SKP1-SnRK protein kinase interactions mediate proteasomal binding of a plant SCF ubiquitin ligase

Abstract

Arabidopsis Snf1-related protein kinases (SnRKs) are implicated in pleiotropic regulation of metabolic, hormonal and stress responses through their interaction with the kinase inhibitor PRL1 WD-protein.Here we show that SKP1/ASK1, a conserved SCF (Skp1-cullin-F-box) ubiquitin ligase subunit, which suppresses the skp1-4 mitotic defect in yeast, interacts with the PRL1-binding C-terminal domains of SnRKs. The same SnRK domains recruit an SKP1/ASK1-binding proteasomal protein, a4/PAD1, which enhances the formation of a trimeric SnRK complex with SKP1/ASK1 in vitro. By contrast, PRL1 reduces the interaction of SKP1/ASK1 with SnRKs. SKP1/ASK1 is co-immunoprecipitated with a cullin SCF subunit (AtCUL1) and an SnRK kinase, but not with PRL1 from Arabidopsis cell extracts. SKP1/ASK1, cullin and proteasomal a-subunits show nuclear co-localization in differentiated Arabidopsis cells, and are observed in association with mitotic spindles and phragmoplasts during cell division. Detection of SnRK in puri®ed 26S proteasomes and co-puri®cation of epitopetagged SKP1/ASK1 with SnRK, cullin and proteasomal a-subunits indicate that the observed protein interactions between SnRK, SKP1/ASK1 and a4/ PAD1 are involved in proteasomal binding of an SCF ubiquitin ligase in Arabidopsis.

β-Subunits of the SnRK1 Complexes Share a Common Ancestral Function Together with Expression and Function Specificities; Physical Interaction with Nitrate Reductase Specifically Occurs via AKINβ1-Subunit

“…Recently a major contribution was brought by the work of Baena-González and collaborators using transient expression experiments and transgenic plants. Their data implicate the two Arabidopsis (Arabidopsis thaliana) SnRK1 kinases as central integrators of transcriptional networks in response to stress and energy signaling (Baena-González et al, 2007), thus reinforcing diverse previous reports (Nemeth et al, 1998;Farras et al, 2001;Bradford et al, 2003;Buitink et al, 2003;Hao et al, 2003;Thelander et al, 2004;Gissot et al, 2006;Radchuk et al, 2006).…”

The F-box subunit of the SCF E3 complex is encoded by a diverse superfamily of genes in Arabidopsis

“…This superfamily represents Ϸ2.7% of the predicted genes in Arabidopsis, making it one of the largest so far described in plants. (5,34,35), suggesting that nematodes and plants have evolved an extensive combinatorial system of SCF complexes beyond that used by these other eukaryotes.…”

Cullins 3a and 3b Assemble with Members of the Broad Complex/Tramtrack/Bric-a-Brac (BTB) Protein Family to Form Essential Ubiquitin-Protein Ligases (E3s) in Arabidopsis

“…At2g13690 was first annotated as interacting with the WD protein PRL1, a protein that has pleiotropic effects on Arabidopsis hormone and sugar responses and development (58). PRL1 interacts with SNF1 protein kinases and with the SCF components ASK1 and CUL1 and a 26 S proteasome subunit (13), suggesting that PRL1 has a central role in Ub-mediated events. At5g55000, which is unique in the superfamily because it contains C-terminal pentapeptide repeats (Fig.…”

“…The only apparent exception is the VHL E3 complex where potential orthologs of the Elongin B and C and SOCS box proteins are not evident. Remarkably Arabidopsis encodes almost 700 F-box proteins, 21 SKPs (designated ASKs in Arabidopsis), and two CUL1 orthologs (CUL1 and -2a), indicating that a large array of SCF E3s is assembled in plants (11,13). Sequence and/or functional orthologs of yeast CUL4 and APC2 that should associate with the DDB1/DET1/COP1 and the APC E3 complexes, respectively, are evident in various plant genomes, indicating that these E3 types are present as well (9,12).…”