Abstract 12894: Activation of Intracellular Protein Kinase C (PKC) Alpha by Phorbol 12-Myristate 13-Acetate (PMA) Induces CD36 Ectodomain Phosphorylation
CD36 is a multiligand scavenger receptor expressed in many cell types, including microvascular endothelial cells (MVEC). Binding of thrombospondin-1 (TSP-1) and related peptides to MVEC CD36 initiates a signaling pathway that inhibits angiogenesis and promotes apoptosis. Previous studies reported that CD36 could be phosphorylated on its extracellular domain, and that phosphorylation inhibited binding of TSP-1. The mechanisms of cellular CD36 ectodomain phosphorylation and whether it can be regulated in cells is not known. Using CD36 immunoprecipitation followed by western blot with specific antibodies to phospho-threonine we examined CD36 phosphorylation in a melanoma cell line stably transfected with human CD36 cDNA. Basal levels of phosphorylation were detectable, but low, and treatment of the cells with 100ng/ml PMA for 4hr induced a 6.7 fold increase (p < 0.01). Treatment of the cells with cycloheximide to inhibit mRNA translation or Brefeldin A to inhibit ER to Golgi transport blocked PMA-induced CD36 phosphorylation but had no effect on phosphorylation of other PKC substrates. These data suggest that phosphorylation occurs in an intracellular compartment as CD36 protein is synthesized and processed. The potential physiological importance of CD36 phosphorylation was shown in vivo by demonstrating that TSP-1-mediated CD36 intracellular signaling, detected as the amount of Src family kinase co-precipitated with CD36, was inhibited by PMA treatment to a degree consistent with the degree of CD36 phosphorylation. We also showed that a recombinant CD36 protein containing the consensus PKCα phosphorylation site at Thr92 and the TSP-1 binding domain which lies immediately downstream could be phosphorylated by PKCα in vitro. We estimated using a mass spectrometry based assay that up to 50% of the total CD36 could be phosphorylated by long term exposure to PKC. Binding of CD36 peptides to immobilized TSP-1 or TSP peptides was inhibited by Thr92 phosphorylation and the degree of inhibition correlated closely with the level of p-Thr92. These studies define a novel pathway for CD36 phosphorylation in vivo and suggest a mechanism by which CD36 affinity for TSP-related proteins can be down-regulated to blunt anti-angiogenic signaling.
- © 2011 by American Heart Association, Inc.