A maize cDNA encoding a member of the retinoblastoma protein family: involvement in endoreduplication.

Abstract

Retinoblastoma (RB-1) is a tumor suppressor gene that encodes a 105-kDa nuclear phosphoprotein. To date, RB genes have been isolated only from metazoans. We have isolated a cDNA from maize endosperm whose predicted protein product (ZmRb) shows homology to the "pocket" A and B domains of the Rb protein family. We found ZmRb behaves as a pocket protein based on its ability to specifically interact with oncoproteins encoded by DNA tumor viruses (E7, T-Ag, E1A). ZmRb can interact in vitro and in vivo with the replication-associated protein, RepA, encoded by the wheat dwarf virus. The maize Rb-related protein undergoes changes in level and phosphorylation state concomitant with endoreduplication, and it is phosphorylated in vitro by an S-phase kinase from endoreduplicating endosperm cells. Together, our results suggest that ZmRb is a representative of the pocket protein family and may play a role in cell cycle progression. Moreover, certain plant monopartite geminiviruses may operate similarly to mammalian DNA viruses, by targeting and inactivating the retinoblastoma protein, which otherwise induces G1 arrest.Retinoblastoma (RB-1) is a tumor suppressor gene that encodes a nuclear phosphoprotein with a molecular mass of about 105 kDa (1, 2). Inactivation of the RB-1 gene contributes to both familial and sporadic forms of cancer (3). Retinoblastoma and its related p107 and p130 proteins are among the negative regulators of the cell cycle (4, 5). While hypophosphorylated, the Rb protein exerts a growth suppressive effect and arrests cells in G1 phase.Hyperphosphorylation of Rb, or its interaction with viral oncoproteins, prevents Rb from performing its normal functions at G1 phase and enables cells to begin DNA synthesis (6).

Cyclin-Dependent Kinase Inhibitors in Maize Endosperm and Their Potential Role in Endoreduplication

“…Rb/RBR inhibitory activity can be released either as a consequence of phosphorylation or binding of specific viral proteins, such as RepA (reviewed by Gutierrez, 2000). Since Grafi et al (1996) suggested that ZmRb phosphorylation increases during the period of endoreduplication, it was hypothesized that inactivation of this protein is associated with the polyploidization process.…”

“…To determine whether overexpression of Zeama;KRP;1 and Zeama;KRP;2 can cause endoreduplication in cells in which the activity of the maize RBR is reduced or eliminated, embryonic calli were transformed with gene constructs expressing the wheat dwarf virus RepA protein in the presence and absence of the maize KRPs. RepA was previously shown to interact with the maize RBR protein and stimulate the cell cycle (Grafi et al, 1996;Gordon-Kamm et al, 2002). Maize embryos were transformed by particle bombardment as described by Gordon-Kamm et al (2002), and, after culturing them on selective media, the levels of Zeama;KRPs and RepA transcripts were measured by RT-PCR and the nuclear ploidy of cells was determined by flow cytometric analysis.…”

“…Although Zeama;KRP;1 was found to be associated with the CKI activity described by Grafi and Larkins (1995), it did not account for all of the CDK inhibitory activity present in this fraction. Overexpression of Zeama;KRP;1 in maize embryonic calli that ectopically expressed the wheat dwarf virus RepA protein, which counteracts Rb-related (RBR) protein function (Grafi et al, 1996;Gordon-Kamm et al, 2002), led to an additional round of DNA replication without nuclear division. While a clear role for Zeama;KRP;1 in endoreduplication could not be demonstrated in maize endosperm, indirect evidence suggests this protein could be involved in this process.…”

ArabidopsisRETINOBLASTOMA-RELATED Is Required for Stem Cell Maintenance, Cell Differentiation, and Lateral Organ Production

“…The requirement for RB in cell differentiation therefore extends beyond regulation of cell cycle entry. For example, the loss of RB function in cardioblasts affects cell differentiation by modulating the activity of cardiogenic factors (Papadimou et al, 2005).RB-related genes, termed RETINOBLASTOMA-RELATED (RBR; Ach et al, 1997), are also found in monocots (Grafi et al, 1996;Xie et al, 1996;Ach et al, 1997) and dicots (Nakagami et al, 1999;Kong et al, 2000) and even in unicellular algae (Umen and Goodenough, 2001). Animal pRb and plant RBR are similar in sequence and predicted protein structure and, thus, likely share similar functions (Flemington et al, 1993;Ramirez-Parra et al, 2003;Gruissem, 2007).…”

Promoter activity associated with the intergenic regions of banana bunchy top virus DNA-1 to -6 in transgenic tobacco and banana cells.

“…Further, the predicted gene product encoded by this component contains an LXCXE motif (R. Wanitchakorn, G. J. Hafner, R. M. Harding & J. L. Dale, unpublished) that is associated with retinoblastoma (Rb) binding-like proteins of animal DNA tumour viruses (Picksley & Lane, 1994). A functional Rb-binding motif has been identified in the RepA early gene product of members of the subgroup I geminiviruses (Xie et al, 1995 ;Collin et al, 1996 ;Ach et al, 1997) and evidence exists for a novel plant encoded Rb-like family of regulatory proteins (Grafi et al, 1996 ;Xie et al, 1996 ;Ach et al, 1997). Based on these findings, we hypothesize that following BBTV infection, the BBTV DNA-5 gene product is the first translated and may function in creating a cellular environment permissive for viral DNA replication and transcription.…”