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, alpha4/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 alpha-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 alpha-subunits indicate that the observed protein interactions between SnRK, SKP1/ASK1 and alpha4/PAD1 are involved in proteasomal binding of an SCF ubiquitin ligase in Arabidopsis.

Fig. 1. SKP1/ASK1 interacts with the α4/PAD1 subunit of 20S proteasome and Snf1-related protein kinases AKIN10 and AKIN11 in the two-hybrid system and in vitro, and suppresses the yeast skp1-4 mutation. (A) LacZ filter assays show two-hybrid interactions of GBD–ASK1 with GAD–PAD1 and GAD–AKIN10, as well as GBD–AKIN11 with GAD–PAD1, but no interactions of GBD baits with a control GAD–lamin prey. (B) [³⁵S]methionine-labelled AKIN10 and AKIN11 loaded in equal amounts (supernatant fractions) show specific binding in vitro to GST–ASK1, but not to control GS and GST matrices (bound fractions). In addition to full-size AKIN10 and AKIN11, artificial early termination of in vitro transcription–translation led to the synthesis of smaller truncated proteins (see supernatant fractions). No binding of shorter translation products to GST–ASK1 indicates that they correspond to C-terminal truncated forms of AKIN10 and AKIN11 that lack the SKP1/ASK1-binding site. (C) [³⁵S]methionine-labelled α4/PAD1 is specifically retained on GST–ASK1, GST–AKIN10 and GST–AKIN11 resins, but not on the control GST matrix, in protein-binding assays in vitro. (D) Expression of SKP1/ASK1 by a methionine-repressible Met25 promoter (pMET-ASK1) rescues the growth defect of thermosensitive skp1-4 yeast mutant at non-permissive temperature (37°C) in methionine-free medium (–Met), but not in the presence of 1 mM methionine (+Met). By contrast, SKP1/ASK1 does not suppress the growth defect of yeast skp1-3 mutant. As controls, the skp1-3 and skp1-4 mutants were transformed with an empty p426Met25 vector (pMET; Mumberg et al., 1994).

Fig. 2. Amino acid sequence comparison of yeast Skp1 (Sc) and 19 Arabidopsis SKP1/ASK1 homologues (At1–19). Conserved positions of amino acid residues corresponding to the yeast skp1-3 and skp1-4 mutations are indicated by arrows in the sequence alignment, which includes yeast Skp1 [194 amino acids (aa); AAB64763], and Arabidopsis Skp1/ASK1 sequences corresponding to the following accession Nos, BAC/P1 clones and predicted genes: 1 (160 aa, AAF26761, T4O12_17, At1g75950); 2 (171 aa, BAB08452, MJC20_30, At5g42190); 3 (163 aa, AAD31370, F3N11_15, At2g25700); 4 (163 aa, AAF79899, T20H2_8, At1g20140); 5 (153 aa, CAB75821, F24G16_290, At3g60020); 6 (123 aa, instead of annotated 85 aa, CAB86910, F8J2_230, At3g53060); 7 (125 aa, BAB00221, MSD21_15, At3g21840); 8 (152 aa, BAB00220, MSD21_14, At3g21830); 9 (153 aa, BAB00222, MSD21_16, At3g21850); 10 (152 aa, BAB00223, MSD21_17, At3g21860); 11 (152 aa, CAA17551, F28A23_30, At4g34210); 12 (152 aa, CAA18826, T4L20_50, At4g34470); 13 (154 aa, CAB75820, F24G16_280, At3g60010); 14 (149 aa, AAC34485, T18E12_16, At2g03170); 15 (194 aa, instead of annotated 177 aa, BAB00602, T5M7_7, At3g25650); 16 (170 aa, AAC34483, T18E12_14, At2g03190); 17 (150 aa, AAD24382, T2G17_4, At2g20160); 18 (158 aa, instead of annotated 183 aa, AAD32873, F14N23_11, At1g10230); and 19 (200 aa, AAC34486, T18E12_17, At2g03160). A longer SKP1-related sequence (AAC28530, F4I18_7, At2g45950, 300 aa) was not included in the alignment.

Fig. 3. SKP1/ASK1 and α4/PAD1 interact with C-terminal domains of AKIN10 and AKIN11. In vitro binding of SnRKs to SKP1/ASK1 is competed by PRL1, but enhanced by α4/PAD1, which selectively recruits SKP1/ASK1 and SnRK from Arabidopsis cell extracts. Unlike PRL1, SnRK is co-immunoprecipitated with SKP1/ASK1 and co-purifies with 26S proteasome. (A) Mapping of SKP1/ASK1 and α4/PAD1 binding domains of AKIN10 and AKIN11 by two-hybrid interaction assays. The results of LacZ filter assays (+ or –) indicate interactions of GBD baits, expressing different segments of AKIN10 and AKIN11 (amino acid positions are indicated in subscript), with GAD–ASK1 and GAD–PAD1 preys. Boxes indicate the positions of known SnRK domains. (B) In vitro SnRK-binding assay with SKP1/ASK1 and PRL1. A preformed [³⁵S]AKIN10–GST–ASK1 complex was challenged with increasing amounts of MBP–PRL1. Equal aliquots from each sample were bound to GS (GST pull-down) and amylose–agarose (MBP pull-down) to monitor the amount of [³⁵S]AKIN10 present in complex with GST–ASK1 and MBP–PRL1, respectively. Recruitment of a C-terminally truncated form of AKIN10 by MBP–PRL1 (lower band in MPB pull-down assay), but not by GST–ASK1, indicates that PRL1 can also interact with AKIN10 sequences located upstream of the C-terminal SKP1/ASK1 binding site. (C) In vitro SnRK-binding assay with SKP1/ASK1 and α4/PAD1. [³⁵S]AKIN10 was saturated with GST–ASK1, then increasing amounts of His-α4/PAD1 were added to the samples that were bound to GS. Following SDS–PAGE separation of eluted proteins, the amounts of GST–ASK1-associated [³⁵S]AKIN10 and His-α4/PAD1 proteins were monitored by autoradiography and western blotting with an anti-His₆ antibody, respectively. (D) In vitro kinase competition assay with SKP1/ASK1 and PRL1. Upper panel, phosphorylation of TRX-KD substrate by GST–AKIN10 alone (C) and in the presence of MBP, MBP–PRL1 and GST proteins. Lower panels, GST–AKIN10 was either incubated with increasing amounts of GST–ASK1 (left panel) or pre-incubated with GST–ASK1 followed by addition of increasing amounts of MBP–PRL1 (right panel) before performing the kinase assays with the TRX-KD substrate. (E) Protein extract from Arabidopsis Col-0 cells was bound to immobilized α-ASK1 IgG and protein A–Sepharose resins. Aliquots from the cell extract and proteins eluted from the IgG matrix (IP α-ASK1) and control protein A beads (w/o IgG) were immunoblotted with α-ASK1, α-SnRK and α-PRL1 antibodies and subjected to SnRK kinase assays. (F) Protein extract from Arabidopsis Col-0 cells was bound to His-α4/PAD1 immobilized on Ni-NTA–agarose and to control Ni-NTA-resin. The cell extract (Total) and protein fractions eluted from the His-α4/PAD1 (His-α4 pull-down) and Ni-NTA (Control) beads were immunoblotted with α-SnRK and α-ASK1 antibodies. (G) Purified 26S proteasome separated and stained in a non-denaturing polyacrylamide gel (Native) was eluted for separation of subunits by SDS–PAGE, which was either silver stained (Silver) or immunoblotted with α-20S proteasome and α-SnRK antibodies. Expected molecular masses for SnRK (AKIN10 or AKIN11) and proteasomal α-subunits are indicated.

Fig. 4. SKP1/ASK1, AtCUL1 and proteasomal α-subunits show nuclear co-localization in Arabidopsis roots and shoots. (A) Transverse section through the meristematic zone of root (bars = 10 µm). (B) Longitudinal cross-section of root apex (bars = 10 µm). (C) Longitudinal section through the shoot apex (bars = 25 µm). DAPI, nuclei stained with the DNA dye DAPI (blue). α-ASK1, sections treated with immunoaffinity-purified polyclonal rabbit α-ASK1 antibody and stained with either fluorescein isothiocyanate (FITC)-conjugated (green) or Cy™3-conjugated (red) goat anti-rabbit IgGs. α-20 and α-AtCUL1, the sections were treated with mouse α-20S proteasome and rabbit polyclonal α-AtCUL1 antibodies followed by staining with FITC-conjugated goat anti-mouse and anti-rabbit IgGs, respectively. The first three images in (A) and (B) show identical sections double-stained with α-ASK1 and α-20S antibodies, and counterstained with DAPI. The first and third images in (C) show the same section stained with DAPI and α-20S antibody.

Fig. 5. SKP1/ASK1, AtCUL1 and α-subunits of 20S proteasome are co-localized with mitotic spindles and phragmoplasts during mitosis. (A) Double-staining of cotyledon cells with anti-α-tubulin (green) and α-ASK1 (red) antibodies shows co-localization of SKP1/ASK1 with the mitotic spindle in late metaphase (upper cell), and phragmoplast during telophase (lower cell). (B) Overlapping staining patterns of mitotic spindle in a cotyledon cell stained with anti-α-tubulin (green) and α-AtCUL1 (red) antibodies. (C) Root cells stained with DAPI and α-20S antibody. The lower cell in metaphase shows a dotted staining pattern with the α-20S antibody (green), which marks the position of mitotic spindle flanking the equatorially arranged DAPI-stained chromosomes that are not detected by the α-20S antibody. The upper cell in early telophase shows a dotted α-20S-staining pattern of phragmoplast between the DAPI-stained chromosomes. (D) The α-ASK1 (red) and α-20S (green) antibodies stain the phragmoplast, but not the newly forming daughter nuclei (stained by DAPI in blue) in a root cell during telophase. (E) The α-AtCUL1 antibody (green) stains the phragmoplast in a root cell during telophase. Bars = 5 µm.

Fig. 6. SnRK is associated with SKP1/ASK1, cullin and 20S proteasome α-subunits. (A) SKP1/ASK1 is specifically detected by the polyclonal α-ASK1 antibody. Double-staining of Arabidopsis cells expressing an HA epitope-tagged form of SKP1/ASK1 protein (ASK1-HA) with polyclonal α-ASK1 (red) and monoclonal anti-HA (green) antibodies shows identical images of mitotic spindles in late anaphase (left column) and phragmoplasts in telophase (right column). Chromosomes and daughter nuclei are stained with DAPI (blue). Bars = 5 µm. (B) SKP1/ASK1 is co-immunoprecipitated with SnRK and cullin. Protein extract prepared from Arabidopsis cells expressing ASK1-HA was bound to immobilized anti-HA.11 IgG. The crude extract (T) and proteins eluted from the α-HA IgG matrix (IP) were separated by SDS–PAGE and immunoblotted with α-HA, α-SnRK and α-AtCUL1 antibodies. A control immunoprecipitation experiment was performed under identical conditions with a protein extract prepared from a cell line expressing an HA epitope-tagged β-glucuronidase enzyme (HA-GUS). (C) Purification of 20S proteasome–SCF complex. 20S proteasomal fractions co-purifying with cullin, SnRK and ASK1-HA on DEAE-Affi-Gel blue and Sephacryl S-400 were immunoaffinity purified on an anti-HA.11 IgG column. Proteins eluted with HA-peptide were separated by SDS–PAGE and immunoblotted with α-20S proteasome and α-HA antibodies. (D) Immunaffinity binding to α-HA and α-SnRK IgGs destabilizes the SCF complex. A Sephacryl S-400-purified 20S proteasome–SCF fraction (TOTAL) was immunoprecipitated using immobilized α-AtCUL1, α-SnRK and α-HA IgG antibodies, separated by SDS–PAGE and immunoblotted with α-HA, α-AtCUL1 and α-SnRK antibodies.

Fig. 7. Schematic model showing protein interactions in the proteasomal–SCF complex. (A) The α4/PAD1 proteasome subunit interacts with Snf1-related Arabidopsis protein kinases AKIN10 and AKIN11 in the yeast two-hybrid system and in vitro. SnRK protein kinase is detected in association with purified 26S proteasomes. (B) Epitope-tagged SKP1/ASK1 co-purifies and co-immunoprecipitates with SnRK, cullin and 20S proteasome α-subunits. SnRKs interact with the C-terminus of the α4/PAD1 proteasome subunit, which carries characteristic KEKE motifs present in several other α-subunits. The α4/PAD1 and SKP1/ASK1 proteins interact with each other and bind to the C-terminal regulatory domains of SnRKs. The activity and interaction of SnRKs with SKP1/ASK1 are inhibited by the PRL1 WD-protein.