functioning as both a ligand and a receptor for Plexin-A1.
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, we show that deficiency of c-Abl and deficiency of ATM differentially altered cell responses to oxidative stress by induction of antioxidant protein peroxiredoxin I (Prx I) via Nrf2 and cell death, both of which required protein kinase C (PKC) delta activation and were mediated by reactive oxygen species. c-abl-/- osteoblasts displayed enhanced Prx I induction, elevated Nrf2 levels, and hypersusceptibility to arsenate, which were reinstated by reconstitution of c-Abl; Atm-/- osteoblasts showed the opposite. These phenotypes correlated 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 could be mimicked by overexpression of PKC delta in normal cells and impeded by inhibition of PKC delta, and diminished responses of Atm-/- cells could be rescued by PKC delta overexpression, indicating that PKC delta mediated the effects of c-Abl and ATM in oxidative stress response. Hence, our results unveiled a previously unrecognized mechanism by which c-Abl and Atm participate in oxidative stress response.
The c-abl tyrosine kinase is the proto-oncogene of the v-abl oncogene of the Abelson murine leukemia virus. Although mutational variants of c-Abl can exhibit gain of function and can produce a transformed phenotype, the function of c-Abl in transformation remained unclear. Here, we report that the loss of c-abl facilitates transformation. c-abl-knockout mouse embryonic fibroblasts (MEFs) immortalized by SV40 T antigen acquired anchorage-independent growth, and by constructing mutational variants of T antigen we showed that binding of large T antigen to p53 and RB was necessary to induce anchorage-independent growth. Although c-abl/p53 double-knockout MEFs did not undergo anchorage-independent growth, those expressing human papilloma virus 16 E7, which mainly inactivates RB, did. Our results show that the loss of c-abl facilitates anchorage-independent growth in the context of p53 and RB deficiency, and suggest that loss of function of c-abl facilitates some types of transformation.
It is becoming increasingly clear that alternative splicing enables the complex development and homeostasis of higher organisms. To gain a better understanding of how splicing contributes to regulatory pathways, we have developed an alternative splicing library approach for the identification of alternatively spliced exons and their flanking regions by alternative splicing sequence enriched tags sequencing. Here, we have applied our approach to mouse melan-c melanocyte and B16-F10Y melanoma cell lines, in which 5,401 genes were found to be alternatively spliced. These genes include those encoding important regulatory factors such as cyclin D2, Ilk, MAPK12, MAPK14, RAB4, melastatin 1 and previously unidentified splicing events for 436 genes. Real-time PCR further identified cell line-specific exons for Tmc6, Abi1, Sorbs1, Ndel1 and Snx16. Thus, the ASL approach proved effective in identifying splicing events, which suggest that alternative splicing is important in melanoma development.
The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.
Filopodia are dynamic F-actin structures that cells use to explore their environment. c-Abl tyrosine kinase promotes filopodia during cell spreading through an unknown mechanism that does not require Cdc42 activity. Using an unbiased approach, we identified Dok1 as a specific c-Abl substrate in spreading fibroblasts. When activated by cell adhesion, c-Abl phosphorylates Y361 of Dok1, promoting its association with the Src homology 2 domain (SH2)/SH3 adaptor protein Nck. Each signaling component was critical for filopodia formation during cell spreading, as evidenced by the finding that mouse fibroblasts lacking c-Abl, Dok1, or Nck had fewer filopodia than cells reexpressing the product of the disrupted gene. Dok1 and c-Abl stimulated filopodia in a mutually interdependent manner, indicating that they function in the same signaling pathway. Dok1 and c-Abl were both detected in filopodia of spreading cells, and therefore may act locally to modulate actin. Our data suggest a novel pathway by which c-Abl transduces signals to the actin cytoskeleton through phosphorylating Dok1 Y361 and recruiting Nck.
Bcr-Abl kinase is known to reverse apoptosis of cytokine-dependent cells due to cytokine deprivation, although it has been controversial whether chronic myeloid leukemia (CML) progenitors have the potential to survive under conditions in which there are limited amounts of cytokines. Here we demonstrate that early hematopoietic progenitors (Sca-1(+) c-Kit(+) Lin(-)) isolated from normal mice rapidly undergo apoptosis in the absence of cytokines. In these cells, the expression of Bim, a proapoptotic relative of Bcl-2 which plays a key role in the cytokine-mediated survival system, is induced. In contrast, those cells isolated from our previously established CML model mice resist apoptosis in cytokine-free medium without the induction of Bim expression, and these effects are reversed by the Abl-specific kinase inhibitor imatinib mesylate. In addition, the expression levels of Bim are uniformly low in cell lines established from patients in the blast crisis phase of CML, and imatinib induced Bim in these cells. Moreover, small interfering RNA that reduces the expression level of Bim effectively rescues CML cells from apoptosis caused by imatinib. These findings suggest that Bim plays an important role in the apoptosis of early hematopoietic progenitors and that Bcr-Abl supports cell survival in part through downregulation of this cell death activator.
The vertebrate retina is comprised of seven major cell types that are generated in overlapping but well-defined intervals. To identify genes that might regulate retinal development, gene expression in the developing retina was profiled at multiple time points using serial analysis of gene expression (SAGE). The expression patterns of 1,051 genes that showed developmentally dynamic expression by SAGE were investigated using in situ hybridization. A molecular atlas of gene expression in the developing and mature retina was thereby constructed, along with a taxonomic classification of developmental gene expression patterns. Genes were identified that label both temporal and spatial subsets of mitotic progenitor cells. For each developing and mature major retinal cell type, genes selectively expressed in that cell type were identified. The gene expression profiles of retinal Muller glia and mitotic progenitor cells were found to be highly similar, suggesting that Muller glia might serve to produce multiple retinal cell types under the right conditions. In addition, multiple transcripts that were evolutionarily conserved that did not appear to encode open reading frames of more than 100 amino acids in length ("noncoding RNAs") were found to be dynamically and specifically expressed in developing and mature retinal cell types. Finally, many photoreceptor-enriched genes that mapped to chromosomal intervals containing retinal disease genes were identified. These data serve as a starting point for functional investigations of the roles of these genes in retinal development and physiology.