TISSUE TRANSGLUTAMINASE-DEPENDENT POSTTRANSLATIONAL MODIFICATION OF THE RETINOBLASTOMA GENE-PRODUCT IN PROMONOCYTIC CELLS UNDERGOING APOPTOSIS

The retinoblastoma gene product (pRB) plays an important role in contr olling both cell release from the G(1) phase and apoptosis, We show he re that in the early phases of apoptosis, pRB is posttranslationally m odified by a tissue transglutaminase (tTG)-catalyzed zed reaction, In fact, by employing a novel haptenized lysis synthetic substrate which allows the isolation of glutaminyl-tTG substrates in vivo, we identifi ed pRB as a potential tTG substrate in U937 cells undergoing apoptosis , In keeping with this finding, we showed that apoptosis of U937 cells is characterized by the rapid disappearance of the 105,000- to 110,00 0-molecular-weight pRB forms concomitantly with the appearance of a sm ear of immunoreactive products with a molecular weight of greater than 250,000, The shift in pRB molecular weight was reproduced by adding e xogenous purified tTG to extracts obtained from viable U937 cells and was prevented by dansylcadaverine, a potent enzyme inhibitor. The effe ct of the pRB posttranslational modification during apoptosis was inve stigated by determining the E2F-1 levels and by isolating and characte rizing pRB-null clones from U937 cells, Notably, the lack of pRB in th ese U937-derived clones renders these p53-null cells highly resistant to apoptosis induced by serum withdrawal, calphostin C, and ceramide, Taken together, these data suggest that tTG, acting on the PRE protein , might play an important role in the cell progression through the dea th program.