The serine/threonine kinase Akt plays a central role in cell survival and proliferation. Its activation is linked to tumorigenesis in several human cancers. Although many Akt substrates have been elucidated, the Akt-binding proteins that regulate Akt function remain unclear. We report herein having identified casein kinase 2-interacting protein-1 (CKIP-1) as an Akt pleckstrin homology (PH) domain-binding protein with Akt inhibitory function. CKIP-1 formed a complex with each Akt isoform (Akt1, Akt2, and Akt3) via its NH 2 terminus. Dimerization of CKIP-1 via its leucine zipper (LZ) motif at the COOH terminus was found to be associated with Akt inactivation because deletion of the LZ motif eliminated Akt inhibitory function, although it could still bind to Akt. Expression of the NH 2 terminus-deleted CKIP-1 mutant containing the LZ motif, but lacking Akt-binding ability, induced Akt phosphorylation and activation by sequestering the ability of endogenous CKIP-1 to bind to Akt. Stable CKIP-1 expression caused Akt inactivation and cell growth inhibition in vitro. In addition, the growth of stable CKIP-1 transfectants xenografted into nude mice was slower than that of mock transfectants. These results indicate that CKIP-1, a novel Akt PH domain-interacting protein, would be a candidate of tumor suppressor with an Akt inhibitory function. [Cancer Res 2007;67(20):9666-76]

Human ATP-binding cassette transporter ABCB11 (SPGP/BSEP) mediates the elimination of bile salts from liver cells and thereby plays a critical role in the generation of bile flow. In the present study, we have developed in vitro high-speed screening and quantitative structure-activity relationship (QSAR) analysis methods to investigate the interaction of ABCB11 with a variety of drugs. Plasma membrane vesicles prepared from insect cells overexpressing human ABCB11 were used to measure the ATP-dependent transport of [14C]taurocholate. Over 40 different drugs and natural compounds were tested to evaluate their interaction with ABCB11-mediated taurocholate transport. On the basis of the extent of inhibition, we have analyzed the QSAR to identify one set of chemical fragmentation codes closely associated with the inhibition of ABCB11. This approach can be used to predict compounds with a potential risk of drug-induced intrahepatic cholestasis.

3-Phosphoinositide-dependent protein kinase-1 (PDK1) is a key regulator of cell proliferation and survival signal transduction. PDK1 is known to be constitutively active and is further activated by Srcmediated phosphorylation at the tyrosine-9, -373, and -376 residues. To identify novel regulators of PDK1, we performed E. coli-based two-hybrid screening and revealed that tumor suppressor candidate 4 (TUSC4), also known as nitrogen permease regulator-like 2 (NPRL2), formed a complex with PDK1 and suppressed Src-dependent tyrosine phosphorylation and activation of PDK1 in vitro and in cells. The NH 2 -terminal 133 amino acid residues of TUSC4 were involved in binding to PDK1. The deletion mutant of TUSC4 that lacked the NH 2 -terminal domain showed no inhibitory effects on PDK1 tyrosine phosphorylation or activation. Thus, complex formation is indispensable for TUSC4-mediated PDK1 inactivation. 3-Phosphoinositide-dependent protein kinase-1 (PDK1) was originally identified as a protein serine/threonine kinase that could phosphorylate Akt at the Thr 308 residue in its activation loop.(1,2) Later studies have shown that PDK1 is not only an Akt kinase but also a master regulator of a group of protein kinases known as the AGC (cAMP-dependent, cGMP-dependent, and protein kinase C) family, including p70 ribosomal protein S6 kinase (S6K), serum-and glucocorticoid-inducible kinases (SGKs), protein kinase A (PKA), protein kinase C (PKC) isoforms, and p90 ribosomal protein S6 kinases (RSKs) at the equivalent residues of Thr 308 in Akt (T-loop).(3) Therefore, PDK1 functions as a pivotal molecule for activation of a number of signaling pathways involved in proliferation and cell survival. Many components involved in the downstream of PDK1 have been elucidated, though the regulatory mechanism to control PDK1 activity is still controversial.It has long been thought that auto-phosphorylation at the Ser 241 residue in the activation loop is sufficient for PDK1 activation.Thus, PDK1 is thought to be constitutively active in resting cells and not further activated by growth factor stimulation.However, recent reports suggest that PDK1 activity and stability are regulated by interaction with other proteins. (6)(7)(8)(9)(10)(11) For example, PDK1 binds to Hsp90, and its binding protects PDK1 from proteasome-dependent degradation.(6) PDK1 also binds to 14-3-3 through the residues surrounding the auto-phosphorylation site Ser 241 and its binding decreases PDK1 kinase activity.Moreover, Src kinase directly phosphorylates PDK1 at Tyr 9 Tyr 373 , and Tyr 376 leading to an increase in PDK1 kinase activity. (8)(9)(10)(11) It has also been proposed that Abl, RET/PTC, Pyk2, insulin receptor, and Hsp90 were candidates for PDK1 activation.(11) To identify novel regulators of PDK1, we performed E. coli-based two-hybrid screening using the Pleckstrin homology (PH) domain of human PDK1 as bait. We were fortunate to identify the tumor suppressor candidate 4 (TUSC4), also known as nitrogen permease regulatorlike 2 (NPRL2), as a novel PDK1-interacting...

Drug-induced intrahepatic cholestasis is one of the major causes of hepatotoxicity, which often occur during the drug discovery and development process. Human ATP-binding cassette transporter ABCB11 (sister of P-glycoprotein/bile salt export pump) mediates the elimination of cytotoxic bile salts from liver cells to bile, and, therefore, plays a critical role in the generation of bile flow. The authors have recently developed in vitro high-speed screening and quantitative structure-activity relationship analysis methods to investigate the interaction of ABCB11 with a variety of compounds. Based on the extent of inhibition of the bile salt export pump, the authors analysed the quantitative structure-activity relationship to identify chemical groups closely associated with the inhibition of ABCB11. This approach provides a new tool to predict compounds with a potential risk of drug-induced intrahepatic cholestasis.