Ze-Yi Lim

PtdIns(3)P Regulates the Neutrophil Oxidase Complex by Binding to the PX Domain of p40phox

The production of reactive oxygen species (ROS) by neutrophils has a vital role in defence against a range of infectious agents, and is driven by the assembly of a multi-protein complex containing a minimal core of five proteins: the two membrane-bound subunits of cytochrome b558 (gp91phox and p22phox) and three soluble factors (GTP–Rac, p47phox and p67phox (refs 1, 2). This minimal complex can reconstitute ROS formation in vitro in the presence of non-physiological amphiphiles such as SDS. p40phox has subsequently been discovered as a binding partner for p67phox (ref. 3), but its role in ROS formation is unclear. Phosphoinositide-3-OH kinases (PI(3)Ks) have been implicated in the intracellular signalling pathways coordinating ROS formation but through an unknown mechanism. We show that the addition of p40phox to the minimal core complex allows a lipid product of PI(3)Ks, phosphatidylinositol 3-phosphate (PtdIns(3)P), to stimulate specifically the formation of ROS. This effect was mediated by binding of PtdIns(3)P to the PX domain of p40phox. These results offer new insights into the roles for PI(3)Ks and p40phox in ROS formation and define a cellular ligand for the orphan PX domain.

Identification of ARAP3, a novel PI3K effector regulating both Arf and Rho GTPases, by selective capture on phosphoinositide affinity matrices

We show that matrices carrying the tethered homologs of natural phosphoinositides can be used to capture and display multiple phosphoinositide binding proteins in cell and tissue extracts. We present the mass spectrometric identification of over 20 proteins isolated by this method, mostly from leukocyte extracts: they include known and novel proteins with established phosphoinositide binding domains and also known proteins with surprising and unusual phosphoinositide binding properties. One of the novel PtdIns(3,4,5)P3 binding proteins, ARAP3, has an unusual domain structure, including five predicted PH domains. We show that it is a specific PtdIns(3,4,5)P3/PtdIns(3,4)P2-stimulated Arf6 GAP both in vitro and in vivo, and both its Arf GAP and Rho GAP domains cooperate in mediating PI3K-dependent rearrangements in the cell cytoskeleton and cell shape.

Differential Binding of Traffic-related Proteins to Phosphatidic Acid- or Phosphatidylinositol (4,5)- Bisphosphate-coupled Affinity Reagents*

Phosphatidic acid (PA) is an important bioactive lipid, but its molecular targets remain unknown. To identify such targets, we have synthesized and coupled PA to an agarose-based matrix, Affi-Gel 10. Using this matrix as an affinity reagent, we have identified a substantial number of potential PA-binding proteins from brain cytosol. One class of such proteins is known to be involved in intracellular traffic and it included coatomer, ADP-ribosylation factor (Arf), N-ethylmaleimide-sensitive factor (NSF), and kinesin. Binding of these proteins to PA beads was suppressed by soluble PA, and it occurred preferentially over binding to beads coupled to phosphatidylinositol (4,5)-bisphosphate. For coatomer, Arf, and NSF, we verified direct binding to PA beads using purified proteins. For recombinant Arf1 and Arf6, binding to PA required myristoylation. In addition, for NSF and Arf6, an ATPase and a GTPase, respectively, binding to PA beads was extremely sensitive to the nucleotide state of the protein. Binding to PA may be a property linking together distinct participants in one complete round of membrane transport from a donor to an acceptor compartment.

Synthesis and biological evaluation of a PtdIns(4,5)P2 and a phosphatidic acid affinity matrix

Analogues of dipalmitoyl phosphatidic acid (PA), dilauroyl PA and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] were synthesised and immobilised onto a solid support, Affi-Gel 10. Using them as affinity matrices, a number of known proteins as well as a set of novel proteins were found to bind specifically to PA.

Synthesis and biological evaluation of aPtdIns(3,4,5)P3 affinity matrix

New PtdIns(3,4,5)P3 binding proteins have been identified utilising PtdIns(3,4,5)P3 modified affinity matrix 1 which was synthesised from myo-inositol derivative 2, phosphoramidite 9 and an agarose based solid support.