A screen for phosphatidylinositol 3,4-bisphosphate and other 3-phosphoinositide effector proteins

Class I phosphoinositide (PI) 3-kinases exert profound effects on cell growth, division, motility and metabolism via their primary lipid product phosphatidylinositol 3,4,5- trisphosphate (PtdIns(3,4,5)P3) and a metabolite of this, phosphatidylinositol 3,4- bisphosphate (PtdIns(3,4)P2). Many effector proteins for PtdIns(3,4,5)P3 are well recognised but by contrast, few molecular targets for PtdIns(3,4)P2 have been identified. This study describes a screen to identify PI 3-kinase-responsive proteins that is selective particularly for these. The approach features a unique three-tier affinity approach and incorporates a primary recruitment of target proteins to membranes of intact cells, selectively enriched in PtdIns(3,4)P2. In addition, this screen utilises stable isotope labelling with amino acids in cell culture (SILAC) to differentially label cells stimulated in the absence and presence of the PI 3-kinase inhibitor wortmannin. The integration of these techniques provides a ratio-metric readout, allowing authentically 3- phosphoinositide (3-PI) responsive components to be distinguished from the co-purifying background proteins. The identification of tandem pleckstrin homology domain containing protein-1 (TAPP-1) and protein kinase B (PKB) among a multitude of proteins expressing known lipid binding domains (LBDs) demonstrates the utility of this strategy. Analysis of other similarly, isotopically enriched candidate 3-PI interacting proteins yielded two novel lipid binding proteins, PARIS-1 (prostate antigen recognised and identified by SEREX 1) and IQGAP1 (IQ motif containing GAP1). The concentration dependent interaction of PARIS-1 and IQGAP1 with PtdIns(3,4,5)P3 was confirmed by an in vitro, SPR based assay. Intriguingly, IQGAP1, a potential tumour promoter, lacks a currently established LBD and may therefore exemplify an entirely novel 3-PI selective binding domain.

Name dc:type.qualificationname

Doctor of Philosophy

Level dc:type.qualificationlevel

Doctoral Thesis

Grantor dc:publisher.institution

University of Dundee

Year dc:date.issued

2010

Author dc:creator

• Dixon, Miles

Language dc:language

eng

Identifier URI

https://discovery.dundee.ac.uk/files/1937248/Supplementary_Table_2.xml
https://discovery.dundee.ac.uk/files/1937249/Supplementary_Table_3.xml

Identifier

oai:discovery.dundee.ac.uk:studenttheses/374a230d-78ec-49e5-a52a-ac3f46082cdc
https://discovery.dundee.ac.uk/en/studentTheses/374a230d-78ec-49e5-a52a-ac3f46082cdc

OAI identifier oai:identifier

oai:discovery.dundee.ac.uk:studenttheses/374a230d-78ec-49e5-a52a-ac3f46082cdc