Sep 22, 1993 - mediated gene transfer, suppresses the tumorigenicity of ... differential phosphorylation during the cell cycle (6-8). Dur- ing G1, Rb is ...
Proc. Natl. Acad. Sci. USA Vol. 91, pp. 418-422, January 1994
Cell Biology
The retinoblastoma gene product is a cell cycle-dependent, nuclear matrix-associated protein (cell regulation/tumor suppression/lamins/nuclear structure/protein interaction)
MICHAEL A. MANCINI*, BEI SHAN*, JEFFREY A. NICKERSONt, SHELDON PENMANt, AND WEN-HWA LEE* *Center for Molecular Medicine, Institute of Biotechnology, The University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245; and tThe Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139
Contributed by Sheldon Penman, September 22, 1993
in the nucleus is architecturally organized on the matrix and includes DNA replication (17-19), transcription (20), and RNA processing (21-23). Several cellular proteins have been shown to associate with hypophosphorylated Rb including RBP-1 and RBP-2 (24), the transcription factor E2F (25-27), c-myc and N-myc proteins (28), p46 (29), several cyclins, and other proteins (25). Previous reports have demonstrated that a portion of hypophosphorylated Rb resists low-salt/detergent extraction only during early G1 (30). We hypothesize that the interaction of regulatory factors with Rb has important physiological consequences and that this interaction may take place on the nuclear matrix. We report here that Rb associates with the nuclear matrix, in a cell cycle-specific manner. In addition, it will be important to identify nuclear matrix proteins that interact with Rb on the matrix. We have identified several cellular gene products that interact with Rb by directly screening expression libraries with purified Rb protein as a probe (25) or by using the yeast two-hybrid system (31). Molecular characterization reveals that three of these genes encode nuclear matrix-associated proteins. One such gene shows complete identity with lamin C (25). We also present further characterization of Rb-lamin A/C binding in vitro and colocalization of both proteins at the peripheral nuclear matrix.
ABSTRACT The retinoblastoma gene product (Rb) has been established as a tumor suppressor and cell cycle regulator, although its mechanism of action remains obscure. The observations that several Rb-binding viral oncoproteins al associate with the nuclear matrix suggest that these interactions may occur on this structure. To determine whether Rb itself is a component of the matrix, we extracted synchronized cultured ceUls to isolate matrix proteins while preserving nuclear architecture. Immunoblot and immunolabeling data show that a significant portion of hypophosphorylated Rb associates with the matrix only during early Gi. Mutant Rb in tumor cells did not associate with the matrix, whereas Rb-reconstituted cells contained abundant matrix-bound Rb. Rb is distributed widely throughout the matrix, particularly concentrated at the nuclear periphery and in nucleolar remnants. Core filaments of the matrix contained no detectable Rb. Our screening of expression libraries for potential Rb-associated proteins has identified several that are part of the matrix. Specifically, the peripheral matrix proteins lamin A and C bound Rb in vitro. We therefore suggest that Rb interactions with the nuclear matrix may be important for its ability to regulate cell cycle progression.
The retinoblastoma gene product (Rb) is a 110-kDa, tumor suppressor protein that is absent or mutated in many human tumors (1). Expression of wild-type Rb, via retrovirusmediated gene transfer, suppresses the tumorigenicity of several neoplastic cell lines that contain Rb mutations, thereby establishing its tumor suppressive function in vivo (2-4). Although suppression of tumor formation by Rb is poorly understood, it may involve regulation of the cell cycle. In this vein, microinjection of Rb into synchronized cells inhibits S-phase progression (5). Rb, a DNA-binding nuclear phosphoprotein, undergoes differential phosphorylation during the cell cycle (6-8). During G1, Rb is predominantly hypophosphorylated and becomes increasingly phosphorylated through the rest of the cell cycle. Marked dephosphorylation occurs during late mitosis (9). Phosphorylation may regulate Rb function by altering its ability to bind other regulatory proteins. Only hypophosphorylated Rb binds to several viral oncoproteins including simian virus 40 large tumor antigen (TAg) (10), adenovirus ElA protein (11), and the human papilloma viral protein E7 (12). Interestingly, these Rb-binding viral oncoproteins are all associated with the nuclear matrix (13-15), suggesting that some of the important molecular interactions with Rb might occur on the matrix. The nuclear matrix is the nonchromatin structure of the nucleus that survives the experimental removal of chromatin. Structurally, the nuclear matrix consists of the nuclear lamina and a web of internal fibers connected to the lamina (16). Nucleic acid metabolism
MATERIALS AND METHODS Nuclear Matrix Preparation. The procedure of He et al. (16) was followed as described except that antipain, leupeptin, and aprotinin (each at 1 ,ug/ml) and sodium fluoride (5 mM) were added in all extraction solutions. Cells were arrested in early G1 with a 48-hr exposure to lovastatin followed by a 2-hr release with mevalonic acid (32). A double block of hydroxyurea was used to arrest cells at the G1/S boundary and was followed by a 4-hr release to obtain cells in mid-S phase (5). Cells were fixed in 4% formaldehyde in CSK buffer (16) for 30 min at ambient temperature before immunolabeling. Immunocytochemistry and Immunoblotting. Monoclonal antibodies (mAbs) to Rb (11D7, 7F12, 4H6, and lE5; Canji, San Diego) were combined (each at 10 ,ug/ml) for immunocytochemistry. For double labeling, whole mouse antiserum raised against FLAG-lamin C (see below) was used (1:2000) simultaneously with rabbit antibody 0.47 against Rb. Fluorescence-conjugated secondary antibodies were preabsorbed with transblotted core filament nuclear matrix proteins and used at 1:100 dilution. Anti-mouse or anti-rabbit goldconjugated secondary antibodies were used at 1:10 dilution. Nuclear matrix preparations were embedded in diethylene glycol distearate, sectioned, and dewaxed as described (33). Supernatants and extracted cell pellets were dissolved in SDS
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: Rb, retinoblastoma gene product; TAg, simian virus 40 large tumor antigen; mAb, monoclonal antibody; MBP, maltosebinding protein; GST, glutathione S-transferase.
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sample buffer, boiled for 5 min, and then separated by SDS/PAGE. Immunoblotting was performed as described
(6).
Confocal Microscopy and Volume Rendering. Confocal images were obtained with a Zeiss LSM 310 equipped with argon and HeNe lasers. Digitized images of confocal optical sections were volume rendered with VoxelView software (Vital Images, Fairfield, IA) and recorded directly onto 35-mm color slide film. cDNA Library Screening for Rb-Associated Proteins. An extensive description of direct Rb library screening is reported elsewhere (25). Fusion Protein Construction and in Vitro Binding Assays. The cDNA insert from clone RbAp6 (lamin C) was subcloned in frame into a fusion plasmid expressing the maltose-binding protein (MBP; New England Biolabs), and the expressed fusion protein was purified by amylose affinity chromatography. The same cDNA insert was also used in an inverted orientation to serve as a control for binding assays. Bacterially expressed, purified pllORb, or MOLT-4 cell lysate, was mixed with MBP-lamin C beads and incubated at 4°C for 60 min. After the beads were washed, bound proteins were analyzed by immunoblot with mAb 11D7. The cDNA insert was also subcloned into the pFLAG fusion plasmid (IBI); pFLAG-BAP (bacterial alkaline phosphatase fused to pFLAG) was used as a control plasmid. The bacterial lysates were incubated with a p56Rb crosslinked gel matrix, and the bound proteins were analyzed by immunoblot using antiFLAG mAb (IBI). Glutathione S-transferase (GST) fusion proteins Rbs-(377-928), Rbs3-(377-611), and Rbs4-(611-928) (B.S. and W.-H.L., unpublished data), or GST alone, were incubated with 35S-labeled, in vitro transcribed and translated full-length lamin A; bound proteins were examined by SDS/ PAGE and autoradiography.
RESULTS Rb Is in the Nuclear Matrix During Early Gi. It has been shown previously that hypophosphorylated Rb resists lowsalt/detergent extraction only during early G1 (30). To investigate the possibility that Rb might bind to the nuclear matrix during this part of the cell cycle, we synchronized CV-1 cells to G, or S phase and isolated the nuclear matrix by the sequential extraction method of He et al. (16). Supernatants containing released proteins from each extraction step, and the final insoluble nuclear matrix pellet, were dissolved in SDS sample buffer, separated by SDS/PAGE, and examined by immunoblotting. During early G1, hypophosphorylated Rb was found in two fractions (Fig. 1). Most hypophosphorylated Rb was released with soluble proteins, whereas the remainder associates with the nuclear matrix core filaments (Fig. 1). During S phase, when Rb is predominantly hyperphosphorylated, all Rb is released in the first Triton X-100 CSK extraction, which removes the soluble cell proteins (data not shown). In cells
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FIG. 2. Hypophosphorylated Rb is found in the core filament nuclear matrix during early G, in Rb reconstituted WERI retinoblastoma cells. As with the CV-1 cells, hypophosphorylated Rb is released in the Triton X-100 fraction (lane 1) but not in the 0.25 M (NH4)2SO4 (lane 2) or 2 M NaCl (lane 3) fractions. A strongly labeled band of hypophosphorylated Rb is found in the core filament nuclear matrix fraction. Equivalent cell numbers were loaded into lanes 1-3; lane 4 was loaded with 2 times the number of cells.
containing a mutated RB gene (Saos2, Du145, H209), all Rb was removed during the CSK extraction, even in early G1 (data not shown), consistent with low-salt/detergent extraction experiments (30, 34). Rb Is in the Nuclear Matrix in RB-Reconstituted Retinoblastoma Cells. Expression of wild-type Rb in tumor cells containing inactivated Rb results in suppressed tumorigenicity in vivo (2-4). To determine whether an association exists between this suppression and the presence of Rb on the nuclear matrix, Rb-reconstituted and parental WERI retinoblastoma cells were extracted as described above. Fig. 2 shows a Western blot of nuclear matrix proteins from Glarrested Rb+ WERI cells. In Gl-arrested Rb+ WERI cells, a significant portion of hypophosphorylated Rb was contained in the core filament nuclear matrix fraction. Spatial Distribution of Rb in the Nuclear Matrix. Extracting CV-1 cells to reveal core filament nuclear matrix resulted in a markedly different pattern of Rb immunolocalization compared to whole cells. The subnuclear distribution of Rb associated with the nuclear matrix, seen by volume rendering a series ofconfocal images, is presented in Fig. 3. The nucleolar remnant is labeled and a delicate speckled pattern is observed in the nuclear interior. The nuclear lamina was also decorated by Rb mAb. Our direct visualization of Rb at the nuclear
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FIG. 1. Hypophosphorylated Rb is found in the core filament nuclear matrix during early Gi. Lovastatin-arrested CV-1 cells were processed to obtain nuclear matrix. Supernates or pellets from equivalent numbers of cells were solubilized in SDS sample buffer, separated by PAGE, and immunoblotted with Rb mAb 11D7. Whole cell extracts in early G1 contain predominantly the faster-migrating pRb (lane 1). A sizable portion of Rb is released by Triton X-100 (lane 2). No Rb is released by 0.25 M (NH4)2SO4 (lane 3) or by 2 M NaCl (lane 4). A significant portion of hypophosphorylated Rb remains associated with the core filament nuclear matrix fraction (lane 5).
FIG. 3. Three-dimensional view of Rb in the nuclear matrix. A series of confocal images were volume rendered to illustrate the localization of Rb in the core filament-nuclear matrix fraction of CV-1 cells. A pseudocolor table represents fluorescence intensity as low (blue) to high (red). Note the amount of Rb at the nuclear periphery and in the nucleolar remnant. Contrast, opacity, and background color were adjusted to accentuate the delicate pattern of Rb staining. (Bar = 4 ,um.)
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periphery supports work by Templeton (35), shown by biochemical means, that Rb associates with a nuclear envelope fraction. As expected from the biochemical data, no Rb labeling was observed in core filament nuclear matrix preparations from cells during S phase (data not shown). The nuclear matrix cannot be imaged by conventional embedded-section electron microscopy; however, its ultrastructure is clearly imaged with resinless sections. Immunogold labeling of resinless sections has previously been used to localize specific nuclear matrix components (36). Fig. 4 is a panel of resinless-section photomicrographs of GI-arrested CV-1 cells that were labeled before embedment with mAbs to Rb. Fig. 4A is a low-magnification view that shows the ultrastructure after matrix extraction. In a high-magnification view, numerous clusters of 10-nm gold beads decorate the nuclear lamina (Fig. 4B). Fig. 4C shows that fibrogranular masses and the nucleolar remnant are also labeled (double arrowheads). The 10- to 12-nm core filaments that supported these granular structures were themselves unlabeled. Cells in mid-S phase did not show Rb-specific immunogold labeling in the nuclear matrix (data not shown). These data corroborate the confocal microscopy results described above. Cytoplasmic structures were not labeled, indicating a low background staining. Collectively, our
biochemical and immunocytochemical data demonstrated the resistance of a significant portion of hypophosphorylated Rb to extraction with detergent, nuclease, and high salt, indicating that it is firmly bound to the nuclear matrix during G1. Rb Binds to Lamin A/C in Vitro. Direct screening of human cDNA libraries with functional, N-terminally truncated p56Rb has identified nine Rb-associated proteins (25). Significantly, antisera against fusion proteins derived from this group indicate that all localize to the nucleus (M.A.M. and W.-H.L., unpublished data). One such protein, AP 6, is identical to lamin C, an integral nuclear matrix protein (25). Localization of Rb at the nuclear periphery in core filament matrix preparations is consistent with a Rb-lamin C interaction in vivo. To further establish this association, we performed in vitro binding experiments between lamin fusion proteins and Rb. Both purified bacterially expressed, full-length Rb (Fig. 5A) and hypophosphorylated Rb from an unsynchronized MOLT-4 cell lysate (Fig. SB) bound to an immobilized MBP-lamin C column. Furthermore, FLAG-lamin C was shown to bind a p56Rb-crosslinked gel matrix (Fig. SB). As lamins A and C are alternatively processed products of the same gene, differing only in the length of the C terminus,
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FiG. 5. Lamin A/C binds Rb in vitro. (A) Bacterially expressed and purified pllORb (lanes 1 and 2), or MOLT-4 cell lysate (lanes 3 and 4), were mixed with a MBP-lamin C fusion protein bound to amylose beads (lanes 1 and 3) or control MBP-Cnimal (inverted lamin C) beads (lanes 2 and 4). The protein complex retained on the beads was assayed by immunoblot analysis using Rb mAb 11D7. Only hypophosphorylated Rb from the MOLT-4 lysates bound the column. (B) Bacterial lysate containing pFLAG-lamin C (lane 1) or pFLAG-BAP (lane 2) fusion protein was incubated with a p56 Rb-crosslinked gel matrix. Bound protein was assayed by immunoblot using anti-FLAG mAb (IBI). (C and D) In vitro translated, 35S-labeled, full-length lamin A was mixed with bacterially expressed and purified GST (Gex) fused to an N-terminal truncated Rb (lane 1; Rbs), a fragment of Rb containing the first TAg-binding domain (lane 2; Rbs3), a portion of Rb containing the second TAg-binding domain plus C terminus (lane 3; Rbs4), or Gex alone (lane 4). Bound proteins were then analyzed by SDS/PAGE and autoradiography.
35S-labeled in vitro translated lamin A was used to bind GST-Rb deletion mutants (Fig. 5D). Fig. SC shows that pGEXRbs-(377-928) (lane 1) and pGEXRbs4-(611-928) (lane 3) bound to full-length lamin A. pGEXRbs3-(377-611) (lane 2) containing the first TAg-binding domain and the GST control (lane 4) did not bind, indicating that Rb-lamin A binding was not dependent on the integrity of both TAgbinding domains. Our in vitro Rb-lamin A/C-binding study likely identifies a component of the nuclear lamina shells that bound purified Rb (35). Spatial Organization of Rb and Lamin A/C in the Nucleus. Fig. 6 shows an unextracted, Gl-arrested CV-1 cell immunolabeled with anti-Rb and anti-lanin A/C antibodies. The pattern seen in this tangential optical section reiterates the nonhomogeneous intranuclear pattern of Rb and, furthermore, shows that lamin A/C is present both at the nuclear periphery
FIG. 6. Rb and lamin A/C colocalize at the nuclear periphery and internal foci. In this somewhat tangential optical section of a whole CV-1 cell in G1, Rb labeling is green and lamin A/C labeling is red. Spatial overlap of these two proteins is represented as yellow. Note the significant colocalization of Rb and lamin A/C at the nuclear periphery and internal foci. (Bar = 5 pm.)
and in Gl-specific structures within the nuclear interior (37). Colocalization (yellow) is observed at the periphery, yet it is clear that most of the Rb does not overlap with lamin A/C. As a significant fraction of Rb is bound to the nuclear matrix, additional nuclear matrix proteins capable of binding Rb are likely to exist within the internal nuclear matrix.
DISCUSSION The nuclear matrix is the nonchromatin substructure of the nucleus, which is experimentally seen after removal of the nucleoplasm and chromatin by detergent extraction, nuclease digestion, and salt extraction. Proteins that remain with the nuclear structure after these treatments are defined as being nuclear matrix associated. Using this criterion, we have shown in this report that a significant fraction of Rb is nuclear matrix associated during the G1 stage of the cell cycle. At this stage of the cycle, Rb exists predominantly in a hypophosphorylated form, the isoform thought to be biologically active in suppressing cell growth, and it is this form that is matrix associated. No matrix association was observed with several naturally occurring TAg-binding mutants of Rb, previously shown to be entirely soluble after low-salt/detergent extractions (30, 34). Numerous reports have indicated that hypophosphorylated Rb specifically interacts with several classes of proteins including viral oncoproteins (which are known to be nuclear matrix associated), regulatory factors (E2F, ATF-2), and enzymes (cdc2 kinase, protein phosphatase 1). However, the nuclear site(s) of these interactions remains unknown. Inasmuch as most nuclear functions, including replication and RNA processing, are associated with the nuclear matrix, it is intriguing to speculate that Rb may exert its influence on the cell cycle within a nuclear matrix environment. Furthermore, Rb-mediated suppression of tumorigenicity can be correlated with the presence of nuclear matrix-associated Rb in reconstituted Rb cells. The mechanistic relationship between tumor suppression and Rb association with the nuclear matrix, however, will require additional study. The outer shell of the nuclear matrix is the nuclear lamina, which is made, in most cells, predominantly from three proteins: lamins A, B, and C. The nuclear lamina is attached to both intermediate filaments of the cytoskeleton and to the internal nuclear matrix, integrating them into a single cell-wide structure, the nuclear matrix-intermediate filament scaffold (33). Lamin C has been identified as an Rb-associated protein (25) based on its binding to pS6Rb. We have presented here
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additional in vitro binding and colocalization data indicating that lamin A/C is a nuclear matrix protein that binds hypophosphorylated Rb. Of particular interest is a recent report (37) that identifies lamin A/C within the interior ofthe nucleus only during early G1 (see Fig. 6). It is possible that matrixbound Rb could also interact with internal lamins before either completely integrates into the lamina. The functional significance of potential Rb-lamin A/C interactions in vivo remains obscure. However, as dephosphorylation of Rb begins during metaphase, Rb-lamin A/C interactions may be important for events from midmitosis through early G1. While some Rb colocalized with the nuclear lamina, additional labeling was present on granular masses that are enmeshed in the 10-nm core filaments of the nuclear matrix (16). It is likely that these structures have a complex protein composition. We infer that some of these nuclear matrix proteins will have Rb-binding activity and that they may serve as "docking sites" for Rb in the fibrogranular core filament structure. An 84-kDa protein that binds the N terminus of hypophosphorylated Rb in several different binding assays has been characterized as a candidate internal nuclear matrixdocking protein for Rb (T. Durfee, M.A.M., and W.-H.L., unpublished data). Antibodies against Rb also labeled regions of the nuclear matrix presumed to be nucleolar remnants. This is consistent with the identification of UBF, a ribosomal gene transcription factor, as a Rb-binding protein (25). Based on our observations, we propose that the nuclear pool of Rb is in dynamic, cell cycle-dependent equilibrium between a soluble (nucleoplasmic) and insoluble (nuclear matrix) state, dependent on phosphorylation. The Rb that is released from the nucleus during matrix isolation could represent a transitional, less tightly bound form that is minimally phosphorylated. Perhaps only subtle phosphorylation is necessary to dissociate Rb from the nuclear matrix, changes not readily identifiable by PAGE. We have previously suggested that Rb may function in a cell cycle-dependent fashion by sequestering (inhibiting) growth-promoting factors (38). As a result of our current study, we further suggest it is the nuclear matrix-associated Rb that architecturally sequesters growth regulatory proteins during G1, probably in association with Rb-binding nuclear matrix proteins. Concomitantly, Rb could also serve to concentrate some transcription factors, thus facilitating expression of certain genes. Recently, it was shown that transcription factors required for osteocalcin activation are sequestered in the nuclear matrix precisely when the gene is turned on (39). Indeed, during G1, Rb may serve as a biophysical interface between well-studied soluble signals (such as transcription factors) that influence the cell cycle and proliferation and the more poorly understood signals from tissue and cellular architecture (40, 41). Cell cycledependent phosphorylation and subsequent disassembly of the matrix-associated Rb complex might release factors causing cell cycle progression. The Rb. mutations that frequently accompany malignancy would prevent matrix-associated complex formation. The binding of matrix-associated oncoproteins to Rb may interfere with signaling without necessarily disrupting its matrix association. In either case, the consequence would be loss of cell cycle control. We thank Gabriela Krockmalnic for technical expertise and Drs. Z. Dave Sharp, D. Goodrich, R. E. Hollingsworth, and D. Riley for their critical reading of the manuscript. M.A.M. is a recipient of a postdoctoral fellowship from the National Cancer Institute (lF32CA60435). This work was supported by National Cancer Institute Grant 5ROlCA58318-02 (W.-H.L.) and National Institutes of
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