The integrin-linked kinase (ILK) is critical for anchorage-dependent cell growth and survival, cell cycle progression, epithelial to mesenchymal transition, cell motility, contractility and early development. ILK is also required for cardiac, vascular, brain, kidney, muscle, skin, platelet, chondrocyte and T cell function and plays important roles in tumor angiogenesis. There are multiple signaling pathways downstream of integrins but many of these pathways require the formation of a heterotrimeric complex between ILK, PINCH and parvin (IPP). This IPP complex serves as a hub in integrin-actin and integrin-signaling networks, and in mammalian systems IPP complex formation precedes and is required for its correct targeting to adhesions. There are currently significant deficiencies in our understanding of how the IPP complex forms, how it interacts with integrins, and whether it is enzymatically competent.
We aim to improve the understanding of integrin signaling by the IPP complex using a structure-directed functional approach and to resolve the key functional question of whether ILK is catalytically active.
In Aim 1 we will determine crystal structures and conduct binding studies to provide a comprehensive molecular description of the interaction between ILK and PINCH. These biophysical studies will allow us to rationally investigate the cellular effects of targeted interruption of this interaction.
In Aim 2 we will provide structural, enzymatic and functional analyses of the ILK kinase domain. The catalytic competence of this pseudokinase remains the subject of much controversy, but is reported to be critical for IPP-mediated integrin signaling. We will solve crystal structures of the ILK kinase domain and assess its catalytic activity to provide structural evidence for the molecular basis of ILK catalytic competence. Potential substrate specificities will be studied as will the role of intramolecular interactions on kinase activity. Using structure-guided site-directed mutagenesis we will investigate the functional role of ILK catalytic activity in cells.
In Aim 3 we will determine the molecular basis for ILK interactions with parvin and whether ILK directly interacts with integrin 2 subunit cytoplasmic tails. If ILK binds integrin 2 tails we will determine the structural basis for this interaction and investigate the functional effects of its targeted disruption.
In Aim 4 we will determine the molecular architecture of the complete IPP complex. This long-term goal will provide a structural description of how the IPP complex forms and will allow targeted functional analysis of its cellular role. The studies proposed will provide answers to some of the critical unresolved questions regarding integrin signaling by the IPP complex. They may also facilitate the design of targeted anti-ILK or anti-IPP therapeutics relevant to the treatment of cancer, cardiovascular and inflammatory and kidney diseases. Our results will significantly enhance the molecular, enzymatic and functional understanding of a critical integrin signaling complex.

Public Health Relevance

The project aims to enhance our molecular and functional understanding of integrin-linked kinase-PINCH- parvin (IPP) complex. This complex is essential for embryonic development, tissue maintenance and repair, host defense and hemostasis. We will determine crystal structures, conduct functional studies and describe the effects of mutations in cells. The proposed studies will help us understand how the IPP complex mediates its critical cellular roles.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM088240-01
Application #
7696817
Study Section
Intercellular Interactions (ICI)
Program Officer
Flicker, Paula F
Project Start
2009-08-17
Project End
2013-07-31
Budget Start
2009-08-17
Budget End
2010-07-31
Support Year
1
Fiscal Year
2009
Total Cost
$347,550
Indirect Cost
Name
Yale University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Iwamoto, Daniel V; Calderwood, David A (2015) Regulation of integrin-mediated adhesions. Curr Opin Cell Biol 36:41-7
Lee, Monica Y; Skoura, Athanasia; Park, Eon Joo et al. (2014) Dynamin 2 regulation of integrin endocytosis, but not VEGF signaling, is crucial for developmental angiogenesis. Development 141:1465-72
Huet-Calderwood, Clotilde; Brahme, Nina N; Kumar, Nikit et al. (2014) Differences in binding to the ILK complex determines kindlin isoform adhesion localization and integrin activation. J Cell Sci 127:4308-21
Stiegler, Amy L; Grant, Thomas D; Luft, Joseph R et al. (2013) Purification and SAXS analysis of the integrin linked kinase, PINCH, parvin (IPP) heterotrimeric complex. PLoS One 8:e55591
Liu, Weizhi; Draheim, Kyle M; Zhang, Rong et al. (2013) Mechanism for KRIT1 release of ICAP1-mediated suppression of integrin activation. Mol Cell 49:719-29
Brahme, Nina N; Harburger, David S; Kemp-O'Brien, Karl et al. (2013) Kindlin binds migfilin tandem LIM domains and regulates migfilin focal adhesion localization and recruitment dynamics. J Biol Chem 288:35604-16
Calderwood, David A; Campbell, Iain D; Critchley, David R (2013) Talins and kindlins: partners in integrin-mediated adhesion. Nat Rev Mol Cell Biol 14:503-17
Yates, Luke A; Lumb, Craig N; Brahme, Nina N et al. (2012) Structural and functional characterization of the kindlin-1 pleckstrin homology domain. J Biol Chem 287:43246-61
Bouaouina, Mohamed; Goult, Benjamin T; Huet-Calderwood, Clotilde et al. (2012) A conserved lipid-binding loop in the kindlin FERM F1 domain is required for kindlin-mediated ?IIb?3 integrin coactivation. J Biol Chem 287:6979-90
Stiegler, Amy L; Draheim, Kyle M; Li, Xiaofeng et al. (2012) Structural basis for paxillin binding and focal adhesion targeting of ?-parvin. J Biol Chem 287:32566-77

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