SKP2A protein, an F-box that regulates cell division, is degraded via the ubiquitin pathway

Abstract

The ubiquitin pathway is emerging as a powerful system that controls the stability of key regulatory proteins. In plants, this pathway plays an important role in controlling several developmental processes, responses to environmental changes and also cell division. Arabidopsis SKP2A is an F-box protein that regulates the stability of the E2FC-DPB transcription factor, a repressor of cell proliferation. Although the function of SKP2A is to recruit targets for degradation, we have shown that SKP2A is also degraded through the Ub/26S pathway and, interestingly, auxin stimulates such degradation. Overexpression of SKP2A positively regulates cell division, increasing the number of cells in G₂/M, reducing the level of ploidy and developing higher number of lateral root primordia. In addition, we showed in this report that overexpression of SKP2A increased the survival of Arabidopsis plants when they grown on a medium with high levels of sucrose, likely by maintaining cell division active. Thus, it is likely that SKP2A connects cell division with stress responses.

Addendum to: Jurado S, Díaz-Triviño S, Abraham Z, Manzano C, Gutierrez C, Del Pozo C. SKP2A, an F-box protein that regulates cell division, is degraded via the ubiquitin pathway. Plant J 2008; 53:828-41.

The Ubiquitin Pathway and Cell Division in Plants

In the last years, ubiquitination has emerged as a powerful mechanism that regulates the activity of multiple proteins in plants.1–5 Ubiquitin (Ub) is attached to target proteins in a sequential biochemical cascade that involves an E1, an E2 and an E3 enzyme. Primarily, Ub is activated by an E1 in an ATP dependent manner and then the Ub moiety is transferred to an E2 enzyme (also called Ubiquitin conjugating enzyme). The last step might be divided in two parts: first the target protein is recognized by the E3, recruiting it to the proximity of the E2, and second, the Ub moiety is attached to the target.6 Among the different types of E3 described, the SCF complex is one of the most studied. The SCF complex is composed of 4 protein subunits, CUL1, RBX, ASK1 and an F-box. The first three forms a structural core in which different F-box proteins are assembled to generate diverse types of SCF complexes that might recruit different targets.

The completion of the Arabidopsis and rice genome sequences has revealed that these species have a large number of proteins involved in the Ub-pathway (approximately 5% of the total protein), suggesting that a large number of proteins might be regulated through ubiquitination.7,8 In plants, the Ub-pathway regulates the majority of biological processes, such as plant hormones signaling, light responses, circadian clock regulation or pathogen responses (reviewed in refs. 1, 2, 4 and 9). In addition to this, the Ub-pathway also regulates cell division by degrading key regulatory proteins. We have identified two F-box proteins, SKP2A and SKP2B, which share homology with human SKP2, a key regulator of cell division in humans. SKP2A targets two cell division transcription factors E2FC and DPB, which negatively regulates cell proliferation.10,11 In addition, SKP2B also seems to regulate the stability of cell cycle proteins. Recently, it has been shown that SKP2B and the RING Protein RKP degraded the CDK inhibitor KRP1.12 Although SKP2A and SKP2B share a high homology at the level of protein sequence, it has been shown that they have different target specificity since SKP2A did not target KRP112 and SKP2B did not promote DPB degradation.11

F-box protein represent one of the largest family with more than 700 members in Arabidopsis and rice.7,8,13 However, it is not clear whether all of these predicted F-box proteins are able to function as E3 ligases of Ub. Recently, we have developed a biochemical assay to test this activity of F-box protein. Essentially, the SCF^MYC-SKP2A is immunopurified from transgenic plant protein extract and then mixed with recombinant E1 and E2 and Ub in the presence of ATP. This assays showed that SKP2A forms an SCF complex with E3 ubiquitin ligase activity, since SCF^SKP2A is able to label different proteins with ubiquitin.5

Auxin Regulates the Stability of Skp2A

We have found that SKP2A is also regulated by degradation through the Ub/26S pathway. Interestingly, we found that auxin regulates the stability of SKP2A since when Arabidopsis seedlings are treated with auxin the amount of MYC-SKP2A protein is reduced in an Ub-dependent manner.5 To corroborate this biochemical data with genetic evidences, we crossed the MYC-SKP2A^OE plants with mutants that have affected the auxin response (axr1; axr2 and axr3). Immunoblotting analyses showed that SKP2A slightly accumulated in the axr1 mutant, but surprisingly its level was reduced in the axr2 and axr3 mutants.5 This finding might have different explanations. One might be that SKP2A is only degraded when assembled into a SCF complex. It is well known that CUL1 is not modified with RUB in the axr1 mutants,14 what might limit the assembly of SKP2A into a functional SFC, and therefore, SKP2A is not degraded. On the other hand, axr2 and axr3 mutants might alter the auxin response homeostasis or auxin concentration in the plant, what may affect SKP2A degradation. Something else to consider is that we have found that addition of auxin to the in vitro degradation reaction stimulates the degradation of SKP2A (Jurado S and del Pozo C, unpublished). This result suggests that auxin might directly regulate the stability of SKP2A. However, whether this degradation is similar to that found for the IAA/Aux proteins15,16 need to be tested.

Skp2A Increases Tolerance to Osmotic Stress

An expression analyses search using public databases revealed that SKP2A is highly induced upon osmotic stress (Fig. 1). To test whether SKP2A has a role during the response to this stress, Arabidopsis wild type and MYC-SKP2A overexpressing seedlings (MYC-SKP2A^OE) were grown in a MS medium with increased levels of sucrose. We found that MYC-SKP2A^OE plants showed a higher percentage of survival than wild type when grown in a MS medium containing 5 or 7% of sucrose (Fig. 2). This result suggests that SKP2A might have a role during osmotic stress and/or by a stress caused by an excess of sugar. It is known that high level of sucrose in the medium inhibited cotyledon expansion and leaf and root growth.17 We have found that SKP2A overexpression stimulates cell division in the root and shoot.5 Opposite, when Arabidopsis seedlings grown in a medium with high level of sucrose or mannitol, cell division is reduced (our observations). Thus, it is likely that overexpression of SKP2A maintains cell division active longer periods of time that wild type seedlings when they are grown on high amount of sucrose, and therefore SKP2A^OE are able to grow more effectively, surviving higher percentage than wt seedlings.

This data and the results published by Jurado and collaborators,5 indicate that SCF^SKP2A complex regulates positively cell division by degrading cell cycle repressors. In addition, the fact that MYC-SKP2A^OE plants are able to survive better to stresses (high sucrose or phosphate starvation, Jurado S and del Pozo C, unpublished) suggests that SKP2A regulates the programmed cell division but also it might connect cell division to stress responses.

Figures and Tables

Figure 1 SKP2A expression level is increased during osmotic stress. Taken from http://www.bar.utoronto.ca/.18

Figure 2 SKP2A overexpression increased the percentage of plants that survived when grown in high concentration of sucrose. Control and SKP2A^OE plants were grown on MS medium with different concentration of sucrose during 14 days. SKP2A overexpression increased plant survival when they are grown in a MS medium with 5% or 7% of sucrose. All plants survived when they were grown in MS medium with 1% or 3% (data not shown).

Addendum to: