Lamin A/C binding protein LAP2alpha is required for nuclear anchorage of retinoblastoma protein.
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) 2alpha, a binding partner of lamins A/C, bound strongly to pocket C and weakly to pocket B. When LAP2alpha 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 LAP2alpha, provided that pocket C was present in the GFP chimeras. On redistribution of endogenous lamin A/C and LAP2alpha into nuclear aggregates by overexpressing dominant negative lamin mutants in tissue culture cells, Rb was also sequestered into these aggregates. In primary skin fibroblasts, LAP2alpha is expressed in a growth-dependent manner. Anchorage of hypophosphorylated Rb in the nucleus was weakened significantly in the absence of LAP2alpha. Together, these data suggest that hypophosphorylated Rb is anchored in the nucleus by the interaction of pocket C with LAP2alpha-lamin A/C complexes.
Markiewicz
Ewa
E
Department of Biological Sciences, The University of Durham, Durham DH1 3LE, United Kingdom.
Dechat
Thomas
T
Foisner
Roland
R
Quinlan
Roy A
RA
Hutchison
Christopher J
CJ
D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D017353:Gene Deletion; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D034904:Lamin Type A / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D034904:Lamin Type A; D008164:Luminescent Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; C440308:lamin C; C093413:lamina-associated polypeptide 2; D049452:Green Fluorescent Proteins