The broad long-term objective of this proposal is to understand the molecular mechanisms of cell adhesion and signaling. The cell adhesion or cell-extracellular matrix (ECM) interaction is mediated primarily by integrins, a group of heterodimeric (alpha/beta) transmembrane receptors. Upon activation, integrins bind to ECM proteins via their extracellular tomains, which triggers conformational changes and clustering of the receptors, leading to the generation of large intracellular protein complexes called focal adhesions that link to the cytoskeleton. Such integrin-mediated physical linkage between extracellular and intracellular compartments allows dynamic regulation of many cellular processes including cell shape change, migration, proliferation and differentiation. Understanding how mechanical and chemical information flow along this linkage for the regulation of cellular functions is of fundamental interest in cell biology and signal transduction. Although significant progress has been made in the molecular understanding of integrin activation and subsequent ECM-integrin interaction, little is known about early intracellular events following the ECM-integrin binding, e.g., upon ECM engagement, how is the integrin cytoplasmic domain connected to the focal adhesion sites and to the cytoskeleton? Further, how is the connection regulated during cell adhesion and migration? We wish to address the issues here by focusing on the integrin-linked kinase (ILK), a key component involved in this early intracellular event. ILK is a highly conserved 50kDa protein that contains an N-terminal ankyrin repeat (ANK) domain, a short pleckstin homology (PH) domain, and a C-terminal serine/threonine kinase (KD) domain. ILK has low basal kinase activity but is activated and recruited to focal adhesions upon ECM engagement. The activated ILK phosphorylates integrin beta cytoplasmic domains and multiple downstream signaling/cytoskeletal proteins, thereby regulating cascades of integrin-mediated cellular processes such as focal adhesion formation, gene expression, and cytoskeleton reorganization. ILK is being pursued as a """"""""hot"""""""" therapeutic spot since its dysfunction leads to numerous disease states such as cardiac hypertrophy, atherosclerosis, and cancer. This proposal seeks to dissect the molecular mechanism of ILK at three crucial steps: (a) ILK subcellular localization, (b) ILK activation, and (c) ILK phosphorylation on the key target, integrin. Vigorous structural approaches combined with biochemical methods will be used to address the following specific aims:
Aim 1 -To elucidate structural basis of ILK localization to focal adhesions;
Aim 2 -To investigate auto-regulatory mechanism of ILK activation;
and Aim 3 - To examine structural and functional effects of ILK-mediated phosphorylation on the key target, integrin beta cytoplasmic domain. The study is expected to provide a detailed molecular understanding of ILK signaling in cell adhesion and migration. The results may also contribute to the development of therapeutic agents for ILK-related diseases.
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