Identification and functional characterization of kindlin-3 phosphorylation and its role in integrin regulation in mice

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Integrins are a family of transmembrane proteins that anchor cells within their ambient extracellular matrix. Some integrins expressed on blood cells bind to surface receptors of other cells such as endothelial cells, enabling leukocyte adhesion and transmigration through the vascular wall into the tissue. Integrins mediate strong binding to their ligands by increasing their ligand binding affinity and by their assembly into complex adhesion structures that are strongly linked to the cytoskeleton. These functions depend on the two key integrin regulatory proteins, talin and kindlin, which bind to the cytoplasmic domain of integrins and control both integrin conformation and organization into adhesion complexes. Deciphering the essential role of talin and kindlin in integrin regulation has been a central focus of integrin research over the past 10-20 years. Recently, the regulation of these two integrin regulators has begun to be elucidated in more detail. In particular, the regulation of kindlins by post-translational modifications has been poorly understood and is central to the research project proposed here. Mass spectrometry studies of kindlin-3, the member of the kindlin family formed exclusively in hematopoietic cells, have allowed us to identify several phosphorylation sites. Here, we plan to investigate their role in regulating integrin function in different hematopoietic cell populations. In preliminary work, we have shown that phosphorylation of serine 8 increases integrin-mediated cell adhesion in static and dynamic adhesion studies. To this end, we have generated genetically modified Hoxb8 cell lines in which the serine 8 of the kindlin-3 protein has been replaced by alanine or glutamic acid. In comprehensive biochemical and cell biological studies on neutrophils and macrophages differentiated from these cell lines, we aim to investigate the impact of this modification on (i) cell adhesion, regulation of integrin function and activity, and formation of integrin adhesion sites. (ii) In addition, we have already generated two genetically modified mouse lines carrying these mutations in the kindlin-3 gene to investigate the relevance of serine 8 phosphorylation for the regulation of integrin-mediated processes in vivo. (iii) Furthermore, we will elucidate the mechanism underlying the enhanced integrin-dependent cell adhesion due to kindlin-3 serine 8 phosphorylation. (iv) Accompanying this work, we will characterize additional phosphorylation sites we have identified in the kindlin-3 protein with respect to their impact on the regulation of integrin function in the Hoxb8 cell system.