title: Phosphorylation negatively regulates exosome mediated secretion of cryAB in glioma cells.
abstract: Exosomes mediate secretion of crystallin alphaB (cryAB), a well characterized molecular chaperone with anti-apoptotic activity. However, the mechanisms governing its packaging and secretion remained unexplored. In glioma cells, notwithstanding extensive phosphorylation of cryAB at Ser59 followed by Ser45 (Ser19 is largely unphosphorylated), we discovered that the majority of secreted exosomal cryAB is nonphosphorylated. Transient ectopic expression of a yellow fluorescent protein (YFP) tagged triple phosphomimic (3-SD) cryAB construct in cryAB absent glioma cells led to the formation of large cytosolic inclusions. Our findings demonstrate that mimicking phosphorylation significantly reduces cryAB secretion via exosomes. Moreover, decreased colocalization of 3-SD YFP-cryAB with multivesicular endosome (MVE) and exosome marker, CD63 or Rab27, a small GTPase regulating exocytosis of MVEs, suggests that phosphorylation deters packaging of cryAB in vesicles bound for secretion as exosomes. Additionally, we found that preventing O-GlcNAcylation on cryAB also curtailed its colocalization with CD63 and Rab27 resulting in reduced exosomal secretion. Thus, our study points to O-GlcNAcylation and lack of phosphorylation as being the selective processes involved in the packaging and secretion of cryAB via exosomes.
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
authors.forename: Rajshekhar A
authors.initials: RA
authors.lastname: Kore
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. Electronic address: AbrahamEdatharaC@uams.edu.
authors.forename: Edathara C
authors.initials: EC
authors.lastname: Abraham
mesh_terms: D015153:Blotting, Western; D045744:Cell Line, Tumor; D055354:Exosomes / Q000378:metabolism* / Q000648:ultrastructure; D005910:Glioma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D006031:Glycosylation; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D057146:Multivesicular Bodies / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D060149:Tetraspanin 30 / Q000378:metabolism; D038203:alpha-Crystallin B Chain / Q000235:genetics / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D000075745:rab27 GTP-Binding Proteins
chemical_list: C554586:CD63 protein, human; D008164:Luminescent Proteins; D060149:Tetraspanin 30; D038203:alpha-Crystallin B Chain; D000075745:rab27 GTP-Binding Proteins; D012694:Serine; C066694:RAB27A protein, human; D020691:rab GTP-Binding Proteins
keywords: Exosomes; Glioblastoma multiforme; Glycosylation; Phosphorylation; cryAB