PRB AND P107 REGULATE E2F ACTIVITY DURING LENS FIBER CELL-DIFFERENTIATION

During growth arrest and differentiation, activity of the E2F family o f transcription factors is inhibited by interactions with pRb and the related proteins, p107 and p130. To determine which members of the E2F and pRb families may contribute to growth arrest as lens epithelial c ells differentiate into fiber cells, we examined the expression of ind ividual E2F species and characterized the E2F protein complexes formed in rat lens epithelia and fibers. RT/PCR detected all five known memb ers of the E2F family in lens epithelial cells, but only E2F-1, E2F-3, and E2F-5 in fiber cells. Proteins extracted from lens epithelia of n ewborn rats formed at least two specific complexes with an E2F consens us oligonucleotide. Proteins from lens fiber cells formed three specif ic complexes, one of which comigrated with an epithelial cell complex. Incubation of epithelial and fiber cell extracts with an antibody spe cific for p107 demonstrated that two fiber cell complexes and one epit helial cell complex contained p107. Although the remaining fiber cell complex did not react with antibodies to pRb or p130 in this assay, a strong reaction with pRb antibody was observed when the electromobilit y shifted complexes were subsequently immunoblotted (shift/Western ass ay). Immunocytochemistry confirmed that pRb protein is present in the nuclei of both epithelial cells and fiber cells. Immunoblotting of who le cell extracts with pRb antibody showed multiple, phosphorylated for ms of pRb in the epithelial cells, but predominantly hypophosphorylate d pRb in the fiber cells. None of the complexes formed with E2F were r ecognized exclusively by the p130 antibody, although the previously id entified p107 complexes reacted weakly. The absence of p130/E2F comple xes was correlated with the presence of multiple ubiquitinated forms o f p130, especially in the fiber cells. Thus, although p130/E2F complex es are implicated in the terminal differentiation of many cell types, in differentiating lens fiber cells pRb and p107 seem to be the primar y regulators of E2F activity.