title: Epithelial membrane protein 2 regulates sphingosylphosphorylcholine-induced keratin 8 phosphorylation and reorganization: Changes of PP2A expression by interaction with alpha4 and caveolin-1 in lung cancer cells.
abstract: Sphingosylphosphorylcholine (SPC) is found at increased in the malignant ascites of tumor patients and induces perinuclear reorganization of keratin 8 (K8) filaments that contribute to the viscoelasticity of metastatic cancer cells. However, the detailed mechanism of SPC-induced K8 phosphorylation and reorganization is not clear. We observed that SPC dose-dependently reduced the expression of epithelial membrane protein 2 (EMP2) in lung cancer cells. Then, we examined the role of EMP2 in SPC-induced phosphorylation and reorganization of K8 in lung cancer cells. We found that SPC concentration-dependently reduced EMP2 in A549, H1299, and other lung cancer cells. This was verified at the mRNA level by RT-PCR and real-time PCR (qPCR), and intracellular variation through confocal microscopy. EMP2 gene silencing and stable lung cancer cell lines established using EMP2 lentiviral shRNA induced K8 phosphorylation and reorganization. EMP2 overexpression reduced K8 phosphorylation and reorganization. We also observed that SPC-induced loss of EMP2 induces phosphorylation of JNK and ERK via reduced expression of protein phosphatase 2A (PP2A). Loss of EMP2 induces ubiquitination of protein phosphatase 2A (PP2A). SPC induced caveolin-1 (cav-1) expression and EEA1 endosome marker protein but not cav-2. SPC treatment enhanced the binding of cav-1 and PP2A and lowered binding of PP2A and alpha4. Gene silencing of EMP2 increased and gene silencing of cav-1 reduced migration of A549 lung cancer cells. Overall, these results suggest that SPC induces EMP2 down-regulation which reduces the PP2A via ubiquitination induced by cav-1, which sequestered alpha4, leading to the activation of ERK and JNK.
authors.affiliation: BK21PLUS R-FIND team, College of Pharmacy, Dongguk University, Seoul 100-715, Republic of Korea.
authors.forename: Eun Ji
authors.initials: EJ
authors.lastname: Lee
authors.affiliation: BK21PLUS R-FIND team, College of Pharmacy, Dongguk University, Seoul 100-715, Republic of Korea.
authors.forename: Mi Kyung
authors.initials: MK
authors.lastname: Park
authors.affiliation: BK21PLUS R-FIND team, College of Pharmacy, Dongguk University, Seoul 100-715, Republic of Korea.
authors.forename: Hyun Ji
authors.initials: HJ
authors.lastname: Kim
authors.affiliation: BK21PLUS R-FIND team, College of Pharmacy, Dongguk University, Seoul 100-715, Republic of Korea.
authors.forename: Eun Ji
authors.initials: EJ
authors.lastname: Kim
authors.affiliation: BK21PLUS R-FIND team, College of Pharmacy, Dongguk University, Seoul 100-715, Republic of Korea.
authors.forename: Gyeoung-Jin
authors.initials: GJ
authors.lastname: Kang
authors.affiliation: BK21PLUS R-FIND team, College of Pharmacy, Dongguk University, Seoul 100-715, Republic of Korea.
authors.forename: Hyun Jung
authors.initials: HJ
authors.lastname: Byun
authors.affiliation: BK21PLUS R-FIND team, College of Pharmacy, Dongguk University, Seoul 100-715, Republic of Korea. Electronic address: uatheone@dongguk.edu.
authors.forename: Chang Hoon
authors.initials: CH
authors.lastname: Lee
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D015153:Blotting, Western; D051242:Caveolin 1 / Q000378:metabolism*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D053533:Keratin-8 / Q000378:metabolism*; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D018832:Molecular Chaperones; D010766:Phosphorylation / Q000187:drug effects; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D054648:Protein Phosphatase 2 / Q000378:metabolism*; D034622:RNA Interference; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D054875:Ubiquitination / Q000187:drug effects
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051242:Caveolin 1; C486403:EMP2 protein, human; C490467:IGBP1 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D053533:Keratin-8; D008562:Membrane Glycoproteins; D018832:Molecular Chaperones; C005356:sphingosine phosphorylcholine; D010767:Phosphorylcholine; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D054648:Protein Phosphatase 2; D013110:Sphingosine
keywords: Alpha 4; Epithelial membrane protein 2; Keratin 8 phosphorylation; Keratin 8 reorganization; Protein phosphatase 2A; Sphingosylphosphorylcholine