Protein kinase B (PKB/Akt) is a regulator of cell survival and apoptosis. To become fully activated, PKB/Akt requires phosphorylation at two sites, threonine 308 and serine 473, in a phoshpatidylinositol (PI) 3-kinasedependent manner. The kinase responsible for phosphorylation of threonine 308 is the PI 3-kinase-dependent kinase-1 (PDK-1), whereas phosphorylation of serine 473 has been suggested to be regulated by PKB/Akt autophosphorylation in a PDK-1-dependent manner. However, the integrin-linked kinase (ILK) has also been shown to regulate phosphorylation of serine 473 in a PI 3-kinase-dependent manner. Whether ILK phosphorylates this site directly or functions as an adapter molecule has been debated. We now show by in-gel kinase assay and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry that biochemically purified ILK can phosphorylate PKB/Akt directly. Co-immunoprecipitation analysis of cell extracts demonstrates that ILK can complex with PKB/Akt as well as PDK-1 and that ILK can disrupt PDK-1/PKB association. The amino acid residue serine 343 of ILK within the activation loop is required for kinase activity as well as for its interaction with PKB/Akt. Mutational analysis of ILK further shows a crucial role for arginine 211 of ILK within the phosphoinositide phospholipid binding domain in the regulation of PKB-serine 473 phosphorylation. A highly selective small molecule inhibitor of ILK activity also inhibits the ability of ILK to phosphorylate PKB/Akt in vitro and in intact cells. These data demonstrate that ILK is an important upstream kinase for the regulation of PKB/Akt.Interaction of cells with the extracellular matrix results in the suppression of apoptosis and promotes cell cycle progression (1-4). The molecular basis for this anchorage-dependent cell growth and survival is an intensive area of study, since oncogenically transformed cells very often grow in an anchorage-independent manner. Alterations in anchorage-dependent signaling pathways are also likely to be of importance in tumor progression leading to metastasis. The integrin-linked kinase (ILK) 1 is an intracellular protein kinase that couples integrins and growth factors to downstream signaling pathways involved in the suppression of apoptosis and in promoting cell cycle progression. Cell-extracellular matrix interactions stimulate two major signaling pathways, leading to the regulation of cell cycle progression and cell survival. These are the Ras/Raf-MAP kinase pathway, and the protein kinase B/Akt (PKB/Akt) cell survival pathway (5). ILK regulates both the cell cycle, by stimulating the expression of cyclin D1 (6, 7) and cyclin A (7), and the activity of PKB/Akt, by stimulating the phosphorylation of PKB/Akt on serine 473 in a PI-3 kinase-dependent manner (8, 9), a requirement for full activation of this enzyme. Overexpression of ILK suppresses anoikis by activating PKB/ Akt (10), and ILK activity is constitutively up-regulated in tumor cells lacking expression of the PI(3,4,5)P 3 phosphatase tumor suppress...

tumor suppressor ͉ phospholipid phosphatase ͉ cell adhesion ͉ serum T he PTEN͞MMAC͞TEP1 tumor suppressor gene located on chromosome 10q23 (1-3) is mutated at high frequency in a wide variety of human cancers, including glioblastoma, melanoma, and carcinomas of the prostate, breast, endometrium, lung, and head and neck (1, 2, 4, 5-11). Germ-line mutations in the PTEN gene are associated with the development of Cowden's disease and Bannayan-Zonana syndrome (12-15). In addition, the phenotype of PTEN-null mice supports the conclusion that PTEN functions as a tumor suppressor gene (16,17). The homozygous disruption of PTEN results in early embryonic lethality, and heterozygous mice display hyperplastic changes in the prostate, skin, and colon similar to those seen in Cowden's disease. The PTEN gene product shares sequence identity with the protein tyrosine phosphatase family and chicken tensin (18). Recombinant PTEN is capable of dephosphorylating both phosphotyrosine and phosphothreonine, but it also can dephosphorylate phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ], the product of phosphatidylinositol 3-kinase (PI3-kinase) (19,20). Because many of the cancer-related mutations have been mapped to the phosphatase catalytic domain, it has been suggested that the phosphatase activity of PTEN is required for its tumor suppressor function. Overexpression of PTEN can suppress growth in soft agar and tumor formation in nude mice (21,22) and also inhibit focal adhesion kinase (FAK), leading to inhibition of cell adhesion and migration (23). However, targeted disruption of PTEN and PTEN mutations in glioblastoma and prostate carcinoma cells result in the serum-and anchorage-independent activation of protein kinase B (PKB)͞Akt probably because of increased levels of PI(3,4,5)P 3 (16,(24)(25)(26). PKB͞Akt suppresses apoptosis via several possible downstream effectors, including phosphorylation and inactivation of BAD (27-29), inactivation of caspase-9 (30), and repression of the forkhead transcription factor (31). Consistent with this, the disruption of PTEN leads to the suppression of apoptosis (16) but also to accelerated cell cycle progression (25,26). Reconstitution with wild-type (WT) PTEN restores apoptotic sensitivity and induces cell cycle arrest (16,26). These new insights into the tumor-suppressive effects of PTEN recently have been reviewed by Cantley and Neel (32).The activation of PKB͞Akt is regulated in a complex manner via phosphorylation of PKB͞Akt on Thr-308 and Ser-473 (33). Although PDK-1 has been shown to phosphorylate Thr-308, it is not clear whether there is a distinct kinase that exclusively phosphorylates Ser-473. It recently has been proposed that PDK-1 acquires Ser-473 phosphorylation activity in the presence of PRK2 peptide (34). However, as yet, there is no evidence that PDK-1 is the only kinase capable of doing this, and the physiological relevance of the conversion of PDK-1 activity toward Ser-473 phosphorylation by the PRK2 peptide has yet to be demonstrated. On the other hand, the int...

Increased synthesis of Apolipoprotein A-I (ApoA-I) and HDL is believed to provide a new approach to treating atherosclerosis through the stimulation of reverse cholesterol transport. RVX-208 increases the production of ApoA-I in hepatocytes in vitro, and in vivo in monkeys and humans, which results in increased HDL-C, but the molecular target was not previously reported. Using binding assays and X-ray crystallography, we now show that RVX-208 selectively binds to bromodomains of the BET (Bromodomain and Extra Terminal) family, competing for a site bound by the endogenous ligand, acetylated lysine, and that this accounts for its pharmacological activity. siRNA experiments further suggest that induction of ApoA-I mRNA is mediated by BET family member BRD4. These data indicate that RVX-208 increases ApoA-I production through an epigenetic mechanism and suggests that BET inhibition may be a promising new approach to the treatment of atherosclerosis.

Disruption of integrin-extracellular matrix interactions in normal epithelial cells induces apoptosis, a process termed anoikis. Reduced sensitivity to anoikis appears to be an important hallmark of oncogenic transformation, particularly in the process of metastasis. Several pathways have been implicated in the suppression of anoikis, however, the events which take place proximal to the integrin receptors remain unclear. Integrin-linked kinase (ILK) is an integrin-interacting protein kinase which has been identi®ed as a potential PDK-2, as it is capable of phosphorylating PKB/Akt on Ser-473, and stimulating its activity. Here, we show that ILK activity is stimulated upon adhesion of SCP2 mouse mammary epithelial cells to ®bronectin, and inhibited in suspended cells. Overexpression of ILK in the anoikis-sensitive SCP2 cells results in a profound inhibition of anoikis, as determined by annexin V binding and activation of caspases 8 and 3. This eect is reversible by the transfection and expression of a dominant-negative, kinase de®cient ILK (ILK KD), as well as by a dominant negative PKB/Akt (PKB AAA). On the other hand, transfection of a dominant negative form of FAK (FRNK) failed to reverse the suppression of anoikis by ILK. Furthermore, inhibition of ILK activity induced anoikis in two anoikis-resistant human breast cancer cell lines. These ®ndings suggest that ILK plays a major role in the suppression of anoikis. Oncogene (2000) 19, 3811 ± 3815.