Please use this identifier to cite or link to this item: http://hdl.handle.net/2440/51019
Type: Thesis
Title: Ca²⁺ and phosphoinositides regulations in α-actinin -4 F-actin binding.
Author: Chen, Huang-Hui
Issue Date: 2008
School/Discipline: School of Molecular and Biomedical Science : Biochemistry
Abstract: α-actinin-4 is a non-muscle isoform of α-actinin that belongs to the spectrin superfamily. It comprises three functional regions: an N-terminal actin-binding region that consists of two calponin homology (CH) domains, a central region that consists of four copies of the spectrin-like repeat domain and a C-terminal calmodulin-like domain that is predicted to bind Ca²⁺. α-actinin-4 is organised as an antiparallel homodimer formed by the interaction of four spectrin-like repeats between the two monomers, giving a rod-like shape, with actin binding regions at both ends. α-actinin-4 is an abundant actin-bundling protein, which provides a direct link between actin filaments and integrins, and is believed to play an important role in stabilising cell shape and adhesion and regulating cell migration. It also acts as a tumor suppressor and influences the metastatic potential and invasiveness in human cancers. A cluster of three actin binding motifs have been identified in the CH domains (2X CH) from other members of the spectrin superfamily, utrophin and dystrophin. Two of them reside in the CH1 domain and the third resides in the first α-helix of the CH2 domain. In addition, a PIP2 binding site has been mapped on a region adjacent to actin-binding site-3. These observations imply the F-actin binding activity would be regulated by phosphoinositides. Five mutations of α-actinin-4, K122N, an alternative splice variant, K255E, T259I and S263P, have been reported to be involved in three human diseases, non-small lung cancer (NSCLC), small cell lung cancer (SCLC) and focal segmental glomerulosclerosis (FSGS). The mutation site within these mutants is located on the actin binding region. Therefore, the actin binding region is presumed to be associated with the progression of human disease. The aims of this thesis focused on the regulation of the F-actin binding activity of α-actinin-4 by phosphoinositides (PIP2 and PIP3), the calmodulin-like domain and Ca²⁺ , determination of the three-dimensional structure of the CH2 domain in solution and identification of the phosphoinositide binding site on the CH2 domain. In order to investigate the F-actin binding activity quantitatively, a novel in vitro F-actin binding assay (solid phase) was established to replace the semi-quantitative actin bundling assay. Using this novel solid phase F-actin binding assay, Ca²⁺ was shown to enhance the F-actin binding activity of α-actinin-4 in a concentration-dependent manner. The presence of 10 mM Ca²⁺ results in a two-fold increase in the F-actin binding activity. Both PIP2 and PIP3 inhibited the F-actin-binding activity of α-actinin-4 in a concentration-dependent manner with an approximate IC₅₀ of 75 and 45 μM, respectively. In order to characterise how phosphoinositides regulated the F-actin binding activity of α-actinin-4, the solution structure of α-actinin-4 CH2 domain was determined and the phosphoinositide binding residues within the CH2 domain were identified using NMR spectroscopy. The solution structure of α-actinin-4 CH2 domain contained six α-helices and was similar to that of other spectrin superfamily members. The strategy used in identification of the phosphoinositide binding site was an NMR-based 2D ¹H-¹⁵N HSQC ligand titration assay to replace the traditional semi-quantitative protein-lipid overlay assay. Using the NMR-based ligand titration assay, the recognition site for the inositol head group resides in residues Trp 172, Tyr 265 and His 266 and the binding region of acyl chains resides in the first α-helix structure which is one of the putative F-actin binding sites. In order to examine the interaction of phosphoinositides with this site, Y265A and H266E mutants of α-actinin-4 CH2 domain were generated using site-directed mutagenesis and verified the interaction with phosphoinositides and the inositol head group using an NMR-based ligand titration assay. These results confirmed the phosphoinositide binding site on the CH2 domain and residues, Tyr 265 and His 266, are critical for interacting with phosphoinositides. Wildtype and mutants (Y265A and H266E) of α-actinin-4 were expressed in mammalian cells as EGFP-fusion proteins. Wildtype α-actinin-4 was shown to be co-localised with focal adhesions and actin stress fibres. However, Y265A and H266E mutants of α-actinin-4 were co-localised with actin stress fibres but poorly co-localised with focal adhesions. Moreover, both Y265A and H266E mutants of α-actinin-4 were co-localised with actin in the cytoplasm rather than localised along the cell membrane after EGF stimulation for 30 minutes. These results suggested that PIP2 assists the co-localisation of α-actinin-4 with focal adhesions. Taken together, the results described in this thesis concluded that Ca²⁺ enhanced the F-actin binding activity of α-actinin-4 in vitro. However, phosphoinositides (PIP2 or PIP3) inhibited the F-actin binding activity in vitro. Moreover, the results described in this thesis provided a phosphoinositide binding site on α-actinin-4 CH2 domain. Binding to PIP2 is important to the localisation of α-actinin-4 in focal adhesions.
Advisor: Booker, Grant William
Dissertation Note: Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008
Keywords: alpha-actinin-4; calponin homology-2; domain; phosphoinositides; calcium
Provenance: This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exception. If you are the author of this thesis and do not wish it to be made publicly available or If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at: http://www.adelaide.edu.au/legals
Appears in Collections:Research Theses

Files in This Item:
File Description SizeFormat 
01front.pdf62.84 kBAdobe PDFView/Open
02whole.pdf5.97 MBAdobe PDFView/Open


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.