Oncogene (2000) 19, 562 ± 570 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc Glutamic acid mutagenesis of retinoblastoma protein phosphorylation sites has diverse e€ects on function

Sylvia Barrientes1, Carolyn Cooke1 and David W Goodrich*,1

1Department of Cancer Biology, Box 108, University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, Texas, TX 77030, USA

The retinoblastoma tumor suppressor gene (Rb) has 1997), to repress transcription (Bremner et al., 1995; many functions within the cell including regulation of Sellers et al., 1995), and to associate with a number of transcription, di€erentiation, apoptosis, and the cell cellular proteins including the E2F family of transcrip- cycle. Regulation of these functions is mediated by tion factors (Suzuki-Takahashi et al., 1995; Knudsen phosphorylation at as many as 16 cyclin-dependent and Wang, 1997). The kinases responsible for pRb kinase (CDK) phosphorylation sites in vivo. The phosphorylation in vivo likely include members of the contribution of these sites to the regulation of the cyclin-dependent kinase (CDK) family. Phosphoryla- various Rb functions is not well understood. To tion of pRb coincides with the activation of CDKs in characterize the e€ect of phosphorylation at these sites, vivo (Chen et al., 1989; DeCaprio et al., 1989; we systematically mutagenized the serines or threonines Buchkovich et al., 1989; Mihara et al., 1989), and to glutamic acid. Thirty-®ve mutants with di€erent pRb serves as an ecient substrate for CDKs in vitro combinations of modi®ed phosphorylation sites were (Taya et al., 1989; Lees et al., 1991; Lin et al., 1991; assayed for their ability to arrest the cell cycle and for Kato et al., 1993; Knudsen and Wang, 1996; Connell- their potential to induce di€erentiation. Only the most Crowley et al., 1997). Since CDKs can phosphorylate highly substituted mutants failed to arrest cell cycle pRb in vitro on most, if not all, of the sites that are progression. However, mutants with as few as four modi®ed in vivo, they probably play a major role in the modi®ed phosphorylation sites were unable to promote regulation of pRb. di€erentiation. Other mutants had increased activity in Phosphorylation of pRb is complex. There are 16 this assay. We conclude that modi®cation of Rb consensus CDK phosphorylation sites distributed phosphorylation sites can increase or decrease protein throughout pRb (Figure 1) and most of these are activity, that di€erent Rb functions can be regulated utilized in vivo (Lees et al., 1991). Seven consensus sites independently by distinct combinations of sites, and that are clustered in the C-terminal domain, two sites reside the e€ects of modi®cation at any one site are context in the spacer region between the minimal domains dependent. Oncogene (2000) 19, 562 ± 570. required for SV40 T antigen binding, and the remaining sites are in the N-terminal third of the Keywords: retinoblastoma; phosphorylation; cyclin- protein. Attempts have been made to unravel the dependent kinase; mutagenesis regulation of pRb function by blocking phosphoryla- tion at consensus CDK phosphorylation sites by alanine substitution (Hamel et al., 1992). Since Introduction replacement of serine or threonine with alanine prevents phosphorylation, mutants are assayed for Mutational inactivation of the retinoblastoma tumor resistance to inactivation by phosphorylation either in suppressor gene is the cause of the pediatric cancer vitro or in cells that eciently phosphorylate pRb. retinoblastoma, and deregulation of the Rb-mediated Through this type of analysis, the seven sites in the C- growth control pathway may contribute to the genesis terminal domain, the two sites in the spacer region, and of most human tumors (Goodrich and Lee, 1990; some N-terminal sites have all been implicated as being Sherr, 1996). The Rb protein (pRb) functions to important for regulation of pRb-mediated function restrict transit of the cell cycle, particularly during (Lukas et al., 1997; Knudsen and Wang, 1997; Chew et the initial stages of G1 (Goodrich et al., 1991; Qin et al., 1998; Brown et al., 1999). Phosphorylation of one al., 1992; Sherr, 1996) and possibly during S phase of the C-terminal sites in vitro, serine 795, is required (Knudsen et al., 1998; Chew et al., 1998). This pRb- to inhibit pRb-mediated G1 arrest upon microinjec- mediated block to cell proliferation is thought to be a tion, although other sites may be required as well central component of the restriction point and, there- (Connell-Crowley et al., 1997). Phosphorylation of fore, important for normal growth and di€erentiation either the seven C-terminal sites or the two spacer (Weinberg, 1990). sites is sucient to inhibit E2F-1 binding in vitro The function of pRb is regulated by phosphoryla- (Knudsen and Wang, 1997). Phosphorylation of S807 tion. Phosphorylation of pRb inhibits its ability to and/or S811 is required for inhibition of pRb/Abl restrict cell cycle progression (Hinds et al., 1992; Ewen binding in vitro while T821 and/or T826 phosphoryla- et al., 1993; Dowdy et al., 1993; Connell-Crowley et al., tion is necessary to prevent pRB/SV40 T antigen association in vitro (Knudsen and Wang, 1996). The contribution of individual phosphorylation sites to regulation of Rb function is less clear since, with the *Correspondence: DW Goodrich Received 31 August 1999; revised 25 October 1999; accepted 27 exception of S795, no single site has been demonstrated October 1999 to be required for regulation of pRb function in cells. Phosphorylation site mutagenesis alters Rb function S Barrientes et al 563 An alternative approach to phosphorylation site analysis is glutamic acid substitution mutagenesis. The rationale for this analysis is based on the assumption that glutamic acid mimics the structure of a phosphorylated serine or threonine residue. In contrast to alanine substitution, glutamic acid substitu- tion has the potential to directly assess the e€ects of modi®cation at particular sites without the need for additional phosphorylation in vitro or in cells. We have systematically mutated serine or threonine residues at consensus CDK phosphorylation sites to glutamic acid. A collection of 35 mutants with varying combinations of modi®ed phosphorylation sites has been tested in two assays of Rb function. We con®rm the importance of multiple C-terminal and spacer region sites for Rb- induced cell cycle arrest as ®rst determined by alanine substitution mutagenesis. Unexpectedly, the ability of pRb to induce di€erentiation is inactivated by as few as four modi®ed sites and mutants containing as many as six modi®ed sites have increased activity. These ®ndings raise the possibility that phosphorylation may activate, as well as inactivate, pRb function and that di€erent functions of pRb may be regulated indepen- dently by phosphorylation of particular combinations of sites.

Results Figure 1 Glutamic acid substitution mutations made in Rb phosphorylation sites. A schematic of pRb is presented at the top Rb glutamic acid substitution mutants indicating the extent of the A, B, and C domains. Wild-type pRb contains 16 consensus proline-directed phosphorylation sites The Rb protein contains 16 consensus CDK phosphor- indicated by arrowheads. The amino acid residue and number ylation sites (Figure 1). Seven of these sites are of each phosphorylation site is listed. The phosphorylation site contained within a 50 amino acid stretch within the mutants described in this study are shown, with the particular sites mutated to glutamic acid in each mutant indicated by a ®lled C-terminal domain of the protein. This domain is square. The names of each mutant indicate the total number of sucient for c-Abl binding (Welch and Wang, 1993) glutamic acid substitutions contained in that mutant. Mutants are and is required for association with many non-LXCXE constructed as described in Materials and methods. All mutants, containing pRb-associated proteins like E2F-1 (Huang except 56M10, are expressed as full-length proteins. Translation et al., 1992; Qin et al., 1992). Two sites are located in of 56M10 likely begins M379 producing a protein that lacks the N-terminal third of wild-type Rb the spacer region between the A and B domains. The A and B domains comprise the minimal sequence required for association with SV40 T antigen (Huang et al., 1990; Hu et al., 1990). One site, S563, is located within the A domain but its phosphorylation in vivo is This observation may indicate that regulation of pRb uncon®rmed. The remaining sites are distributed occurs by accumulation of a threshold quantity of throughout the N-terminal third of the protein. The phosphorylated residues (Brown et al., 1999). Alter- function of the N-terminal region is unknown since the natively, qualitative di€erences in the e€ects of A, B, spacer, and C domains are sucient for activity phosphorylation at individual sites may go unnoticed in most assays of pRb function. This region is, because of potential complications in alanine substitu- however, required for normal biological function in tion experiments. For example, phosphorylation may vivo (Riley et al., 1997). Regions of this N-terminal not uniformly decrease protein activity. Phosphoryla- domain are required for association with three known tion at some sites may activate, rather than inhibit, a cellular proteins whose signi®cance for Rb function are particular pRb function. Also, blocking phosphoryla- uncharacterized (Durfee et al., 1994; Inoue et al., 1995; tion at one site may a€ect the eciency with which Sterner et al., 1998). remaining wild-type sites are phosphorylated in vitro or We chose to focus our mutagenesis on 12 of the in cells. Alanine substitution at S795, for example, consensus sites including the seven C-terminal sites, the alters the sites phosphorylated by cyclin D1/CDK4 in two spacer region sites, and three sites within the N- vitro (Connell-Crowley et al., 1997). The ability of the terminal domain. These sites were chosen based on the remaining, wild-type sites to be phosphorylated may criteria that they are con®rmed phospho-acceptors in also vary depending on the cell line or kinase vivo. Using site-directed mutagenesis 35 mutants were preparation used. Finally, the e€ects of phosphoryla- created with various combinations of the serines or tion at a particular site may depend on the context of threonines substituted with glutamic acid (Figure 1). The other modi®ed sites. The impact of modi®cation at one name of each of the mutants was based on the total site, therefore, may be revealed under some circum- number of mutated phosphorylation sites with di€erent stances but not others. combinations of the same number of sites designated by

Oncogene Phosphorylation site mutagenesis alter Rb function S Barrientes et al 564 A, B, C, etc. The mutants were all expressed in the same Since the various mutant expression vectors were vector under control of the cytomegalovirus promoter identical, excepting the base changes at the mutated and the small T antigen polyadenylation site. All of the codons, we considered the possibility that lack of mutants, except 56M10, were expressed as full-length expression in some mutants was due to inecient proteins. The 56M10 mutant expresses an N-terminally transcription or translation unlikely. However, lack of truncated form of the M10 mutant that retains the A, detectable protein expression from such mutants could spacer, B, and C domains. be due to acquisition of inadvertent mutations. To rule out this possibility, we sequenced the entire Rb cDNA from the M5C mutant and found that its nucleotide Expression of mutant Rb proteins sequence was identical to the sequence of the wild-type Extracts containing equivalent amounts of total cell pRb cDNA, excepting the changes at the altered protein prepared from transfected 293 cells (Figure 2) phosphorylation sites (S Barrientes, 1999, unpublished were resolved by SDS ± PAGE. Rb protein was observations). detected by Western blotting and staining with an anti-pRb rabbit polyclonal antibody. Although 293 Rb protein function is inhibited by glutamic acid cells expressed endogenous pRb, under the conditions substitution at phosphorylation sites used endogenous protein was undetectable. Most of the mutants expressed protein at a level approximately A number of the mutants have been analysed for their equal to that of wild-type pRb. activity in the `¯at cell' assay upon transfection of the Although most of the mutants expressed similar indicated Rb expression vectors and pEGFP-C1 into levels of protein, there were some notable exceptions. SAOS-2 cells. In this assay the appearance of isolated, The M10, M9 and M3 mutants expressed lower levels non-dividing cells with a characteristic ¯at morphology of pRb. Several mutants did not express any detectable is counted after selection of successfully transfected protein. These mutants included M6A, M5C, M4B, cells by growth in the presence of G418 for 2 weeks. M4C, and M4D. Even upon Western analysis of These ¯at cells typically express markers indicative of greater amounts of total protein where endogenous di€erentiation, hence the assay is a measure of the pRb was detectable, no increase in pRb above ability of pRb to promote di€erentiation and is distinct endogenous levels could be detected in cells transfected from the ability of pRb to arrest G1 cell cycle with the M5C mutant (D Goodrich, 1999, unpublished progression (Sellers et al., 1998). Since activity in this observations). The failure of the M5C mutant to make assay is directly proportional to transfection eciency, a detectable level of full-length protein was reproduci- we determined transfection eciency for each culture 1 ble in multiple, independent transfections. Qualitatively day post-transfection by counting the percentage of similar results were observed in SAOS-2 cells, although GFP-positive cells (data not shown). Transfection the general level of protein expression detected was eciencies ranged from 8 ± 10% in di€erent experi- lower, likely because of lower transfection eciencies ments, but there was no consistent di€erence among (D Goodrich, 1999, unpublished observations). the various mutant plasmids that would potentially bias the results. Several of the mutants tested were de®cient in ¯at cell formation with activity indistinguishable from the empty vector control (Figure 3). While several of the most highly substituted mutants like M12, M8, and M7 were inactive, there was no correlation between the total number of sites mutated and activity. For example, M10 demonstrated activity comparable to

Figure 2 Expression of pRb in 293 cells transfected with mutant Figure 3 Flat cell assay of Rb phosphorylation site mutants. expression plasmids. Protein extracts from cells transfected with The indicated mutant expression plasmids were assayed for ¯at the indicated expression plasmids were analysed for Rb protein by cell formation as indicated in Materials and methods. The SDS ± PAGE and Western blotting as described in Materials and number of ¯at cells per microscope ®eld for each mutant is methods. The numbers on the left show the position of molecular expressed as a percentage of the value for wild-type Rb. The weight standards of the indicated molecular mass while the values are the mean of at least three independent experiments position of full-length Rb protein is shown at right with error bars equal to one standard deviation from the mean

Oncogene Phosphorylation site mutagenesis alters Rb function S Barrientes et al 565 pRb while M4E had no detectable activity above the G1 cells about 10% compared to a typical increase of empty vector control. Several of the mutants showed 27% for pRb. Many of the mutants, including M12, activity that was signi®cantly greater than pRb, in M8, M7, M5B, and M4E, had no detectable ¯at cell particular mutants M6 and M2B. These mutants activity yet did demonstrate signi®cant cell cycle arrest consistently generated threefold the number of ¯at activity. The M6 mutant, on the other hand, generated cells generated by pRb. Other mutants, such as M3 three times more ¯at cells than wild type yet had no and M3D, generated a quantity of ¯at cells inter- greater a€ect on G1 arrest. mediate between wild-type pRb and empty vector The di€erences in the behavior of particular mutants control, although M3 activity may be low because of in each of the assays may, in part, re¯ect the sensitivity low protein expression (see above). As expected, of the di€erent assays to a given amount of pRb. In mutants that failed to produce detectable Rb protein, this case, mutants with diminished activity might go including M5C, were also unable to eciently produce undetected if the amount of protein expressed was in ¯at cells in this assay. vast excess of that required to elicit a response. To address this possibility, we determined the sensitivity of the cell cycle arrest assay to the amount of Rb plasmid Cell cycle arrest induced by pRb mutants DNA used in the transfection. Both the ¯at cell and Knudsen and Wang (1996, 1997) have demonstrated cycle arrest assays were performed with 25 ± 27 mgof that modi®cation of certain subsets of pRb phosphor- Rb DNA in a total of 30 mg DNA per transfection. ylation sites di€erentially a€ects binding to various While keeping the total amount of DNA constant, we pRb-associated proteins. Using naturally occurring, titrated the amount of wild-type Rb expression plasmid non-phosphorylation site Rb mutants, Sellers et al. DNA used and measured the increase in percentage of (1998) have shown that di€erent functions of pRb, G1 cells in transfected SAOS-2 cells. A maximum such as induction of cell cycle arrest or di€erentiation, increase in the percentage of G1 cells was achieved are independent of one another. Based on these ®ndings, we wished to determine if modi®cation of phosphorylation sites could di€erentially regulate distinct pRb functions. We tested each of the mutants for their ability to induce G1 arrest upon transfection into SAOS-2 cells. Successfully transfected cells were analysed for their DNA content to determine their cell cycle phase distribution. Cells successfully transfected with pRb contained a signi®cantly higher percentage of cells with G1 DNA content than untransfected cells, indicative of its ability to induce a G1 phase cell cycle arrest. In contrast, cells transfected with the empty vector had a slightly lower percentage of G1 cells relative to untransfected cells. Excepting the mutants that fail to express detectable protein, most of the mutants caused a signi®cant increase in the percentage of G1 cells (Figure 4). Although most of the mutants demonstrated statistically signi®cant activity above the empty vector control, some of the mutants generated smaller changes in cell cycle distribution than pRb. For example, M12 consistently increased the percentage of

Figure 5 G1 phase cell cycle arrest assayed at low ratio of Rb to total DNA. (a) The Rbwt expression plasmid was assayed for G1 phase cell cycle arrest as described in Materials and methods using the indicated amount of expression plasmid DNA in a transfection containing 30 mg total DNA. The increase in the percentage of G1 cells in transfected cells relative to untransfected cells is shown. The values are normalized to the CMV control which was set to zero. The results are the mean of at least three Figure 4 Cell cycle arrest assay of Rb phosphorylation site independent experiments with error bars of one standard mutants at high ratios of Rb to total DNA. The indicated mutant deviation from the mean. (b) The indicated mutant expression expression plasmids were assayed for G1 phase cell cycle arrest plasmids were assayed for G1 phase cell cycle arrest activity as activity as described in Materials and methods using 27 mgof described in Materials and methods using 2 ± 3 mg of expression expression plasmid DNA in a transfection containing 30 mg total plasmid DNA in a transfection containing 30 mg DNA. The DNA. The increase in the percentage of G1 cells in transfected increase in the percentage of G1 cells in transfected cells relative cells relative to untransfected cells is shown. The values are to untransfected cells is shown. The values are normalized to the normalized to the CMV negative control which was set to zero. CMV negative control which was set to zero. The results are the The results are the mean of at least three independent experiments mean of at least three independent experiments with error bars of with error bars of one standard deviation from the mean one standard deviation from the mean

Oncogene Phosphorylation site mutagenesis alter Rb function S Barrientes et al 566 Table 1 Oligonucleotides used for site-directed mutagenesis Mutation Oligonucleotides 788E CATTCCTCAgagCCTTAC 795E TTTCCTAGTgaaCCCTTACGG 821E GTCTGCCAgaaCCAACAAAAATG 612E CTCCTGTAAGAgagCCAAAGAAAAAGG 608E+612E GCAGATATGTATCTTgagCCTGTAAGAgagCCAAAGAAAAAAGG 373E CTCCACACgagCCAGTTAGG S788 CATTCCTCGAagcCCTTAC S795 GTTTCCTAGTtcaCCCTTACGG S608+612E GCAGATATGTATCTTtctCCTGTAAGAgagCC 608E+S612 gagCCTGTAAGAtctCCAAAGAAAAAAGG 807E+811E GGGAACATCTATATTtaaCCCCTGAAGgagCCATATAAAATTTC 807E+S811 gaaCCCCTGAAGagtCCATATAAAATTTC S807+811E GGGAACATCTATATTtcaCCCCTGAAGgagCC 780E CCCTACCTTGgaaCCAATACCTC 826E CCAACAAAAATGgagCCAAGATCAAG 249E+252E CCCATTAATGGTgaaCCTCGAgaaCCCAGGCGAGG The sequence of the oligonucleotides used as primers to create the listed base changes at the indicated phosphorylation sites are presented. For example, 788E introduces a glutamic acid at amino acid 788 of the pRb sequence. The lower case bold face sequence indicates the position and sequence of the mutated codons

Table 2 Construction of pRb phosphorylation site mutants control. Other mutants behaved similarly to wild-type Mutanta Oligonucleotideb Template usedc pRb, including mutant M7 and M4E, both of which 788 788E Wild-type expressed stable protein but were inactive in the ¯at 795 795E Wild-type cell assay. Hence, some pRb mutants were clearly 821 821E Wild-type M2 821E 795 active for cell cycle arrest at minimum threshold levels M2A 788E 795 of transfected DNA while they were inactive in M2B 821E 788 inducing ¯at cells at high levels of transfected DNA. M2C 608E+612E Wild-type Other mutants, like M12, M9 and M8, had partially M2D 612E 821 compromised cell cycle arrest activity and undetectable M2E 612E 788 M2F 612E 795 ¯at cell activity. Most of the remaining mutants, M3 788E M2 including mutants containing one or two phosphoryla- M3A 821E M2C tion site alterations, were as active as wild-type Rb M3B 612E M2B protein in both ¯at cell and cell cycle arrest assays. A M3C 612E M2 M3D 612E M2A summary of the results is presented in Table 3. M4 373E M3 M4B S788 M5C M4C S795 M5C Discussion M4D S608+612E M5C M4E 608E+S612 M5C M5 807E+811E M3 Interpretation of the experiments presented here is M5A 373E M4A based on the assumption that glutamic acid substitu- M5B 807E+S811 M4A tion mimics the phosphorylated state of the native M5C 608E+612E M3 serine or threonine residues. Since most of the mutants M6 373E M5C M6A 807E+S811 M5C did retain some activity, it is unlikely that the glutamic M6B 807E+811E M4A acid substitutions drastically altered the structure of M6C 373E M5 the protein. Previous work has shown that Rb mutants M7 608E+612E M5 substituted with other amino acids, including alanine, M8 373E M7 arginine, and proline, at phosphorylation sites also M9 826E M8 M10 780E M9 retain activity (Knudsen and Wang, 1997; Brown et al., M12 249E+252E Wild-type 1999). Flexibility in the structure of the protein in aThe Rb mutant as diagrammed in Figure 1. bThe oligonucleotide regions containing phosphorylation sites is also con- used for mutagenesis as listed in Table 1. cThe mutant single stranded sistent with the available crystallographic data (Lee et M13 DNA used as template for mutagenesis al., 1998). The assumption also requires that the negative charge introduced by glutamic acid substitu- tion has e€ects similar to phosphorylation of the native with as little as 2 ± 3 mg of pCMVRbwt DNA in a serine or threonine residues. There is ample precedent transfection using a total of 30 mg DNA (Figure 5a). for this in the literature for a number of proteins We then tested selected mutants for cell cycle arrest (Ducommun et al., 1991; Maciejewski et al., 1995; using 2 ± 3 mg of Rb expression vector in a transfection Pearson et al., 1995; Engel et al., 1995; Knauf et al., containing 30 mg total DNA. Under these conditions, 1996; Yang et al., 1998; Chen et al., 1999). The fact mutants M12, 56M10, M9, and M8 all caused a that our results are consistent, in general, with previous smaller increase in the percentage of G1 cells than ®ndings using alternative approaches suggests that this wild-type pRb (Figure 5b). However, with the possible is also true for pRb. exception of M12 they still caused an increase in the The seven C-terminal sites and the two spacer region percentage of G1 cells relative to the empty vector sites have previously been implicated by alanine

Oncogene Phosphorylation site mutagenesis alters Rb function S Barrientes et al 567 Table 3 Summary of the e€ects of phosphorylation site mutations phosphorylation is required for inactivation of pRb on pRb function function in a microinjection assay and S788 as a Rb mutanta Protein Flat cellsc G1 arrestd preferred substrate for both cyclin D1/Cdk4 and cyclin Rb wild-type ++ ++ ++/++ A or E/Cdk2 in vitro. The data presented here also M12 ++ 7 +/7 M10 + ++ ++/++ con®rm the ®nding that N-terminal sites can in¯uence 56M10 N.D. N.D ++/+ the regulation of Rb function (Brown et al., 1999). For M9 + ++ ++/+ example, M10 demonstrates activity in both the ¯at cell M8 ++ 7 ++/+ and cell cycle arrest assays. Addition of the N-terminal M7 ++ 7 ++/++ modi®cations S249E and S252E to M10 results in M12 M6 ++ +++ ++/++ M6A 7 N.D. 7/7 that is de®cient in both ¯at cell and cell cycle arrest M6B N.D. N.D. N.D./++ assays. M6C ++ ++ ++/++ While the data con®rmed the importance of C- M5 ++ N.D. ++/++ terminal, spacer region, and N-terminal sites, several M5A N.D. N.D. ++/++ M5B ++ 7 N.D./++ unexpected observations were made as well. First, there M5C 7 7 7/7 was no strict correlation between the number of sites M4 + ++ ++/++ modi®ed and the activity of the protein. Several M4A ++ ++ ++/++ mutants with a large number of modi®ed sites had M4B 7 7 7/7 greater activity than mutants with fewer phosphoryla- M4C 7 7 +/7 M4D 7 7 +/7 tion site modi®cations. For example, M10 with ten M4E + 7 ++/++ modi®ed sites had greater ¯at cell activity than M4E M3 + + ++/++ containing four modi®ed sites. M10 contained all the M3A ++ ++ ++/N.D. modi®cations in M4E as well as six others. M8 was M3B ++ N.D. ++/N.D. M3C ++ ++ ++/++ de®cient in ¯at cell activity but adding the T826E M3D ++ ++ ++/N.D. modi®cation to create M9 resulted in a protein with M2 N.D. ++ ++/N.D. signi®cant ¯at cell activity. The M9 mutant was M2A N.D. +++ ++/N.D. de®cient in cell cycle arrest activity at low levels of M2B N.D. +++ ++/N.D. transfected DNA. Addition of the S780E modi®cation M2C ++ ++ ++/N.D. M2D ++ ++ ++/N.D. to create M10 increased cell cycle arrest activity to M2E + N.D. ++/N.D. wild-type pRb levels. These ®ndings suggest that M2F ++ N.D. ++/N.D. phosphorylation of Rb protein can increase, as well 788 N.D. N.D. ++/N.D. as decrease, activity. Second, the e€ects of modi®cation 795 N.D. N.D. ++/N.D. 821 N.D. N.D. ++/N.D. at any one site were context dependent. For example, addition of the S780E modi®cation to M9 creates M10 aThe Rb mutant as diagrammed in Figure 1. bFrom Figure 3. ++, 50 ± 100% expression relative to Rbwt: +, less than 50% expression with greater cell cycle arrest activity. However the relative to Rbwt: 7, no detectable expression: N.D., not determined. addition of S780E had no e€ect on a mutant protein cFrom Figure 2. +++, greater than 150% Rbwt activity: ++, 50 ± lacking the N-terminus (56M10) or on a full-length 150% Rbwt activity: +, less than 50% Rbwt activity, but greater protein containing the S249E/S252E modi®cations in than CMV control: 7, less than or equal than CMV control: N.D., not determined. dFrom Figures 4/6. ++, greater than 15% increase the N-terminus (M12). Knudsen and Wang (1997) also in % G1 cells: +, 5 ± 15% increase in % G1 cells: 7, less than 5% observed variable e€ects of C-terminal phosphorylation increase in % G1 cells: N.D., not determined site modi®cation depending on the status of the N- terminus. Finally, exogenous protein expression could not be detected by Western blot analysis of extracts substitution mutagenesis as being important for from cells transfected with the M6A, M5C, M4B, regulation of pRb function. A cumulative e€ect of M4C, and M4D mutants. DNA sequencing and the these sites on E2F regulation and cell cycle arrest has fact that several plasmids derived from these mutant been observed (Knudsen and Wang, 1997; Brown et vectors did express Rb protein ruled out inadvertent al., 1999). C-terminal sites are also important for mutation as being responsible for this observation. binding c-Abl as well as proteins that utilize the Another possibility was that the mutations introduced LXCXE motif, like SV40 T antigen (Knudsen and destabilized Rb protein. If true, phosphorylation of Wang, 1996). Our results also indicate that C-terminal particular sites on Rb may target it for proteolysis. and spacer region sites are important for regulation of There is precedent for this since phosphorylation Rb function. For example, multiple phosphorylation targets the Rb family member p130 for destruction site modi®cations in the C-terminus and spacer region by the 26S proteasome (Smith et al., 1998). Interest- are required to compromise the ability of Rb to arrest ingly, all ®ve of these mutants contained the S612E and cell cycle progression. Only the most highly substituted T821E mutations and at least two of the S608E, mutants, such as M12, M9, and M8, have decreased S788E, or S795E mutations. Mutants containing ability to arrest SAOS-2 cells in G1. Further, the modi®cations in addition to these, such as M6, minimum number of modi®cations necessary to expressed detectable Rb protein. compromise ¯at cell activity among the mutants tested Sellers et al. (1998) has observed that some non- is four (M4E), and these four include both spacer phosphorylation site mutants are defective for G1/S region and C-terminal sites. Of these four, S788E and control yet are able to promote di€erentiation. Our S795E are the only modi®cations conserved in all of results extend these ®ndings by characterization of the mutants with compromised ¯at cell activity, mutants that are active in G1/S control yet are suggesting that these two sites may be particularly de®cient in promotion of di€erentiation. M4E and important for the regulation of this function. Connell- M7, for example, are as active as wild-type Rb in Crowley et al. (1997) also de®ned S795 as a site whose arresting cell cycle progression yet are inactive in the

Oncogene Phosphorylation site mutagenesis alter Rb function S Barrientes et al 568 ¯at cell assay. These results demonstrate that pRb has passage 1 : 4 one day prior to use and a change to fresh media multiple, independent functional activities. Our results 2 h before transfection. Transfections were performed using further extend this ®nding by suggesting that these the calcium phosphate precipitate method essentially as independent functional activities can be regulated described (Wigler et al., 1979). Brie¯y, 30 mg of DNA total independently by targeting phosphorylation to speci®c was used to prepare the DNA precipitate that was incubated with cells for 16 h (SAOS-2) or for 4 ± 6 h (293). Precipitate subsets sites. Rb protein, therefore, may coordinate cell was removed and the cells were washed 16 with phosphate growth by integrating di€erent regulatory pathways bu€ered saline (PBS), 16 brie¯y with PBS+2.5 mM EDTA, whose signals terminate in site-speci®c pRb phosphor- and 16 PBS before being re-fed with fresh media. The ylation. EDTA was omitted for transfections with 293 cells. Is phosphorylation of a threshold number of qualitatively similar sites important for pRb regulation, Expression plasmids and mutagenesis or are the e€ects of phosphorylation at each site qualitatively distinct? The answer seemingly depends The full-length human Rb coding sequence with a BamHI site on the particular function of pRb assayed. Our results engineered at the 5' end and a HindIII site engineered at the are consistent with previous studies in suggesting that 3' end was subcloned into pCMV, a plasmid containing the an accumulation of modi®ed sites with qualitatively cytomegalovirus early promoter and the small T antigen polyadenylation signal. The EcoRI-HindIII restriction frag- indistinguishable e€ects is important for regulating the ment containing 10 of the 12 sites analysed, was subcloned ability of pRb to bind E2F-1 and inhibit G1 phase cell into M13 mp18 and the resulting plasmid used to generate cycle progression (Knudsen and Wang, 1997; Brown et single-stranded DNA for mutagenesis. For modi®cation of al., 1999). On the other hand, the e€ects of modi®ca- sites S249 and T252, an M13 clone containing the N-terminal tion at particular sites on ¯at cell activity are clearly Rb sequences was used (Leng et al., 1997). Mutagenesis was qualitatively di€erent. Of the mutants analysed in this performed using the Sculptor In Vitro Mutagenesis Kit study, a minimum of four phosphorylation site (Amersham Pharmacia Biotech, Piscataway, NJ, USA) as modi®cations is required to abolish ¯at cell activity. directed by the manufacturer. The oligonucleotide primers However, many mutants with four or more modi®ed used for mutagenesis are listed in Table 1. The template and sites retain or have increased ¯at cell activity. Further, primer used for the construction of each particular mutant is listed in Table 2. DNA sequencing of the M13 various clones some modi®cations seem to increase ¯at cell activity con®rmed the presence of the desired mutations. Expression rather than decrease activity. Hence the identity of the vectors containing these various mutants were constructed by sites modi®ed is more important than the number of inserting the EcoRI-HindIII restricted 1.8 kbp fragment from sites modi®ed. It is apparent in both assays that the the successfully mutated, double-stranded M13 DNA into e€ect of modi®cation at any one particular site is EcoRI-HindIII restricted pCMVRbwt. The M4A mutant was dependent on the context of other modi®ed sites and made by inserting the EcoRI-MluI fragment from M2C into the presence of the N-terminus. EcoRI-MluI restricted M2A. The 56M10 mutant expression We conclude that phosphorylation of multiple C- vector was cloned by inserting the EcoRI-HindIII C-terminal terminal and spacer region sites is important for mutant fragment directly into the EcoRI-HindIII restricted regulation of the ability of pRb to inhibit cell cycle pCMV. This vector expresses a protein that likely begins at the ®rst in-frame methionine at amino acid residue 379 of the progression, con®rming previous work utilizing alanine wild-type protein. The M12 mutant was generated using the substitution mutagenesis. These results are consistent N-terminal Rb M13 clone as template and the 249E+252E with a quantitative model of Rb regulation by oligonucleotide as primer. The successfully mutated N- phosphorylation. In contrast, regulation of the ability terminal fragment was subcloned with the EcoRI-HindIII of pRb to promote di€erentiation can be regulated by C-terminal fragment from the M10 mutant into pCMV. as few as four speci®c sites and some modi®cations increase protein activity indicating that the e€ect of Rb assays phosphorylation at di€erent sites is qualitatively distinct. We conclude that the e€ect of phosphoryla- Cell cycle analysis was performed on one 10 cm dish of tion at any one site is likely to be dependent on the SAOS-2 (approximately 36106 cells) cells for each sample function being assayed as well as the context of other cotransfected as above with a Rb expression plasmid (2 ± 27 mg), pCMVCD20 (3 mg), and pCMV to 30 mg total DNA. modi®ed sites. Our results also place into question the Two days following transfection, cells were removed from widely held assumption that highly phosphorylated, dishes by treatment with 2.5 mM EDTA and cell suspensions `hyperphosphorylated,' pRb that is typically recognized stained for CD20 by incubation for 30 min with 20 mlof by altered electrophoretic mobility is completely undiluted FITC-conjugated anti-CD20 (Becton-Dickinson, inactive. Finally, our results suggest that functions of Franklin Lakes, NJ, USA) on ice. Cell suspensions were pRb that operate independently of one another (Sellers washed with PBS and ®xed by addition of ice-cold 95% et al., 1998), can also be regulated independently by EtOH. Cells were stored in EtOH at 7208C until processing phosphorylation. for ¯ow cytometry. EtOH ®xed cells were washed in PBS, resuspended in PBS+10 mg/ml propidium iodide and 250 mg/ ml RNAse A, and incubated for 30 min at 378C prior to ¯ow cytometry. Flow cytometry was performed using a Coulter EPICS Pro®le instrument. At least 2000 FITC positive cells Materials and methods were analysed. The cell cycle pro®les were determined by analysis of the cytometry data with Multicycle (Phenix Flow Cell culture and transfections Systems, San Diego, CA, USA). Flat cell assays were SAOS-2 human osteogenic sarcoma cells and 293 human performed by cotransfecting SAOS-2 cells on 10 cm plates embryonic kidney cells were obtained from the American with 25 mg of Rb expression vector and 5 mg of pEGFP-C1 Type Culture Collection and were cultured in DMEM (Clontech, Palo Alto, CA, USA). Stable transfectants were containing 10% fetal bovine serum (FBS) and antibiotics at selected by growth in regular media plus 500 mg/ml G418 for 2 weeks. The total number of GFP-positive ¯at cells was 378C with 5% CO2. Cells were prepared for transfection by

Oncogene Phosphorylation site mutagenesis alters Rb function S Barrientes et al 569 counted from at least twenty randomly selected 1006 by Bradford assay according to manufacturer's instructions microscopic ®elds under a ¯uorescence microscope and the (BioRad, Hercules, CA, USA). Twenty mg of total soluble number of ¯at cells per ®eld determined. protein for each sample, normalized for transfection eciency, was loaded on 7.5% SDS-polyacrylamide gel. After electrophoresis, the proteins were transferred to DNA sequencing nitrocellulose and the blot blocked with a solution of 5% DNA sequencing for con®rmation of Rb mutants was dried milk powder in TTBS (100 mM Tris pH 7.5, 150 mM performed using the Thermo Sequenase kit (Amersham NaCl, 0.1% Tween 20) for 1 h at room temperature. The blot Pharmacia Biotech, Piscataway, NJ, USA) according to was incubated with primary antibody diluted in fresh TTBS manufacturer's recommendations. Automated sequencing of for 1 h at room temperature of 48C overnight. Primary the entire coding sequence of the M5C Rb mutant was antibody was detected using a peroxidase-conjugated second- performed by the MD Anderson DNA sequencing core ary antibody and enhanced chemiluminescence (ECL) as facility by dye terminator cycle sequencing and analysis using described by the manufacturer (Amersham Pharmacia a Biomeck 2000 instrument. Biotech, Piscataway, NJ, USA). Under the conditions used, endogenous Rb protein in 293 cells was undetectable. Western blotting 293 cells were transfected with 27 mg Rb expression vector DNA and 3 mg pEGFP-C1 DNA. Transfection eciency was checked the following day by determining the percentage of Acknowledgments GFP positive cells by counting under ¯uorescent microscopy. We thank Dr Wen-Hwa Lee and for Rb antibodies and the Transfection eciencies ranged from 25 ± 35%. Two days pCMV vector. We also thank Dr J Wade Harper for the N- following transfection, cells were collected and resuspended in terminal Rb M13 clone used for mutagenesis. We acknowl- lysis bu€er containing 50 mM Tris, pH 7.4, 250 mM NaCl, edge other members of the Goodrich laboratory for helpful 5mM EDTA, 0.1% NP-40, 50 mM NaF, 1 mM PMSF, 1 mg/ discussions. This work was supported by the National ml leupeptin. Cells were lysed by three cycles of freezing and Institutes of Health (CA-70292-01 to DW Goodrich) and thawing. Cell debris was pelleted by centrifugation and the the MD Anderson Physicians Referral Service (DW Good- total protein concentration of the soluble extract determined rich).

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Oncogene