Spatiotemporal regulation of the kinase Mst1 by binding protein RAPL is critical for lymphocyte polarity and adhesion Koko Katagiri 1 , Masashi Imamura 2 & Tatsuo Kinashi 1 RAPL, a protein that binds the small GTPase Rap1, is required for efficient immune cell trafficking. Here we have identified the kinase Mst1 as a critical effector of RAPL. RAPL regulated the localization and kinase activity of Mst1. ‘Knockdown’ of the gene encoding Mst1 demonstrated its requirement for the induction of both a polarized morphology and integrin LFA-1 clustering and adhesion triggered by chemokines and T cell receptor ligation. RAPL and Mst1 localized to vesicular compartments and dynamically translocated with LFA-1 to the leading edge upon Rap1 activation, suggesting a regulatory function for the RAPL- Mst1 complex in intracellular transport of LFA-1. Our study demonstrates a previously unknown function for Mst1 of relaying the Rap1-RAPL signal to induce cell polarity and adhesion of lymphocytes. The immune system requires considerable trafficking ability of immune cells; lymphocytes recirculate in the blood and peripheral lymphoid tissues, searching for foreign antigens carried by migrating antigen-presenting dendritic cells. Dynamic modulation of integrin adhesive function is critical during such trafficking 1,2 . After stimulation with chemokines or antigens, lymphocyte integrin adhesion is tran- siently upregulated with little change in surface expression. The intracellular signaling triggered after stimulation that mediates this change, called ‘inside-out signaling’, is ultimately thought to control integrin conformation 3 and surface distribution 4,5 . Cellular and genetic studies have shown that immune cell adhesion mediated by b 1 and b 2 integrins is modulated by protein and lipid kinases, small GTPases of the Ras and Rho families, and their regulators and adaptor proteins 6,7 . Integrin-mediated attachment facilitates lymphocyte transendothe- lial and interstitial migration. Cell migration is a coordinated process: asymmetrical cell morphology develops, forming a protrusion and uropod, and new adhesion sites form at the leading edge and detach at the rear, pulling the cell body in the direction of cell migration 8,9 . Cell polarization and regulated adhesion are induced by extrinsic and intrinsic cues, which prompt cytoskeletal reorganization and vesicle transport, including endocytosis and recycling of integrins 10–14 . Understanding of the molecular network that orchestrates activation of integrin and cell migration, however, remains incomplete. The small GTPase Rap1 is an important inside-out signal in controlling cell adhesion through modulating the affinity and/or spatial organization of integrins 15 . Stimulation with chemokines or antigen stimulation through the T cell antigen receptor (TCR) activates Rap1, which is required for transmigration of lymphocytes through endo- thelial cells under flow and immunological synapse formation with antigen-presenting cells 16,17 . Rap1 activation has been found to be defective in human leukocyte adhesion deficiency 18 . Rap1A-deficient T cells and B cells show impaired b 1 and b 2 integrin clustering and adhesion 19 . Those studies support the idea that Rap1 is a crucial inside- out signaling molecule in vivo. RAPL is a Rap1-binding effector protein that mediates Rap1-dependent affinity and spatial distribution of the integrin LFA-1 (ref. 20). Lymphoid tissues are enriched for RAPL 20 and Nore1, an alternatively spliced product of the gene encoding RAPL (Rassf5). Targeted deletion of the locus encoding RAPL has demon- strated defective adhesion and migration of lymphocytes and dendritic cells 22 . After Rap1 activation, RAPL forms a complex with LFA-1 and relocates to the leading edge and also accumulates at immunological synapses. A patch-like distribution of LFA-1 at the leading edge is critical for triggering adhesion and motility 20 . Rap1- and RAPL- triggered changes in integrin spatial distribution in concert with the induction of cell polarity result in robust cell motility. To explore further the molecular mechanisms by which Rap1 and RAPL regulate integrin functions and cell polarity, we have used yeast two-hybrid screening for RAPL-associated molecules with an activated RAPL mutant protein as the ‘bait’. This analysis identified the kinase Mst1 as a ‘downstream’ effector molecule of RAPL. In drosophila, the Mst ortholog Hipo has many crucial functions in the regulation of cell growth and apoptosis 23 . In mammals, Mst1 is also associated with apoptosis, although its physiological function remains unclear 24 . Here we demonstrate a previously unknown function for Mst1 in the regulation of lymphocyte polarization and adhesion ‘downstream’ of Rap1 and RAPL. Received 30 March; accepted 12 July; published online 6 August 2006; doi:10.1038/ni1374 1 Department of Molecular Genetics, Institute of Biomedical Science, Kansai Medical University, Osaka 570-8506, Japan. 2 Center Research Institute, Ishihara Sangyo Kaisha, Kusatsu, Shiga 525-0025, Japan. Correspondence should be addressed to T.K. (kinashi@takii.kmu.ac.jp). NATURE IMMUNOLOGY VOLUME 7 NUMBER 9 SEPTEMBER 2006 919 ARTICLES © 2006 Nature Publishing Group http://www.nature.com/natureimmunology