Protein-tyrosine kinase signalling pathways in normal hematopoiesis and leukemogenesis

The reversible phosphorylation of proteins on tyrosine residues modulates protein function and is central to the regulation of hematopoiesis. Consistent with the emerging role of phosphotyrosine in normal hematopoietic processes, gain- or loss-of-function mutations in genes encoding protein-tyrosine kinases (PTKs) have been identified in a number of human hematological disorders. A well characterized example of an activating mutation is fusion of the ' abl' and 'bcr' genes which produces a chimeric Bcr-Abl protein with deregulated Abl kinase activity. The 'bcr-abl' fusion gene is the result of a reciprocal t(9;22) translocation and occurs in chronic myelogeneous leukemia and a subset of acute lymphoblastic leukemia. Identification of the targets of normal and transforming PTKs is necessary for understanding both normal signal transduction pathways and the means by which these pathways are deregulated during cellular transformation. In this thesis, proximal targets of the Bcr-Abl oncoproteins are characterized. Src homology 2 (SH2) and Src homology 3 (SH3) domains are protein interaction domains which recognize phosphotyrosine-containing and proline-rich motifs respectively, and are present in an otherwise diverse group of signalling molecules. In Chapters 2 and 3, a number of SH2/SH3-containing enzymes and adaptor molecules, along with their associated proteins, are implicated in ' bcr-abl'-mediated transformation. These include the 120 kDa Ras GTPase activating protein (p120-Gap), the Gap-associated proteins p62 and p190, Grb2, mSos1, Shc, Shc-associated p140/p145, Vav, the p85 regulatory subunit of phosphatidylinositol 3'-kinase and phospholipase C-ã1. Subsets of these proteins are substrates of Bcr-Abl (or a downstream PTK) and/or form stable complexes with Bcr-Abl 'in vivo'. A number of the identified targets are potential regulators of Ras and provide mechanisms whereby Bcr-Abl may be directly linked to Ras signalling pathways. These data are consistent with a prominent role for Ras in PTK-mediated transformation. In Chapter 4, some of these proteins, notably Shc, Shc-associated p140/p145, Grb2 and Vav, are demonstrated to participate in cytokine-stimulated pathways which control the survival, proliferation or activation of normal primary hematopoietic cells. Taken together, these data support the notion that leukemogenesis involves the constitutive stimulation of signalling molecules that are normally transiently activated in cells responding to cues within the hematopoietic microenvironment. The structural basis for the interaction of PTKs with some of their targets was also investigated. These data suggest that SH2-mediated interactions are important in hematopoietic cell signalling. In Chapter 3, the binding of Grb2 to Bcr-Abl is demonstrated to be mediated by the Grb2 SH2 domain which recognizes an autophosphorylated sequence, p.Tyr177-Val178-Asn 179-Val180, located within the N-terminal ' bcr'-encoded sequence common to all isoforms of Bcr-Abl. The Bcr element of the Bcr-Abl fusion proteins thus contributes to the target specificity of the activated Abl kinases. This is a novel function of the Bcr moiety which has not previously been defined, and provides new insight into the potential mechanisms whereby fusion of the 'abl' and 'bcr' genes is oncogenically activating.