Extracellular signals transduced to the cell interior are often encoded by complex temporal and spatial changes in intracellular Ca2+ and by equally dynamic changes in the phospholipid composition of intracellular membranes. Decoding of such messages require signaling proteins to interpret these signals and transmit them further, to downstream effector molecules. A host of different protein domains receive Ca2+ and phospholipid signals and are found in many signaling proteins.
The calcium-dependent lipid-binding domain (CaLB or C2 domain) is one such protein domain that responds to Ca2+ signals and confers Ca2+ sensitivity its host protein. C2 domains have been identified in more than 1000 known and hypothetical proteins and are found in virtually all eukaryotes, suggesting that the C2 domain is a highly conserved and evolutionarily ancient signaling motif. The best-characterized function of C2 domains is in the Ca2+–dependent binding to phospholipid membranes, and many C2 domains involved in signal transduction, including those from protein kinase C (PKC) and cytoplasmic phospholipase A2 (cPLA2) facilitate docking of the host protein to membrane, where it exerts biological activity.
The pleckstrin homology (PH) domain is another protein domain that specifically binds to different phosphoinositide (PI) species in cell membranes. Like the C2 domain, it is found in hundreds of proteins, including many important signaling proteins such as Akt/PKB and PLCd1. PH domains exhibit remarkable PI specificity, and individual PH domains often recognize a single PI species. This specificity is translated into the specific targeting of PH domain host proteins to membranes where the cognate PI species is generated.
The overall aim of this research is to investigate the biology of C2 and PH domains and their host proteins. A special emphasis in the laboratory is investigation of Ca2+ and PI signaling in innate immunity response, specifically the chemotactic and phagocytic responses of macrophages to different physiological agonists. Complex Ca2+ and PI signaling accompany the processes of chemotaxis and phagocytosis; however, how these signals control these processes are largely unknown.
The laboratory uses a multi-disciplinary approach to investigating Ca2+/C2 domain and PI/PH domain signaling, including live cell multispectral fluorescence imaging of fluorescently tagged proteins, live cell fluorescence-based Ca2+ imaging, and in vitro protein/membrane binding techniques.
Evans, J.H. and Falke, J.J. (2007) Calcium influx is an essential component of the positive feedback loop that maintains leading edge structure and activity in macrophages. (in press, PNAS)
Corbin, J.A, Evans, J.H., Landgraf, K.E., and Falke, J.J. (2007) Mechanism of Specific Membrane Targeting by C2 Domains: Localized Pools of Target Lipids Enhance Ca2+ Affinity. Biochemistry 46: 4322-36.
Evans, J.H., Murray, D., Leslie, C.C., and Falke, J.J. (2006) Specific Translocation of PKCa to the Plasma Membrane Requires Both Ca2+ and PIP2 Recognition by Its C2 Domain Mol. Biol. Cell 17:56-66.
Girotti, M., Evans, J.H., Burke, D. and Leslie, C.C. (2004) Cytosolic phospholipase A2 translocates to forming phagosomes during zymosan phagocytosis in mouse macrophages J. Biol. Chem. 279:19113-21.
Pettus, B., Bielawska, A., Subramanian, P., Wijesinghe, D.S., Mayceka, M., Leslie, C.C., Evans, J.H., Freiberg, J., Roddy, P., Hannun, Y.A., Chalfant, C.E. (2004) Ceramide-1-phosphate is a direct activator of cytosolic phospholipase A2. J. Biol. Chem. 279:11320-6.
Evans, J.H. and Leslie, C.C. (2004) The cytosolic phospholipase A2 catalytic domain modulates association and residence time at Golgi membranes J. Biol. Chem. 279: 6005-6016.
Evans, J.H., Gerber, S.H., Murray, D., and Leslie, C.C. (2004) The Ca2+-binding loops of the cytosolic phospholipase A2 C2 domain specify Golgi and ER targeting Mol. Biol. Cell 15: 371-383.
Evans, J.H., Fergus, D.J., and Leslie, C. C. (2003) Regulation of cytosolic phospholipase A2 translocation. Adv. Enzyme Regul. 43:229-244.
Evans, J.H., Fergus, D.J., and Leslie, C.C. (2002) Inhibition of the MEK/ERK pathway reduces arachidonic acid independently of cPLA2 phosphorylation and translocation BMC Biochem. 3:30.
Evans, J.H., Spencer, D.M., Zweifach, A., and Leslie, C.C. (2001) Calcium requirements for cytosolic phospholipase A2 translocation to internal membranes. J. Biol. Chem. 276: 30150-30160.
Fry, T, Evans, J.H. and Sanderson, M.J. (2001) Propagation of intercellular calcium waves in C6 glioma cells transfected with connexins 43 or 32. Microsc. Res. Tech. 52:289-300.
Evans, J.H. and Sanderson, M.J. (1999) Ciliary beat frequency of airway epithelial cells is regulated by the frequency of intracellular calcium oscillations. Cell Calcium 26: 103-110.
Evans, J.H. and Sanderson, M.J. (1999) Intracellular calcium oscillations induced by ATP in airway epithelial cells. Am. J. Physiol. 277:L30-41.