Internalized group V secretory phospholipase A2 acts on the perinuclear membranes.
Mammalian secretory phospholipases A(2) (sPLA(2)) have been implicated in cellular eicosanoid biosynthesis but the mechanism of their cellular action remains unknown. To elucidate the spatiotemporal dynamics of sPLA(2) mobilization and determine the site of its lipolytic action, we performed time-lapse confocal microscopic imaging of fluorescently labeled sPLA(2) acting on human embryonic kidney (HEK) 293 cells the membranes of which are labeled with a fluorogenic phospholipid, N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-1-hexadecanoyl-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphoethanolamine. The Western blotting analysis of HEK293 cells treated with exogenous sPLA(2)s showed that not only the affinity for heparan sulfate proteoglycan but also other factors, such as sPLA(2) hydrolysis products or cytokines, are necessary for the internalization of sPLA(2) into HEK293 cells. Live cell imaging showed that the hydrolysis of fluorogenic phospholipids incorporated into HEK293 cell membranes was synchronized with the spatiotemporal dynamics of sPLA(2) internalization, detectable initially at the plasma membrane and then at the perinuclear region. Also, immunocytostaining showed that human group V sPLA(2) induced the translocation of 5-lipoxygenase to the nuclear envelope at which they were co-localized. Together, these studies provide the first experimental evidence that the internalized sPLA(2) acts on the nuclear envelope to provide arachidonate for other enzymes involved in the eicosanoid biosynthesis.
Kim
Young Jun
YJ
Department of Chemistry, University of Illinois at Chicago, Chicago, Illinois 60607, USA.
Kim
Kwang Pyo
KP
Rhee
Hae Jin
HJ
Das
Sudipto
S
Rafter
John D
JD
Oh
Youn Sang
YS
Cho
Wonhwa
W
D001094:Arachidonate 5-Lipoxygenase / Q000032:analysis; D016718:Arachidonic Acid / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D006868:Hydrolysis; D009685:Nuclear Envelope / Q000201:enzymology*; D010741:Phospholipases A / Q000032:analysis / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism
D010743:Phospholipids; D016718:Arachidonic Acid; D001094:Arachidonate 5-Lipoxygenase; D010741:Phospholipases A; D054467:Phospholipases A2