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Iir 3.O3 Llt iir 3.o3 llt CRUCE t Pleckstrin Homology and Tec Homology Domains Link Tec Kinase Signalling to the Cytoskeleton Anita Merkel A thesis submitted in fulfilment of the requirements for the degree of Doctor of PhilosoPhY Biochemistry DisciPline Department of Molecular Biosciences The University of Adelaide Adelaide, Australia August 2002 Summary The Tec family of non-receptor protein tyrosine kinases is involved in the progression of intracellular signalling pathways that involve changes in calcium influx and cellular gene expression, cell growth and differentiation, and cytoskeletal remodelling. The five members of the Tec-family of kinases (Btk, Itk, Bmx, Txk and Tec) are expressed primarily in haematopoietic cells and are responsible for the transmission of signals from a variety of cell surface receptors. This includes antigen receptors such as the B cell, T cell and Fc-y receptors, cytokine receptors, receptor tyrosine kinases, G-protein-coupled receptors and integrin adhesion receptors. Tec-family kinases are biologically important molecules, since, lack of functional Btk causes the severe human immunodeficiency disease X-linked agammaglobulinemia. The constituent modular domains of Tec-family tyrosine kinases are responsible for the allosteric regulation of the kinase domain catalytic activity and subcellular targeting. Both intermolecular (with proteins and 3'-phosphatidylinositol lipids) and intramolecular interactions play an important role in this process. Like Src-family kinases, Tec-family kinases contain carboxyl terminal Src homolgy-3 (SH3), Src homolgy-2 (SH2) and tyrosine kinase domains. However, Tec-family kinases also contain amino terminal pleckstrin homology (PH) and Tec homology (TH) domains that make them unique amongst tyrosine kinase families. There is increasing evidence that links the signalling of Tec-family kinases to actin cytoskeleton remodelling. The cytoskeleton provides a dynamic structural framework within cells. As a key part of the contractile apparatus, it plays fundamental roles in the maintenance of cell morphology, cell-cell and cell-matrix adhesion, cell division and phagocytosis. The PH domain plays an integral role in this cytoskeletal connection through interaction with proteins such as filamentous actin (F-actin), focal adhesion kinase and integrin-associated signalling complexes. PH domains, in general, function to target the activated protein to the correct cellular site, such as the cytoskeleton or the cell membrane. The primary objective of this work was to identiff protein ligands of the PH and TH domain pair (PHTH domain) of Tec kinase and to better understand the role of the PHTH domain in Tec signalling pathways. A yeast two-hybrid assay was performed in which the Tec PHTH domain was used as a bait to screen allbrary of potential protein ligands expressed in human liver tissue; this tissue was chosen since Tec was first identified as a tyrosine kinase expressed in hepatocellular carcinoma. Actinin-4 was identified as a novel ligand of Tec. The ut bulk of the work presented in this thesis involves characterisation of the Tec:Actinin-4 interaction using biochemical, molecular biology and cell biology techniques. Actinin-4 is a newly identified non-muscle s-actinin isoform that belongs to the spectrin-family of proteins. ø-actinins are described as actin-bundling proteins that determine the mechanical properties of the actin filament network. They form anti-parallel homodimers with an actin-binding domain at each end, dynamically cross-linking actin filaments by simultaneously binding to adjacent F-actin strands through their distinct actin binding sites. Regulation of F-actin binding by cr-actinins plays an important role in cell morphology change. Actinin-4 has been implicated in the metastatic potential of human cancers and is, therefore, an attractive target for anti-cancer therapies. g-actinins are composed of modular domains; these include dual N-terminal calponin homology (CH) domains, a central rod region that has four copies of the spectrin repeat domain and dual C-terminal calmodulin-like EF-hand domains that are predicted to bind Ca2n and confer sensitivity to intracellúar C** concentrations. The F-actin binding region is adjacent to a phosphatidylinositol 4,5-bisphosphate binding site and is contained within the CH domains. In the homodimer, these are juxtaposed against the EF-hand domains of the other molecule. The spectrin repeat domains contain the intermolecular dimerisation interface and contact protein ligands. The Tec binding region of Actinin-4 includes the third spectrin repeat domain and flanking sequences that confer dimerisation. Co-immunoprecipitation experiments utilizing mammalian cell expressed Tec and Actinin-4 proteins confirmed the interaction in a cellular context. Site-directed mutagenesis was used to create variants for probing the involvement of specific residues predicted to affect the binding of the two proteins. Targeted residues included critical actin binding determinants of the Tec PH domain: K18A/K194/IO0A; as well as regulatory residues predicted to endorse activity-dependent conformations of the Tec molecules and, thus, availability of activation-dependent ligand binding sites: Y187E, K3978 and R29C. In Actinin-4, a substitution mutation of a candidate PH-domain ligand known as a HIKE-like motif was created: H602NI6034/K6044. None of the analysed mutations appeared to abrogate binding. The subcellular localisations of endogenous Tec and Actinin-4 proteins were investigated using indirect immunofluorescence microscopy. Extensive colocalisation in an ultrafine filamentous network as well as in juxtanuclear regions of the cell were detected. The subcellular localisation of enhanced green fluorescent protein (EGFP)-tagged Tec protein variants, including wild-type Tec as well as the substitution mutants described above, were lv investigated using direct fluorescence microscopy of transiently transfected COS-I cells' Interestingly, they have distinct as well as overlapping subcellular localisation patterns compared with endogenous Tec. These overexpressed fusion proteins were also used in co-immunoprecipitation experiments. Both Triton-X-l0O soluble and insoluble pools of the various EGFP-Tec proteins were identified. This was also the case for endogenous Tec and Actinin-4 proteins. Potentially, the insoluble fraction represents cfloskeletal-translocated protein and suggests that insoluble cytoskeletal proteins are targets of tyrosine kinase regulation. While phosphorylation is a major regulator of Tec-family kinase activation and function, future experiments will be required to address whether or not this is also the case for a-actinins. Given that Tec is implicated in actin reaffangement during phagocytosis, it would be attractive to consolidate the physiological relevance of the Tec:Actinin-4 interaction using phagocytosis experiments. Since change in cell morphology is a key aspect of oncogenesis, it is important to understand and characterise the cytoplasmic signals that regulate cytoskeletal architecture' Modulation of these signals can be used to combat aberrant or inappropriate signalling that arises in disease states. In an effort to elucidate the Tec binding site of Actinin-4 and, thus, provide a potential target site for chemical interference of the Tec:Actinin-4 interaction, recombinant protein encompassing the third spectrin repeat of Actinin-4 was prepared for structural studies. Preliminary nuclear magnetic resonance spectroscopy studies were performed and indicate that this protein is amenable to structure determination. It is envisaged that the Actinin-4 residues of the interaction interface will be identified by comparison of spectra obtained from PHTH domain bound and unbound samples of the Actinin-4 third spectrin repeat. This suggests that a template for design of modulators of Actinin-4 function, through the interference of Tec binding, can be obtained and is a major step toward dissecting the signalling pathway involving Tec and Actinin-4 proteins. Taken together, the results presented here provide valuable information conceming a potential direct link between Tec tyrosine kinase and regulation of cytoskeletal architecture. Tec could be involved in cell restructuring through interaction with and phosphorylation of cytoskeletal components in processes such as cell adhesion, migration and phagocytosis' Since tyrosine kinase signalling pathways are activated immediately downstream of cellular receptors, this helps to explain the concomitant swiftness of cytoskeletal reorganisation' Lastly, this research has identified new targets for cancer therapies and provided a reference point to manipulate signalling pathways involved in tumour metastasis. v Acknowledgments Thankyou to Prof Graham Mayrhofer and Prof Peter Rathjen for providing me the opportunity to study a doctorate degree in the Department of Molecular Biocsciences and Department of Biochemistry at the University of Adelaide. I am grateful for the Australian postgraduate Award Scholarship that gave me financial support throughout my PhD. I am sincerely grateful to Dr Grant Booker for his supervision and support throughout these studies. My dear friends Ines Atmosukarto and Sharon Pursglove were invaluable in their guidance in the laboratory and willingness to discuss experimental results, among (lots of) other things. Gavin Chapman (Harem Master), Trish Pelton and, more recently, Carlie Delaine, Sue Fowler and Kasper Kowalski also provided invaluable friendship
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