Navegando por Palavras-chave "β-actina"

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    Caracterização da presença de AKT no núcleo de células de melanoma e identificação de proteínas associadas por espectrometria de massas
    (Universidade Federal de São Paulo, 2016-08-16) Coa, Larissa Leggieri [UNIFESP]; Machado Junior, Joel [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    The serine threonine kinase AKT/PKB is a critical regulator of various essential physiological cellular processes including cell proliferation, survival, motility and size, metabolism and differentiation. Deregulation of this pathway has been implicated in many diseases including cancers. Despite AKT action is acknowledged to function mainly in the cytoplasm, where most of its known substrates are located, AKT has been reported to translocate to the nucleus of various cell types. Therefore, AKT functions may be achieved through distinct subcellular compartments, providing spatial specificity in the mediation of biological effects. However, very little is known about the mechanism required for the nuclear import of AKT. Also, the interaction of AKT with proteins that reside in the nucleus has not been well explored as well as how its kinase activity modulates signaling in this compartment. Thus, identification of additional targets or binding proteins of nuclear AKT may provide a better understanding of it functions in the nucleus. In the present study we characterized the presence of endogenous nuclear AKT in two human melanoma cell lines harboring distinct genetic backgrounds (A2058, PTEN mutant/deleted; Mewo, PTEN wild-type). Our results showed that either phosphorylated and non-phosphorylated forms of AKT are present in melanoma cells nuclei, suggesting that phosphorylation of AKT is not required for its nuclear localization. Using co-immunoprecipitation (Co-IP) combined with crosslinking and mass spectrometry we identified a series of putative protein partners of nuclear AKT. Functional groups of proteins with known nuclear role emerged, such as RNA-binding proteins involved in transcription and pre-mRNA processing [e.g. heterogeneous nuclear ribonucleoprotein (hnRNP) and Serine/arginine-rich splicing factors (SRSF)], proteins required for ribosome biogenesis and rRNA processing (Ribosomal proteins), as well as cytoskeleton proteins (e.g. Actin, Vimentin and F-capping subunit ?). We validated ?-actin as a bona fide nuclear AKT-interacting partner. We also provided evidences that proteins such as cofilin and RNA polymerase II, known to interact and work in concert with ?-actin in the nucleus, also couple with nuclear AKT. Considering that nuclear actin has been described to have a key role regulating nuclear process such as chromatin remodeling, transcription, RNA processing and export, our findings provide new insights into the possible direct involvement of AKT in the transcriptional network of nuclear actin.