Lamin A/C Binding Protein LAP2α Is Required for Nuclear Anchorage of Retinoblastoma Protein

Abstract

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.

Stabilization of the Retinoblastoma Protein by A-Type Nuclear Lamins Is Required for INK4A-Mediated Cell Cycle Arrest

“…Previous studies have shown that lamin A/C and pRB interact in vitro, leading to speculation that the nuclear structural component regulates pRB activity (41,42,51). We have reported that immortalized mouse Lmna Ϫ/Ϫ fibroblasts have fivefold reduced pRB levels (27).…”

“…They are hypothesized to affect gene expression at both peripheral and perinucleolar sites due to interactions with transcription factors involved in differentiation, such as the retinoblastoma protein (pRB) (26,41,42,44,51,59,67). Previously, it has been shown that pRB is tethered to the nuclear substructure through the action of lamin A/C (66).…”

Aging of Hutchinson–Gilford progeria syndrome fibroblasts is characterised by hyperproliferation and increased apoptosis

“…This process could be mediated by growth regulators known to interact with lamin-containing structures such as pRb (Markiewicz et al, 2002;Ozaki et al, 1994). Alternatively, since there is a strong link between genome organisation and structures at the nuclear envelope (Bridger and Bickmore, 1998; Holaska et al, 2002) then a compromised lamina organisation may result in aberrant gene expression leading to deregulated growth.…”

Proteins that associate with lamins: Many faces, many functions

“…Since overexpression of emerin inhibited Lmo7, the Lmo7-emerin interaction is tightly regulated by feedback pathways. Finally, LAP2α-lamin A complexes in the nuclear interior bind the tumor supressor retinoblastoma (Rb) [93], which is a negative regulator of E2F-dependent transcription controlling the balance between proliferation and differentiation [94]. Accordingly LAP2 can associate with E2F/Rbresponsive promoters and impair expression of E2F-target genes upon cell cycle exit and differentiation [95].…”