Cell cycle-dependent phosphorylation of pRb-like protein in root meristem cells of Vicia faba

Abstract

The retinoblastoma tumor suppressor protein (pRb) regulates cell cycle progression by controlling the G1-to-S phase transition. As evidenced in mammals, pRb has three functionally distinct binding domains and interacts with a number of proteins including the E2F family of transcription factors, proteins with a conserved LxCxE motif (D-type cyclin), and c-Abl tyrosine kinase. CDK-mediated phosphorylation of pRb inhibits its ability to bind target proteins, thus enabling further progression of the cell cycle. As yet, the roles of pRb and pRb-binding factors have not been well characterized in plants. By using antibody which specifically recognizes phosphorylated serines (S807/811) in the c-Abl tyrosine kinase binding C-domain of human pRb, we provide evidence for the cell cycle-dependent changes in pRb-like proteins in root meristems cells of Vicia faba. An increased phosphorylation of this protein has been found correlated with the G1-to-S phase transition.

Fig. 1

Immumoblotting analysis of pRb-like proteins in root meristems of V. faba using anti-phospho-Rb (S807/811) antibody; lysates of the control cells (a) and after partial synchronization with 1.25 mM HU, followed by 4 h recovery—S phase, 6 h recovery—G2 phase, 8 h recovery—G2/M/G1 phase, and 10 h recovery—G1/S phase (b). Lane I—molecular weight marker, lane II—SDS-Nu-PAGE electrophoresis and Coomasie staining, lanes III and IV—Western blots of separated and electrotransferred proteins performed using primary rabit anti-phospho-Rb (Ser 807/811) IgG fraction and secondary goat anti-rabbit IgG antibody conjugated with peroxidase, lane IV—cell lysate treated with calf intestinal alkaline phosphatase before electrophoresis

Fig. 2

Negative control sections incubated with nonimmune serum (instead of primary antibody) and with FITC-conjugated secondary antibody (a). The same cells stained with DAPI are shown in blue (b). Bar = 10 μm

Fig. 3

Successive phases of the cell cycle in root meristems of V. faba: DAPI staining—blue fluorescence; immunocytochemical identification of RBR-like protein using anti-human phospho-Rb (S807/811)—green fluorescence (FITC). Description in the text. Bar = 10 μm

Fig. 4

Mean intensity of fluorescence (MIF) of proteins detected by anti-phospho-Rb (S807/811)-FITC in the double-stained (FITC/DAPI) root meristem cells of V. faba during successive phases of the cell cycle; G1, S, G2, P prophase, M metaphase, A anaphase, T telophase, PT post-telophase. Pixel value = MIF/pel ± SD

Fig. 5

a–d Selected immunofluorescence linescans for double-stained nuclei [phospho-Rb (S807/811)-FITC—green line and DNA-DAPI—blue line) in V. faba root meristem cells; green and blue arrows indicate plot lines