The goal of the proposed research is to understand how adhesion regulates cell proliferation. The focus will be on the Abl tyrosine kinase as a transducer of the growth regulatory signal. The ubiquitous c-Abl is localized in the cytoplasm and the nucleus. The nuclear c-abl binds DNA and regulates transcription. The cytoplasmic c-abl interacts with both G and F-actin. Recently, a collaboration between two units of this program has demonstrated that the kinase activity and the subcellular localization of c-Abl is regulated by the adherent status of a cell. In addition, overexpression of a kinase-defective c-Abl can interfere with adhesion-mediated activation of MAP kinase. The c-Abl kinase is constitutively activated by fusion with Bcr sequences in the Bcr-Abl protein produced from the Philadelphia chromosome in chronic myelogenous leukemia (CML). The targets of transformation in CML are the pluripotent stem cells which are adherent to the bone marrow stroma. The CML stem cells show reduced adhesion and this defect is correlated with the diseased state. In a fibroblast model we have developed, Bcr-Abl is also found to affect the adhesive function. Moreover, Bcr-Abl can abrogate the adhesion requirement to promote anchorage-independent growth, but does not function as a mitogen. Taken together, these observations suggest a model in which regulated activation of c-Abl tyrosine kinase transduces adhesion signal to promote normal growth, and a deregulated Bcr-Abl signals independently of adhesion to cause transformation. To test this hypothesis, we propose to pursue four specific aims.
In Aim 1, the functional domains required for c-Abl to respond to adhesion will be identified and the abl+/+ and abl-/- cells will be compared for their response to adhesion.
In Aim 2, the mechanism by which Bcr-Abl affects adhesion will be investigated.
In Aim 3, downstream targets of c-Abl and Bcr-Abl in the adhesion-regulated pathways will be identified, in particular the pathways regulated by Rho and the activation of MAP kinase. We have shown p130cas to be a potential substrate of Bcr-Abl, and will identify others. We will also isolate genes that are commonly regulated by adhesion and by Bcr-Abl. The goal is to obtain nuclear targets with which to better understand the adhesion-regulated pathways.
In Aim 4, we will examine the function of Abl targets in the regulation of cell proliferation. Results and reagents obtained from the experiments conducted with the fibroblast model will be extended to hematopoietic cells and utimately in long- term bone marrow culture to study the Abl tryosine kinase in the control of stem cell proliferation.
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