CD36 ectodomain phosphorylation blocks thrombospondin-1 binding: structure-function relationships and regulation by protein kinase C. Arterioscler Thromb Vasc Biol 2012 Mar;32(3):760-7
Date
01/17/2012Pubmed ID
22247259Pubmed Central ID
PMC3298081DOI
10.1161/ATVBAHA.111.242511Scopus ID
2-s2.0-84857648664 (requires institutional sign-in at Scopus site) 32 CitationsAbstract
OBJECTIVE: CD36 phosphorylation on its extracellular domain inhibits binding of thrombospondin-1. The mechanisms of cellular CD36 ectodomain phosphorylation and whether it can be regulated in cells are not known. We determined structure-function relationships of CD36 phosphorylation related to thrombospondin-1 peptide binding in vitro and explored mechanisms regulating phosphorylation by protein kinase C (PKC) in melanoma cells.
METHODS AND RESULTS: Phosphorylation of CD36 peptide on Thr92 by PKCĪ± suppressed binding of thrombospondin-1 peptides in vitro, and the level of inhibition correlated with the level of phosphorylation. Basal phosphorylation levels of CD36 in vivo in platelets, endothelial cells, and melanoma cells were assessed by immunoprecipitation and immunoblot and were found to be very low. Treatment of CD36-transfected melanoma cells with phorbol 12-myristate 13-acetate (PMA), a PKC activator, induced substantial CD36 phosphorylation and decreased ligand-mediated recruitment of Src-family proteins to CD36. PMA treatment did not induce detectable extracellular or cell surface-associated kinase activity, and both cycloheximide and brefeldin A blocked CD36 phosphorylation.
CONCLUSION: New protein synthesis and trafficking through the Golgi are required for PMA-induced CD36 phosphorylation, suggesting that phosphorylation probably occurs intracellularly. These studies suggest a novel in vivo pathway for CD36 phosphorylation that modulates cellular affinity for thrombospondin-related proteins to blunt vascular cell signaling.
Author List
Chu LY, Silverstein RLAuthor
Roy L. Silverstein MD Professor in the Medicine department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Blotting, WesternCD36 Antigens
Cell Line, Tumor
Endothelial Cells
Enzyme Activation
Enzyme Activators
Golgi Apparatus
Humans
Immunoprecipitation
Melanoma
Phosphorylation
Protein Binding
Protein Conformation
Protein Interaction Domains and Motifs
Protein Interaction Mapping
Protein Kinase C-alpha
Protein Synthesis Inhibitors
Protein Transport
Structure-Activity Relationship
Tetradecanoylphorbol Acetate
Thrombospondin 1
Time Factors
Transfection
src-Family Kinases
