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

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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, α4/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 α‐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 purified 26S proteasomes and co‐purification of epitope‐ tagged SKP1/ASK1 with SnRK, cullin and proteasomal α‐subunits indicate that the observed protein interactions between SnRK, SKP1/ASK1 and α4/PAD1 are involved in proteasomal binding of an SCF ubiquitin ligase in Arabidopsis .