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AKT and 14-3-3 regulate Notch4 nuclear localization.

ABSTRACT: Members of the Notch family of transmembrane receptors, Notch1-4 in mammals, are involved in the regulation of cell fate decisions and cell proliferation in various organisms. The Notch4 isoform, which is specific to mammals, was originally identified as a viral oncogene in mice, Int3, able to initiate mammary tumors. In humans, Notch4 expression appears to be associated with breast cancer stem cells and endocrine resistance. Following ligand binding, the Notch4 receptor undergoes cleavage at the membrane and the Notch4-intracellular domain (ICD), translocates to the nucleus and regulates gene transcription. Little is known on the mechanisms regulating Notch4-ICD and its nuclear localization. Here, we describe the identification of four distinct AKT phosphorylation sites in human Notch4-ICD and demonstrate that AKT binds Notch4-ICD and phosphorylates all four sites in vitro and in vivo. The phosphorylation in cells is regulated by growth factors and is sensitive to phosphatidyl inositol-3 kinase (PI3K) inhibitors. This phosphorylation generates binding sites to the 14-3-3 regulatory proteins, which are involved in the regulation of nucleocytoplasmic shuttling of target proteins, restricting phosphorylated Notch4-ICD to the cytoplasm. Our findings provide a novel mechanism for Notch4-ICD regulation, suggesting a negative regulatory role for the PI3K-AKT pathway in Notch4 nuclear signaling.

SUBMITTER: Ramakrishnan G

PROVIDER: S-EPMC4350099 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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AKT and 14-3-3 regulate Notch4 nuclear localization.

Ramakrishnan Gopalakrishnan G Davaakhuu Gantulga G Chung Wen Cheng WC Zhu He H Rana Ajay A Filipovic Aleksandra A Green Andrew R AR Atfi Azeddine A Pannuti Antonio A Miele Lucio L Tzivion Guri G

Scientific reports 20150305

Members of the Notch family of transmembrane receptors, Notch1-4 in mammals, are involved in the regulation of cell fate decisions and cell proliferation in various organisms. The Notch4 isoform, which is specific to mammals, was originally identified as a viral oncogene in mice, Int3, able to initiate mammary tumors. In humans, Notch4 expression appears to be associated with breast cancer stem cells and endocrine resistance. Following ligand binding, the Notch4 receptor undergoes cleavage at th ...[more]

PMID: 25740432

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Project description:Background: Maspin (SERPINB5) is a potential tumor suppressor gene with pleiotropic biological activities, including regulation of cell proliferation, death, adhesion, migration and gene expression. Several studies indicate that nuclear localization is essential for maspin tumor suppression activity. We have previously shown that the EGFR activation leads to maspin nuclear localization in MCF-10A cells. The present study investigated which EGFR downstream signaling molecules are involved in maspin nuclear localization and explored a possible role of cell-cell contact in this process. Methods: MCF-10A cells were treated with pharmacological inhibitors against EGFR downstream pathways followed by EGF treatment. Maspin subcellular localization was determined by immunofluorescence. To investigate the role of cell-cell contact these cells were either treated with chelating agents or plated on different cell densities. Maspin and E-cadherin subcellular localization was determined by immunofluorescence. In addition, proteomic and interactome analyses were conducted in order to identify molecular partners which interact with maspin in EGF-treated cells only. Results: EGFR activation regulates cell behavior via de extracellular signal–related kinase (ERK)/MAP kinase, phosphatidyl-inositol-3 (PI 3) kinase- AKT and Jak-STAT pathways. Using pharmacological inhibitors against key components of these signaling pathways we found that PI3K-AKT and Jak-STAT, but not MAP kinase pathway, regulate EGF-induced maspin nuclear accumulation in MCF-10A cells. Interestingly, we observed that maspin is predominantly nuclear in sparse cell culture, but it is redistributed to the cytoplasm in confluent cells even in the presence of EGF. Proteomic and interactome results suggest a role of maspin on post-transcriptional and translation regulation, protein folding and cell-cell adhesion.Conclusions: Maspin nuclear accumulation is determined by an interplay between EGFR (via PI3K-AKT and Jak-STAT pathways) and cell-to-cell contact.

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AKT and 14-3-3 regulate Notch4 nuclear localization.

Publications

AKT and 14-3-3 regulate Notch4 nuclear localization.

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