Phagocytic leukocytes (monocytes, macrophages, and neutrophils) perform essential functions in the immune response to microbial infection, but also contribute to host tissue damage in both infectious and non-infectious inflammatory disorders. Therefore, definition of the biochemical mechanism that regulate the activation of phagocytes is required for development of improved therapies to augment antimicrobial defenses and limit deleterious consequences to host tissues. Phospholipase D (PLD) is an important signal-transducing enzyme that regulates both antimicrobial and host-damaging functions of phagocytes. However, the biochemical mechanisms that regulate the assembly and activation of a functional PLD complex at the membrane substrate, remain incompletely defined. A common feature of the leukocyte effector functions regulated by PLD is their absolute dependence on the actin cytoskeleton. We recently demonstrated that stimulation of PLD1 in U937 promonocytes is accompanied by its stable association with a detergent-insoluble fraction containing F-actin and other cytoskeletal proteins. Currently unknown are: (1) the physical basis for association of PLD1 and the w cytoskeleton, and (2) its consequences for regulation of PLD activity. Our general hypothes' is that interactions between PLO and the actin cytoskeleton regulate the assembly and activation of a multi-component PLD complex at the membrane interface. The proposed studies test the novel specific hypotheses that (a) PLD1 binds to membrane-associated actin filaments and monomeric actin, and (b) these physical interactions between PLD1 and actin regulate PLD activity. We will utilize both highly purified in vitro systems for biochemical analysis as well as physiologic in vivo studies of phagocyte functional responses to evaluate these hypotheses in the context of pursuing three Specific Aims: (1) Characterize the physical association of PLD1 with actin filaments (2) Define the binding of PLD1 to G-actin, and (3) Determine whether association with actin modulates PLD activity. These studies will further both our specific knowledge of signal transduction mechanisms that regulate inflammation as well as our more general understanding of the spatial and temporal determinants of cellular activation and motility.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM062302-04
Application #
6795493
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Marino, Pamela
Project Start
2001-08-01
Project End
2006-07-31
Budget Start
2004-08-01
Budget End
2005-07-31
Support Year
4
Fiscal Year
2004
Total Cost
$169,111
Indirect Cost
Name
University of Iowa
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
062761671
City
Iowa City
State
IA
Country
United States
Zip Code
52242
Iyer, Shankar S; Kusner, David J (2009) Coordinate regulation of sphingosine kinase and actin dynamics. Methods Mol Biol 531:347-61
Kusner, David J; Thompson, Christopher R; Melrose, Natalie A et al. (2007) The localization and activity of sphingosine kinase 1 are coordinately regulated with actin cytoskeletal dynamics in macrophages. J Biol Chem 282:23147-62
Herrmann, Tara L; Agrawal, Reitu S; Connolly, Sean F et al. (2007) MHC Class II levels and intracellular localization in human dendritic cells are regulated by calmodulin kinase II. J Leukoc Biol 82:686-99
Iyer, Shankar S; Agrawal, Reitu S; Thompson, Christopher R et al. (2006) Phospholipase D1 regulates phagocyte adhesion. J Immunol 176:3686-96
Iyer, Shankar S; Kusner, David J (2006) Assay of phospholipase D activity in cell-free systems. Methods Mol Biol 332:281-98
Herrmann, Tara L; Morita, Craig T; Lee, Kelvin et al. (2005) Calmodulin kinase II regulates the maturation and antigen presentation of human dendritic cells. J Leukoc Biol 78:1397-407
Thompson, Christopher R; Iyer, Shankar S; Melrose, Natalie et al. (2005) Sphingosine kinase 1 (SK1) is recruited to nascent phagosomes in human macrophages: inhibition of SK1 translocation by Mycobacterium tuberculosis. J Immunol 174:3551-61
Iyer, Shankar S; Barton, James A; Bourgoin, Sylvain et al. (2004) Phospholipases D1 and D2 coordinately regulate macrophage phagocytosis. J Immunol 173:2615-23
Malik, Zulfiqar A; Thompson, Christopher R; Hashimi, Samad et al. (2003) Cutting edge: Mycobacterium tuberculosis blocks Ca2+ signaling and phagosome maturation in human macrophages via specific inhibition of sphingosine kinase. J Immunol 170:2811-5
Kusner, David J; Barton, James A; Qin, Chunbo et al. (2003) Evolutionary conservation of physical and functional interactions between phospholipase D and actin. Arch Biochem Biophys 412:231-41

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