Gi Cyclins Control the Retinoblastoma Gene Product Growth Regulation Activity via

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1 Vol. 6, , April 1995 Cell Growth & Differentiation 395 Gi Cyclins Control the Retinoblastoma Gene Product Growth Regulation Activity via Upstream Mechanisms1 Lynn E. Horton, Yongyi Qian, and Dennis I. Templeton2 Institute of Pathology and Program in Cell Biology, Case Western Reserve University School of Medicine, Cleveland, OH Abstract Inactivation of the retinoblastoma gene produd (prb) occurs concomitant with the appearance of its hyperphosphorylated form in mid to late G1. Multiple cyclin/cdk complexes are implicated in the cell cycle phosphorylation of prb. Using in vivo expression systems, we show that cyclins A, E,, D2, and D3 each function to phosphorylate and inactivate prb. In vivo, Gi cyclin/kinase complexes enhance the phosphorylation of prb, and these effeds of cyclin/kinases on prb can be overcome by the addition of p21, a wide spedrum inhibitor of Gi kinases. Kinases associated with cyclins A, E, and Dl phosphorylate prb indistinguishably in vivo, according to proteoiytic maps. Although cyclin Dl has been reported to bind to prb diredly, requiring the prb-binding motif LXCXE, a mutant Dl lacking the prb-binding motif remains able to phosphorylate prb in vivo and in vitro and is also able to reverse the growth-inhibitory properties of prb in intad cells. Finally, coexpression of Gi cyclins and kinases represses prb-mediated growth inhibition in Saos-2 cells. The multiplicity of mechanisms for prb phosphorylation and inadivation suggests that several pathways exist for the regulation of prb by phosphorylation. Introdudion The retinoblastoma susceptibility gene product (prb) is a Mr 110,000 nuclear phosphoprotein (1-4) that is ubiquitously expressed in tissues (5) and functions as a cell cycle regulatory protein (reviewed in Refs. 6 and 7). Mutations and deletions ofthe retinoblastoma susceptibility gene RB-i are found in a subset of tumors including retinobiastomas and other tumor cell types (1,8, 9). A model for prb function is that prb holds cell cycle progression in check, allowing for regulated, normal growth. Introduction of the wild-type RB-i gene into tumor cells can lead to inhibition of cell cycle progression and growth arrest (10). Subtle mutations of prb identified in tumors have lost this ability to cause growth arrest (1 1, 1 2). Despite the fact that prb synthesis is constant throughout the cell cycle (4), prb apparently exerts its growth inhibi- Received 9/16/94; revised 1/14/95; accepted 1/27/95. 1 This work was supported by Grant CA-557i9 from the NIH. L. E. H. is supported by an NIH training grant to the Cell and Molecular Biology Program at Case Western Reserve University. 2 To whom requests for reprints should be addressed, at Case Western Reserve University School of Medicine, Institute of Pathology and Program in Cell Biology, E. Euclid Avenue, Cleveland, OH tion only in G1 (1 3). Phosphorylation of prb varies throughout the cell cycle; prb is underphosphorylated at G0-G1 and hyperphosphorylated on serine and threonine residues during S and G2 phases (3, 4, 14, 15). Dephosphorylation of prb occurs at M phase, and evidence points to the role of protein phosphatase 1 as a prb phosphatase (16). prb phosphorylation is coincident with the activity of a variety of cyclins and cell cycle kinases. Several CDKs3 including CDC2, CDK2, CDK4, and CDK5 are present through the cell cycle (1 7-20) but are activated by complex formation with a regulatory subunit (termed cyclin; reviewed in Ref. 21 ). Expression of dominant negative mutants of CDC2 and CDK2, but not CDK4, cause a block in the cell cycle at G1 as determined by flow cytometry (22). prb can be phosphorylated in vitro by CDC2, CDK2, and CDK4, consistent with the observation that prb contains several consensus phosphorylation sequences for the CDKs (23-27). prb can apparently bind to both CDC2 and CDK2 (28, 29). As the name implies, cyciins are expressed variably throughout the cell cycle. Cyclins fail into three major subgroups: the Gi cyclins including Dl,D2, D3 and E; the S phase cyclin A; and a G2-M cyclin, cyclin B (21). Different cyclins complex with and activate different CDKs, creating a complex system of timed, cell cycle-dependent phosphorylations (30, 31). Recent experiments show that prb can be phosphorylated by cyclins A and E in conjunction with their partner CDKs in vivo and in vitro, whereas prb has been shown to be phosphorylated by the D-type cyclins plus CDK4 in vitro (26, 32-34). This phosphorylation of prb correlates with the relief of growth suppression of Saos-2 cells (32). These data concur with mounting evidence that phosphorylation of prb negates the growth-regulatory activity of prb: (a) prb hyperphosphorylation coincides with the exit of the cell from G1 and entry into S phase (4, 34); (b) SV4O large T antigen binds exclusively to the underphosphorylated form of prb, and other viral oncoproteins show similar binding specificity (1 2, 35, 36); and (c) transcriptional activation by E2F is repressed more efficiently by a form of prb refractory to phosphorylation than by wild-type prb (37). This evidence, along with the fact that cell cycle kinase/cyclin complexes can phosphorylate and block the growth suppressive function of prb, provides support for the hypothesis that prb is inactivated by phosphorylation. Recent reports conflict as to whether the G1 phase D-type cyclins can inactivate prb, through upstream regulation either by phosphorylation or through direct prb binding, or whether the D-type cyclins are themselves inhibited by direct binding to prb. All three D-type cyclins bind to prb, although the interaction of Dl and prb is significantly lower than that observed with D2 or D3 and prb (33, 38). 3 The abbreviations used are: CDK, cyclin-dependent kinase; SDS-PAGE, SDS-polyacrylamide gel electrophoresis.

2 396 Upstream Regulation of prb Activity by Gi Cyclins Immune complexes of cyclins Dl, D2, or D3 with CDK4 expressed either in insect or mammalian cells contain activities that can phosphorylate a prb-gst fusion protein in vitro (26, 27, 33). Unlike cyclins A and E, when cyclin Dl is expressed in cells alone, it is unable to phosphorylate prb; however, it has been shown to regulate prb activity in vivo. (32) This observation has been used to argue that the D-type cyclins do not inactivate prb through phosphorylation, despite the fact that prb is phosphorylated in vitro by the D-type cyclins in conjunction with their kinase CDK4 (38). We examined the function of G1 cyclins in an in vivo system to clarify the role of G1 cyclins in the regulation of prb function. The data we present here indicate that, like cyclins A and E, the D-type cyclins operate upstream of prb by directly phosphorylating and inactivating prb. Mutants ofcyclin Dl lacking the putative prb-binding domain function identically to the wild-type cyclin Dl,suggesting that D1-pRb binding is unimportant for the regulation of prb by cyclin Dl. Because of close correlation of: (a) cyclin D expression with prb phosphorylation; (b) the ability of cyclin D/CDK4 to phosphorylate prb in vitro indistinguishably from cyclins A/CDK2 or E/CDK2 complexes; and (C) the ability of all cyclin/cdk combinations to block prb activity in vivo, we conclude that the cyclin/cdk complexes directly phosphorylate and inactivate prb. We also conclude that Gi cyclins operate upstream of prb, as regulators of prb activity, rather than as downstream effectors of prb signaling. Results 4 L. E. Horton, unpublished results. Vaccinia Virus System. The vaccinia virus expression system used in our experiments provides us with a way to overexpress prb and other proteins in mammalian cells. Cells are first infected with a recombinant vaccinia virus (v-tf7-3; Ref. 39), which encodes the bacteriophage T7 R polymerase, and then the cells are lipofected with the plasmid vector ptm1 containing the gene of interest under the control of the T7 promoter. In preliminary experiments, greater than 70% of cells expressed f3-galactosidase using this vector system (data not shown). When prb was expressed using this system in the prb-deficient cell line Saos-2 (40), the exogenous prb failed to become hyperphosphorylated (Fig. 1,Lane 1; Ref. 32). Thus, this system allows us to measure the effects of coexpressed kinases and kinase regulators on prb hyperphosphorylation. Use of the Saos-2 cells provides an in vivo system to reconstitute the pathway that leads to the hyperphosphorylation of fulllength prb. We also used this overexpression system to purify prb and cyclin proteins for in vitro experiments. We modified the genes encoding prb and the cyclins to express a protein which contains 1 1 additional amino acids, either at the carboxy terminus (for prb) or at the amino terminus (for each cyclin). The added epitope (termed EE, see Materials and Methods ) is recognized by a monoclonal antibody (termed anti-ee) and provides for simple purification of the epitope-tagged protein as well as recognition by Western blot. For immunopurification, proteins were immunoprecipitated using the EE monoclonal antibody and eluted from the antibody-bead complex by incubation with an excess of EE cognate peptide. Vectors encoding CDKs were constructed to express native proteins without epitope tags, since early experiments showed that such tags reduced the kinase activity of some recombinant kinases. Cyclins and Cyclin/Kinase Combinations Cause the Phosphorylation of prb in Vivo. To help define the pathways leading to prb phosphorylation in vivo, we tested the effect of coexpression of cyclins, CDKs, and cyclin/cdk combinations on prb phosphorylation in vivo. We transfected Saos-2 cells with prb expression plasmids alone or with prb vectors, together with vectors encoding kinases or cyclins or cyclins plus kinases. Equivalent amounts of lysates from these cells were analyzed by Western blot using anti-prb antibody (Fig. 1A). Expression of prb alone gave a characteristic Mr 1 1 0,000-band of underphosphorylated prb (Fig. 1A, Lanes i and 18). A mobility shift of prb consistent with hyperphosphorylation was seen when prb was cotransfected with cyclin A (Fig. 1 A, Lane 3) or cyclin E (Fig. 1 A, Lane 5), and to a much lesser extent CDK2 (Fig. 1A, Lane 2), CDK4 (Fig. 1A, Lanes io and i7) and Dl (Fig. 1A, Lane 1 1). Cotransfection of CDC2 (Fig. 1A, Lane 7) orcyclins B (Fig. 1A, Lane8), D2 (Fig. 1A, Lane 13), or D3 (Fig. 1A, Lane 15) caused no change in the pattern of prb phosphorylation. In summary, as described above, none of the cyclins or kinases expressed alone caused a complete shift to the slowest migrating Mr 1 1 6,000 hyperphosphorylated form of prb that is seen in cycling cells. In contrast, expression of several combinations of cyclins and CDKs induced quantitative phosphorylation of prb (Fig. 1A), where prb is represented only by a single, slowly migrating band of Mr 116,000. Cyclin A/CDK2 (Fig. 1A, Lane 4) and E/CDK2 (Fig. 1 A, Lane 6) combinations caused an increase in the mobility of prb, consistent with the hyperphosphorylation of prb, which was enhanced over that seen with either cyclin A or E alone. Cyclins Dl, D2, and D3 also caused an increase of phosphorylation of prb when cotransfected with CDK4 (Fig. 1A, Lanes 12, 14, and 16, respectively), identical to that seen with cyclins A/CDK2 and E/CDK2. Cotransfection ofcyclin B plus CDC2 caused only a minor shift of prb to the hyperphosphorylated form (Fig. 1 A, Lane 9). In addition to cooperation with CDK2, cyclins A and E could also Cooperate with CDC2 to cause the hyperphosphorylation of prb (data not shown; see also Ref. 33). Expression of the cyclins was detected by labeling cells with [35Sjmethionine, followed by immunoprecipitation with the anti-ee antibody (Fig. 1 B). Equivalent levels of each of the cyclins was immunoprecipitated, showing that differential abilities of the cyclins to phosphorylate prb is not due to underexpression. The exception to this is cyclin B/CDC2 plus prb (Fig. 1 B, Lane 9). Coexpression of cyclin B/CDC2, but not cyclin B alone, causes a reproducible decrease in the expression level of both cyclin B and prb, whereas prb expression is constant throughout the rest of the experiment (see Fig. 1A and [35S]methionine labeling; data not shown). This low level expression ofcyclin B could account for the weak hyperphosphorylation of prb by cydin B/CDC2. Because of the problems caused by this decreased expression of both prb and cyclin B when coexpressed with CDC2, we excluded cyclin B/CDC2 from further coexpression studies performed with the TM1 system. To determine if both CDK4 and CDK2 could be activated in vivo with the D-type cyclins, we compared the ability of CDK2 and CDK4 to cooperate with the D-type cyclins on prb phosphorylation (Fig. 1 C). When expressed alone, the

3 Cell Growth & Differentiation 397 A CDK: O - /Q#{176}i?r1,,Q4?,Q - Cyclin: - - A A E E - B B - Dl Dl D2 D2 D3 D B Cyclin: CDK: 0 - C, C, C, - I Ii 1 - b t,i -C, C) C) Cyclin:, Cyclin Dl Cyclin D2 Cyclin D3 c,,i. Kinase: - c 0, C bj CJ P:: Fig. 1. Phosphorylation of the retinoblastonia gene product (prh( in vivo by cyclins, CDKs, and cyclin/cdk combinations. prb was detected using Western blotting against prb. A, prb was expressed in Saos-2 cells either alone (Lanes 1 and 18); or together with cyclins A (Lane 3), E (Lane 5), B (Lane 8), Dl (Lane 11), D2 (Lane 13), or D3 (Lane 15); or kinases CDK2 (Lane 2), CDC2 (Lane 7), or CDK4 (Lanes 10 and 17); or cyclin/kinase combinations of A/CDK2 (Lane 4), E/CDK2 (Lane 6), B/CDC2 (Lane 9), D1/CDK4 (Lane 12), D2/CDK4 (Lane 14), or D3/CDK4 (Lane 16). Hyperphosphorylation of prb is seen in cultures coexpressing cyclins together with CDKs and, to a lesser extent, in those expressing cyclin A or E singly. B, prb was expressed in Saos-2 cells alone (Lane 1); with individual cyclins (Lanes 3, 5, 8, 1 1, 13, and 15) or individual kinases (Lanes 2, 7, and 10); or cyclin/kinase combinations (Lanes 4, 6, 9, 12, 14, and 16); and the cells were labeled with I 5Slmethionine. Except for cyclin B/CDC2, expression levels of each of the cyclin proteins is equivalent. C, prb was expressed in Saos-2 cells, together with cyclins Dl (Lane 1), D2 (Lane 4), or D3 (Lane 7); or cyclin D/kinases combinations of D1/CDK2 (Lane 2), D1/CDK4 (Lane 3), D2/CDK 2 (Lane 5), D2/CDK4 (Lane 6), D3/CDK2 (Lane 8), or D3/CDK4 (Lane 9). All three D cyclins cooperate with CDK4 to phosphorylate PRb. Cyclins D2 and Dl also cooperate with CDK2, but not cyclin D3. prb, underphosphorylated prh; pprb, hyperphosphorylated prb.

4 398 Upstream Regulation of prb Activity by Gi Cyclins D cyclins caused little if no change in the phosphorylation of prb (Fig. 1 C, Lanes i, 4, and 7). Both cyclin Dl and D2 increased the phosphorylation of prb in conjunction with either CDK2 or CDK4 (Dl : Fig. 1 C, Lanes 2 and 3; D2: Fig. 1 C, Lanes 5 and 6). Activation of cyclin D3, however, was specific to CDK4, since cotransfection of D3, together with CDK2, did not increase the phosphorylation of prb relative to transfection of D3 alone (Fig. 1 C, compare Lanes 7, 8, and 9). The D-type cyclins can bind both CDK4 and CDK2 and may be active with either kinase (33, 41, 42). The D-type cyclins did not cooperate with CDC2 to enhance prb phosphorylation (data not shown). These data support a pathway involving the D-type cyclins through either CDK2 or CDK4, leading to the phosphorylation of prb. Different Cyclins Phosphorylate prb Indistinguishably. To determine if each cyclin/cdk combination results in similar phosphorylation events on prb, we purified 32Plabeled prb from cells transfected with different cyclin! kinase combinations. The radiolabeled prb was subjected to proteolysis with CNBr and analyzed on a high-concentration SDS-polyacrylamide gel (Fig. 2A). 32P-Iabeled prb was also subjected to proteolysis by chymotrypsin and analyzed by electrophoretic separation in one dimension, followed by chromatography in a second dimension (Fig. 2B). Phosphopeptide patterns from prb phosphorylated by all cyclin combinations were identical using either onedimensional or two-dimensional separation techniques, suggesting identical sites of phosphorylation by all kinases. prb expressed without cyclin/cdk (Fig. 2A, Lane i) displayed a different pattern, possibly representing basally phosphorylated prb (1 2). p21 Blocks Phosphorylation of prb by All Cyclins in Vivo. Recently, an inhibitor of cyclin/kinases (p21 sdil/ wafi/cipi/pici) has been cloned and shown to inhibit the phosphorylation of prb by cyclins A, E, and Dl kinase complexes in vitro (43). To determine if p21 would inhibit the phosphorylation of prb by all cyclin/kinase combinations in vivo as it does in vitro, we cotransfected prb and cyclin/kinase combinations with and without the p21 expression plasmid and analyzed the prb phosphorylation state by Western blot (Fig. 3). If the D-type cyclins are inhibited by prb in vivo, then the addition of p21 to the experiment should have no further effect on the activity of the D-type cyclins. However, if the D-type cyclins function upstream of prb to cause the phosphorylation of prb, then p21 should block this activity. When coexpressed with all cyclin/cdk combinations tested (A/CDK2, E/CDK2, Dl! CDK4, D2/CDK4, and D3/CDK4), p21 dramatically increased the amount of the underphosphorylated form of prb (Fig. 3, compare Lanes 2 and 3, Lanes 4 and 5, Lanes 6and 7, Lanes8and 9, and Lanes ioand ii). We conclude fromthisthatp2l functions in vivoto inhibitthefunction of the cyclin/kinase complexes to phosphorylate prb. Because p21 can inhibitthe kinasefunction ofthea/cdk2, E/CDK2, and D/CDK4 complexes towards prb both in vitro and in vivo, we present this as further evidence that all the Gl cyclins act upstream of prb to cause the phosphorylation of prb. Biological Inadivation of prb by Cyclins and Cyclin/ Kinase Combinations. We have previously described an assay (1 2, 44) that measures facets of prb biofunction including growth inhibition, induction of cell morphology changes, and stabilization of plasmid expression within transfected cells. In this assay, prb is transfected into prbdeficient Saos-2 cells, together with a puromycin-resistance A ac? (Q Cyclin/Kinase: +0 ((> Qb. 43Kd 29Kd - l8kd- 4 l4kd- 6Kd 3Kd < $ C C.) > C.) LU C C.) >1 C) lii7 C C.) + 0. Fig. 2. Partial proteolytic phosphopeptide maps of in vivo 32P-labeled prb proteins cleaved with CNBr or chymotrypsin. A, prb expressed in CV1 cells alone (Lane 1) or together with cyclin A/CDK2 (Lane 2), E/CDK2 (Lane 3), or D1/CDK4 (Lane 4) was immunoprecipitated using anti-ee epitope antibody from cells radiolabeled with 2PO4. prb isolated from these cells was treated with 50 mg/mi CNBr and run on a 16.5% SDS PAGE gel. prb phosphorylation by any of the three cyclin/cdk combinations was indistinguishable and distinct from that of prb expressed alone. Exposure time was 36 h with intensifying screen. B, partial proteolytic phosphopeptide maps of in vivo 32P-labeled prb proteins cleaved with chymotrypsin. prb expressed in Saos-2 cells, together with cyclin A/CDK2, E!CDK2. or D1/CDK4, was immunoprecipitated using anti-ha and prb monoclonal antibodies from cells radiolabeled with 2PO4. prb isolated from these cells was treated with 4 pg chymotrypsin and electrophoresed in the first dimension and chromatographed in the second dimension. prb phosphorylation by any of the three cyclin/cdk combinations was indistinguishable from one another. Exposure time was 4 days with intensifying screen.

5 Cell Growth & Differentiation 399 Cyclin/Kinase: p21: - I 1 1 I I I I I I , pprb1 prb iis- #{149}1 -** ---- :: Fig. 3. Inhibition by p21 of cyclin/cdk phosphorylation of prb in vivo. prb was expressed in Saos-2 cells alone (Lane 1) ortogether with cyclin A/CDK2 (Lanes 2 and 3), cyclin D1/CDK4 (Lanes 4 and 5), D2/CDK4 (Lanes 6 and 7), D3/CDK4 (Lanes 8 and 9), or E/CDK2 (Lanes 10 and 1 1 (. Expression of prb, together with cyclin/cdks, was performed either without p21 (even lanes) or with p21 (odd-numbered lanes, excluding Lane 1(. The phosphorylation state of prh was evaluated by anti-prb Western blot. p21 effectively inhibits phosphorylation of prh induced by all cyclin/cdk complexes. Table 1 Biological activity of prb coexpressed with cyclins and p21 Plasmid Experiment 1 Experiment 2 Experiment 3! Average Y Recovery % SVE-pRb + 58 ± ± ± E1A 0±0 0±0 47.3± cyc A 8 ± ± ± cyc B ND 75.2 ± ± cyc Dl 1 1 ± ± ± cyc D ± ± ± cycd3 52± ± ± cyc E 2 ± ± ± p21 ND 0±0 ND 0 E1A+p21 ND 0±0 ND 0 0 cyc A + p21 52 ± ± 7.3 ND cyc B + p21 ND 79.2 ± 4.92 ND 75.8 cycdl +p2l 49± ±11 ND cyc D2 + p ± ± 6.45 ND 60 cyc D3 + p ± ± 15.7 ND 80.3 cyc E + p21 45 ± ± 7.88 ND , Four fields (at xloo) from 10 transfected cells were counted. Transfected Saos-2 cells were plated, subjected to drug selection, and counted 7-10 days later as described in Materials and Methods. I, Three aliquots from 1 0 transfected cells were counted on a Coulter counter as described in Materials and Methods. Percentage offlat cells from cyclin-prb transfections in Experiments 1 and 2 were subtracted from the percentage offlat cells from cyclin-p21 -prb transfections in Experiments 1 and 2 to obtain the percentage of recovery caused by expression of p21.,1 Average percentage of three experiments.. Average percentage of two experiments.,percentage of one experiment. 5 Control experiments transfecting only cyclin cd vectors resulted in no large cell formation. h ND, not done;, not available. plasmid, to distinguish between transfected and untransfected cells. The cells transiently transfected with prb expression vectors become enlarged (about 5-fold in length) and remain persistently resistant to puromycin, presumably because the cell cycling blockade induced by prb prevents loss of the drug resistance plasmid during cell division. Nearly all cells surviving in puromycin after 10 days are of aberrant morphology and can be enumerated after fixing and staining. Expression of prb is correlated with the arrest in G1 of cells arising in this assay (32). We used this assay to test whether cotransfection of plasmids encoding cell cycle kinases or cyclins, together with prb expression vectors, could eliminate the prb-mediated growth suppression effect. We also used this assay to determine whether p21 could override the cyclin and cyclin/kinase inhibition of prb growth suppression. Table 1 summarizes these results. Cotransfection of prb with cyclins A, E, and Dl expression vectors inhibited prb

6 400 Upstream Regulation of prb Activity by Gi Cyclins Table 2 Biological activity of pr b coexpressed with cyclin s, kinases, and p21 SvE-pRb+ Plasmid Experiment 1 a Experiment 2 Experiment 3 Average %C Recovery % E1A cyc A + cyc B + CDC2 cycdi + CDK4 cyc D2 + CDK4 cyc D3 + CDK4 cyc E + CDK2 cyc A + cdk2 + P! cyc Dl + CDK4 + p21 cyc 02 + CDK4 + p21 cyc D3 + CDK4 + p21 cyc E + CDK2 + p21 58±6.39 0±0 23 ± 39.5 ND 10 ± ± ± ± ± ± ± ± ±4.65 0± ± 10 ND 14.5 ± ± ± ± ± ± ± ± ± ± ±0 9.0 ± ± ± ± ± ± 2.44 NP ND ND ND a Four fields (at xl 00) from 1o transfected cells were counted. Transfected Saos-2 cells were plated, subjected to drug selection, and counted days later as described in Materials and Methods. I, Three aliquots from 1o transfected cells were counted on a Coulter counter as described in Materials and Methods. C Percentage offlat cells from cyclin-prb transfections in Experiments 1 and 2 were subtracted from the percentage offlat cells from cyclin-p21 -prb transfections in Experiments 1 and 2 to obtain the percentage of recovery caused by expression of p21. d Average percentage of three experiments. C Percentage of one experiment. Average percentage of two experiments. I; Control transfections of cyclins and CDKs without prb resulted in no large cell formation. h ND, not done;, not available. ND 100 0d 27#{149}1d d 262d pq.5f activity in this assay (respectively, 84, 83, and 94% fewer aberrant cells compared to prb alone) to an extent similar to that seen with cotransfection of El A expression plasmid with the prb expression vector. Cotransfection of vectors encoding cyclins B, D2, or D3 had little effect on prb growth-suppression activity. However, cyclins D2 and D3 showed an enhanced ability to block prb growth suppression when cotransfected with CDK4, as does cyclin B with CDC2 (Table 2). Cyclins A, E, and Dl show a similar ability to inhibit prb growth suppression when transfected alone or cotransfected with their partner kinase. The ability of the cyclins to abrogate prb-mediated growth inhibition in Saos-2 cells correlates with their ability to phosphorylate prb in vivo. As a control, the cyclins, kinases, and cyclins plus kinases were transfected into Saos-2 cells with the puromycin resistance plasmid but without a prb expression plasmid. None ofthe cyclins, kinases, or cyclin/kinase combinations can elicit the large cell phenotype (data not shown). Because p21 has the ability to block the phosphorylation of prb by the cyclin/kinases, we wanted to test whether p21 is also able to block the cyclin inhibition of prb growth suppression and restore wild-type prb function. To do this, we cotransfected expression plasmids for prb, p21, and either cyclins or cyclins plus kinases into Saos-2 cells. In every case in which a cyclin could inhibit prb growthsuppressive function, p21 could partially restore prb-mediated growth inhibition. This was not due to p21 -mediated growth suppression because p21 itself does not cause growth inhibition in this assay, nor can it block E1A inhibition of prb function (Table 1). p21 also did not have an effect on the cyclin B-pRb, cyclin D2-pRb, or cyclin D3- prb cotransfections, which showed no inhibition of prbmediated growth suppression. In the case of cyclins A, E, and Dl, it restored between 57 and 62% of prb activity. p21 also showed an ability to restore prb growth-suppressive function when cotransfected with the cyclin/kinase combinations, but to a lesser degree (29-52%; Table 2). From this experiment, we conclude that all ofthe Gi cyclin/ kinases function to inactive prb growth-suppressive functions, and this ability is a result of their kinase activity towards prb. Mutation of the LXCXE Region in Cyclin Dl Does Not Compromise Its Ability to Phosphorylate prb in Vivo or in Vitro. prb binds to viral oncoproteins such as large T antigen, E1A, and E7 through a conserved amino acid sequence leucine-x-cysteine-x-glutamic acid (LXCXE), where X is any amino acid (45-47). A mutation of LXCXE to LXGHE in the viral oncoprotein E1A abolishes its ability to bind to prb (48, 49). A similar LXCXE sequence is found in the aminoterminus ofthe D-type cyclins, and prb has been reported to bind cyclin Dl dependent on this sequence (38). To determine if the binding of cyclin Dl to prb through the LLCCE site was essential for cyclin D1/CDK4 phosphorylation of prb, we constructed the corresponding mutation in cyclin Dl of LLCCE to LLGHE (named Dl- 7GH). To test whether cyclin D1-7GH could cause phosphorylation of prb in vivo, we cotransfected cyclin Dl - 7GH, together with CDK4 and prb, and analyzed the results by Western blot as done in Fig. 1. Cyclin Dl-7GH/ CDK4 caused hyperphosphorylation of prb (Fig. 4, Lane 3) similar to that seen with wild-type D1/CDK4 (Fig. 4, Lane 2) or cyclin AJCDK2 (Fig. 4, Lane 4). This result indicates that the putative prb binding motif of cyclin Dl is not required to increase prb phosphorylation in vivo. This in vivo phosphorylation of prb by Dl -7GH/CDK4 can be eliminated by the coexpression of p21,similar to that seen in Fig. 3 (data not shown). To test whether the wild-type or mutant Dl cyclin/kinase pairs could phosphorylate prb in vitro, we used purified prb and cyclins from our overexpression system. We have previously detected a kinase adherent to immunopurified prb (28). To eliminate this kinase from our prb purified for use as a substrate, we washed the immunoprecipitated prb 5 Unpublished data.

7 Cell Growth & Differentiation 401 Kinase: - Cyclin: - Dl 7GH A pprb< prb I ::ii Fig. 4. Phosphorylation of prb in vivo by a mutant cyclin Dl defective in prb binding domain. prb expressed in Saos-2 cells alone (Lane 1) or together with cyclin D1/CDK4 (Lane 2). D1-7GH/CDK4 (Lane 3), or A/CDK2 (Lane 4) was visualized by anti-prb Western blot. The mutant form of cyclin Dl was equivalently able to induce phosphorylation of prb. with 4 M LiCI before the kinase reaction was performed. prb thus washed was without kinase activity and corresponding background 2P incorporation (Fig. 5A, Lane 4; Fig. 58, Lane 1), while prb was still detectable in the sample by Western blot (data not shown). Each purified cyclin was cotransfected with its putative partner kinase. We used the immunopurified cyclin/cdk to phosphorylate purified prb substrate with [y-32piatp (Fig. 5A). Kinases associated with either cyclins A or F strongly phosphorylated prb (Fig. 5A, Lanes i and 2). Cyclin Dl! kinase was less able to phosphorylate prb compared to cyclins A and F (Fig. 5A, Lane 3). The three cyclin Ds were able to phosphorylate prb to an approximately equal amount, with Dl slightly weaker than D2 or D3 (Fig. 58, Lanes 2, 4, and 5). However, the mutant Dl-7GH showed approximately 2- to 3-fold lower phosphorylation of prb than wild-type Dl (Fig. 58, compare Lanes 2 and 3), showing thatthe in vitro kinase activity ofcyclin Dl towards prb is not seriously compromised by the abolishment of its prb binding region. The prb binding region mutation in Dl may be important for efficient binding of prb and Dl in vitro, but the fact that D1-7GH can still phosphorylate prb above background shows that Dl-7GH retains activity towards prb. We conclude from these experiments that the D-type cyclins function upstream of prb to phosphorylate prb and that a mutation that effects the ability of cyclin Dl to bind to prb does not significantly change cyclin Dl/CDK4 phosphorylation of prb. prb Is Phosphorylated Equivalently by Low Levels of Cyclin Dl and D1-7GH. It has been proposed that the prb-cyclin Dl interaction serves to inactivate Dl (38). Although we found that cyclin Dl directly phosphorylates prb (see above), it could be argued that cyclin Dl is only able to phosphorylate prb when the cyclin is overexpressed and thus able to overcome negative effects that prb has on cyclin Dl activity. To test this possibility, we sought means 4 of titrating levels of cyclin Dl expression within the cell. We performed an experiment in which the amount of plasmid encoding prb and CDK4 transfected into Saos-2 cells was held constant while plasmid levels of either Dl or the Dl-7GH mutant were decreased. We labeled the transfected cells with [35S]methionine, immunopurified the prb and Dl cyclins using the anti-ee antibody, and subjected the immunopurified proteins to gel electrophoresis and autoradiography. The amount of prb phosphorylation seen increased proportionally to the amount of cyclin Dl or Dl-7GH expressed (Fig. 6, top panel). However, even undetectable levels of cyclin Dl and Dl-7GH are able to enhance prb phosphorylation (Fig. 6, top panel, Lanes 6 and i2). Importantly, cyclin Dl and Dl-7GH showed indistinguishable levels of prb phosphorylation over the entire titration spectrum, discounting the theory that this mutant is hyperfunctional towards prb due to the lack of its prb-binding/ regulatory region. We conclude that, since such a high ratio of prb to cyclin Dl still results in prb phosphorylation, prb does not inhibit the activity of cyclin Dl and that Dl/CDK4 can function to phosphorylate prb. We also conclude that Dl and Dl-7GH function in the same manner towards prb, because their ability to phosphorylate prb remains identical throughout the titration. An alternative hypothesis that we have not been able to test is that the binding of prb may change the substrate specificity for cyclin Dl,so that the binding of prb to Dl may cause prb phosphorylation but may block the kinase activity of Dl towards other substrates. An experiment to test if prb changes the substrate specificity of cyclin Dl could be done if there were any known physiological substrates for cyclin Dl besides prb, but there are none. Biological Inactivation of prb by Dl and D1-7GH. To further test the ability of the cyclin Dl-7GH mutant to function equivalently to wild-type cyclin Dl,we compared their ability to negate prb function in the biological assay of prb growth suppression as described in Table 1. A plasm Id expressing the mutant Dl-7GH could inhibit prb function equivalently to cyclin Dl when Dl was expressed in that same vector (respectively, 72.5 and 74.3% fewer aberrant cells compared to prb alone; Table 3). In conjunction with CDK4, they both exhibit a similar ability to inactive prb in this assay. p21 can also block the activity of Dl and Dl- 7GH, resulting in the restoration of prb growth suppression. This occurs at equivalent levels to that seen with cyclin A and F and whether the Dl or Dl -7GH is expressed alone or in conjunction with the CDK4 kinase. Thus, mutation of the putative prb binding domain of Dl does not affect biological control of prb by cyclin Dl. Discussion Since prb function is regulated by phosphorylation, the mechanism of prb phosphorylation is a central question in cell growth control. We compare the abilities of cyclins A, F, and the D-type cyclins to phosphorylate and inactivate prb. We show here that the D-type cyclins function to phosphorylate and inactivate prb through mechanisms similar to cyclins A and E. Our studies indicate that a general role for the Cl cyclins is to function as upstream regulators of prb. Previous experiments on cyclin Dl have focused on the ability of cyclin Dl to function as a promoter of the cell cycle. Cyclin Dl was initially cloned as the potential on-

8 402 Upstream Regulation of prb Activity by G1 Cyclins A Cyclin: A E Dl Rb: pprb B + bi CPb b bi Cyclin/kinase: \ # A1 (, \ S QvQ;F js Rb: pprb Fig. 5. Phosphorylation of prb in vitro by purified cyclin/cdk combinations. A, immunopurified prb was reacted with cyclin A/CDK2 (Lane 1), E/CDK2 (Lane 2), D1/CDK4 (Lane 3), or a mock extract (Lane 4) in the presence of l y-32p]atp. Exposure time was 30 mm plus intensifying screen. B, immunopurified prb was reacted with mockextract(lane 1), cyclin D1/CDK4(Lane2), cyclin D1-7GH/CDK4(Lane3), cyclin D2/CDK4 (Lane4), cyclin D3/CDK4(Lane5(, orcyclin A/CDK2 (Lane 6) in the presence ofly-32piatp. Cyclin D1/CDK4 (Lane 7) and cyclin D1-7GH/CDK4 (Lane 8) were reacted in the presence of l y-32piatp without the addition of prb. Exposure time was 1 6 h. CycllnIklnas.: - r Dl/CDK4 Ir Dl-7GH/CDK4 jtg cyclin D: 4 % - *b s , Cyclin Dl- /. _ Fig. 6. Effect oftitration ofcyclin Dl and D1-7GH on prb phosphorylation. A composite picture from the same autoradiogram showing prb (toppanel, Lanes 1-14), cyclin Dl (bottom panel, Lanes 3-8), and cyclin D1-7GH (bottom panel, Lanes 9-14) immunopurified from cells labeled with l35slmethionine. prb was expressed alone (Lane 1), with CDK4 (Lane 2), or with CDK4 and decreasing amounts oftransfected cyclin Dl (Lane 3, 5 pg; Lane 4, 1 pg; Lane 5, 0.5 pg; Lane 6, 0.1 pg; Lane 7, 0.05 pg; and Lane 8, 0.01 pg) or with CDK4 plus decreasing amounts oftransfected cyclin D1-7GH (Lane 9, 5 pg; Lane 10, 1 pg; Lane 11, 0.5 pg; Lane 12, 0.1 pg; Lane 13, 0.05 pg; and Lane 14, 0.01 pg(. Cyclins Dl and Di-7GH phosphorylate prb equivalently over the entire titration and retain their ability to phosphorylate prb, even at undetectable cyclin levels. Exposure time was 1 6 h. cogene PRAD-i, and cyclin Dl has been demonstrated to be overexpressed in breast and squamous and esophageal carcinomas (50-53). Overexpression of cyclin Dl can decrease the time a cell spends in C1 (54, 55). Dl is also essential for progression through G1, but in cells containing mutations inactivating the prb protein, Dl function in G1 is no longer required (56, 57). Both wild-type cyclin Dl and the LLGHE mutant Dl can cooperate with an E1A domain 2 mutation in a cell transformation assay, indicating cyclin Dl can function to inactivate prb, an essential step in E1A cell transformation (58). Investigation into the role of D cyclins in prb regulation has resulted in mixed conclusions. Three hypotheses have been proposed to explain the interaction of prb with the

9 Cell Growth & Differentiation 403 Table 3 Biological activity of prb coexpre ssed with wild-type or mutant cyclin Dl and p21 SVE-pRb E1A cyc cyc Dl Di-7GH cyc Dl + p21 cyc Di-7GH + p21 cyc Dl + CDK4 cyc D1-7GH + CDK4 Plasmid Experiment 1 Experiment 2 Average 0/,, Recovery b cyc Dl + CDK4 + p21 cyc D1-7GH + CDK4 + p ± 6.4 0± ± ± ± ± ± ± ± ± ± 6.4 0± ± ± ± ± ± ± ± ± 8.34 a Four fields (at xloo) from 10 transfected cells were counted. Transfected Saos-2 cells were plated, subjected to drug selection, and counted 7-10 days later as described in Materials and Methods. b Percentage offlat cells from cyclin-prb transfections in Experiments 1 and 2 were subtracted from the percentage offlat cells from cyclin-p21 -prb transfections in Experiments 1 and 2 to obtain the percentage of recovery caused by expression of p D-type cyclins. A preliminary hypothesis raised in Dowdy et a!. (38) is that prb is inactivated by binding to the D-type cyclins, but prb is not phosphorylated by cyclin Dl. A similar model has been used to explain the interaction of prb with id-2 (59). The binding of prb to Dl has been reported to occur through the classic prb-binding sequence LXCXE found in the D-type cyclins and in all viral oncoproteins that bind prb (45-47). When the LLCCE sequence found in Dl is mutated to LLGHE, cyclin Dl loses its ability to bind prb (38). Our data refutes the hypothesis that prb is inactivated through binding to cyclin Dl since the Dl- 7GH mutant retains an equivalent ability to repress prbmediated growth inhibition and demonstrates equivalently the same ability to phosphorylate prb in vivo and in vitro. If binding of prb to cyclin Dl is required for cell cycle control, then Dl-7GH should not have any of these abilities. The second hypothesis presented in Dowdy et a!. (38) and Hinds et al. (58) is based on the model of prb-e2f interaction wherein prb binds to E2F and inhibits the activity of E2F (60). In this model, rather than being phosphorylated by cyclin Dl,pRb binds to cyclin Dl and inhibits the cyclin D-associated kinase function. Other groups have reported that coexpression of the LLGHE cyclin Dl mutation, and the corresponding mutant in D2, with prb in Saos-2 cells results in a greater inactivation of prb growthsuppressive function than does coexpression of wild-type cyclin Dl or D2 and prb (33, 38). The conclusion drawn by the authors from the Dl experiment is that prb inhibits the function of wild-type Dl but not the function of D1-7GH (38). This model proposes a role for D-type cyclins which is distinct from other members of the cyclin family, that the D-type cyclins function downstream of prb,as targets for prb regulation,rather than as upstream regulators of prb. In contrast to the two models above, our data support a third model, that the function of prb is directly regulated by D-type cyclins via phosphorylation, similar to the regulation of prb by cyclins A and E. This model has been proposed by others and accompanied by conclusive demonstrations that prb can be phosphorylated by the D-type cyclins in vitro (26, 27, 33). A recent study also shows that the Saccharomyces cerevisiae Dl -related cyclin, CLN3, is required to cause the hyperphosphorylation of prb in S. cerevisiae (61 ). Support for this hypothesis also comes from our observations that: (a) the D-type cyclins can phosphorylate and inactivate prb in vivo; (b) minute quantities of Dl expressed in cells induces hyperphosphorylation of prb; (c) the CDK inhibitor p21 can block the phosphorylation and the inactivation of prb growth-suppressive activities caused by cyclins A, E, and Dl ; (e) the LLGHE mutant of Dl retains activity towards prb quantitatively similar to that of wildtype Dl ; and ( 1) cyclins A, E, and Dl phosphorylate prb on similar sites in vivo. We investigated the effect of coexpression of the cyclins and the cyclins plus their partner kinases with prb in the context of a whole cell. In our system, coexpression of prb and the D-type cyclins with CDK4 results in the phosphorylation of prb at levels equivalent to those seen by A/CDK2 and E/CDK2. Expression of cyclins A, E, and, to a lesser extent, Dl alone caused a minor shift of prb to its hyperphosphorylated form. In addition, the ability of each of the cyclins or cyclin!kinases to phosphorylate prb correlates with their ability to repress prb-mediated growth arrest. We observed that the Dl-7GH and wild-type cyclin Dl have similar abilities to inhibit prb-mediated growth arrest in Saos-2 cells. This result is in contrast to the work of Dowdy et a!. (38), who noted that, in one assay, wild-type cyclin D had minimal effect on large cell induction by prb, while a cyclin Dl LXCXE mutant had strong inhibitory activity (38). We cannot reconcile these contrasting results but note that an earlier report (32) from this group detected strong inhibition of prb induction of large cells using wildtype cyclin Dl, similar to the effect we observed. Our interpretation has focused on the similar ability of mutant and wild-type cyclin D to phosphorylate prb. We also observe that cyclins Dl!CDK4 and Dl-7GH! CDK4 phosphorylate prb to a similar degree in our coexpression system. This correlates to their equivalent ability to negate prb growth suppression. Phosphorylation of prb by mutant and wild-type Dl!CDK4 is apparent, even in transfections including 50-fold less cyclin than the prb plasmid. In addition, if Dl-7GH were able to overcome prb inhibition better than wild-type Dl, then Dl-7GH!CDK4 should be significantly more able to phosphorylate prb at low expression levels than wild-type Dl. Instead of this difference, we observe a uniform and equivalent decrease in the amount of hyperphosphorylated prb when either Dl and Dl -7GH levels are reduced, supporting the model that cyclin Dl exerts a catalytic rather than stoichiometric effect on prb function. It is possible that prb changes the substrate specificity of cyclin Dl (i.e., blocking cyclin Dl from recognizing anything but bound prb); however, this hypothesis cannot be tested until other substrates besides prb are found for cyclin Dl.

10 404 Upstream Regulation of prb Activity by Gi Cyclins p21 has been shown to be a universal inhibitor of Gl cyclin-associated kinase activities (62, 63). Inhibition by p21 ofcyclin D1-induced prb phosphorylation, together with restoration by p21 of the prb growth-suppressive effects in Saos-2 cells, strengthens the case that prb inactivation occurs by D/CDK kinase activity on prb. The effect of p21 on the cyclin D/CDK4 complexes is equivalent to the effect p21 has on the cyclin/kinase complexes which contain cyclins A and E. This data indicates that, even if prb is not directly phosphorylated by the cyclin/kinases, the pathway involving the cyclin/kinases does lead to prb phosphorylation and regulation. The inhibition of prb phosphorylation and rescue of prb growth suppression by p21 is equivalent for both Dl and D1-7GH, indicating that both function in a similar manner to cause the phosphorylation and inactivation of prb. Because p21 has been shown to be an inhibitor of kinase activity and does not inhibit binding of cyclins to kinase (42,63),we therefore conclude that the function of p21 in our assays is to block the kinase activity of the Gl cyclins towards prb. We also conclude that the ability of Dl to repress prb-mediated growth suppression is a function of the kinase activity of D1/CDK4 towards prb. Throughout our studies, the effect of A, E, and the D-type cyclins on prb have proved equivalent. In vivo-iabeled prb resulting from coexpression with cyclin A/CDK2, cyclin E/CDK2, or cyclin Dl/CDK4 combinations produce similar one-dimensional and two-dimensional prb phosphopeptide maps, suggesting similar hyperphosphorylation of prb. Like cyclins A and E, the D-type cyclins also phosphorylate prb in vitro. We conclude that, similar to cyclins A and E, the D-type cyclins function upstream of prb to cause its phosphorylation and inactivation. Our data suggest that mammalian cells have evolved redundant means of regulating the inhibitory activity of prb by upstream mechanisms. In this regard, it should be remembered that the cyclin D family was identified as representative of a class of proteins whose expression is induced by mitogenic stimuli (64). Such mitogenically stimulated kinase complexes could represent a mechanism of prb regulation alternate to regulation of prb during the cell division cycle of normally growing cells. prb phosphorylation has been shown to be initiated upon mitogenic stimulation of cells (65-67), as well as during G1 progression of cycling cells (1 4). These two pathways of prb phosphorylation might involve distinct kinases, yet result in functionally indistinguishable effects on prb. The multiple mechanisms for prb phosphorylation lead to the hypothesis that phosphorylation of prb at different times during the cell cycle, by different cyclin, kinases, resuits in modification of unique sites on the prb molecule (67). This has also been proposed as a reason why cyclin D phosphorylation of prb is barely detectable in vivo (38). Under this model, the D-type cyclins may be responsible for a low level of prb phosphorylation at one or a few sites, which may be required for initial progression into or through early G1. In support of this is the data that a Dl -related activity, in conjunction with a second cyclinrelated activity, is required for the hyperphosphorylation of prb in S. cerevisiae (61). However, the view that different cyclins cause the different hyperphosphorylation patterns of prb is unsupported by our prb phosphopeptide maps, which show similar phosphopeptides resulting from each of the cyclin/kinase combinations used. Since the cyclins direct prb phosphorylation activity to similar sites and the temporality of prb phosphorylation during the cell cycle (late G i to M phase) is similar to that covered by the activity ofthe cyclin/kinases (beginning with the D-type cyclins and ending with cyclin A), we suggest that the cyclin/kinases function cooperatively throughout the cell cycle to keep prb in a phosphorylated state. Such cooperativity could prevent premature inhibition of cell cycle progression by prb should dephosphorylation of prb, or de novo synthesis of prb, occur after the expression of the initial kinase. Circumstantial support for this view comes from a system in which prb is induced to overexpress in S phase (68). This mis-timed overexpression of prb results in cell cycle arrest in G2, possibly because insufficient inactivating kinases are present at this time. Continual hyperphosphorylation and inactivation of prb in normal cells could be required for smooth transition through the cell cycle. Thus, the G1 through S phase cyclins may cooperatively inactivate prb and allow cells to progress through the cell cycle. Materials and Methods Cell Culture. Saos-2 cells (obtained from American Type Culture Collection) were maintained in high-glucose DMEM supplemented with l0% fetal bovine serum and penicillin/streptomycin. CV1 cells were maintained in DMEM with 1 0% calf serum. All cells were grown at 37#{176}C in 5% CO2. Plasmids. Construction of the RB-i expression vectors prb-3ha-sve and prb-3ha-tm1 is described elsewhere (12). cd clones for human cyclins A and B were from J. Pines (The Wellcome/CRC Institute, University of Cambridge, Cambridge, UK), human cyclin E was from S. Reed (Scripps Research Institute, La Jolla, CA) and human cyclin Dl was from A. Arnold (Massachusetts General Hospital, Boston, MA). Murine cyclins D2 and D3 cd clones were from C. Sherr (St. Judes Childrens Research Hospital, Memphis, TN). A CDK2 cd clone was obtained from E. Harlow (Massachusetts General Hospital, Charlestown, MA), one for human CDC2 was from P. Nurse (Oxford University, Oxford, UK), and one for CDK4 was from S. Hanks (Vanderbilt University, Nashville, TN). All were cloned into the polylinker of the psve vector. A cd clone for p21 (waf-i) was obtained from B. Vogelstein (Johns Hopkins University, Baltimore, MD). T7-based expression vectors were constructed using the vector ptmi (39). Coexpression of an epitope-tagged version of prb using this vector has been described previously (69). For this study, we used a synthetic epitope, termed FE, derived from the medium T antigen of polyoma virus (70). The FE epitope was ligated into the 3 -end of the prb coding region using synthetic oligonucleotides, resulting in a fusion protein ending with a protein sequence of LDGP- GEEEEYMPME-term, substituted for the natural sequence prb FEK-term. Vectors expressing CDK cds were constructed by ligation into the polylinker of ptm1, in all cases using the Ncol site of TM1 blunted with mung bean nuclease to ensure translation from the authentic initiator codon. Cyclin cds were expressed using an epitope-tagged variant of the ptmi vector constructed by Mark Esser in our laboratory, wherein coding sequence for the peptide MHEEEEYMPMEGP- precedes the ATG present within the Ncol site at the 5 -end of the polylinker. In the cases of

11 Cell Growth & Differentiation 405 cyclins A, B, and Dl,cDs were ligated into the Ncol site of ptml,resulting in expression of a fusion protein encoding this sequence. For cyclins D2, D3, and p21,an NcoI site was introduced at the initiator ATG by PCR, and the modified fragment was subsequently fused by ligation into the epitope-encoding vector. An FE epitope-tagged cyclin E was constructed by blunt ligation of a restriction site preceding the initiator codon into a blunted Ncol site of FE TM1, resulting in an epitope with sequence MHEFEEYMP- MEGPMGINSRDAKERDT preceding the coding region of cyclin F. The Di-7GH mutant was created by site-directed mutagenesis using a variation of the mega primer mutagenesis (71 ). The correct mutation was confirmed by sequencing. In some experiments, a cytomegalovirus vector containing the amino terminal coding sequence for the peptide MHEEEEYMPME was used.6 p21 (Tables 1, 2, and 3), cyclin Dl (Table 3 only) and cyclin Dl-7GH (Table 3) were fused by ligation into the epitope-containing vector. Saos-2 Growth Inhibition Assay. For assays counted on a Coulter counter, about i0 Saos-2 cells on a 9-cm tissue culture dish were transfected by the calcium phosphate transfection protocol according to Chen and Okayama (72). Transfections included mixtures of: (a) 20 ig prb plasmid together with (b) 10 pg competitor plasmid encoding either a cyclin, cyclin plus kinase E1A, or psve empty vector plasmid; and (c) 1 pg of puromycin resistance plasmid pbabfpuro (73). After incubation at 2.5% CO2 for 6 to 10 h, the medium was removed and replaced with DMEM plus 1 0% FBS. Twenty-four h after transfection, the cells were trypsinized and replated onto new 9-cm plates. Forty-eight h after transfection, cells were treated with medium contaming 1 mg/mi puromycin (Sigma Chemical Co.) and then refed at 3-day intervals for 7 to 10 days. To remove dead cells from the plate, cultures were washed in HFPFS-buffered saline and then trypsinized and allowed to reattach. Twenty-four h after reattachment, the cells were again trypsinized and counted using a Coulter counter, with amperage set at 4, threshold at 10, and aperture current at 8. For assays counted by microscope, about 1 0 cells on a 24-well dish were transfected as above using 1 0-fold less amounts of plasmid in the transfections. Transfections without prb mixes included 2 pg cyclin or kinase with 1 pg kinase or psvf empty vector plasmid. Cells were treated identically until the day of counting, except for treatment with trypsin 1 day before counting. The day of counting, the plates were rinsed four times with 1 00% methanol and stained with 0.1 % methylene blue. Four fields at X 1 00 from each well were then counted and averaged. Expression of prb in Saos-2 Cells and Western Blot Analysis. Approximately 5 X 1 0 Saos-2 cells plated on a 35-mm dish the night before were infected with vaccinia virus vector vtf7-3 (39) encoding the phage T7 R polymerase. The cells were subsequently lipofected (74) for 3-4 h with 5 pg of ptml expression plasmids. For cotransfection of cyclins and/or kinases and/or p21 and RB-i,5 pg of each plasmid D was used, except where noted. For Western blots shown in Figs. 1, 3, and 4, 24 h after lipofection, cells were lysed in MLB (25 mti morpholinepropanesulfonic acid (ph 7.0), 250 mi NaCI, 5 mist EDTA, 0.1% NP4O, 1 mm DTT, 1 mg/mi aprotinin, 1 mg/mi 6 D. J. Templeton, unpublished observation. leupeptin, and 50 mg/mi phenylmethylsulfonyl fluoride). Lysates were clarified by centrifugation, and 1 % of the supernatant was run on 6% SDS-PAGE. After electrophoresis and transfer to Immobilon-P filters (Millipore), prb was detected with mab24s (PharMingen). In all experiments, equal fractions of each transfection (representing protein recovered from the same number of cells) were analyzed. In Vivo 32P-Iabeling of prb in Saos-2 Cells for CNBr Cleavage Analysis. Approximately 5 X 1 0 Saos-2 cells were infected/transfected as described above. After 24 h, cells were starved for 4 h in phosphate-free medium supplemented with 2% dialyzed serum and then labeled for 4 more hours using 0.5 mci [32P]P1 in 0.5 ml medium. They were then lysed in 1 X MLB [as above plus phosphatase inhibitors (1 0 mist -glyceroi phosphate, 1 0 mist para-nitrophenyiphosphate, 4 mist sodium PP. 1 mist sodium orthovanadate and 20 mist sodium fluoride)], clarified by centrifugation, and immunoprecipitated using 40 p1 of a 1 :1 slurry of FF antibody conjugated to agarose beads [5 mg EE antibody/mi of Affigel 10 (BRL); anti-ee beads ]. Samples were washed three times in 1 X MLB, once in 50 mist Tris (ph 7.4), 1 mm DTT (TD), and analyzed on a 6% SDS/ PAGE. CNBr Cleavage Analysis of in Vivo-labeled prb. Radioactive prb bands from the above gel were excised and treated with 50 mg/mi cyanogen bromide in 70% formic acid at room temperature for 1 h before analysis on a 16.5% SDS-PAGE using the Tricine buffer system (75). In Vivo 32P-labeling of prb in Saos-2 Cells for Chymotryptic Cleavage Analysis. Approximately 1 0 Saos-2 cells were infected/transfected as described above, except that the prb was tagged with the epitope HA. After 24 h, cells were starved for 4 h in phosphate-free medium supplemented with 2% dialyzed serum and then labeled for 4 more hours using 1.5 mci [32P]P1 in 0.5 ml medium. They were then lysed in 1 X MLB [as above plus phosphatase inhibitors (1 0 mm n-glycerol phosphate, 1 0 mist para-nitrophenylphosphate, 4 mri sodium PP1, 1 mist sodium orthovanadate, and 20 mist sodium fluoride)], clarified by centrifugation, and immunoprecipitated using 40 p1 protein A beads and 2 p1 each 21 C9 (anti-prb) ascites, 12CA5 (anti-ha) ascites, and Rb-02 (PharMingen). Samples were washed three times in 1 X MLB, once in 50 misi Tris (ph 7.4), and 1 mm DTT (TD) and analyzed on a 6% SDS-PAGE. Chymotryptic Cleavage Analysis of in Vivo-labeled prb. Radioactive prb bands from the above gel were excised and soaked in 0.5% PVP-360 (polyvinylpyrrolidone; Mr 360,000) in 1 00 mm acetic acid for 30 mm at 37#{176}C. The membrane was washed four times in water and once in 0.05 M ammonium bicarbonate. Proteins were digested with 2 pg chymotrypsin (Boehringer-Mannheim) overnight at 37#{176}C, vortexed, and digested with an additional 2 pg for 2 more hours at 37#{176}C. Samples were then dried on a speed vac and then oxidized by treatment with performic acid for 1.5 h. Samples were again dried on a speed vac, resuspended in 5 p1 water, and loaded at or 2500 cpm/tlc plate. Samples were electrophoresed at 1 kv for 27 mm in ph 8.9 buffer (i% w/v ammonium carbonate) and chromatographed for 6 h in Phosphochromo buffer (37.5% v/v n-butanol, 25% v/v pyridine, and 7.5% v/v acetic acid). In vitrokinaseassay. Approximately i0 CV1 cells were infected/transfected as described above with either EFtagged expression vectors (for cyclins), HA-tagged expression vectors (for prb), or untagged expression vectors (for kinases). Cells were lysed in MLB, and proteins were rn-

12 406 Upstream Regulation of prb Activity by Gi Cyclins munoprecipitated as described above. The protein A-HA antibody-prb complex was washed three times in 4 M lithium chloride, once in MLB, once in TD, and resuspended in TD. The anti-ee-bead-cyclin/kinase complexes were washed in MLB only (omitting the lithium chloride washes) and resuspended in TD; then proteins were eluted into 25 p1 TD containing 0.1 mg/rnl ofthe peptide cognate to the FE antibody (EEEEYMPME), 1 0 mist 3-glyceroI phosphate, 1 mist sodium orthovanadate, and 20% glycerol. Equal aliquots of purified prb were mixed with 2 p1 of cyclins A or F or D-type cyclins in a final reaction mixture of 20 p1 containing TD, 10 mm MgCl2, and 20 pci of [ y-32p]atp (6000 Ci/rnmol) per reaction. Samples were run and analyzed by 6% SDS/PAGF, transferred to lmmobilon-p filters (MilIipore), and analyzed by autoradiography. In Vivo [35SjMethionine Labeling of prb and Cyclins in Saos-2 Cells. Approximately 5 X 1 0 Saos-2 cells were infected/transfected as described above with either all cyclin/kinase combinations plus prb (Fig. 1 B) or with Dl! CDK4 or D1-7GH/CDK4 plus prb (Fig. 6). After 24 h, cells were starved for 4 h in methionine-free medium supplemented with 2% dialyzed serum and then labeled for 4 more hours using 20 pci [35S]methionine in 0.5 ml medium. They were then lysed in 1 X MLB (as above), clarified by centrifugation, and immunoprecipitated using 20 p1 of anti-fe beads (as described above). Samples were washed three times in 1 X MLB, once with TD, and electrophoresed on a 12.5% SDS/PAGE. This gel was then subjected to fluorography by incubation in l00% glacial acetic acid for 30 mm, followed by incubation in PPO for 40 mm. The gel was then rinsed for 1 5 mm in lukewarm water four times. The gel was analyzed by autoradiography. Acknowledgments We thank Mark Esser, Jeremy Sanford, Carol Luckey, Jeff Stear, and Margaret Lewis for technical assistance; Minhong Yan for help with peptide mapping; and Fred Moeslein for critical reading of the manuscript. We also thank Kathy O Connor and Dr. Lloyd CuIp for the suggestion and use of their Coulter counter. We also thank Drs. Jonathan Pines, Steve Reed, Andrew Arnold, Charles Sherr, Ed Harlow, Paul Nurse, Steven Hanks, and Burt vogelstein for providing useful clones and reagents. References 1. Lee, W. H., Shew, J. Y., Hong, F. 0., Sery, T. W., Donoso, 1. A., Young, L. I., Bookstein, R., and Lee, E. Y. The retinoblastoma susceptibility gene encodes a nuclear phosphoprotein associated with D binding activity. Nature (Lond.), 329: , Horowitz, J. M., Park, S. H., Bogenmann, E., Cheng, J. C., Yandell, 0. W., Kaye, F. J., Minna, J. 0., Dryja, T. P., and Weinberg, R. A. Frequent inactivation of the retinoblastoma anti-oncogene is restricted to a subset of human tumor cells. 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