Effect of pharmacologic manipulation of protein kinase activity on CD33 phosphorylation.

(A) U937 cells were labeled with [³²P]-orthophosphate for 1 hour in the absence or presence of 2 μM BIM or 2 μM staurosporine for 60 minutes. Subsequently, PMA was added to 18 nM, or dibutryl cAMP to 1 mM, as indicated. Following 12 minutes at 37°C, the cells were chilled and lysed, and CD33 isolated and analyzed. PMA treatment leads to an increase of CD33 phosphorylation, which is inhibited by pretreatment with BIM or even further reduced by pretreatment with staurosporine. Dibutryl cAMP does not influence the level of CD33 phosphorylation. (B) Phosphorylation of CD33 in transiently transfected COS cells is enhanced by PMA, but not by dibutryl cAMP. COS cells, transfected with pCD33 48 hours earlier, were labeled with [³²P]-orthophosphate for 60 minutes followed by 12 minutes treatment with PMA (18 nM) or dibutryl cAMP (1 mM).