T-Loop Phosphorylation ofArabidopsisCDKA;1 Is Required for Its Function and Can Be Partially Substituted by an Aspartate Residue

Abstract

As in other eukaryotes, progression through the cell cycle in plants is governed by cyclin-dependent kinases. Phosphorylation of a canonical Thr residue in the T-loop of the kinases is required for high enzyme activity in animals and yeast. We show that the Arabidopsis thaliana Cdc2 1 /Cdc28 homolog CDKA;1 is also phosphorylated in the T-loop and that phosphorylation at the conserved Thr-161 residue is essential for its function. A phospho-mimicry T161D substitution restored the primary defect of cdka;1 mutants, and although the T161D substitution displayed a dramatically reduced kinase activity with a compromised ability to bind substrates, homozygous mutant plants were recovered. The rescue by the T161D substitution, however, was not complete, and the resulting plants displayed various developmental abnormalities. For instance, even though flowers were formed, these plants were completely sterile as a result of a failure of the meiotic program, indicating that different requirements for CDKA;1 function are needed during plant development.

Evolution of networks and sequences in eukaryotic cell cycle control

“…Loss-of-function analysis (by dominant-negatives, by siRNA, or by identification and testing of T-DNA insertion null alleles) of the homologues provides complementary functional information (e.g. requirement for cdka;1, the most likely Cdc2 homologue in Arabidopsis, for early cell cycles in gametogenesis [77]). …”

Control of Cell Proliferation, Organ Growth, and DNA Damage Response Operate Independently of Dephosphorylation of the Arabidopsis Cdk1 Homolog CDKA;1

“…CDKA;1 is also up to now the only CDK from Arabidopsis that could complement yeast cdc2/cdc28, while CDKB1;1 (formerly known as Cdc2bAt) was reported to fail so (Ferreira et al, 1991;Hirayama et al, 1991;Imajuku et al, 1992). Mammalian, yeast, and plant PSTAIRE kinases share the key regulatory sites in the T-and P-loops (Dissmeyer et al, 2007;Harashima et al, 2007; see Supplemental Figure 1 online). As shown in other organisms, phosphorylation of the T-loop of the Arabidopsis CDKA;1 is required for proper function of this enzyme (Dissmeyer et al, 2007;Harashima et al, 2007).…”

“…Mammalian, yeast, and plant PSTAIRE kinases share the key regulatory sites in the T-and P-loops (Dissmeyer et al, 2007;Harashima et al, 2007; see Supplemental Figure 1 online). As shown in other organisms, phosphorylation of the T-loop of the Arabidopsis CDKA;1 is required for proper function of this enzyme (Dissmeyer et al, 2007;Harashima et al, 2007). However, phosphorylation of the T-loop is not thought to be regulatory since no indication of a change in phosphorylation state during cell cycle progression was found in S. cerevisiae Cdc28p (Hadwiger and Reed, 1988).…”

The regulatory network of cell-cycle progression is fundamentally different in plants versus yeast or metazoans

“…23 Similarly, phosphorylation of the T-loop, a highly conserved domain in the activation segment of CDKs, was found to be needed in Arabidopsis as in animals to achieve high levels of kinase activity. 36,37 Moreover, it seems more than likely that also Arabidopsis CDKA;1 operates as a biological switch and that the system of CDK regulation results in hysteresis.…”