Enhanced expression of GTP-binding proteins in differentiated U937 monocytic cells: Possible involvement of tyrosine kinase and protein kinase C

Monocytic U937 cells were differentiated into mature macrophages in the presence of 100 nM phorbol 12-myristate 13-acetate (PMA) for 24 h at 37°C. We investigated the alterations in the expression of GTP-binding proteins that take place during differentiation of these cells. A 40 KDa α-subunit of the inhibitory G-protein was identified by specific antibodies to G_iα-1/2 and G_iα-3 on Western blots and also by ADP-ribosylation catalyzed by pertussis toxin. The expression of the 40 KDa G_iα subunit was increased 3.4 fold in differentiated cells. The expression of a 43 kDa G_sα subunit identified by Western blotting using specific antibody to G_sα and by ADP-ribosylation in the presence of cholera toxin was increased approximately 2 fold in differentiated cells. A faintly recognizable 46 KDa G_sα subunit was also increased but to a lesser extent (1.3 fold). Small molecular weight GTP-binding proteins identified by [³⁵S]GTPγS binding on nitrocellulose blots were also increased significantly. The PMA-induced expression of G_iα-1/2 and G_sα subunits was blocked to control level by both genistein and staurosporine, inhibitors of protein tyrosine kinase and protein kinase C, respectively. However, staurosporine was unable to block the PMA-induced expression of G_iα-3; this was blocked only by genistein. These data suggest a role for tyrosine kinase and protein kinase C in the expression of G-proteins during differentiation of U937 cells.