Cell, Vol. 73, 499-511, May 7, 1993, Copyright 0 1993 by Cell Press Physical Interaction of the Retinoblastoma Protein with Human D Cyclins Steven F. Dowdy,* Philip W. Hinds,’ Kenway Louie,’ into Rb- tumor cells by microinjection, viral infection, or Steven I. Reed,t Andrew Arnold,* transfection can lead to the growth arrest of these recipient and Robert A. Weinberg” cells (Huang et al., 1988; Goodrich et al., 1991; Templeton *The Whitehead Institute for Biomedical Research et al., 1991; Hinds et al., 1992). and Department of Biology Oncoproteins specified by the SV40, adenovirus, and Massachusetts Institute of Technology papilloma DNA tumor viruses have been shown to associ- Cambridge, Massachusetts 02142 ate with pRb in virus-transformed cells (Whyte et al., 1988; tThe Scripps Research Institute DeCaprio et al., 1988; Dyson et al., 1989). Oncoprotein Department of Molecular Biology binding of pRb is presumed to lead to its sequestration 10666 North Torrey Pines Road and functional inactivation. Conserved region II mutants La Jolla, California 92037 of adenovirus ElA, SV40 large T antigen, human papil- *Endocrine Unit loma E7 viral oncoproteins that have lost their ability to and Massachusetts General Hospital Cancer Center bind pflb, and other pRb-related proteins exhibit signifi- Massachusetts General Hospital cantly reduced transforming potential (Moran et al., 1986; and Harvard Medical School Lillie et al., 1987; Cherington et al., 1988; DeCaprio et al., Boston, Massachusetts 02114 1988; Moran, 1988; Smith and Ziff, 1988; Whyte et al., 1989). This suggests that binding of pRb and related pro- teins by these oncoproteins is critical to their transforming abilities. The detailed study of pRb structure and function has The retinoblastoma protein (pRb) functions as a regu- revealed two sequence segments that together form the lator of cell proliferation and in turn is regulated by domain responsible for its ability to bind the various viral cyclin-dependent kinases. Cyclins Dl and D3 can form oncoproteins. This domain, termed the pRb “pocket,” has complexes with pRb that resemble those formed by been defined experimentally as the minimal region of pRb several viral oncoproteins and are disrupted by the required for viral oncoprotein binding (Hu et al., 1990; Hu- adenovirus El A oncoprotein and derived peptides. ang et al., 1990; Kaelin et al., 1990). pRb also uses this These cyclins contain a sequence motif similar to the pocket to bind a series of cellular proteins, such as the pRb-binding conserved region II motif of the viral on- E2F transcription factor, a group of proteins whose genes coproteins. Alteration of this motif in cyclin Dl pre- have been isolated by affinity cloning, an unidentified nu- vents formation of cyclin Dl -pRb complexes while en- clear structure, and the MyoD and myogenin myogenic hancing the biological activity of cyclin Dl assayed in factors (Chellappan et al., 1991; Bandaraand LaThangue, vivo. We conclude that cyclins Dl and D3 interact with 1991; Chittenden et al., 1991; Kaelin et al., 1991; Huang pRb in a fashion distinct from cyclins A and E, which et al., 1991; DefeoJoneset al., 1991; Mittnacht and Wein- can induce pRb hyperphosphorylation, and that cyclin berg, 1991; Gu et al., 1993). These observations suggest Dl activity may be regulated by its association with that the viral oncoproteins may be structural mimics of pRb. these cellular proteins. This mimicry may enable the El A, T antigen, and E7oncoproteins to occupy the pRb pocket, Introduction thereby preempting interaction of pRb with its normal cel- lular partners. The product of the retinoblastoma susceptibility gene pRb has been shown to be phosphorylated at serine (pflb) appears to be an important regulator of cell prolifera- and threonine residues present in sequence motifs remi- tion. This gene product, a 110 kd nuclear phosphoprotein, niscent of those modified by the cyclin-dependent kinases is expressed in a wide variety of cell types (Lee et al., (cdks) (Lees et al., 1991; Lin et al., 1991). These kinases, 1967; Friend et al., 1987). However, mutation of the Rb acting with associated cyclins, form the machinery regulat- gene is associated with only a narrow subset of tumors, ing cell cycle progression (see Hunt, 1989; Nurse, 1990; including retinoblastomas, osteosarcomas, and small cell Mailer, 1991; Hunter and Pines, 1991; Reed, 1991; Mur- and some nonsmall cell lung, bladder, breast, and cervical ray, 1992). The phosphorylation of pRb is modulated dur- carcinomas (for a review see Weinberg, 1992). Several ing the cell cycle, in that pRb is present in a hypophosphor- lines of evidence have converged on the model that the ylated state in the Go and early G, phases of the cell cycle Rb gene product acts in normal cells to constrain growth and becomes hyperphosphorylated in late G1. This hyper- and that its loss permits the unconstrained growth charac- phosphorylated state is maintained through S, GP, and teristic of cancer cells. Thus, a number of mutant Rb alleles most of M phase (Lee et al., 1987; Buchkovich et al., 1989; have been isolated from these tumors, and all appear to DeCaprio et al., 1989, 1992; Chen et al., 1989; Mihara et have suffered loss-of-function mutations (Friend et al., al., 1989). Together, these facts suggest that pRb is a 1987; Harbour et al., 1988; Shew et al., 1989; Varley et direct substrate of cdks. al., 1989; Horowitz et al., 1989, 1990; Furukawa et al., A body of experimental evidence suggests that the hypo- 1991). Furthermore, theintroductionofwild-typeRballeles phosphorylated form of pRb is active in growth restraint, while the hyperphosphorylated form is inactive. This belief The in vitro mixing experiments were repeated using is supported primarily by the observation that the El A, equivalent amounts of either the wild-type pRb or the mu- T antigen, and E7 oncoproteins specifically bind to the tant A22 pRb (Figure 1C). Both cyclins Dl and D3 showed hypophosphorylated form of pRb, ignoring the hyperphos- clear binding to wild-type pRb (Figure 1C, lanes 1 and 3). phorylated forms (Ludlow et al., 1989; Templeton et al., However, neither bound to the A22 mutant protein (Figure 1991; lmai et al., 1991; Mittnacht et al., 1991). By seques- lC, lanes 2 and 4). These results suggest that D cyclins tering hypophosphorylated pRb, these viral proteins are bind to pRb via its pocket domain. thought to reduce or eliminate the pool of pRb molecules that are active in growth regulation. Moreover, the ability Mechanism of pRb Binding by D-type Cyclins of pRb to arrest the growth of human osteosarcoma cells The binding of the D cyclins to the pRb pocket raised can be reversed by overexpressed cyclin A or E that the possibility that D cyclins may associate with pRb in a causes its hyperphosphorylation (Hinds et al., 1992). Fi- fashion similar to the mechanism used by the viral onco- nally, hypophosphorylated pRb can bind and apparently proteins. This possible functional analogy caused us to regulate the activity of the E2F transcription factor (Chel- examine the sequences of D cyclins for structural similari- lappan et al., 1991; Shirodkar et al., 1992; Weintraub et al., 1992). The apparent importance of pRb phosphorylation leads in turn to the notion that the cdks can regulate pRb function by promoting its phosphorylation. We report here that cer- tain cyclins interact directly with pRb, but in a dramatically different fashion. The present results suggest that pRb may be regulated by certain cyclins that modulate its state of phosphorylation and may in turn regulate the activity of yet other cyclins through direct binding. Results In Vitro Association of pRb and Cyclins To uncover functional interactions between cyclins and pRb, we first determined whether cyclin proteins can form B aRB complexes with pRb. To do so, we studied the interac- r tions of these proteins in vitro, using human pRb purified from recombinant baculovirus-infected insect cells and 35S-labeled reticulocyte lysate-expressed human cyclins A, Bl, 82, C, Di , D3, and E. Complex formation between pRb and the various cyclins was assessed by measuring the ability of the cyclins to coimmunoprecipitate with pRb following addition of an anti-pRb monoclonal antibody. We 29 performed the mixing experiments with relatively small 1 2 3 4 5 6 7 R 9 amounts of the purified pRb (50 ng per 500 PI reaction) to minimize nonspecific aggregation driven by high pRb C aRB concentrations. Analysis of the immune precipitates (Figures 1A and 1 B) demonstrated that cyclin Dl specifically associates with pRb, while cyclins A, Bl, and E did not do so under these conditions. We did note a barely detectable signal with cyclins 82 and C. We have not determined whether this weak signal represents nonspecific aggregation of these proteins to pRb or a bonafide low affinity interaction. In our further work, we focused on the avidly binding D cyclins. Because a number of viral and cellular proteins associ- Figure 1. Complex Formation between Human Cyclins and pRb In ate with pRb via its pocket domain, wedetermined whether Vitro the D cyclins also exploit this pocket to bind to pRb. To (A and B) In vitro translated (IVT) human cyclins A, Bl, 82, C, Dl, and E (10 ul) were mixed with 50 ng of purified insect cell-produced address this possibility, we studied a mutant form of pRb human pRb or buffer (control) in a 1 ml reaction. The resulting com- bearing a defective pocket. This pRb variant, termed A22, plexes were immunoprecipitated with anti-pRb monoclonal antibodies is derived from the human small cell lung carcinoma 592 (a mixture of 21C9 and XZ55) and resolved by PAGE. In vitro translated cell line and is unable to bind viral oncoproteins or tether (IVT) cyclin reactions (1 ~1) were separated on the gels as controls.