Molecular Biology of the Cell Vol. 13, 4401–4413, December 2002
The phosphorylation-dependent anchorage of retinoblastoma protein Rb in the nucleus is essential
for its function. We show that its pocket C domain is both necessary and sufficient for nuclear
anchorage by transiently expressing green fluorescent protein (GFP) chimeras of Rb fragments in
tissue culture cells and by extracting the cells with hypotonic solutions. Solid phase binding assays
using glutathione S-transferase-fusion of Rb pockets A, B, and C revealed a direct association of
lamin C exclusively to pocket C. Lamina-associated polypeptide (LAP) 2 , a binding partner of
lamins A/C, bound strongly to pocket C and weakly to pocket B. When LAP2 was immunoprecipitated
from soluble nuclear fractions, lamins A/C and hypophosphorylated Rb were
coprecipitated efficiently. Similarly, immunoprecipitation of expressed GFP-Rb fragments by
using anti-GFP antibodies coprecipitated LAP2 , provided that pocket C was present in the GFP
chimeras. On redistribution of endogenous lamin A/C and LAP2 into nuclear aggregates by
overexpressing dominant negative lamin mutants in tissue culture cells, Rb was also sequestered
into these aggregates. In primary skin fibroblasts, LAP2 is expressed in a growth-dependent
manner. Anchorage of hypophosphorylated Rb in the nucleus was weakened significantly in the
absence of LAP2 . Together, these data suggest that hypophosphorylated Rb is anchored in the
nucleus by the interaction of pocket C with LAP2 –lamin A/C complexes.
