RESUMO
Engagement of cell surface receptor tyrosine kinases by insulin and growth factors activates phosphatidylinositol 3-kinase (PI3K) and generates the second messenger, phosphatidylinositol 3,4,5-trisphosphate. This second messenger leads to the recruitment of 3-phosphoinositide-dependent protein kinase-1 (PDK1) to the proximal side of the plasma membrane, which results in the activation of AKT kinase. In addition, PDK1 can phosphorylate numerous other kinases, including p90RSK, a kinase downstream of mitogen-activated protein kinase (MAPK) that is important for cell proliferation and survival. Previous studies have shown that the loss of PDK1 sensitizes tumor cells to chemotherapeutic agents and radiation but have not focused on delineating the contribution of PDK1 to pathway-specific mutations associated with various cancers other than the PI3K/AKT pathway. In this study, we show that the reduction of PDK1 by RNAi in melanoma and colon cancer cell lines activated in the MAPK pathway results in significant cell growth inhibition and apoptosis. Furthermore, PDK1 reduction in tumor cells resulted in impaired PAK kinase signaling, altered actin polymerization, and reduced cell migration. These studies show that PDK1 plays a pivotal role in MAPK and PI3K signaling in tumor cells.
Assuntos
Proliferação de Células , Neoplasias/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Apoptose/genética , Carcinoma/enzimologia , Carcinoma/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Neoplasias do Colo/enzimologia , Neoplasias do Colo/genética , Regulação para Baixo/genética , Ativação Enzimática/genética , Regulação Enzimológica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Sistema de Sinalização das MAP Quinases/genética , Melanoma/enzimologia , Melanoma/genética , Neoplasias/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA/fisiologiaRESUMO
AKT is a key serine/threonine kinase in the PTEN/PI3K/AKT pathway(1) and activationof AKT is often observed in human cancers. To explore the role of AKT in cell survival in different tumor cells, we tested 20 human tumor cell lines for response to knockdown of AKT by small interference RNA (siRNA) and/or a kinase-dead mutant AKT. siRNA-mediated knockdown of all three AKT isoforms in tumor cell lines led to a reduction of phosphorylation of AKT substrates. Knockdown of AKT resulted in apoptosis in six out of 11 tumor cells with activated AKT. In contrast, knockdown of AKT induced apoptosis in three out of nine cell lines with a low level of active AKT. The responsiveness of the cells to knockdown of AKT was not affected by mutational status of p53 but appeared correlated with overexpression of HER2. To assess the role of individual AKT isoforms, five of the cell lines responsive to knockdown of AKT were further characterized. In ZR-75 cells, AKT1 is the predominant isoform responsible for cell proliferation and survival. Conversely, in IGROV1 cells, AKT2 plays a major role in cell proliferation, but no single isoform is essential for cell survival. Thus, the relative importance of the AKT isoforms is cell line-specific. Our data suggest that inhibiting all three AKT isoforms is necessary to elicit maximal apoptotic response in tumor cells, and the level of activated AKT is a favorable but not always reliable biomarker for preselection of responsive tumor cell lines to AKT inhibitors.
Assuntos
Apoptose/genética , Inativação Gênica/fisiologia , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Adenoviridae/genética , Western Blotting , Proliferação de Células , Sobrevivência Celular/fisiologia , Humanos , Neoplasias/genética , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Most of the protein kinase inhibitors being developed are directed toward the adenosine triphosphate (ATP) binding site that is highly conserved in many kinases. A major issue with these inhibitors is the specificity for a given kinase. Structure determination of several kinases has shown that protein kinases adopt distinct conformations in their inactive state, in contrast to their strikingly similar conformations in their active states. Hence, alternative assay formats that can identify compounds targeting the inactive form of a protein kinase are desirable. The authors describe the development and optimization of an Immobilized Metal Assay for Phosphochemicals (IMAP)-based couple d assay using PDK1 and inactive Akt-2 enzymes. PDK1 phosphorylates Akt-2 at Thr 309 in the catalytic domain, leading to enzymatic activation. Activation of Akt by PDK1 is measured by quantitating the phosphorylation of Akt-specific substrate peptide using the IMAP assay format. This IMAP-coupled assay has been formatted in a 384-well microplate format with a Z' of 0.73 suitable for high-throughput screening. This assay was evaluated by screening the biologically active sample set LOPAC trade mark and validated with the protein kinase C inhibitor staurosporine. The IC(50) value generated was comparable to the value obtained by the radioactive (33)P-gamma-ATP flashplate transfer assay. This coupled assay has the potential to identify compounds that target the inactive form of Akt and prevent its activation by PDK1, in addition to finding inhibitors of PDK1 and activated Akt enzymes.
