The research in my laboratory is focused on elucidation of cellular signal transduction mechanisms involved in the activation of NADPH oxidase, an enzyme responsible for the generation of toxic oxygen radicals in many cell types. NADPH oxidase is found in high levels in phagocytic cells, like neutrophils and monocytes, where the elicited production of oxygen radicals plays critical roles in both protection against microbial infections and in damage to tissue in a variety of diseases, including degenerative neurological diseases, arthritis, stroke, and myocardial infarction. Homologs of NADPH oxidase have now been discovered in many cell types, including smooth muscle cells, kidney cells, thyroid cells, endothelial cells, and epithelial cells.  These enzymes are present at low levels (compared to phagocytic cells) and may be involved in both host defense and cell signaling by reactive oxygen species.  Thus, a mechanistic understanding of the regulation of NADPH oxidase could ultimately be used for design of therapeutic approaches to control the activity of this enzyme in a wide variety of disease situations. Mechanisms involved in NADPH oxidase activation include phosphorylation of components, lipid binding to components, activation of GTP-hydrolyzing proteins, and assembly of enzyme components from various intracellular compartments to form an active complex. We are exploring these mechanisms using site-directed mutagenesis of NADPH oxidase components and various analytical and functional assays.  Our current focus is on dissecting the structure/function relationship of protein phosphorylation and lipid binding events in the regulation of two NADPH oxidase components – the soluble p47^phox and the integral membrane protein flavocytochrome b₅₅₈.  A working model depicting the phosphorylation events and the lipid-binding interactions we believe are taking place is shown below.  In addition, a new project is studying the interaction between neutrophils and pathogens, to determine the biochemical mechanisms by which pathogens alter neutrophil function during host defense and inflammation.  Techniques used in my laboratory include: mutagenesis, expression of recombinant proteins, Western blotting, lipid-binding assays, protein:protein interaction assays, cell culture and blood cell isolation, flow cytometry, transfection of cultured cells, enzymatic assays (NADPH oxidase, protein kinase, phospholipase D), and functional assays (chemotaxis, degranulation, bactericidal activity).

Recent publications:

Chial, H.J., Wu, R., Ustach, C. V., McPhail, L. C., Mobley, W.C., and Chen, Y. Q.  Membrane Targeting by APPL1 and APPL2: Dynamic scaffolds that oligomerize and bind phosphoinositides.  Traffic, in press (2008).

Lehman, N., Ledford, B., Di Fulvio, M., Frondorf, K., McPhail, L.C., and Gomez-Cambronero, J.  Phospholipase D2-derived phosphatidic acid binds to and activates ribosomal p70 S6 kinase independently of mTOR.  FASEB J. 21:1075-1087 (2007).

Taylor, R.M., Lord, C.I., Gripentrog, J.M., Riesselman, M.H., Leto, T.L., McPhail, L.C., Berdichevsky, Y., Pick, E., and Jesaitis, A.J.  Characterization of surface structure and p47^phox SH3 domain-mediated conformational changes in human neutrophil flavocytochrome b.  Biochemistry 46:14291-14304 (2007).

Taylor RM, Foubert TR, Burritt JB, Baniulis D, McPhail LC, and Jesaitis AJ.  Anionic amphiphile and phospholipid-induced conformational changes in human neutrophil flavocytochrome b observed by fluorescence resonance energy transfer.  Biochim. Biophys. Acta 2004 1663:201-213.

Price MO, McPhail LC, Lambeth JD, Han C-H, Knaus UG, and Dinauer MC.  Creation of a genetic system for analysis of the phagocyte respiratory burst:  high level reconstitution of the NADPH oxidase in a non-hematopoietic system.  Blood 2002 99:2653-2661.

Sergeant S, Waite KA, Heravi J, and McPhail LC.  Phosphatidic acid regulates tyrosine phosphorylating activity in human neutrophils: Enhancement of Fgr activity.  Published, JBC Papers In Press (11/14/00). J. Biol. Chem. 2001 276:4737-4746.

Palicz A, Foubert TR, Jesaitis AJ, Marodi L, and McPhail LC.  Phosphatidic acid and diacylglycerol directly activate NADPH oxidase by interacting with enzyme components.  Published, JBC Papers In Press (11/01/00).  J. Biol. Chem. 2001 276:3090-3097.

Karlsson A, Nixon JB, McPhail LC.  Phorbol myristate acetate induces neutrophil NADPH-oxidase activity by two separate signal transduction pathways: dependent or independent of phosphatidylinositol 3-kinase.  J Leukoc Biol. 2000 Mar;67(3):396-404. 

Regier D S, Greene DG, Sergeant S, Jesaitis AJ, and McPhail LC.  Phosphorylation of p22^phox is mediated by phospholipase D-dependent and -independent mechanisms: correlation of NADPH oxidase activity and p22^phox phosphorylation.  J. Biol. Chem. 2000 275:28406-28412.

McPhail LC, Waite KA, Regier DS, Nixon JB, Qualliotine-Mann D, Zhang WX,   Wallin R, Sergeant S.   A novel protein kinase target for the lipid second messenger phosphatidic acid.  Biochim Biophys Acta. 1999 Jul 30;1439(2):277-90. Review. 

Nixon JB, McPhail LC.  Protein kinase C (PKC) isoforms translocate to Triton-insoluble fractions in stimulated human neutrophils: correlation of conventional PKC with activation of NADPH oxidase.  J Immunol. 1999 Oct 15;163(8):4574-82. 
Regier DS, Waite KA, Wallin R, McPhail LC.  A phosphatidic acid-activated protein kinase and conventional protein kinase C isoforms phosphorylate p22(phox), an NADPH oxidase component.  J Biol Chem. 1999 Dec 17;274(51):36601-8.