The cdc2Ms Kinase Is Differently Regulated in the Cytoplasm and in the Nucleus

Abstract

To study a cyclin-dependent kinase (CDK) from alfalfa (Medicago sativa L.), an antibody was raised against the C-terminal 16 amino acids of the protein cdc2aMs. l h e cdc2Ms protein was immunopurified with this antibody and its histone kinase activity was measured. l h e cdc2Ms kinase is activated at the Cl/S transition when phosphate-starved cells from the CO phase re-enter the cell cycle and remain active as cells transit the S, C2, and M phases, indicating that the same CDK regulates all of these phases in alfalfa. In contrast, when cdc2Ms kinase was purified by binding to p13""", it was active only in the C 2 and M phases. In immunoblots the C-terminal antibody detected an equal amount of the cdc2Ms protein in the cytoplasm and in the nucleus. By indirect immunofluorescence, however, the cytoplasmic form of cdc2Ms could not be found in the S phase of the cells, indicating that the epitope for the cdc2 antibody is not accessible. Binding of putative inhibitor proteins to cdc2 was shown by inactivation of purified plant CDK when cell extracts were added. Furthermore, purified CDK inhibitors, such as the mouse ~2 7~' p ' and the yeast p40""', blocked the purified plant CDK activity.By irradiation of root meristems it was found that the two active phases of the cell cycle, the DNA synthesis (S) phase and segregation of chromosomes (M) phase, are each preceded by a regulatory phase called the G1 and G2 gap phases, respectively (Howard and Pelc, 1953). Within the gap phases the cell cycle can be interrupted at specific points at which developmental signals, the accomplishment of previous events in the cell cycle, as well as DNA

Control of the AtMAP65-1 interaction with microtubules through the cell cycle

“…Kinases were precipitated using Suc1 beads, anti-cdc2a, anti-cdc2b, anti-MPK4 and anti-MPK6 as described by Bögre et al (Bögre et al, 1997). Histone 1 (5 g) was used as a positive control for the phosphorylation reactions with cyclindependent kinases, and myelin basic protein (MBP; 5 g) was used as a positive control for phosphorylation reactions with mitogen-activated protein kinases.…”

“…Protein was extracted from BY-2 cells and phosphorylation assays were setup as described previously (Bögre et al, 1997). Each phosphorylation reaction contained 5 g of either recombinant AtMAP65-1, one of its fragments, or mutated AtMAP65-1 and 1 g of total protein extract where appropriate.…”

A plant cyclin B2 is degraded early in mitosis and its ectopic expression shortens G2-phase and alleviates the DNA-damage checkpoint

“…Immunostaining and microscopy For indirect immunofluorescence, cells were fixed and stained as previously described (Bögre et al, 1997). The HA.11 monoclonal antibody (BabCO, Richmond, California) was used in a 1:1000 dilution and with the secondary anti-mouse Cy3-conjugated antibody (Sigma) at a dilution of 1:200.…”

“…Proteins on western blots were detected by using anti-rabbit PSTAIRE antibody (Bögre et al, 1997), the HA.11 monoclonal antibody (BabCO, Richmond, California) or the polyclonal rabbit anti Nterminal cyclin B1;1 antibody (kindly provided by Pascal Genschik, Strasbourg, France). Cdk activities were measured in the samples after immunoprecipitation with the HA.11 monoclonal antibody or after binding to p13 suc1 , as described previously (Bögre et al, 1997).…”

“…The following drug concentrations were used in these experiments: 10 µM amiprophos methyl, 50 µM taxol, 10 µM propyzamide, 100 µM roscovitine (a gift from M. Strnad, Olomouc, Czech Republic), 100 µM MG 132 (Calbiochem), 5 µM lactacystin (Affinity, UK) and 1 µM epoxomicin (Affiniti, UK). To follow cell cycle progression, flow cytometric analysis was performed as described (Bögre et al, 1997) using a PAS2 flow cytometer (Partec, Münster, Germany). For mitotic index, cells were fixed in a 3:1 (v/v) ethanol/acetic acid mixture, then washed with 70% (v/v) ethanol.…”