Markiewicz, E. and Dechat, T. and Foisner, R. and Quinlan, R. A. and Hutchison, C. J. (2002) 'Lamin A/C binding protein LAP2 alpha is required for nuclear anchorage of retinoblastoma protein.', Molecular biology of the cell., 13 (12). pp. 4401-4413.
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.
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|Publisher Web site:||http://dx.doi.org/10.1091/mbc.E02-07-0450|
|Publisher statement:||© 2002 by The American Society for Cell Biology|
|Date accepted:||No date available|
|Date deposited:||01 October 2008|
|Date of first online publication:||December 2002|
|Date first made open access:||No date available|
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