CD36 clustering induces protein tyrosine and MAPK phosphorylation.

Monocyte surface antigen was ligated with primary murine anti-CD36 FA6-152 antibody and cross-linked with the addition of goat antimouse F(ab′)₂ fragments. Ligation and cross-linking were performed at 4°C, and cellular reactions were initiated by bringing the cells to 37°C in a 5% CO₂incubator. Western blot analysis was performed after lysis of the cells at the times indicated. (A) Western blot staining for tyrosine-phosphorylated proteins after simple antibody ligation of surface CD36 or clustering of CD36 by antibody cross-linking. Note the more intense pattern of tyrosine phosphorylation that follows cross-linking; the secondary F(ab′)₂ alone does not induce intracellular tyrosine phosphorylation. (B) Western blot staining for tyrosine-phosphorylated proteins after cross-linking of CD36 in the presence or absence of genistein (10 μg/mL). Note the time course of phosphotyrosine accumulation, which peaks 10 to 20 minutes after cross-linking. (C) Accumulation of dually phosphorylated forms of the ERK and p38 MAPKs after cross-linking of surface CD36. In both cases, phosphorylated forms can be appreciated 60 to 120 minutes after cross-linking. Pretreatment of monocytes with genistein abolishes the induction of ERK phosphorylation and inhibits p38 MAPK phosphorylation. (D) Effect of monocyte pretreatment with PD98059 (50 μmol/L) or SB203580 (30 μmol/L) on the induction of tyrosine phosphorylation following cross-linking of CD36. Cells were lysed after 10 minutes of incubation at 37°C, 5% CO₂. (E) Effect of monocyte pretreatment with PD98059 (50 μmol/L) or SB203580 (30 μmol/L) on CD36-dependent induction of ERK and p38 MAPK phosphorylation. Cells were lysed after a 20-minute incubation at 37°C, 5% CO₂. All studies are representative of results obtained on at least 3 separate occasions. Although only data with the FA6-152 clone are presented, similar results were obtained with the OKM5 antibody.