Characterization of Hck signalling networks in hematopoietic cells, with a focus on a novel, transforming Cbl mutant

Abstract

Src family kinases were first identified as transforming alleles captured in the genome of retroviruses almost three decades ago. These tyrosine kinases integrate and relay information to negotiate appropriate biological outcomes in response to a multitude of extracellular cues. Their inappropriate activation has been associated with a number of pathophysiologies, including cancer, autoimmune, and inflammatory diseases. Therefore, strict spatiotemporal control of these kinases is vital for the maintenance of cellular homeostasis and tissue integrity. However, very little is known with respect to the normal signalling pathways with which they serve. The overriding theme of this thesis was to describe and dissect the elaborate circuitry within which these kinases act, in order to enhance our understanding of Src family biology. Hematopoietic cell kinase (Hck) is one such Src family kinase that is expressed in the myelomonocytic lineage and B-cells. In these cells, Hck serves multiple signalling capacities, and its deregulation has been implicated in several disease states. In the current study, the signalling networks involving Hck are investigated. Using GST-Hck fusion constructs, the association of this kinase with numerous signalling molecules in unstimulated and activated hematopoietic cells was established. This led to the biochemical characterization of a tyrosine kinase activity likely corresponding to Syk; a novel putative effector of Hck; the c-cbl proto-oncogene product; and a novel, transfonning mutant of c-Cbl, termed p95Cbl. Protein ubiquitination has emerged as an important regulatory modification in diverse cellular processes, including tyrosine kinase signalling. c-Cbl acts as an adaptor protein and as an E3 ubiquitin ligase, directing ubiquitination and the ensuing destruction protein and as an E3 ubiquitin ligase, directing ubiquitination and the ensuing destruction of signalling molecules. This investigation also includes the detailed characterization of the herein identified p95Cbl mutant, which was found to harbour an internal deletion removing the RING fmger, thus crippling its ubiquitin ligase activity, while retaining all of its protein-protein interaction domains. p95Cbl displays aberrant subcellular localization and tyrosine phosphorylation, and potently transforms NIH3T3 fibroblasts and BalF3 hematopoietic cells in culture. In addition, a pronounced, selective sensitivity of mutant c-Cbl proteins to the antileukemic compound piceatannol was observed, thus demarcating c-Cbl mutants as potential therapeutic targets. A possibility for alterations to c-Cbl in human malignancies is discussed.