5, D and E). site-specific phosphorylation of the integrin cytoplasmic domains is important for the dynamic regulation of these complex receptors in cells. Introduction The heterodimeric cell surface receptors called integrins are exceptional in that they can function as bidirectional signaling devices, regulating cell adhesion and migration after so-called inside-out signaling, and they can also signal into the cell to regulate growth, differentiation, and apoptosis after ligand binding (Giancotti and Ruoslahti, 1999; Hynes, 2002). The relatively small intracellular domains of integrins are involved in regulating signaling functions. Recently, separation of integrin cytoplasmic domains has been postulated as a mechanism of regulating integrin bidirectional signaling (Vinogradova et al., 2002; Kim et al., 2003; Tadokoro et al., 2003). Proximal events in the regulation of integrin activation and outside-in signaling presumably involve the binding of cytoplasmic molecules to the intracellular tails (Calderwood, 2004). Dynamic adhesion is especially important in the immune system, where cells need to attach and detach continuously. The leukocyte function-associated antigen-1 (LFA-1) integrin (L2 or CD11a/CD18) is expressed exclusively in leukocytes and is of fundamental importance to the function of the immune system (Springer, 1990; Gahmberg, 1997). LFA-1 mediates cell adhesion under various conditions, e.g., during immunological synapse formation between the T cell and the antigen-presenting cell and during leukocyte Mouse monoclonal to IFN-gamma emigration from the bloodstream into tissues. Whereas T cell receptor (TCR)Cmediated adhesion is slow and sustained, chemokine-induced adhesion is fast and rapidly reversible. Both affinity-dependent and -independent mechanisms have been postulated as being important in the regulation of integrin activation (van Kooyk and Figdor, 2000; Carman and Springer, 2003; Calderwood, 2004). These mechanisms are not mutually exclusive, and different modes of integrin activation may involve different mechanisms working alone or together. For example, TCR-induced activation of LFA-1 has not been shown to involve affinity regulation (conformational changes) in the integrin, but instead has been closely correlated with the spreading phenotype of T cells and actin cytoskeleton rearrangements (Stewart et al., 1996, 1998). In contrast, chemokines mediate rapid conformational changes in LFA-1, TGR-1202 as measured by activation epitope expression with mAbs and the measurement of soluble ligand binding to the integrin (Weber et al., 1999; Constantin et al., 2000). Chemokine-induced adhesion also involves the clustering of integrins (Constantin et al., 2000). Ligands can also induce conformational changes and clustering of integrins (Cabanas and Hogg, 1993; Li et al., 1995; Kotovuori et al., 1999; Kim et al., 2004). Phosphorylation is a common mechanism for the regulation of surface receptor function and has also been reported in integrins, but its role in integrin regulation has remained only partially understood (Fagerholm et al., 2004). LFA-1 is phosphorylated on both the and chains, with the chain being constitutively phosphorylated, whereas chain phosphorylation becomes detectable after inside-out stimulation of TGR-1202 the integrin (Hara and Fu, 1986; Chatila et al., 1989; Valmu and Gahmberg, 1995). The chain phosphorylation sites have not been mapped, and their functions are completely unknown. In contrast, the chain phosphorylation sites are known (Hibbs et al., 1991; Fagerholm et al., 2002b; Hilden et al., 2003). The main phosphorylation site after phorbol ester stimulation of cells is Ser756, but this site is not involved in regulating adhesion (Hibbs et al., 1991). The threonine triplet (Thr758C760) in the 2 2 chain is important for adhesion, interactions with the actin cytoskeleton, and modulation TGR-1202 of cell spreading (Hibbs et al., 1991; Peter and O’Toole, 1995). Interestingly, threonine phosphorylation of the chain has been reported (Valmu and Gahmberg, 1995) and threonine-phosphorylated integrins distribute preferentially to the actin cytoskeleton in cells (Valmu et al., 1999a). Additionally, it has been shown that 14-3-3 proteins from cell lysates interact with a Thr758-phosphorylated 2 integrin peptide in vitro (Fagerholm et al., 2002b), but whether the interaction occurs in vivo or plays a role in adhesion has not been discovered. In this study, we investigated the role of both and chain phosphorylations in the regulation of LFA-1Cmediated adhesion. Results L is phosphorylated on Ser1140.