Proc. Natl. Acad. Sci. USA Vol. 93, pp. 8962-8967, August 1996

A maize cDNA encoding a member of the retinoblastoma family: Involvement in endoreduplication (/RepA/wheat dwarf virus) GIDEON GRAFI*t#, RONALD J. BURNETTr*, TIM HELENTJARIS*, BRIAN A. LARKINS*§, JAMES A. DECAPRIOt, WILLIAM R. SELLERSt, AND WILLIAM G. KAELIN, JR.t *Department of Sciences, University of Arizona, Tucson AZ 85721; and tDana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02115 Contributed by Brian A. Larkins, May 14, 1996

ABSTRACT Retinoblastoma (RB-1) is a tumor suppres- We identified a partial maize cDNA (ZmRB) that is pre- sor that encodes a 105-kDa nuclear phosphoprotein. To dicted to encode a protein with homology to members of the date, RB have been isolated only from metazoans. We pocket protein (Rb) family. Here we provide evidence that have isolated a cDNA from maize whose predicted ZmRb is a member of the retinoblastoma protein family and protein product (ZmRb) shows homology to the "pocket" A demonstrate its ability to bind the replication-associated pro- and B domains of the Rb protein family. We found ZmRb tein WDV RepA and its involvement in the process of DNA behaves as a pocket protein based on its ability to specifically endoreduplication during maize endosperm development. interact with oncoproteins encoded by DNA tumor viruses (E7, T-Ag, E1A). ZmRb can interact in vitro and in vivo with MATERIALS AND METHODS the replication-associated protein, RepA, encoded by the wheat dwarf virus. The maize Rb-related protein undergoes Plant Materials and Chemicals. Maize (Zea mays L.) ker- changes in level and phosphorylation state concomitant with nels from various developmental stages of an inbred developed endoreduplication, and it is phosphorylated in vitro by an from pool 33 QPM were obtained from grown at the S-phase from endoreduplicating endosperm cells. To- University of Arizona. DNA and protein-modifying enzymes gether, our results suggest that ZmRb is a representative ofthe were purchased from BRL, New England Biolabs, or Boehr- pocket protein family and may play a role in cell cycle inger Mannheim, unless otherwise indicated. progression. Moreover, certain plant monopartite geminivi- cDNA Library and Plasmids. An endosperm (18 days after ruses may operate similarly to mammalian DNA viruses, by pollination) cDNA library in A-ZAPII was screened with the targeting and inactivating the retinoblastoma protein, which Rb-related clone 6C02C02 (14) to isolate ZmRB. ZmRB, otherwise induces G1 arrest. which encodes the pocket region of the maize protein (1-458, see Fig. 1A), was amplified by PCR with a sense primer Retinoblastoma (RB-1) is a tumor suppressor gene that en- containing a BamHI site and in frame ATG start codon codes a nuclear phosphoprotein with a molecular mass of (5'-GCCGGATCCCATATGCCGTGGGCAGAAGCCAG-3') about 105 kDa (1, 2). Inactivation of the RB-1 gene contributes and an antisense primer containing an EcoRI site (5'- to both familial and sporadic forms of cancer (3). Retinoblas- GGAATTCGCCTTCACCAATGCATGC-3') using Pfu toma and its related p107 and p130 are among the DNA polymerase (Stratagene). The amplified DNA fragment negative regulators of the cell cycle (4, 5). While hypophos- was subcloned into the BamHI/EcoRI sites of either pBlue- phorylated, the Rb protein exerts a growth suppressive effect script SKI (Stratagene), pGEX-2TK (Pharmacia; ref. 15), or and arrests cells in . pSG5 (Invitrogen) modified by Ralph Scully (Dana-Farber Hyperphosphorylation of Rb, or its interaction with viral Cancer Institute) to contain the hemagglutinin epitope with oncoproteins, prevents Rb from performing its normal func- Ncol, HindIII, SmaI, and Sall upstream and BamHI, EcoRI, tions at G1 phase and enables cells to begin DNA synthesis (6). EcoRV, andXhol restriction sites downstream. Introduction of One function of Rb appears to be an interaction with and a C to G to generate ZmRB706G was performed inactivation of transcriptional factors, such as the protein with a mutagenic primer (CGATCAACTTATCCTTgGCTG- family, that are required for the expression of genes involved TCTTTATGGTGTTGC) using a Transformer Site Directed in G1-S transition (7). The product of the RB gene, pRb, as Mutagenesis Kit (Clontech) and confirmed by DNA sequenc- well as its related p107 and p130 proteins, contains a "pocket" ing. HuZmRB is a hybrid molecule containing the human Rb region required for binding with cellular proteins, as well as A domain (379-602) and the maize Rb B domain (251-458; with oncoproteins encoded by DNA tumor viruses (8, 9). The see Fig. 1A). To generate the hybrid molecule, the human RB pocket region contains two functional domains, A and B, with cDNA corresponding to the large pocket region (379-928) in an intervening spacer. Certain cellular proteins, as well as viral pBluescript SKI was cleaved with BglII and EcoRI to remove oncoproteins, interact with Rb through a domain containing the B domain and C-terminal region. The ZmRB B domain the core sequence LxCxE (10). This binding motif is found in was amplified by PCR using 5'-AGGGCCGGATCCATGG- plant D-type (B-cyclins; refs. 11 and 12) and in several AGTCCACATTTGCAAG-3' as the sense BamHI primer and replication-associated proteins encoded by single-stranded the above antisense EcoRI primer. The PCR product was DNA monopartite geminiviruses (13). Among them is the digested with BamHI and EcoRI and subcloned into the RepA protein encoded by wheat dwarf virus (WDV), which BglII/EcoRI site of pBluescript carrying the human Rb A has recently been shown to interact with the Rb-related p130 protein. The LxCxE motif in RepA was found to be required Abbreviations: WDV, wheat dwarf virus; GST, glutathione-S- for efficient replication of the virus (13). transferase; CDK, -dependent kinase; DAP, day after pollination. Data deposition: The sequence reported in this paper has been deposited in the GenBank data base (accession no. U52099). The publication costs of this article were defrayed in part by page charge VPresent address: Department of Plant Genetics, The Weizmann payment. This article must therefore be hereby marked "advertisement" in Institute of Science, Rehovot 76100, Israel. accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 8962 Downloaded by guest on September 27, 2021 Cell Biology: Grafi et al. Proc. Natl. Acad. Sci. USA 93 (1996) 8963 domain. To generate pGEM-RepA, an NcoI-NdeI fragment and B domains (Fig. 1B). Furthermore, four conserved amino containing the WDV RepA coding region and part of the acid residues in ZmRb are also sites of naturally occurring pRb N-terminal region of the WDV RepB protein was excised from (arrowheads Fig. 1B, Ala-562, Ser-567, Arg-661, and pWI-II (kindly provided by J. Messing; ref. 16) and inserted Cys-706; refs. 22-25). Therefore, it appeared that ZmRB might into the NcoI-NdeI sites of the pGEM-5Zf vector (Promega) also encode a pocket protein. The ZmRB gene maps to the long and used as a template in a coupled /translation arm of chromosome 2, and its simple hybridization pattem reaction (TNT; Promega). To produce glutathione-S-transferase suggests that there is only one gene (data not shown). (GST)-RepA protein, the RepA DNA fragment was also inserted ZmRb Specifically Interacts with Oncoproteins Encoded by into BamHI-SmaI sites of pGEX-2TK. The human RB small Mammalian DNA Tumor Viruses. A diagnostic feature of pocket (379-792; HuRBsp) and human RB point mutant (C706 pocket proteins is their ability to interact with viral oncopro- to F, HuRB706F) were previously described (8). teins via a short motif with the core sequence LxCxE (10). We Antibodies, Immunodetection and Immunoprecipitation. A investigated whether ZmRb has properties of a pocket protein monoclonal antibody (mAb) was raised against bacterially by testing its ability to interact with the E7 oncoprotein produced GST-ZmRb by standard procedures (17). Superna- encoded by papillomavirus. Various plasmids were con- tants from growing hybridoma cell cultures were the source for structed (Fig. 2A): pZmRB contains the wild-type sequence ZmRb mAb (2E5) and that for the hemagglutinin epitope and pZmRB706G is a mutated form in which the cysteine at (12CA5) (18). Kernels were homogenized with NETN (20 mM the position corresponding to residue 706 of human Rb was Tris HCl, pH 8.0/100 mM NaCl/1 mM EDTA/0.5% Nonidet converted to glycine. A naturally occurring mutation at this P-40) buffer containing 87 mM NaF, 2 mM sodium orthovana- position renders human Rb incapable of interacting with date, 5 ,tg of aprotinin per ml, and 5 ,ug of leupeptin per ml. oncoproteins (23). pHuZmRB is a hybrid molecule containing Samples were cleared by centrifugation, aliquotted and stored the human Rb A domain and ZmRb B domain which was at -80°C. The various RB and RepA constructs were used as constructed to address the question of evolutionary conser- templates in the TNT system and proteins were labeled with vation between plant and animal pocket proteins. As a positive [35S]methionine. Two microliters of each TNT reaction, or control we used the pocket region of human Rb (HuRBsp) total protein extract (1 mg), was mixed with 50 ,ul of protein which is known to interact with E7, and as a negative control A-Sepharose (50% slurry) and 100 gl (tissue culture super- we used the mutant HuRB706F (Cys-706 -> Phe; ref. 23). natant) of the indicated mAb and incubated in NETN buffer at Plasmids were used as templates in a coupled transcription/ 4°C for 2-12 h with gentle shaking. The Sepharose was washed translation system to produce the various 35S-labeled proteins five times with NETN buffer, and bound proteins were either (Fig. 2B, lanes 1-5). As shown in Fig. 2B, ZmRb, HuZmRb, directly resolved by 6-10% SDS/PAGE or treated with Lambda and HuRbsp all bound specifically to the papillomavirus E7 protein (New England Biolabs, according to the protein. This interaction was dependent on the LxCxE, as no manufacturer's protocol) before loading on the gel. Gels were interaction was evident with E7ADLYC, which lacks the either treated with EN3HANCE (DuPont), dried, and exposed to Rb-binding motif (Fig. 2B lanes 16-20). Both HuRb7O6F (lane x-ray film, or subjected to immunoblotting with 2E5 mAb. 15) and ZmRb7O6G (lane 12) did not bind E7 protein. Thus, In Vitro Binding Assay. Glutathione-Sepharose containing ZmRb behaves as a pocket protein. ZmRb also bound specif- GST alone, or various GST-fusion proteins prepared as de- ically to simian virus-40 large tumor-antigen and to the ade- scribed (19), was mixed with 2 ,ul of each TNT reaction, or total novirus ElA protein (data not shown). protein extract (1 mg), and incubated in NETN buffer at 40C In Vitro and in Vivo Interaction of ZmRb with the Wheat for 2-12 h with gentle shaking. The agarose beads were washed Dwarf Virus RepA Protein. To gain insight into a possible five times with NETN buffer and treated as described for functional activity of ZmRb in plants, we searched the Gen- immunoprecipitation. Bank data base for plant proteins carrying an LxCxE motif. Two Hybrid System. For analyzing in vivo binding of Several cellular proteins as well as bacterial and viral proteins ZmRb to RepA, we used the yeast two hybrid system (ref. 20; were identified (Fig. 3A). Among them are the Arabidopsis a gift from S. J. Elledge). ZmRB and ZmRB706G (NcoI- thaliana cyclin A1-3 proteins (11), Agrobacterium tumefaciens EcoRV fragments from pSG5L constructs) were inserted into VirD4 (26), Agrobacterium rhizogenes RolD (27), and several the NcoI-SmaI sites of the pAS2 vector containing the Gal4 replication-associated proteins encoded by single-stranded DNA-binding domain. The NcoI-BamHI fragment of pWI-II DNA monopartite geminiviruses (13). The RepA protein (containing both RepA and RepB sequences) was inserted into encoded by WDV was of particular interest, since it has been the NcoI-BamHI sites of the pACT2 vector containing the shown to interact with the human Rb-related protein, p130, Gal4 activation domain. A pACT2-RepA plasmid was trans- and its LxCxE motif was found to be required for efficient viral formed into yeast cells expressing the DNA-binding domain DNA replication in plant cells (13). Fig. 3B shows that fused to ZmRB or ZmRB706G. Interaction of the DNA- GST-ZmRb and GST-HuRbsp, but not their mutated forms binding fusion protein and the transactivating fusion protein (GST-ZmRb7O6G and GST-HuRb7O6F), specifically bound resulted in transcriptional activation from promoters contain- RepA. By using the yeast two hybrid system, we demonstrated ing Gal4-binding sites: the upstream activating sequence from the in vivo binding of ZmRb (but not ZmRb7O6G) to RepA GAL] (UASG), lacZ, and HIS3 genes. Transformants were protein. Binding of ZmRb to RepA in ZmRb/RepA transfor- analyzed for ,B-galactosidase activity (21) and their ability to mants was shown by the activation of 13-galactosidase, as well grow on medium lacking histidine and containing 25-35 mM as by the ability of cells to grow on medium lacking histidine 3-aminotriazole. and containing 3-aminotriazole (Fig. 3C). The Level and the Phosphorylation State of ZmRb Are Altered During Endoreduplication in Maize Endosperm. Dur- RESULTS ing endoreduplication, cells undergo multiple rounds of DNA Isolation of a Maize cDNA Encoding a Member of the synthesis in the absence of (28). This results as a Pocket Protein Family. We previously identified a maize consequence of altering the normal cell cycle, which is com- cDNA that is predicted to encode a protein with homology to posed of Gl, S, G2, and M phases, into one containing only S members of the Rb protein family (14). Using this clone as a and Gap phases. In maize endosperm, this process initiates probe, we obtained a partial cDNA of about 1.8 kb (ZmRB). 10-12 days after pollination (DAP) and it involves a substan- Alignment of the predicted amino acid sequence of ZmRB tial increase in level and activity of S-phase cyclin-dependent (Fig. 1A) with several members of the Rb protein family (CDKs) (29). Since Rb exerts its effect at the G1 phase showed that the homology is primarily confined to the pocket A of the cell cycle, endoreduplication should be a suitable Downloaded by guest on September 27, 2021 8964 Cell Biology: Grafi et al. Proc. Natl. Acad. Sci. USA 93 (1996)

A 1 LDLNCANAFD IPWAEARKVE ASKLYYRVLE

51 NERFHRCLIA CSADLVLATH KTVIMMFPAV LESTGLTAFD LSKIIENFVR

101 HEETLPRELK PSVASEINRL

151 GLLAEPMPSL DDLVSRQNVR IEGLPATPSK KRAAGPDDNS DPRSPKRSCN

201 EFRNTVVERN LQTPPPKQSH MVSTSLKAKC HPLQSTFASP TVCNPVGGNE

251 KCADVTIHIF FSKILKFPAI RIRNLCERVQ CVEQTERVYN VFKQILEQQT

301 TLFFNRHIDQ LILCCLYGVA KVCQLELTFR EILNNYKREA QCKPEVFSSI

351 YIGSTNRNGV LVSRHVGIIT FYNEVFVPAA KPFLVSLISS GTHPEDKKNA

401 SGQIPGSPKP SPFPNLPDMS PKKVSASHNV YVSPLRQTKL DLLLHQVPGV

451 FMHALVKAPM LNRAHLRIWL L* B Zm 11 IPWAEARKVEASKff VLIICRA.LQNSNVNNLTPL~SNER CZI.SADL~LATHK 72 107 321 IDFAVNRLKLAEI'j.KILTrVMVQ TRRLHGMDMSVL~EQDIWRS.LS CLEIgLFAYS 382 130 438 KEIASKHFRFAE KVL]SVIEQ QKRLGDMDLSGIWEQDAJRS[L.MCLEVgTFSYK 488 Rb 451 VEIGSQRYKLGVRi VM SMLKS EERLSIQNFSKL NDNI SfLgALEV 4ATY 498 Mo 445 VDIGVQRYKLGVRGRVMRSMLKSWEERLSIQNFSKLNDNIjSLALEVMATY 492 GG 441 AEIGSQRYQLGVRJRVMUSMLKKSEERLSVHNFSKL NDNI TSLALEEIgATY 488 Xn 440 AEIGYQRYKLGVC gVM-ILKT[EERLSVHNFSKLrNDI WC ^ ZVEVUASY 499 cC Zm 77 iWAVLESTGLTAFDLS3INFVRBHETPRELKRHNSLWEQLLESMEKGS SL 143 107 386 WI IEQILESL»SHDWAIWEAL 452 0 IEVLNLQPFYFYWVWVVIRSWEGBSRDMVKH[NS n 130 493 [lFITEIFDVPLYHFFYYV FIRAWGCREVVKHQIEQILDHL~KPEPEKI 552 Rb 514 ]WILNVLNLKAFDFYIVwSFIKAwGNIrREMIKHERCwHRIMESLLLSDwPMFD 571 Mo 508 WiWILNVLNLKAFDFYVSFIKVWANREMIREKHWERCWHRIMESL LSDSPWFD 565 GG 506 §WILNVFDLKAFDFYwVSwSFIKV1PSrRRDMIKHwERCIRIMESLmQSD P FD 563 Xn 515 .WILRAFEIKAFDFYgVfCFIKAPSjTSNIKYWERCWHQIMECLCQSDPD 572 E Zm 257 IHIWFSWILKFPAIWIRNICERVQCVEQTER- -VYNVFKQILEQQTTIFFNaIIs_LILMCLYG 318 107 655 LALjYRRiVYHLASVWLRDJCLKLDVSNELRRK- IWTCFEFTLVHCPDJMKD=LiJeLLL[LAFYI 717 130 779 LSLWFRWVYHLAAVWLRDtCAKLDISDELRKK-IWTCFEFSIIQCPEIMDkLiJLaILLM AIYV 841 Rb 647 LSLWYKVYRLAYLWLNTICERLLSEHPELEHIIWTLFQHTLQNEYErMRDLLIIMMHSMYG 710 Mo 639 LALIYKVYRLAYLWLNTACARLLSDHPELEHIIWTLFQHTLQNEYEr4RD ial*IMMHSMYG 702 GG 639 LSLUYKWVFRLAYLWLHTlFFRLLSEHPDLEPLIWTLQHTLQNESEWRD1 LgIIMMlSMYG 702 Xn 648 LSLtJK{VYLLAYKISSSICSSLLSDHPELEQVIWTLLQHTLQQEYES4RDLia_l]IMMrSMYG 711 Oco Zm 319 VAiVCQLELTiREuLNIfKREAQCKPEVFSSIYIGSTNRNGVLVSRHVGI iEVFVP 382 0 107 718 MAaVTKEERTWQErMKSURNQPQANSHVYRSV------DLK TIYVGRV S 811 Q 130 842 MAWVTKEDKSWQNJMRCURTQPQARSQVYRSV------DLCIJIYIKQITr 962 Rb 711 IC0VKNIDLKijKI VTAjKDLPHAVQETFKRVLIKEEEYD ---- SIUv7 SVFMQRLWr 766 Mo 703 IiqVKNIDLKWKIiVfTAjKDLPHAAQETFKRVLIREEEFD SI*f SVFMQRL6T 758 GG 703 ICJVKNVDLRRKTUVSAuSKELPNTNQETFKRVLIREEQYD - ----SI V LVFMQKLWT 758 Xn 712 ICTAKNIDLRKTUVTAVTKGLTNTNQETFKHVLIRDGQHD ---- SI LVFMQKLi 767

FIG. 1. Predicted amino acid sequence of ZmRb and alignment with members of the Rb protein family. (A) Amino acid sequence of the pocket region of ZmRb. The underlined sequence indicates the putative A domain and the double underlined sequence is the B domain. (B) Alignment of ZmRb with several members of the Rb protein family. Identical amino acids are highlighted. Arrowheads indicate residues that have been mutated in human cancer cells. Horizontal bars represent gaps introduced to align the sequences. Zm, maize; 107, human p107; 130, human p130; Rb, human Rb-1; Mo, mouse; GG, chicken; Xn, frog. process in which to study G1-S transition and ZmRb function. gene product. This protein could be recovered by GST-RepA We therefore examined ZmRb level and phosphorylation state only from endoreduplicating endosperm (data not shown). We during endosperm development (Fig. 4). A monoclonal anti- do not know the relationship, if any, of the 130-kDa protein body raised against ZmRb (2E5) specifically recognized its in and the doublet proteins to ZmRb. Treatment with A-phos- vitro translation product (data not shown) and polypeptides phatase confirmed that both phosphorylated and hypophos- from maize endosperm (Fig. 4 A and B). As shown by phorylated forms of ZmRb protein associated with 2E5 mAb immunoprecipitation and western analyses, a protein of about and GST-RepA (Fig. 4C). Fig. 4D shows that ZmRb can be 130 kDa (asterisks in Fig. 4 A-C), a diffuse protein band of phosphorylated in vitro by an E2F1-associated CDK from about 110 kDa (brackets in Fig. 4 Band C), and doublet endoreduplicating (12 and 14 DAP) endosperm (29). proteins (arrows) between 90-100 kDa (Fig. 4 A-C) were detected. While the 130-kDa protein was found in and DISCUSSION endosperm from all developmental stages tested, appearance of a diffuse protein band (about 110 kDa) and a protein Retinoblastoma has been thought to be a late evolutionary doublet coincided with the initiation of endoreduplication development as homologs have, until the present, only been (29). GST-RepA and GST-E7 were found to interact with the identified in metazoans. Here we report the isolation of a diffuse protein band, but not the doublet or the 130-kDa cDNA from maize endosperm encoding a member of the Rb protein, whereas GST-E7ADLYC and GST alone did not (Fig. protein family. This clone appears to contain the functional 4B). Thus, the diffuse protein band appears to be the ZmRB pocket region of the Rb protein family. The homology is Downloaded by guest on September 27, 2021 Cell Biology: Grafi et al. Proc. Natl. Acad. Sci. USA 93 (1996) 8965

A T7 Barr Hi A A~~~~~- SP B Eco RI SV-40 T-ag NEEE ZmRB HPV-16 E7 ETTE AdS ElA EVIE I "706C" to G T7 Bam Hi A ZmRB706G - A. thaliana Cyc 61 NDMr IID E I _0 A. thaliana Cyc 62 LLDA HuRB ZmRB A. thaliana Cyc 63 MAEN I. Y L S P V RS M E S T F A. tunmefaciens VirD4 VAGI -I IIAICTTTCTCC AGATCC iTG A. rhizogenes RolD ENKH I, WDV RepA PTES T7 Bam Hi Eco RI HuZmRB ZmRB B (9 U- co CD0.co coQ0 CI of a rlof H- E E :I B < N N I I a) cn u) U) en E) c o o o (c IVT GST GST-E7 GST-E7ADLYC

U I 0 U RepA _- 30-- _E."' e3 co E mn00cCO CO CD om lrE m: M Em)a x E x E xf E N a: Ir E N sr wE~ N a: w X NJ tr a: N N : 1: zrX NzNz N N x NN4r C

..... - ~~~~~~~~~~~~~~...... His SNF1/SNF4

......

...... i ZmRB ZmRB/RepA ~~~~~~~~~~~~~~~~~~~~.*~ .:!.. ,,.,...... *~~~~~~~~~~~~~~~~~~~~~~~~.,, .t..3.3. ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...t~~~~~~~~~~~~~~~~~.'"r3Z '.:....:. ..::.' ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...... ZmRB706G ZmRB706G/RepA 1 4 ...... :: 2 3 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 RepA FIG. 2. ZmRb interacts specifically with the papillomavirus E7 protein. (A) Schematic representation of the constructs employed. FIG. 3. In vitro and in vivo interaction of ZmRb with the replication ZmRB encodes the pocket region of the maize protein (1-458; see Fig. associated protein, RepA, encoded by the single-stranded DNA WDV. 1A). ZmRB706G is a Cys to Gly mutation at a position corresponding (A) Comparison of the Rb-binding motifs (LxCxE) in plant cellular to residue 706 of human Rb. HuZmRB is a hybrid molecule containing proteins and plant bacterial and viral proteins with those of oncopro- the human Rb A domain (379-602) and the maize Rb B domain teins encoded by animal DNA tumor viruses. The core sequence (L, (251-458; see Fig. LA).* A,,'~~~~~~~~~~~~~~~~~~~~~~~~~~~.:.-.SP, and B indicate the A domain,''....the.. spacer C, and E) is highlighted, and the adjacent acidic residues are in region, and the B domain, respectively. The predicted amino acid boldface type. (B) In vitro interaction of ZmRb, ZmRb7O6G, HuRbsp, sequence at the junction of human RB and ZmRB is shown. The boxed and HuRb7O6F with RepA protein. Glutathione-agarose containing M and E are extra amino acids incorporated during the cloning the indicated GST-fusion proteins (see legend to Fig. 2) was mixed process. (B) In vitro translated maize Rb binds specifically to the with 2 ,ul of 35S-labeled RepA protein and treated as described in Fig. papillomavirus E7 protein in an LxCxE-dependent manner. The 2B. RepA IVT indicates 1 j,l of the input TNT reaction. Molecular size various Rb constructs, including the human Rb pocket region marker and the position of RepA protein are indicated on the left. (C) (HuRBsp) and the mutant HuRB706F (see Materials and Methods) Interaction of ZmRb with RepA protein in yeast by the two hybrid were translated in vitro with [35S]methionine by using the coupled system. pACT2-RepA was transformed into yeast cells expressing transcription translation system (TNT; Promega). IVT (lanes 1-5) ZmRB or ZmRB706G. Transformants were restreaked on glucose- indicates 1 ,ul of TNT reaction. Two microliters of each reaction were containing plates lacking histidine and containing 25 mM 3-amin- mixed with glutathione-Sepharose containing GST alone (lanes 6-10), otriazole and tested for 13-galactosidase activity. As a positive control, GST-E7 (lanes 11-15), and GST-E7ADLYC (lacks the Rb-binding we used SNF4 in pACT and SNF1 in pAS1 (20). motif, lanes 16-20) and incubated in NETN buffer at 4°C for 2 h. The proteins were resolved by 10% SDS/PAGE and the gel was dried and Does ZmRb function similarly to mammalian Rbs in con- exposed to x-ray film. trolling GI progression? The first clue for the existence of an Rb checkpoint control in plants came with the discovery of confined to the pocket A and B domains, which enable Rb to D-type cyclins (6-cyclins) in Arabidopsis thaliana (11) and in its function as a supressor and to form perform growth alfalfa (12). The mammalian D-type cyclins are regulatory complexes with oncoproteins (8, 9). Four conserved amino subunits of CDK4 and CDK6 and bind to Rb through an acid residues within the pocket region of ZmRb are also sites of naturally occurring pRb mutations (22-25). Our results N-terminal LxCxE motif, directing the CDKs to their sub- strates A similar motif was found at the N-terminal demonstrated that ZmRb behaves as a conventional pocket (31, 32). protein; it not only specifically binds the E7 oncoprotein in an region of the plant D-type cyclins. A second clue was provided LxCxE-dependent manner, but like human Rb, a mutation of by Xie et al. (13), who established the requirement of an LxCxE Cys-706 rendered it incapable of interacting with E7. More- motif in the WDV replication associated protein, RepA, for over, a hybrid molecule containing the human Rb A domain both interaction with the human Rb-related protein, p130, and and ZmRb B domain is fully active in binding to E7, suggesting efficient replication of the virus in plant cells. that the pocket domains have been functionally highly con- In the present report we show the existence of an Rb-like served during evolution. protein in maize and demonstrate its biochemical properties Downloaded by guest on September 27, 2021 8966 Cell Biology: Grafi et al. Proc. Natl. Acad. Sci. USA 93 (1996)

A IP/we tern C IL z 0- 12CA5 2E5 - IP + M 10 14 5 8 10 14 18- DAP a)~ a) Cl) U) cn) r p 1 ;?.,-PPase *...... :3 a- CL +1" -0(14 125-- _ kDa M 14 14 14 14 14 14 14 14 -DAP 79. 228 - .wXs.i.i.F - i]IgG

B ......

L-J - CD 1 25- mi, 2E52E5 co(DDUCO) Cl)CDCDcl) L 5 8 12 14 14 14 14 14 DAP kDa M 1 2 3 4 5 6 7 8 9 10

228- D 0-

CL L " Lf

o 0 I 1 8 12 14-' DAP

125- ..~ T.. ZmRB ::.:.4: .....

~~~~~ of FIG. 4. ZmRb level and phosphorylation state is altered during endosperm development.Y (A) Immunoprecipitation/Western detection proteins in developing endosperm that react with ZmRb mAb (2E5). 12CA5 is a hemagglutinin mAb used as a negative control (see Materials and Methods). Proteins (1 mg) from developing endosperm (5-18 DAP) were immunoprecipitated with 2E5 or 12CA5 and, following 10% SDS/PAGE, the proteins were detected by immunoblotting with 2E5. Specific proteins interacting with 2E5 are indicated by arrows and an asterisk. The position of IgG is labeled on the right of the figure and molecularweight markers are on the left. (B) ZmRb from endosperm can be precipitated with GST-E7 and GST-RepA. Proteins from developing endosperm (5-14 DAP) or maize seedlings (L) were precipitated by 2E5 or glutathione-agarose containing GST alone or various GST fusion proteins (see legend to Fig. 2B; GST-RepA is described in Materials andMethods). Following separation by 6% SDS/PAGE, proteins were detected by immunoblotting with 2E5. Specific proteins detected by 2E5 are identified by arrows, a bracket, and an asterisk. (C) Phosphorylated and hypophosphorylated forms of ZmRb associate with 2E5 and GST-RepA. Protein precipitates obtained either with 2E5 or with GST-RepA from 14-DAP endosperm extract were subjected to in vitro dephosphorylation using lambda-phosphatase (A-PPase) after which the proteins were resolved by 6% SDS/PAGE and immunoblotted with 2E5. Lanes: 1, molecular size markers; 2 and 7, untreated samples; 3, A-PPase buffer only; 4, A-PPase with 10 mM NaF (phosphatase inhibitor); 5 and 8, A-PPase treated samples; 6 and 9, GST-RepA bound proteins; and 10, GST-RepA bound proteins treated with A-PPase. Arrows, bracket, and asterisk indicate the same proteins described in Fig. 4B. The arrowhead in lane 5 indicates the hypophosphorylated form of ZmRb. (D) ZmRb can be phosphorylated in vitro by an E2F-associated CDK from endoreduplicating endosperm. ZmRb was precipitated by 2E5 or GST-RepA and, following treatment with A-PPase, used as a substrate for in vitro phosphorylation. For comparison, histone Hi was used as a substrate. The following were sources of protein kinase: 10 ,ug of 10-or 15-DAP maize endosperm extract, purified Xenopus Cdc2-cyclin B1 kinase (XCDC2) expressed in insect cells (30), partially purified kinase from 15-DAP endosperm (H1K15DAP) that did not bind p13sucl and GST-E2F-associated kinase from 8-14 DAP endosperm (29). The phosphorylated proteins were resolved by 6% SDS/PAGE and detected by autoradiography.

during the course of endoreduplication in maize endosperm. synthesis by targeting and inactivating the retinoblastoma Endoreduplication provides a suitable process to study the protein, which otherwise would cause cell cycle arrest and GI-S transition, as it displays a cycle where cells undergo differentiation. multiple rounds of DNA replication without mitosis. Also, it A common strategy for studying the function of pocket has been previously shown that the level and activity of factors domains has been to characterize naturally occurring mutants regulating G1-S transition, such as S-phase CDKs, increase from tumor cells (22-25, 33, 34). Another approach would be substantially with the initiation of endoreduplication (29). For to compare pocket proteins from different . The latter a cell to enter S-phase, Rb protein must be neutralized either has been limited by the high level of homology among the by phosphorylation, an event mediated by the activity of cyclin Rb-like genes cloned to date. An evolutionary distant relative D-CDKs, or by forming complexes with oncoproteins, such as of Rb, such as ZmRB, provides a good candidate for identi- the adenovirus ElA and the papillomavirus E7 protein (10, 31, fying residuesin the pocket region that may be critical for the 32). The maize Rb-related protein undergoes changes in level ability of Rb to function as a tumor suppressor and as a and phosphorylation state concomitant with endoreduplica- negative regulator of the cell cycle. tion, and it is phosphorylated in vitro by an S-phase kinase from J. for endoreduplicating endosperm cells (29). Moreover, we have We thank K. Munger for the GST-E7 plasmids; Messing providing pWI-II plasmid; S. J. Elledge for the two hybrid system kit; an ZmRb the WDV shown interaction of with RepA protein. A. Kumagai and W. G. Dunphy for providing recombinant baculovi- novel to un- Taken together, these results provide insight ruses; Jennifer Rodgers, Jianmin Gan, and Daniel Roberts for tech- derstanding cell cycle progression in plants and the etiology of nical assistance; D. Shanon, Amy Monighetti, and the members of the plant diseases that arise by single-stranded DNA monopartite Neuroplastic Disease Mechanism Division for their excellent support; geminiviruses. It appears these viruses may operate similarly to and Ms. Ayala Barel for helping G.G. while staying in Boston. This mammalian DNA tumor viruses that induce host cell DNA work was supported by Grant DE-FGO3-95ER20183 from the U.S. Downloaded by guest on September 27, 2021 Cell Biology: Grafi et al. Proc. Natl. Acad. Sci. USA 93 (1996) 8967 Department of Energy (to B.A.L.), by grants from the National 16. Timmermans, M. C. 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