Cell Cycle Regulated D3-type Cyclins form Active Complexes with Plant-specific B-type Cyclin-dependent Kinase in vitro

Abstract

Tobacco (Nicotiana tabacum L.) cv Bright Yellow-2 (BY-2) cells are the most highly synchronizable plant cell culture, and previously we used them to analyze cell cycle regulation of cyclin-dependent kinases (CDKs) containing the cyclin binding motifs PSTAIRE (CDKA) and PPTA/TLRE (CDKB). Here we describe the analysis of tobacco CycD3 cyclins whose transcripts predominantly accumulate during G2 to M phase, which represents a unique feature of this type of cyclin D in plants. Although protein levels of CycD3s fluctuate with different patterns during the cell cycle, kinase assays revealed that the CycD3-associated kinases phosphorylate histone H1 and the tobacco retinoblastoma related protein (NtRBR1) with two peaks at the G1/S and G2/M boundaries. In vitro pull-down assays revealed that cell cycle-regulated CycD3s bind to CDKA, but more weakly than does CycD3;3, and that they also bind to CDKB and the CDK inhibitor NtKIS1a. Mutations in the cyclin box of the CycD3s showed that two amino acids are required for binding with CDKA and NtKIS1a, but no diminished interaction was observed with CDKB. A reconstituted kinase assay was adapted for use with bacterially produced GST-CycD3s, and kinase activity could be activated by incubation of extracts from exponentially growing BY-2 cells. Such activated complexes contained CDKA and CDKB, and the reconstituted GST-CycD3 mutants, retaining binding ability to CDKB, showed kinase activity, suggesting that these cell cycle-regulated CycD3s form active complexes with both A- and B-type CDKs in vitro.

Arabidopsis CYCD3 D-type cyclins link cell proliferation and endocycles and are rate-limiting for cytokinin responses

“…First, CYCD3 could also act as a mitotic cyclin, boosting G 2 /M kinase activity and hence directly enhancing progress from G 2 into mitosis. Although no association between CYCD and mitotic cyclin-dependent kinase has been detected in plant cell extracts using immunoprecipitation or proteomic approaches (32,33), cyclin-dependent kinase can bind Arabidopsis CYCD4;1 and tobacco CYCD3 in vitro (34,35), and histone H1-directed CYCD3 kinase activity is detected in mitotic tobacco suspension cultured BY-2 cells (36). The evidence for CYCD promotion of the G 2 /M transition is equivocal: Antirrhinum CYCD1, when induced at G 2 /M in synchronized BY-2 cells, indeed increased the rate of progression through mitosis (37), and ectopic expression of CYCD3;1 in Arabidopsis trichomes leads to additional mitoses (15).…”

Functional Evolution of Cyclin-Dependent Kinases

“…Phosphorylation of Rb by CDKA-CycD activates the transcription of E2F-regulated genes and S-phase progression [21]. Also, a tobacco CDKB-CycD complex capable of phosphorylating histone H1 and an Rb-related protein was reported recently [22].The sequential expression of the CycD-family, from early G1 to the onset of S-phase [23], is thought to drive the cell through G1 and the G1/S boundary. CycD function in plants may be analogous to the co-operative function of mammalian CycD and CycE, which retain Rb protein in an inactive state up to S-phase initiation (Fig.…”

“…CDK2 seems to be recruited for H1 phosphorylation by Cdc45, a protein required for DNA replication, while chromatin relaxation occurs because the transfer of phosphate groups de-stabilizes the interactions between H1 and chromatin [95][96][97]. In plants, members of the CDKA and CDKB groups have been reported to phosphorylate H1 [17,18,22,[98][99][100][101][102] but their role in regulation of chromatin remodelling remains uncertain.…”

PhyscomitrellaCyclin-Dependent Kinase A Links Cell Cycle Reactivation to Other Cellular Changes during Reprogramming of Leaf Cells

“…Therefore, to determine whether a similar binding happens in P. patens, we examined the interaction between CYCD;1 and CDKA by coimmunoprecipitation using epitope-tagged CYCD;1 ( Figure 5). As a negative control, we prepared a CYCD;1 mutant (CYCD;1KAEA) in which Ala replaced two amino acids (Lys-141 and Glu-171) previously identified in tobacco (Nicotiana tabacum) as being required for CYCD binding CDKA (Kawamura et al, 2006). Constructs containing the coding sequences driven by a heat shock-inducible promoter (HSP; Saidi et al, 2005) and followed by a triple hemagglutinin (HA)-tag were recombined into the PIG1 targeting region (Okano et al, 2009) to obtain the stable HSP:CYCD;1-3HA and HSP:CYCD;1KAEA-3HA transgenic lines (see Supplemental Figure 9 online).…”