Giant obscurins regulate the PI3K cascade in breast epithelial cells via direct binding to the PI3K/p85 regulatory subunit
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Marey Shriver1,#, Saravanakumar Marimuthu1,#, Colin Paul2,3,4,*, Janelle Geist1,*, Tessa Seale1, Konstantinos Konstantopoulos2,3,4, Aikaterini Kontrogianni-Konstantopoulos1,5
1Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA
2Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA
3Johns Hopkins Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD 21218, USA
4Johns Hopkins Physical Sciences-Oncology Center, Johns Hopkins University, Baltimore, MD 21218, USA.
5University of Maryland School of Medicine, Marlene and Stewart Greenebaum National Cancer Institute Cancer Center, Baltimore, MD 21201, USA
#These authors contributed equally to this work
*These authors contributed equally to this work
Aikaterini Kontrogianni-Konstantopoulos, email: [email protected]
Keywords: obscurin, PI3K, PI3K inhibitors, Akt, breast cancer
Received: March 02, 2016 Accepted: May 29, 2016 Published: June 13, 2016
Obscurins are a family of giant cytoskeletal proteins, originally identified in striated muscles where they have structural and regulatory roles. We recently showed that obscurins are abundantly expressed in normal breast epithelial cells where they play tumor and metastasis suppressing roles, but are nearly lost from advanced stage breast cancer biopsies. Consistent with this, loss of giant obscurins from breast epithelial cells results in enhanced survival and growth, epithelial to mesenchymal transition (EMT), and increased cell migration and invasion in vitro and in vivo. In the current study, we demonstrate that loss of giant obscurins from breast epithelial cells is associated with significantly increased phosphorylation and subsequent activation of the PI3K signaling cascade, including activation of AKT, a key regulator of tumorigenesis and metastasis. Pharmacological and molecular inhibition of the PI3K pathway in obscurin-depleted breast epithelial cells results in reversal of EMT, (re)formation of cell-cell junctions, diminished mammosphere formation, and decreased cell migration and invasion. Co-immunoprecipitation, pull-down, and surface plasmon resonance assays revealed that obscurins are in a complex with the PI3K/p85 regulatory subunit, and that their association is direct and mediated by the obscurin-PH domain and the PI3K/p85-SH3 domain with a KD of ~50 nM. We therefore postulate that giant obscurins act upstream of the PI3K cascade in normal breast epithelial cells, regulating its activation through binding to the PI3K/p85 regulatory subunit.
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