A-type lamins regulate retinoblastoma protein function by promoting subnuclear localization and preventing proteasomal degradation - PubMed (original) (raw)

A-type lamins regulate retinoblastoma protein function by promoting subnuclear localization and preventing proteasomal degradation

Brett R Johnson et al. Proc Natl Acad Sci U S A. 2004.

Abstract

The retinoblastoma protein (pRB) is a critical regulator of cell proliferation and differentiation and an important tumor suppressor. In the G(1) phase of the cell cycle, pRB localizes to perinucleolar sites associated with lamin A/C intranuclear foci. Here, we examine pRB function in cells lacking lamin A/C, finding that pRB levels are dramatically decreased and that the remaining pRB is mislocalized. We demonstrate that A-type lamins protect pRB from proteasomal degradation. Both pRB levels and localization are restored upon reintroduction of lamin A. Lmna(-/-) cells resemble Rb(-/-) cells, exhibiting altered cell-cycle properties and reduced capacity to undergo cell-cycle arrest in response to DNA damage. These findings establish a functional link between a core nuclear structural component and an important cell-cycle regulator. They further raise the possibility that altered pRB function may be a contributing factor in dystrophic syndromes arising from LMNA mutation.

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Figures

Fig. 1.

Fig. 1.

Levels of pRB family members in _Lmna_-/- cells. In all blots, -/- indicates the _Lmna_-/- genotype and +/+ indicates the litter-matched Lmna+/+ genotype. Other genotypes are as described. (A) pRB levels in mouse embryo fibroblasts. Antibodies to Rho GDP were used as loading control. (B) pRB levels in immortalized fibroblasts. Antibodies to Rho GDP were used as loading control. (C) Equilibrated extracts from immortalized fibroblasts of indicated genotype were immunoprecipitated with antibodies to p107. Immunoprecipitated proteins were analyzed by Western blots with p107-specific antibodies. The lower band is the heavy chain of the p107 antibodies used for immunoprecipitation. (D) p130 levels in immortalized fibroblasts. Flanking bands are nonspecific. (E) p27KIP1 levels in immortalized fibroblasts. (F) Emerin levels in immortalized fibroblasts.

Fig. 4.

Fig. 4.

A-type lamins regulate pRB stability. (A) Real-time RT-PCR analysis of the ratios of Rb and p107 mRNA in _Lmna_-/- to Lmna+/+ fibroblasts. Rb and p107 mRNA levels were normalized to HPRT RNA levels in each cell type under each condition. A variety of cDNA concentrations were used to ensure consistency of read-outs. See Table 2 for quantitation. (B) The proteasome inhibitor MG132 (25 μM for 6 h) restores pRB stability in immortalized _Lmna_-/- fibroblasts. Actin levels were determined for loading control. For actin blot, extracts were diluted 1/10 relative to blot probed for pRB. -/-, _Lmna_-/- fibroblasts. +/+, Lmna+/+ fibroblasts. (C) GFP-lamin A was introduced by transient transfection into _Lmna_-/- immortalized fibroblasts, and protein levels of pRB were determined by immunoprecipitation and Western analysis. (D) GFP-Lamin A was introduced by transient transfection into _Lmna_-/- immortalized fibroblasts, and immunofluorescence was performed to identify transfected cells and determine pRB levels and localization. In the images, one transfected cell and two untransfected cells are displayed for comparison. Images were collected by CCD microscopy

Fig. 2.

Fig. 2.

Lamin A/C is required for pRB and p107 stability in human osteosarcoma cells. U2OS cells were transfected with siRNA oligos to reduce lamin A/C expression. pRB, p107, or p130 expression was determined by indirect immunofluorescence by using an antibody specific for lamin A/C and an antibody specific for each of the pRB family members. (A) Cells were scored for normal or reduced lamin A/C expression and then for pRB, p107, or p130. Gray bars represent the percentage of cells with normal pRB, p107, or p130 levels within the population of cells with normal lamin A/C expression. Black bars represent the percentage of cells with normal pRB, p107, or p130 levels within the population of cells with reduced lamin A/C expression. Each immunofluorescence experiment was performed in triplicate. (B) Images depict representative cells from the siRNA experiment. Lamin A/C signals are shown in red and pRB family members in green. Images were collected by CCD microscopy. Each field includes both lamin A-down-regulated and unaffected cells.

Fig. 3.

Fig. 3.

pRB and p107 localization in _Lmna_-/- fibroblasts. CCD imaging was used to generate images. -/-, Immortalized _Lmna_-/- fibroblasts. +/+, Litter-matched immortalized Lmna+/+ fibroblasts. Cells were arrested by contact inhibition before indirect immunofluorescence. (A) Exposure times were equilibrated with each fluorophore for images depicting pRB staining in Lmna+/+ and _Lmna_-/- cells and for images depicting p107 staining in Lmna+/+ and _Lmna_-/- cells. All further processing of images was performed identically. (B) Localization of E2F-1 and pRB in Lmna+/+ and _Lmna_-/- fibroblasts. Merging of red and green signals appears yellow.

Fig. 5.

Fig. 5.

_Lmna_-/- fibroblasts have phenotypes resembling _Rb_-/- fibroblasts. (A) Fluorescence-activated cell sorting analysis was performed on proliferating Lmna+/+ and _Lmna_-/- immortalized fibroblasts. See Table 1 for quantification. (B) _Lmna_-/- fibroblasts are smaller than their litter-matched counterparts. Images depict cells maintained at confluence for 2 days. Forward scatter histograms were conducted to determine mean cell size. (C) _Lmna_-/- fibroblasts enter S phase prematurely after arrest in G1. To achieve cell-cycle arrest, cultures were maintained at confluence for a period of 48 h. At this time, >90% of cells have 2N DNA content. (D) _Lmna_-/- fibroblasts have a defective cell-cycle response to DNA damage. Representative data are shown from one of three experiments conducted. Proliferating cells of each genotype were exposed to γ irradiation of the amount indicated. Eight hours after exposure, cells were pulsed with BrdUrd for 15 min, and immunofluorescence was performed to determine the percentage of cells undergoing DNA replication.

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