AKT and 14-3-3 regulate Notch4 nuclear localization.
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.
Cancer Institute, University of Mississippi Medical Center, Jackson, MS 39216.
Gopalakrishnan
G
Ramakrishnan
Cancer Institute, University of Mississippi Medical Center, Jackson, MS 39216.
Gantulga
G
Davaakhuu
Cancer Institute, University of Mississippi Medical Center, Jackson, MS 39216.
Wen Cheng
WC
Chung
Cancer Institute, University of Mississippi Medical Center, Jackson, MS 39216.
He
H
Zhu
Department of Molecular Pharmacology and Therapeutics, Loyola University Chicago, Maywood, IL 60153.
Ajay
A
Rana
Imperial College London, Division of Surgery and Cancer, Department of Oncology, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK.
Aleksandra
A
Filipovic
Department of Histopathology and School of Molecular Medical Sciences, University of Nottingham, Nottingham City Hospital, Nottingham, NG5 1PB, UK.
Andrew R
AR
Green
1] Cancer Institute, University of Mississippi Medical Center, Jackson, MS 39216 [2] Department of Biochemistry, University of Mississippi Medical Center, Jackson, MS 39216.
Azeddine
A
Atfi
1] Cancer Institute, University of Mississippi Medical Center, Jackson, MS 39216 [2] Department of Biochemistry, University of Mississippi Medical Center, Jackson, MS 39216.
Antonio
A
Pannuti
Cancer Institute, University of Mississippi Medical Center, Jackson, MS 39216.
Lucio
L
Miele
1] Cancer Institute, University of Mississippi Medical Center, Jackson, MS 39216 [2] Department of Biochemistry, University of Mississippi Medical Center, Jackson, MS 39216.
Guri
G
Tzivion
D048948:14-3-3 Proteins / Q000378:metabolism*; D021581:Active Transport, Cell Nucleus; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D003593:Cytoplasm; D006801:Humans; D010766:Phosphorylation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D000074562:Receptor, Notch4; D051880:Receptors, Notch / Q000737:chemistry / Q000378:metabolism*
D048948:14-3-3 Proteins; C106075:NOTCH4 protein, human; D011518:Proto-Oncogene Proteins; D000074562:Receptor, Notch4; D051880:Receptors, Notch; D051057:Proto-Oncogene Proteins c-akt