Giant obscurins regulate the PI3K cascade in breast epithelial cells via direct binding to the PI3K/p85 regulatory subunit.
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.
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Marey
M
Shriver
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Saravanakumar
S
Marimuthu
Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.|Johns Hopkins Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD 21218, USA.|Johns Hopkins Physical Sciences-Oncology Center, Johns Hopkins University, Baltimore, MD 21218, USA.
Colin
C
Paul
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Janelle
J
Geist
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Tessa
T
Seale
Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.|Johns Hopkins Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD 21218, USA.|Johns Hopkins Physical Sciences-Oncology Center, Johns Hopkins University, Baltimore, MD 21218, USA.
Konstantinos
K
Konstantopoulos
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine,Baltimore, MD 21201, USA.|University of Maryland School of Medicine, Marlene and Stewart Greenebaum National Cancer Institute Cancer Center, Baltimore, MD 21201, USA.
Aikaterini
A
Kontrogianni-Konstantopoulos
D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D058543:Class Ia Phosphatidylinositol 3-Kinase / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology; D058750:Epithelial-Mesenchymal Transition / Q000502:physiology*; D005260:Female; D006801:Humans; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D064067:Rho Guanine Nucleotide Exchange Factors / Q000378:metabolism*
D058543:Class Ia Phosphatidylinositol 3-Kinase; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D064067:Rho Guanine Nucleotide Exchange Factors; C578224:OBSCN protein, human
Akt; PI3K; PI3K inhibitors; breast cancer; obscurin