title: Phosphoproteins regulated by the interaction of high-density lipoprotein with human skin fibroblasts.
abstract: Interaction of HDL with cells activates protein kinase C (PKC), a process that may be important in stimulating efflux of excess cellular cholesterol. Here we report that HDL treatment of cholesterol-loaded fibroblasts increases 32P labeling of three acidic phosphoproteins. These phosphoproteins, called pp80, pp27, and pp18 based on apparent M(r) in kD, were also phosphorylated by acute treatment of cells with phorbol myristate acetate, suggesting that they are regulated in response to PKC activation. The HDL-stimulated phosphorylation of pp80 and pp18 was significant after only 30 seconds and was sustained for at least 30 and 120 minutes, respectively, while increased phosphorylation of pp27 was transient, reaching a maximum at 10 minutes. Both pp27 and pp18 were phosphorylated on serine/threonine residues, whereas pp80 was phosphorylated on serine/threonine and tyrosine residues. Immunoprecipitation studies suggested that pp80 is the myristoylated alanine-rich C kinase substrate protein, but the identities of pp27 and pp18 are unknown. HDL and trypsin-digested HDL stimulated phosphorylation of pp80 and pp27, while purified apoA-I, apoA-II, or apoE had no stimulatory effects, indicating that the active component in HDL was trypsin resistant and unlikely to be an apolipoprotein. Conversely, HDL, apoA-I, apoA-II, and apoE all stimulated pp18 phosphorylation, while trypsin-digested HDL had less effect, consistent with pp18's being responsive to HDL apolipoproteins. Treatment of cholesterol-depleted cells with apoA-I also stimulated phosphorylation of pp18, but only transiently. These results suggest that HDL interaction with cells activates diverse PKC-mediated pathways that target different phosphoproteins. Of these three phosphoproteins, only pp18 has a phosphorylation response consistent with its being involved in apolipoprotein-mediated lipid transport.
authors.affiliation: Department of Medicine, University of Washington, Seattle 98195-6426, USA.
authors.forename: W S
authors.initials: WS
authors.lastname: Garver
authors.forename: M A
authors.initials: MA
authors.lastname: Deeg
authors.forename: R F
authors.initials: RF
authors.lastname: Bowen
authors.forename: M M
authors.initials: MM
authors.lastname: Culala
authors.forename: E L
authors.initials: EL
authors.lastname: Bierman
authors.forename: J F
authors.initials: JF
authors.lastname: Oram
mesh_terms: D016632:Apolipoprotein A-I / Q000494:pharmacology; D016633:Apolipoprotein A-II / Q000494:pharmacology; D001057:Apolipoproteins E / Q000494:pharmacology; D001692:Biological Transport; D002352:Carrier Proteins; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism*; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008075:Lipoproteins, HDL / Q000494:pharmacology*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000032:analysis; D010769:Phosphothreonine / Q000032:analysis; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D016601:RNA-Binding Proteins; D018110:Receptors, Lipoprotein / Q000187:drug effects* / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects; D012867:Skin / Q000166:cytology
chemical_list: D016632:Apolipoprotein A-I; D016633:Apolipoprotein A-II; D001057:Apolipoproteins E; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008075:Lipoproteins, HDL; D008565:Membrane Proteins; D010750:Phosphoproteins; D011506:Proteins; D016601:RNA-Binding Proteins; D018110:Receptors, Lipoprotein; C031616:high density lipoprotein receptors; D010769:Phosphothreonine; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; C075197:high density lipoprotein binding protein; D010768:Phosphoserine; D002784:Cholesterol; D017346:Protein Serine-Threonine Kinases