Protein-protein interactions mediated by Cys2His2 zinc-fingers

The C2H2 zinc finger motif is a compact ~ 30 amino acid molecular recognition domain that comprises a beta-hairpin followed by an alpha-helix. These domains are typically found as tandem arrays that mediate specific interactions with various macromolecules including DNA, RNA and other proteins. Although very well characterized as a DNA-binding domain, relatively little is currently understood about the molecular details of protein-protein interactions mediated by C2H2 ZFs. The Ikaros and Hunchback transcription factor family provides an ideal model system for studying ZF mediated protein-protein interactions. Ikaros, the founding member of this family is defined as a classical C2H2 ZF protein composed of a cluster of four C2H2 ZFs at the N-terminus and two additional C2H2 ZFs at the C-terminus. While the N-terminal ZFs are involved in specific DNA recognition, the C-terminal domain (termed as Dimerization Zinc Finger or DZF domain) has been shown to mediate the homo- and hetero-typic interactions. In this thesis, the DZF domains found in the Ikaros and Hunchback transcription factor family have been examined using a combination of genetic, biochemical and functional assays. To test if protein-interacting C2H2 ZFs can be used to create novel protein-protein interaction specificities, libraries of synthetic DZFs were constructed by shuffling individual C2H2 ZFs from DZF domains found in the human Ikaros and other related transcription factors. Using a bacterial-based selection system, we identified synthetic heterodimeric DZFs that can mediate activation of a reporter gene in bacterial cells. These protein-protein interaction domains can also be used to reconstitute a synthetic bi-partite activator in the nucleus of a human cell, which results in transcriptional activation of the endogenous VEGF-A gene. In addition, these synthetic two-finger domains can be linked together to create more extended protein-protein interaction interfaces. Analysis of the interaction specificities of these domains led to the discovery of a novel anti-parallel interaction mode for the DZF domain. The homo-typic interaction mediated by different DZF domains was examined in greater detail using mutational analysis. These studies narrowed down residues that are likely to be important for dimerization mediated by the Hunchback DZF domain. To obtain further information about the physical and chemical interaction surface we attempted to purify active peptides consisting of different DZF domains for X-ray crystallography. Although highly purified DZF peptides were obtained, various attempts to refold these peptides into active domains resulted in the formation of aggregates consisting of the various DZFs. Based on findings in the bacterial and cell culture systems, we started exploring if Hunchback dimerizes in Drosophila melanogaster using its DZF domain and if dimerization is essential for the function of the protein. Constructs encoding the full-length Hunchback protein harboring various natural and modified DZF domains were generated and expressed in transgenic flies. These transgenics were used to perform functional in vivo studies of the Hunchback DZF domain in Neuroblast specification during Drosophila melanogaster development. We confirmed previous studies that the C-terminal domain in Hunchback is important for maintaining the function of Hunchback in specifying early-born temporal identity in Drosophila neural stem cell lineages. Importantly, our results indicate that this domain can be functionally replaced with a heterologous (i.e., non fly) DZF domain, suggesting that the importance of the DZF domain is due to its ability to mediate dimerization

The cloning and characterisation of link1: A LIM-domain containing protein kinase

This thesis describes the isolation and cloning of a novel mouse gene, named mLimkl, which exhibits high homology to the human LIMK gene. mLimkl represents a single copy gene and maps to the distal end of mouse chromosome 5. Northern blot analysis showed preferential expression of a 3.5kb message in adult spinal cord and brain. In situ hybridisation studies confirmed high expression levels in the nervous system, particularly in the spinal cord and the cranial nerves and dorsal root ganglia. The amino acid sequence reveals two features which place mLimkl into a novel class of protein kinases. Firstly, although mLimkl contains all motifs found in catalytic kinase domains, amino acids previously described to be diagnostic of either serine/threonine- or tyrosine-kinases are not present. It is demonstrated that mLimkl-fusion protein can autophosphorylate on serine, tyrosine and threonine residues in vitro, and mutation of residue D460 within the IHRDL motif abolishes kinase activity. Secondly, mLimkl has two tandem LIM-domains in the amino-terminal region. These zinc-finger like domains can mediate protein-protein interactions and have been described in transcription factors and cytoskeletal proteins. The combination of LIM- and kinase domains may provide a novel route by which intracellular signaling can be integrated