The Rap1 V12 mutant fails to override stromal inhibition.

(A) Enforced expression in K562 cells of neither Rap1 wild type (wt) nor Rap1 V12 could reverse stromal inhibition of GPA down-regulation. Expression of the indicated YFP-Rap1 fusions, a function of GFP fluorescence, was correlated with GPA expression, a function of PE/red fluorescence. K562 cells transiently transfected with expression vectors for the indicated YFP fusion proteins were induced with or without HESS-5 stromal cells for 48 hours prior to staining with PE-conjugated antibody to GPA. (B) Stromal contact blocked activation of both YFP-Rap1 wt and YFP-Rap1 V12. Similar results were obtained with multiple independently isolated stable transfectants (not shown). Stable transfectants of K562 expressing the indicated YFP fusion proteins were induced with or without stromal cells for 48 hours prior to analysis of Rap1 activation as described in “Materials and methods.” The positions of the YFP-Rap1 fusions, endogenous Rap1, and a background band (Bkrd) are indicated.