2010ParnhamSRPhD.pdf (16.99 MB)
Role of Paxillin and Polyadenylate Binding Protein-1 Complex in mRNA Trafficking During Cell Migration
thesisposted on 2010-07-26, 13:23 authored by Stuart Roy Parnham
Cellular migration is dependent upon the efficient formation of focal adhesions at the point where the cytoplasmic components engage the extracellular matrix via the integrin proteins. The leading edge of the migratory cell is also a site where proteins such as actin are synthesised. In order for these events to take place mRNA transcripts must be transported from the nucleus to the leading edge by a protein or protein:protein complexes that are capable of nucleo-cytoplasmic shuttling. For example paxillin is a scaffold protein that is a central component of focal adhesions and is capable of nucleo-cytoplasmic shuttling. Polyadenylate binding protein-1 was found to be an abundant co-immunoprecipitant of paxillin in lamellipodium formations. Using overlapping PABP-1 and paxillin constructs it was found that one of paxillins N-terminal LD domains (LD1) interacted with PABP-1 RRM (RNA recognition motif) 1 and 2. The NMR derived structures of PABP-1 RRMs 1 and 2 in their unbound forms are presented within this report. Previous reports have indicated the presence of a paxillin binding sub-domain within the PABP-1 RRM 1 domain. NMR titration data, using a synthetic paxillin LD1 peptide, revealed a binding interaction site within PABP-1 RRM 2 and not within the PABP-1 RRM 1 domain. Also reported here are the structural details of the PABP-1 RRM 2/paxillin LD1 complex. The binding interaction appears to be in the fast exchange regime with an estimated Kd of ~211μM. Experimental evidence shows the binding to be electrostatically driven and confined to the four stranded antiparallel β-pleated sheet. The interaction site is shared with the polyadenylated tail of nascent mRNA transcripts. NMR titration data indicates a competition for this site to be biased toward mRNA. This linked with other experimental data, presented here, and would suggest a more complicated picture of binding to include multiple sites of contact.
Supervisor(s)Roberts, Gordon C.K.
Date of award2010-07-07
Awarding institutionUniversity of Leicester