Virtually every cell in our body spends at least a portion of its life time on adhering to the extracellular matrix (ECM). Such cell-to-ECM adhesion is primarily mediated by a class of heterodimeric (?/?) transmembrane receptors, integrins. Upon activation, integrins bind ECM proteins (adhesion) and then transmit signals to intracellular cytoskeleton via large multi-protein complexes called focal adhesions (FAs). This latter step is vital to promote dynamic cell adhesive responses such as cell shape change, cell migration, and proliferation. However, while much has been learned about integrin activation over the decades, the mechanism by which integrin initiates signaling to the cytoskeleton via FAs is still poorly understood. To this end, we have been focusing on integrin-linked kinase (ILK), a nascent mediator of FAs assembly and signaling. For >15 years, ILK was thought to function as a key Ser/Thr kinase to phosphorylate integrin cytoplasmic tail and initiate the receptor signaling. Highly upregulated in many cell adhesion-dependent diseases, ILK was also regarded as a ?hot? kinase target for drug development. However, structural studies from our laboratory uncovered that ILK contains a severely degraded active site incapable of performing kinase catalysis. The finding, corroborated by extensive biochemical and genetic data, led to a conceptual breakthrough as evidenced by dozens of reviews, hundreds of re-themed research articles, and grants. However, a key issue still remains largely unresolved: without kinase function, how does ILK mediate FAs assembly and signaling? This issue has broad relevance as there exist many poorly understood pseudoenzymes including pseudokinases that occupy ~10% of human kinome. In a most recent study, we discovered that by forming a tight complex with FA adaptors PINCH and Parvin (IPP), ILK triggers specific actin filament bundling ? a process known to generate force/mechanical signal to promote cytoskeleton reorganization and dynamic cell adhesion. We further found that such actin bundling is orchestrated by two previously unrecognized actin binding motifs within the ILK-centered IPP, one from PINCH and the other from Parvin. Strikingly, this process is also sensitized by Mg-ATP bound to the pseudoactive site of ILK and impaired by a novel inhibitor we have developed. Our findings thus signify a new milestone towards resolving the mystery of ILK as a pseudokinase in mediating cell adhesion and signaling. We propose to continue our investigation by focusing on three following aims: (i) to resolve a puzzle of how ILK is localized to nascent FAs ? the first step for the ILK action; (ii) to elucidate how ILK acts as a unique protein docking center to mediate different signaling pathways; (iii) to determine detailed molecular basis of non-catalytic roles of ILK-bound MgATP in fine-tuning the ILK-mediated signaling.
These aims reflect a strong momentum of our program, which has reached a critical phase towards establishing a new paradigm in ILK biology and cell adhesion. With newly discovered mechanisms and novel inhibitor for ILK, we believe that these studies have potential to transform the understanding and treatment of ILK-associated diseases such as heart failure and cancer.

Public Health Relevance

ILK-mediated adhesion of cells to extracellular matrix is fundamentally important for many physiological processes such as heart development, tissue regeneration, and wound healing. Dysregulation of ILK has been linked to numerous major diseases such as heart failure and stroke. Our proposal is centered for elucidating the molecular basis of ILK function, which will not only provide mechanistic insight into cell adhesion at normal and disease states of our body but also transform our understanding and treatment of ILK-mediated diseases.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL058758-20
Application #
9903416
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Adhikari, Bishow B
Project Start
1999-01-01
Project End
2023-03-31
Budget Start
2020-04-01
Budget End
2021-03-31
Support Year
20
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Cleveland Clinic Lerner
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
135781701
City
Cleveland
State
OH
Country
United States
Zip Code
44195
Vaynberg, Julia; Fukuda, Koichi; Lu, Fan et al. (2018) Non-catalytic signaling by pseudokinase ILK for regulating cell adhesion. Nat Commun 9:4465
Cai, Gang; Zhu, Liang; Chen, Xing et al. (2018) TRAF4 binds to the juxtamembrane region of EGFR directly and promotes kinase activation. Proc Natl Acad Sci U S A 115:11531-11536
Liu, Caini; Zhu, Liang; Fukuda, Koichi et al. (2017) The flavonoid cyanidin blocks binding of the cytokine interleukin-17A to the IL-17RA subunit to alleviate inflammation in vivo. Sci Signal 10:
Zhu, Liang; Yang, Jun; Bromberger, Thomas et al. (2017) Structure of Rap1b bound to talin reveals a pathway for triggering integrin activation. Nat Commun 8:1744
Bledzka, Kamila; Bialkowska, Katarzyna; Sossey-Alaoui, Khalid et al. (2016) Kindlin-2 directly binds actin and regulates integrin outside-in signaling. J Cell Biol 213:97-108
Plow, Edward F; Qin, Jun (2015) The role of RIAM in platelets put to a test. Blood 125:207-8
Meller, Julia; Rogozin, Igor B; Poliakov, Eugenia et al. (2015) Emergence and subsequent functional specialization of kindlins during evolution of cell adhesiveness. Mol Biol Cell 26:786-96
Qin, Jun; Gronenborn, Angela M (2014) Weak protein complexes: challenging to study but essential for life. FEBS J 281:1948-9
Murphy, James M; Zhang, Qingwei; Young, Samuel N et al. (2014) A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties. Biochem J 457:323-34
Fukuda, Koichi; Bledzka, Kamila; Yang, Jun et al. (2014) Molecular basis of kindlin-2 binding to integrin-linked kinase pseudokinase for regulating cell adhesion. J Biol Chem 289:28363-75

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