SKP1-SnRK protein kinase interactions mediate proteasomal binding of a plant SCF ubiquitin ligase
2001; Springer Nature; Volume: 20; Issue: 11 Linguagem: Inglês
10.1093/emboj/20.11.2742
ISSN1460-2075
AutoresRosa Farrás, Alejandro Ferrando, Jan Jásik, Tatjana Kleinow, László Ökrész, Antonio F. Tiburcio, Klaus Salchert, Juan C. del Pozo, Jeff Schell, Csaba Koncz,
Tópico(s)Fungal and yeast genetics research
ResumoArticle1 June 2001free access SKP1–SnRK protein kinase interactions mediate proteasomal binding of a plant SCF ubiquitin ligase Rosa Farrás Rosa Farrás Max-Planck Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Cologne, Germany Search for more papers by this author Alejandro Ferrando Alejandro Ferrando Max-Planck Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Cologne, Germany Search for more papers by this author Ján Jásik Ján Jásik Max-Planck Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Cologne, Germany Department of Plant Physiology, Comenius University, Mlynska dolina B2, 84215 Bratislava, Slovakia Search for more papers by this author Tatjana Kleinow Tatjana Kleinow Max-Planck Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Cologne, Germany Search for more papers by this author László Ökrész László Ökrész Institute of Plant Biology, Biological Research Center of Hungarian Academy of Sciences, H-6701 Szeged, Temesvári krt. 62, Hungary Search for more papers by this author Antonio Tiburcio Antonio Tiburcio Unitat de Fisiologia Vegetal, Universitat de Barcelona, Diagonal 643, 08028 Barcelona, Spain Search for more papers by this author Klaus Salchert Klaus Salchert Risoe National Laboratory, Plant Biology and Biogeochemistry Department, DK-4000 Roskilde, Denmark Search for more papers by this author Carlos del Pozo Carlos del Pozo Centro de Biologia Molecular 'Severo Ochoa', Cantoblanco, 28049 Madrid, Spain Search for more papers by this author Jeff Schell Jeff Schell Max-Planck Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Cologne, Germany Search for more papers by this author Csaba Koncz Corresponding Author Csaba Koncz Max-Planck Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Cologne, Germany Institute of Plant Biology, Biological Research Center of Hungarian Academy of Sciences, H-6701 Szeged, Temesvári krt. 62, Hungary Search for more papers by this author Rosa Farrás Rosa Farrás Max-Planck Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Cologne, Germany Search for more papers by this author Alejandro Ferrando Alejandro Ferrando Max-Planck Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Cologne, Germany Search for more papers by this author Ján Jásik Ján Jásik Max-Planck Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Cologne, Germany Department of Plant Physiology, Comenius University, Mlynska dolina B2, 84215 Bratislava, Slovakia Search for more papers by this author Tatjana Kleinow Tatjana Kleinow Max-Planck Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Cologne, Germany Search for more papers by this author László Ökrész László Ökrész Institute of Plant Biology, Biological Research Center of Hungarian Academy of Sciences, H-6701 Szeged, Temesvári krt. 62, Hungary Search for more papers by this author Antonio Tiburcio Antonio Tiburcio Unitat de Fisiologia Vegetal, Universitat de Barcelona, Diagonal 643, 08028 Barcelona, Spain Search for more papers by this author Klaus Salchert Klaus Salchert Risoe National Laboratory, Plant Biology and Biogeochemistry Department, DK-4000 Roskilde, Denmark Search for more papers by this author Carlos del Pozo Carlos del Pozo Centro de Biologia Molecular 'Severo Ochoa', Cantoblanco, 28049 Madrid, Spain Search for more papers by this author Jeff Schell Jeff Schell Max-Planck Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Cologne, Germany Search for more papers by this author Csaba Koncz Corresponding Author Csaba Koncz Max-Planck Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Cologne, Germany Institute of Plant Biology, Biological Research Center of Hungarian Academy of Sciences, H-6701 Szeged, Temesvári krt. 62, Hungary Search for more papers by this author Author Information Rosa Farrás1, Alejandro Ferrando1, Ján Jásik1,2, Tatjana Kleinow1, László Ökrész3, Antonio Tiburcio4, Klaus Salchert5, Carlos del Pozo6, Jeff Schell1 and Csaba Koncz 1,3 1Max-Planck Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Cologne, Germany 2Department of Plant Physiology, Comenius University, Mlynska dolina B2, 84215 Bratislava, Slovakia 3Institute of Plant Biology, Biological Research Center of Hungarian Academy of Sciences, H-6701 Szeged, Temesvári krt. 62, Hungary 4Unitat de Fisiologia Vegetal, Universitat de Barcelona, Diagonal 643, 08028 Barcelona, Spain 5Risoe National Laboratory, Plant Biology and Biogeochemistry Department, DK-4000 Roskilde, Denmark 6Centro de Biologia Molecular 'Severo Ochoa', Cantoblanco, 28049 Madrid, Spain *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:2742-2756https://doi.org/10.1093/emboj/20.11.2742 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction AMP-activated protein kinases (AMPKs) modulated by changes in the cellular AMP/ATP ratio are important regulators of metabolic and stress responses in eukaryotes (Hardie and Carling, 1997). Members of the AMPK family recognize similar substrates and consist of homologous core subunits that show a remarkable structural and functional conservation from yeast to humans (Hardie et al., 1998; Kemp et al., 1999; Vaulon et al., 2000). A prototype of AMPKs is the Snf1 (sucrose non-fermenting) protein kinase in budding yeast. Snf1 is required for proper transcriptional control of genes that are repressed when yeast cells grow in the presence of glucose and induced in response to glucose starvation and stress (Carlson, 1998). Snf1 is also implicated in the regulation of key metabolic enzymes and essential cellular processes, including mitochondrial and peroxisome biogenesis, nuclear import, thermotolerance and meiosis (Carlson, 1999). Recently, Snf1 has been identified as a component of Srb/mediator complex of RNA polymerase II (Kutchin et al., 2000). The conservation of the Snf1/AMPK family is illustrated by the observation that the catalytic and activator subunits of type I Snf1-related protein kinases (SnRKs) from higher plants suppress the deficiency of corresponding yeast proteins (Alderson et al., 1991; Muranaka et al., 1994; Jiang and Carlson, 1996, 1997; Kleinow et al., 2000). Despite an involvement of plant SnRKs in the regulation of some rate-limiting metabolic enzymes, their function in signalling is largely unknown (Halford and Hardie, 1998; Smeekens, 1998; Ikeda et al., 2000). Recently, Arabidopsis SnRKs AKIN10 and AKIN11 have been indirectly connected to sugar, hormone and stress signalling through the Pleiotropic Regulatory Locus 1 (PRL1). The prl1 mutation results in transcriptional derepression of many sucrose-regulated genes and causes arrested root elongation, altered leaf development and inhibition of cell elongation. In addition, the prl1 mutation results in hypersensitivity to glucose, sucrose, cold temperature, and the plant hormones cytokinin, auxin, ethylene and abscisic acid (Németh et al., 1998). PRL1 encodes an α-importin-binding nuclear WD-protein that interacts with yeast Snf1 and plant SnRKs in the two-hybrid system. Binding of PRL1 to the C-terminal regulatory domains of kinase catalytic subunits in vitro inhibits the activity of Arabidopsis SnRKs AKIN10 and AKIN11 (Bhalerao et al., 1999). In correlation with the pleiotropic phenotype, an enhanced activation of Arabidopsis SnRKs in the prl1 mutant is postulated to affect several regulators of cellular signalling. Here we show that an Arabidopsis SnRK, corresponding to either AKIN10 or AKIN11, co-purifies in the presence of ATP with the 26S proteasome, which is a large multisubunit complex controlling proteolytic degradation of ubiquitylated intracellular proteins. The 26S proteasome consists of a cylinder-shaped 20S proteolytic core and two 19S regulatory cap particles. The 20S core particle carries two copies each of seven different α and β subunits arranged in four α7β7β7α7 stacked rings (Hochstrasser, 1996; De Mot et al., 1999; Verma and Deshaies, 2000). We have found that Arabidopsis SnRKs interact with the α4/PAD1 subunit of the 20S proteasome (Fu et al., 1998; Rechsteiner, 1998; Voges et al., 1999) and the Skp1/ASK1 subunit of an SCF (Skp1-cullin-F-box) E3 ubiquitin ligase. SCF represents a conserved class of E3 enzymes that consists of core Skp1, F-box protein, Cdc53/cullin and Rbx1/Roc1 subunits, and carries an associated E2 ubiquitin conjugase in eukaryotes (Deshaies, 1999; Tyers and Jorgensen, 2000). SCF-mediated ubiquitylation and subsequent degradation of proteins occur in sequential steps. Following activation by an E1 enzyme, ubiquitin is transferred to an E2 enzyme and then to phosphorylated substrates that are specifically recognized and recruited to SCF by different F-box proteins (Hersko and Ciechanover, 1998; Patton et al., 1998). It is noteworthy that Arabidopsis SnRKs, like many E2 and E3 enzymes and ubiquitin C-terminal hydrolases, carry a ubiquitin- associated UBA domain that is postulated to confer target specificity in protein interactions (Hofmann and Bucher, 1996; Withers-Ward et al., 2000). As in yeast and mammals, SCF components are also implicated in the regulation of essential signalling pathways in plants (del Pozo and Estelle, 1999, 2000). The Arabidopsis SCFTIR1 complex, which carries SKP1/ASK1, AtCUL1 cullin and TIR1 F-box protein subunits, plays a distinguished role in the regulation of growth responses to the plant hormone auxin (Ruegger et al., 1998; Gray et al., 1999). The ask1 mutation causes male sterility due to a chromosome segregation defect during male meiosis, whereas both tir1 and ask1 mutations result in reduction of auxin responses (Gray et al., 1999; Yang et al., 1999). SKP1/ASK1 interacts in the two-hybrid system with several Arabidopsis F-box factors that are not yet characterized in SCF complexes in vivo. These F-box factors include UFO1 (Samach et al., 1999; Zhao et al., 1999) and COI1 (Creelman, 1998; Xie et al., 1998), which control floral organ identity and jasmonate-regulated defence responses, respectively, and several other SKP1/ASK1-interacting proteins (SKIPs) described here. Association of SKP1/ASK1 with an SnRK protein kinase reinforces a key element of the SCF paradigm (Bai et al., 1996; Skowyra et al., 1997), suggesting a requirement for phosphorylation of SCF substrates. In addition, interactions of SnRKs and SKP1/ASK1 with the α4/PAD1 proteasomal subunit, and co-immunoprecipitation of 20S proteasome α-subunits with SnRK, SKP1/ASK1 and cullin, indicate that an SCF ubiquitin ligase forms a proteasomal complex in Arabidopsis. Results Arabidopsis SnRKs AKIN10 and AKIN11 interact with the SCF subunit SKP1/ASK1 and 20S proteasome subunit α4/PAD1 Previous studies demonstrated that the catalytic subunits of Arabidopsis SnRKs AKIN10 and AKIN11 interact through their C-terminal regulatory domains with the yeast Snf1 kinase subunits Snf4 and Sip1, and their plant orthologues in the two-hybrid system (Jiang and Carlson, 1996, 1997; Bouly et al., 1999; Kleinow et al., 2000). The regulatory domains of AKIN10 and AKIN11 were also shown to bind the Arabidopsis PRL1 WD-protein, which is an SnRK inhibitor in vitro. A role for SnRKs in signalling was suggested by genetic analysis of the prl1 mutation, which leads to enhanced activation of SnRKs, transcriptional derepression of sucrose-regulated genes, and hypersensitivity to several plant hormones, including auxin (Németh et al., 1998; Bhalerao et al., 1999). To search for novel SnRK-binding factors, AKIN10 and AKIN11 in fusion with the Gal4 DNA-binding domain (GBD) were used as baits in yeast two-hybrid screens with a pACT2 cDNA library made from cultured Arabidopsis cells (Németh et al., 1998). From 5 × 107 transformants in each screen, 59 clones with AKIN10 and 52 clones with AKIN11 were obtained that grew in the presence of 50 mM 3-aminotriazole (3-AT) HIS3-inhibitor and displayed LacZ reporter enzyme activity (Durfee et al., 1993). Following control self-activation tests and interaction assays with a range of unspecific baits (Németh et al., 1998; data not shown), sequencing of prey cDNAs identified 13 protein classes that showed two-hybrid interactions with both AKIN10 and AKIN11. One class of AKIN-interacting factors described here was represented by five cDNAs that all encoded a full-length Arabidopsis SCF ubiquitin ligase subunit (Gray et al., 1999), SKP1/ASK1 (S phase kinase-associated protein/Arabidopsis Skp1-like; DDBJ/EMBL/GenBank accession No. AF059294), in fusion with the Gal4 activator domain (GAD). Another class included a single cDNA that encoded a peptide carrying 93 C-terminal amino acids from the Arabidopsis 20S proteasome subunit α4/PAD1 (Fu et al., 1998; DDBJ/EMBL/GenBank accession No. AF043522). A previously published full-length α4/PAD1 cDNA (TAS-g64; Genschik et al., 1992) was used to construct a GAD–PAD1 prey. In control two-hybrid assays, GAD–PAD1 showed interaction with GBD–AKIN11 as expected, as well as with a GBD–ASK1 bait, indicating that α4/PAD1 could also bind to SKP1/ASK1. A reversal of bait–prey combination (GBD–ASK1 versus GAD–AKIN10) confirmed interaction of SKP1/ASK1 with AKIN10 (Figure 1A). Figure 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). Download figure Download PowerPoint The specificity of observed protein interactions was tested by in vitro pull-down assays using glutathione S-transferase (GST) fusion proteins. AKIN10 and AKIN11 labelled with [35S]methionine by coupled in vitro transcription–translation were incubated with GST–ASK1 and GST proteins immobilized on glutathione–Sepharose (GS), and with the GS matrix alone. AKIN10 and AKIN11 were specifically retained on GST–ASK1, but failed to bind to the control GST and GS resins (Figure 1B). The α4/PAD1 protein was similarly labelled with [35S]methionine and loaded on GS matrices carrying immobilized GST–ASK1, GST–AKIN10, GST–AKIN11 and GST proteins. α4/PAD1 showed specific binding in vitro to GST–AKIN10, GST–AKIN11 and GST–ASK1, but not to the control GST protein, supporting the results of yeast two-hybrid protein interaction assays (Figure 1B). A two-hybrid screen performed with a GBD–ASK1 bait resulted in the identification of Arabidopsis cDNAs encoding AKIN10. In addition, seven cDNA classes of SKIPs were characterized. Six SKIP cDNAs encoded F-box proteins (Table I). These carried either leucine-rich repeats (SKIPs 1 and 2), or kelch domains (SKIPs 4 and 6; Adams et al., 2000), or no known C-terminal motifs (SKIP3 and 5). SKIP7 corresponded to fibrillarin (AtFIB1), an Arabidopsis orthologue of the nucleolar NOP1 protein (Barneche et al., 2000; Pih et al., 2000). Measurement of LacZ reporter enzyme activity in yeast cells grown under glucose limitation and on 2% glucose (Table I) indicated that all SKIP factors, except for SKIP5, interacted with SKP1/ASK1 in a glucose-regulated fashion, as was previously reported for two-hybrid interactions of yeast SCF components (Li and Johnston, 1997). Table 1. Properties of SKIPs SKIP Interaction with SKP1/ASK1 (β-GAL units) Structural domains Accession No. 0.05% glucose 2% glucose AKIN10 1688.1 ± 56.8 266.1 ± 1.0 SnRK M93023 AKIN11 113.7 ± 14.6 24.4 ± 0.9 SnRK X99279 SKIP1 426.9 ± 33.3 206.4 ± 6.8 F-box, LRR AF263377 SKIP2 91.4 ± 5.3 4.9 ± 1.4 F-box, LRR AF263378 SKIP3 555.2 ± 54.3 58.5 ± 17.5 F-box AF263379 SKIP4 1011.9 ± 56.8 369.1 ± 22.1 F-box, kelch AF263380 SKIP5 125.8 ± 11.0 144.6 ± 37.7 F-box AF263382 SKIP6 481.9 ± 42.5 20.6 ± 0.4 F-box, kelch AF263381 SKIP7 636.5 ± 10.6 366.3 ± 17.1 fibrillarin AF263383 Glucose regulation of bait–prey interactions was assayed by measurement of β-galactosidase enzyme activities (β-GAL units). LRR, leucine-rich repeats; kelch, kelch repeats. SKP1/ASK shared 35% sequence identity with budding yeast Skp1, which reflected a significant conservation of C-terminal protein domains. Amino acid positions corresponding to the yeast skp1-3 (I-172-N) and skp1-4 (L-146-S) mutations (Figure 2), causing G1–S and mitotic defects, respectively (Connelly and Hieter, 1996), were conserved within this region of SKP1/ASK1. Expression of SKP1/ASK1 by a methionine-repressible Met25 promoter (Mumberg et al., 1994) suppressed only the growth defect of the skp1-4, but not that of the skp1-3 thermosensitive yeast mutant, indicating an incomplete functional conservation between yeast Skp1 and Arabidopsis SKP1/ASK1 (Figure 1D). Another functional difference between Skp1 and SKP1/ASK1 was shown by control two-hybrid assays. These failed to reveal interactions between SKP1/ASK1 and yeast Snf1, as well as between yeast Skp1 and Snf1 (data not shown). By analogy, one of the 19 Arabidopsis SKP1/ASK homologues (Figure 2; At5), SKP1_5/ASK5, which carried a unique insertion of five amino acids (SDLLQ) in the C-terminal F-box-binding domain, displayed no interaction with AKIN10 and AKIN11, indicating that not all Arabidopsis SKP1 homologues shared the SnRK-binding property of SKP1/ASK1. Figure 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. Download figure Download PowerPoint SKP1/ASK1 is recruited by the PRL1-binding regulatory domains of SnRKs The SKP1/ASK1-binding regions of SnRKs were mapped by two-hybrid interaction assays with a GAD–ASK1 prey using a series of baits encoding different segments of AKIN10 and AKIN11 (Figure 3A). GAD–ASK1 interacted with homologous C-terminal SnRK peptides, which were located downstream of the UBA (Hofmann and Bucher, 1996) and yeast Snf4-binding regions (Bhalerao et al., 1999), between amino acid positions 349 and 512 in AKIN10, and 399 and 512 in AKIN11. These SKP1/ASK1-binding domains of AKIN10 and AKIN11 were previously observed to interact with the SnRK β and γ subunits (Kleinow et al., 2000; A.Ferrando, unpublished) and kinase inhibitor PRL1 WD-protein (Bhalerao et al., 1999). The mapping data therefore suggested that occupation of the common SnRK-binding sites by either of these factors would exclude binding of another factor to the same regions in AKIN10 and AKIN11. Figure 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. Download figure Download PowerPoint To test this hypothesis, a competitive AKIN10 kinase-binding assay was performed with SKP1/ASK1 and PRL1 (Figure 3B). [35S]methionine-labelled AKIN10 was bound to GST–ASK1, then the complex was challenged with increasing amounts of PRL1 in fusion with a maltose-binding protein (MBP–PRL1). Titration of [35S]AKIN10, using a selective pull-down of GST–ASK1 with GS and MBP–PRL1 with amylose resin, showed that increasing the amount of competitor MBP–PRL1 protein resulted in concomitant loss of AKIN10 from the GST–ASK1-bound fraction and accumulation of AKIN10 in MBP–PRL1-bound form. These data indicated that PRL1 could recruit AKIN10 by disrupting its interaction with SKP1/ASK1. Protein kinase assays performed under similar conditions showed that saturation of GST–AKIN10 with increasing amounts of GST–ASK1, as well as with control MBP and GST proteins, did not alter the AKIN10 kinase activity. By contrast, MBP–PRL1 efficiently inhibited the GST–AKIN10 kinase (Figure 3D). These assays also revealed that neither SKP1/ASK1 nor PRL1 served as substrate for the AKIN10 kinase. Competition of a preformed GST–AKIN10/GST–ASK1 complex with increasing amounts of MBP–PRL1 resulted in a gradual inhibition of the kinase activity, reflecting a recruitment of AKIN10 by PRL1. As PRL1 was also found to inhibit AKIN11 (Bhalerao et al., 1999), these results suggested that the SnRK inhibitor PRL1 WD-protein and SKP1/ASK1 may not occur in common SnRK complexes. To support this conclusion, proteins extracted from cultured Arabidopsis cells were immunoprecipitated with an antibody (α-ASK1) raised against a peptide carrying the last 21 C-terminal amino acids of SKP1/ASK1 (Figure 2). Western blotting of immunoprecipitated proteins with an α-SnRK antibody, recognizing both AKIN10 and AKIN11, showed that SKP1/ASK1 co-immunoprecipitated with a protein kinase that specifically phosphorylated the SnRK substrate peptide TRX-KD derived from sucrose-phosphate synthase (Bhalerao et al., 1999). By contrast, proteins immunoprecipitated with the α-ASK1 antibody did not cross-react with the anti-PRL1 antibody (α-PRL1; Figure 3E). These data were consistent with control experiments showing that the α-PRL1 antibody (Németh et al., 1998) immunoprecipitated an active SnRK, but not SKP1/ASK1, from the same cell extracts (data not shown). SnRK is associated with the 26S proteasome The α4/PAD1-binding SnRK domains were mapped as described above by assaying two-hybrid interactions of a GAD–PAD1 prey with GBD baits encoding different segments of AKIN10 and AKIN11 (Figure 3A). Similarly to SKP1/ASK1, GAD–PAD1 was found to interact with the PRL1-binding regions of SnRKs. To determine how saturation of the SnRK-binding site with SKP1/ASK1 affects subsequent binding of α4/PAD1, a kinase competition assay was performed. [35S]methionine-labelled AKIN10 was saturated by incubation with an excess of GST–ASK1. The preformed [35S]AKIN10–GST–ASK1 complex was then challenged with increasing amounts of His-α4/PAD1 protein, which carried an N-terminal His6 tag. The amount of [35S]AKIN10 in complex with GST–ASK1 was measured by pull-down assays with GS, whereas the amount of His-α4/PAD1 in the GST-bound fractions was monitored by immunoblotting with an α-His antibody (Figure 3C). A gradual increase in the amount of both [35S]AKIN10 and His-α4/PAD1 proteins in the GST–ASK1-bound fractions indicated that the α4/PAD1 protein, unlike PRL1, did not compete with SKP1/ASK1 for binding of the AKIN10 kinase. Rather, increasing the amount of α4/PAD1 proportionally increased the quantity of GST–ASK1-bound AKIN10 kinase. The His-α4/PAD1 protein could thus either recruit some residual free form of [35S]AKIN10 after saturation with GST–ASK1 or, more likely, increase the efficiency of kinase binding by forming a complex with GST–ASK1. To demonstrate the selectivity of α4/PAD1 interactions, protein extract prepared from cultured Arabidopsis cells was subjected to chromatography on Ni-NTA–agarose carrying immobilized His-α4/PAD1 protein. Whereas no specific protein binding was observed to the control empty Ni-NTA matrix, both SnRK
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