TY - JOUR
T1 - Ser-phosphorylation of PCSK9 (Proprotein Convertase Subtilisin-Kexin 9) by Fam20C (Family with Sequence Similarity 20, Member C) kinase enhances its ability to degrade the LDLR (Low-Density Lipoprotein Receptor)
AU - Ben Djoudi Ouadda, Ali
AU - Gauthier, Marie Soleil
AU - Susan-Resiga, Delia
AU - Girard, Emmanuelle
AU - Essalmani, Rachid
AU - Black, Miles
AU - Marcinkiewicz, Jadwiga
AU - Forget, Diane
AU - Hamelin, Josée
AU - Evagelidis, Alexandra
AU - Ly, Kevin
AU - Day, Robert
AU - Galarneau, Luc
AU - Corbin, Francois
AU - Coulombe, Benoit
AU - Çaku, Artuela
AU - Tagliabracci, Vincent S.
AU - Seidah, Nabil G.
N1 - Funding Information:
This work was supported by the Canadian Institutes of Health Research grants Foundation Scheme 148363 and MOP (medical operating grant) 102741, a Canada Research Chair 231335, and a Fondation Leducq grant no. 13CVD03 (to N.G. Seidah) as well as a Welch Foundation Grant I-1911 (to V.S. Tagliabracci). A. Ben Djoudi Ouadda is a recipient of the Montreal Clinical Research Institute Foundation-Jean Coutu fellowship.
Publisher Copyright:
© 2019 American Heart Association, Inc.
PY - 2019/10/1
Y1 - 2019/10/1
N2 - Objective: PCSK9 (proprotein convertase subtilisin-kexin 9) enhances the degradation of the LDLR (low-density lipoprotein receptor) in endosomes/lysosomes. This study aimed to determine the sites of PCSK9 phosphorylation at Ser-residues and the consequences of such posttranslational modification on the secretion and activity of PCSK9 on the LDLR. Approach and Results: Fam20C (family with sequence similarity 20, member C) phosphorylates serines in secretory proteins containing the motif S-X-E/phospho-Ser, including the cholesterol-regulating PCSK9. In situ hybridization of Fam20C mRNA during development and in adult mice revealed a wide tissue distribution, including liver, but not small intestine. Here, we show that Fam20C phosphorylates PCSK9 at Serines 47, 666, 668, and 688. In hepatocytes, phosphorylation enhances PCSK9 secretion and maximizes its induced degradation of the LDLR via the extracellular and intracellular pathways. Replacing any of the 4 Ser by the phosphomimetic Glu or Asp enhanced PCSK9 activity only when the other sites are phosphorylated, whereas Ala substitutions reduced it, as evidenced by Western blotting, Elisa, and LDLR-immunolabeling. This newly uncovered PCSK9/LDLR regulation mechanism refines our understanding of the implication of global PCSK9 phosphorylation in the modulation of LDL-cholesterol and rationalizes the consequence of natural mutations, for example, S668R and E670G. Finally, the relationship of Ser-phosphorylation to the implication of PCSK9 in regulating LDL-cholesterol in the neurological Fragile X-syndrome disorder was investigated. Conclusions: Ser-phosphorylation of PCSK9 maximizes both its secretion and activity on the LDLR. Mass spectrometric approaches to measure such modifications were developed and applied to quantify the levels of bioactive PCSK9 in human plasma under normal and pathological conditions.
AB - Objective: PCSK9 (proprotein convertase subtilisin-kexin 9) enhances the degradation of the LDLR (low-density lipoprotein receptor) in endosomes/lysosomes. This study aimed to determine the sites of PCSK9 phosphorylation at Ser-residues and the consequences of such posttranslational modification on the secretion and activity of PCSK9 on the LDLR. Approach and Results: Fam20C (family with sequence similarity 20, member C) phosphorylates serines in secretory proteins containing the motif S-X-E/phospho-Ser, including the cholesterol-regulating PCSK9. In situ hybridization of Fam20C mRNA during development and in adult mice revealed a wide tissue distribution, including liver, but not small intestine. Here, we show that Fam20C phosphorylates PCSK9 at Serines 47, 666, 668, and 688. In hepatocytes, phosphorylation enhances PCSK9 secretion and maximizes its induced degradation of the LDLR via the extracellular and intracellular pathways. Replacing any of the 4 Ser by the phosphomimetic Glu or Asp enhanced PCSK9 activity only when the other sites are phosphorylated, whereas Ala substitutions reduced it, as evidenced by Western blotting, Elisa, and LDLR-immunolabeling. This newly uncovered PCSK9/LDLR regulation mechanism refines our understanding of the implication of global PCSK9 phosphorylation in the modulation of LDL-cholesterol and rationalizes the consequence of natural mutations, for example, S668R and E670G. Finally, the relationship of Ser-phosphorylation to the implication of PCSK9 in regulating LDL-cholesterol in the neurological Fragile X-syndrome disorder was investigated. Conclusions: Ser-phosphorylation of PCSK9 maximizes both its secretion and activity on the LDLR. Mass spectrometric approaches to measure such modifications were developed and applied to quantify the levels of bioactive PCSK9 in human plasma under normal and pathological conditions.
KW - Cholesterol
KW - Lipoprotein
KW - Mutations
KW - Phosphorylation
KW - Serine
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UR - http://www.scopus.com/inward/citedby.url?scp=85072678773&partnerID=8YFLogxK
U2 - 10.1161/ATVBAHA.119.313247
DO - 10.1161/ATVBAHA.119.313247
M3 - Article
C2 - 31553664
AN - SCOPUS:85072678773
SN - 1079-5642
VL - 39
SP - 1996
EP - 2013
JO - Arteriosclerosis, thrombosis, and vascular biology
JF - Arteriosclerosis, thrombosis, and vascular biology
IS - 10
ER -