The adaptor protein Shb has previously been shown to regulate apoptosis in response to cytokines and inhibitors of angiogenesis although the mechanisms governing these effects have remained obscure. We currently demonstrate interactions between Shb and c-Abl and that Shb regulates c-Abl kinase activity. The data suggest that c-Abl binds to tyrosine phosphorylated Shb via a concerted effort involving both the c-Abl SH3 and SH2 domains. The biological significance of the Shb/c-Abl interaction was presently tested in overexpression experiments and was found to promote hydrogen peroxide-induced cell death. We also show by Shb knockdown experiments that Shb regulates c-Abl activity and modulates cell death in response to the genotoxic agent cisplatin and the endoplasmic reticulum stress-inducer tunicamycin. The findings are in agreement with the notion of Shb playing a pivotal role in modulating c-Abl pro-apoptotic signaling in response to various stress stimuli.
"The SH2 domain of Shb binds to phosphotyrosines on activated receptors such as the platelet derived growth factor receptor (PDGFR), the IL-2 receptor and the T cell receptor (TCR) . Shb’s various signaling domains further recruit intracellular signaling mediators, including focal adhesion kinase (FAK), Src, phosphatidylinositol 3-kinase (PI3K), Vav-1, and c-Abl [24,25], thereby regulating cytoskeletal rearrangements, proliferation as well as apoptosis . "
[Show abstract][Hide abstract] ABSTRACT: Background
The Src homology-2 domain protein B (Shb) is an adapter protein operating downstream of several tyrosine kinase receptors and consequently Shb regulates various cellular responses. Absence of Shb was recently shown to reduce hematopoietic stem cell proliferation through activation of focal adhesion kinase (FAK) and thus we sought to investigate Shb’s role in the progression of leukemia.
Wild type and Shb knockout bone marrow cells were transformed with a retroviral BCR-ABL construct and subsequently transplanted to wild type or Shb knockout recipients. Disease latency, bone marrow and peripheral blood cell characteristics, cytokine expression, signaling characteristics and colony formation were determined by flow cytometry, qPCR, western blotting and methylcellulose colony forming assays.
It was observed that Shb knockout BCR-ABL-transformed bone marrow cells produced a disease with death occurring at earlier time points compared with corresponding wild type controls due to elevated proliferation of transformed bone marrow cells. Moreover, significantly elevated interleukin-6 and granulocyte colony-stimulation factor mRNA levels were observed in Shb knockout c-Kit + leukemic bone marrow cells providing a plausible explanation for the concurrent peripheral blood neutrophilia. Shb knockout leukemic bone marrow cells also showed increased ability to form colonies in methylcellulose devoid of cytokines that was dependent on the concomitantly observed increased activity of FAK. Transplanting BCR-ABL-transformed Shb knockout bone marrow cells to Shb knockout recipients revealed decreased disease latency without neutrophilia, thus implicating the importance of niche-derived cues for the increase of blood granulocytes.
Absence of Shb accelerates disease progression by exerting dual roles in BCR-ABL-induced leukemia: increased cell expansion due to elevated FAK activity and neutrophilia in peripheral blood, the latter dependent on the genetic background of the leukemic niche.
"The C-terminal SH2- domain of Shb  facilitates interactions with receptor tyrosine kinases such as vascular endothelial growth factor receptor-2 (VEGFR-2) , platelet derived growth factor receptor (PDGFR)  and the T cell receptor (TCR)  . Moreover, Shb associates via its other domains with additional signaling elements such as SH2 domain-containing leukocyte protein of 76 kDa (Slp76) , Vav1 , c-Abl , FAK , phospholipase C-γ (PLCγ), Src, and phosphatidylinositol-3 kinase (PI3K), acting as a scaffold in signaling cascades . Several of Shb's known interaction partners, including PDGFR , TCR , Vav1 , and FAK , are established regulators of blood cell development and function. "
[Show abstract][Hide abstract] ABSTRACT: The widely expressed adaptor protein Shb has previously been reported to contribute to T cell function due to its association with the T cell receptor and furthermore, several of Shb's known interaction partners are established regulators of blood cell development and function. In addition, Shb deficient embryonic stem cells displayed reduced blood cell colony formation upon differentiation in vitro. The aim of the current study was therefore to explore hematopoietic stem and progenitor cell function in the Shb knockout mouse. Shb deficient bone marrow contained reduced relative numbers of long-term hematopoietic stem cells (LT-HSCs) that exhibited lower proliferation rates. Despite this, Shb knockout LT-HSCs responded promptly by entering the cell cycle in response to genotoxic stress by 5-fluorouracil treatment. In competitive LT-HSC transplantations, Shb null cells initially engrafted as well as the wild-type cells but provided less myeloid expansion over time. Moreover, Shb knockout bone marrow cells exhibited elevated basal activities of focal adhesion kinase/Rac1/p21-activated kinase signaling and reduced responsiveness to Stem Cell Factor stimulation. Consequently, treatment with a focal adhesion kinase inhibitor increased Shb knockout LT-HSC proliferation. The altered signaling characteristics thus provide a plausible mechanistic explanation for the changes in LT-HSC proliferation since these signaling intermediates have all been shown to participate in LT-HSC cell cycle control. In summary, the loss of Shb dependent signaling in bone marrow cells, resulting in elevated focal adhesion kinase activity and reduced proliferative responses in LT-HSCs under steady state hematopoiesis, confers a disadvantage to the maintenance of LT-HSCs over time.
Experimental Cell Research 03/2013; 319(12). DOI:10.1016/j.yexcr.2013.03.020 · 3.25 Impact Factor
"Several conditions of cell stress induce c-Abl activation, and consequently cell cycle arrest and cytotoxic signaling eventually causing apoptosis ensues [17,18]. We have recently described an interaction between Shb and c-Abl that regulates apoptosis . "
[Show abstract][Hide abstract] ABSTRACT: Induction of apoptosis is one strategy for treatment of prostate cancer. The Shb adapter protein has been found to regulate apoptosis in various cell types and consequently human prostate cancer 3 (PC3) cells were transfected to obtain cells overexpressing Shb in order to increase our understanding of the mechanisms regulating PC3 cell apoptosis.
Human prostate cancer cells (PC3) were transfected with control vector or a vector containing the Shb cDNA. Clones overexpressing Shb were studied with respect to apoptosis (Dapi, M30) and c-Abl activation (Western blot for pY-245-Abl). The cells were exposed to the anti-tumor agent 2-methoxyestradiol (2-ME) and the p38 MAPK and c-Abl inhibitors SB203580 and STI-571, respectively, after which cell death was determined. In vivo tumor growth and tumor cell proliferation (Ki-67 staining) or apoptosis (active caspase 3 staining) were also determined in nude mice.
PC3 cells overexpressing Shb exhibited increased rates of apoptosis in the presence of the anti-tumor agent 2-ME. The Shb cells displayed increased activity of the pro-apoptotic kinase c-Abl. Pre-treatment with p38 MAPK (SB203580) or c-Abl (STI-571) inhibitors completely blocked 2-ME-induced apoptosis, implicating these two pathways in the response. The PC3-Shb cells displayed reduced tumor growth in vivo, an effect occurring as a consequence of increased apoptosis and reduced DNA synthesis.
It is concluded that Shb promotes 2-ME-induced PC3 cell apoptosis by increased pro-apoptotic signaling via the c-Abl pathway and that this causes reduced tumor growth in vivo.
BMC Cancer 02/2007; 7(1):161. DOI:10.1186/1471-2407-7-161 · 3.36 Impact Factor
N Godin-Heymann, S Brabetz, M M Murillo, M Saponaro, C R Santos, A Lobley, P East, P Chakravarty, N Matthews, G Kelly, S Jordan, E Castellano, J Downward
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