Functional molecular imaging of ILK-mediated Akt/PKB signaling cascades and the associated role of b-parvin

Introduction Extracellular signaling is commonly mediated through mechanisms that rely on protein-protein interactions and protein phosphorylation. The dynamics of protein-protein interactions are often dependent on the activation of a particular signal transduction pathway. Even a single protein can affect various cellular functions by interacting with different partners in response to extracellular stimuli. Recent advances in the development of molecular probes have enabled the visualization of many cellular processes and the detection of protein-protein interactions in living cells, advances typified by the fluorescence resonance energy transfer (FRET) system (Förster, 1959), in addition to complementary methods using fluorescence (Hu et al., 2002) or luminescence (Luker et al., 2004). These complementary methods designed to visualize proteinprotein interactions have recently been employed in the screening of interaction partners and the potential semi-high-throughput analysis of small modulator molecules in protein complexes (Kerppola, 2006a; Kerppola, 2006b). In particular, luciferase-based complementation imaging enables sensitive real-time monitoring and quantitative analysis of protein interactions under various cellular conditions (Luker et al., 2004). Integrins and their associated proteins mediate various intracellular signaling pathways involved in cytoskeletal remodeling and cellular processes such as cell proliferation, survival and differentiation. Integrin-linked kinase (ILK), PINCH and parvin(s) form heterotrimeric complexes that function as important regulators of integrin-mediated signaling. The complex comprising ILK, PINCH and parvins has been implicated in mediating intracellular signaling pathways through phosphorylation of the leading downstream molecule Akt (also known as protein kinase B; PKB) (Legate et al., 2006). ILK-dependent target phosphorylation is largely regulated by phosphoinositide 3-kinase (PI3K). Akt/PKB activation requires phosphorylation of Thr308 by PI3K-dependent kinase-1 (PDK1) (Alessi et al., 1997; Williams et al., 2000) and Ser473 by PDK2 [which is also known as hydrophobic motif kinase (HMK)] (Feng et al., 2004; Troussard et al., 2003). ILK is capable of controlling vascular endothelial growth factor (VEGF) transcription through Akt/PKB phosphorylation (Tan et al., 2004). ILK kinase activity is also stimulated by VEGF and other growth factors, as well as by cell adhesion to the extracellular matrix (Attwell et al., 2003). Parvins are a family of proteins involved in linking integrins and their associated proteins with intracellular pathways that regulate actin cytoskeletal dynamics and cell survival. It includes actopaxin/CH-ILKBP/a-parvin (ParvA), affixin/b-parvin (ParvB) and g-parvin (ParvG). ParvB localizes to focal adhesions and is involved in cell adhesion, spreading, motility and survival through interactions with its partners. ParvB accumulates and co-localizes with ILK in heart and skeletal muscle (Bendig et al., 2006; Yamaji et al., 2001). It can inhibit ILK activity and reverse some oncogenic effects in cancer cells (Mongroo et al., 2004). Furthermore, the physiological interaction of ILK with ParvB is thought to be essential in maintaining cardiac contractility (Bendig et al., 2006). Nonetheless, the precise role of ParvB and the mechanism controlling the association of ILK and ParvB remain to be delineated. Functional molecular imaging of ILK-mediated Akt/PKB signaling cascades and the associated role of b-parvin