Arabidopsis protein kinases GRIK1 and GRIK2 specifically activate SnRK1 by phosphorylating its activation loop

Abstract

SNF1-related kinases (SnRK1s) play central roles in coordinating energy balance and nutrient metabolism in plants. SNF1 and AMPK, the SnRK1 homologs in budding yeast (Saccharomyces cerevisiae) and mammals, are activated by phosphorylation of conserved threonine residues in their activation loops. Arabidopsis (Arabidopsis thaliana) GRIK1 and GRIK2, which were first characterized as geminivirus Rep interacting kinases, are phylogenetically related to SNF1 and AMPK activating kinases. In this study, we used recombinant proteins produced in bacteria to show that both GRIKs specifically bind to the SnRK1 catalytic subunit and phosphorylate the equivalent threonine residue in its activation loop in vitro. GRIK-mediated phosphorylation increased SnRK1 kinase activity in autophosphorylation and peptide substrate assays. These data, together with earlier observations that GRIKs could complement yeast mutants lacking SNF1 activation activities, established that the GRIKs are SnRK1 activating kinases. Given that the GRIK proteins only accumulate in young tissues and geminivirus-infected mature leaves, the GRIK-SnRK1 cascade may function in a developmentally regulated fashion and coordinate the unique metabolic requirements of rapidly growing cells and geminivirus-infected cells that have been induced to reenter the cell cycle.

Figure 1.

The GRIKs specifically phosphorylate SnRK1. A, GST-GRIK1 (wt, lanes 1–4) or the kinase-inactive GRIK1(K137A) (m, lanes 5 and 6) were analyzed using in vitro protein kinase assays. B, GST-GRIK2 (wt, lanes 1–4) or the kinase-inactive GRIK2(K136A) (m, lanes 5 and 6) were analyzed in equivalent assays. A and B, The different GRIKs were incubated with kinase-inactive kinase domains of His₆-SnRK1.1(KD, K48A) (lanes 1 and 5), His₆-SnRK1.2(KD, K49A) (lanes 2 and 6), His₆-SnRK3.11(KD, K40A) (lane 4), or kinase-inactive, full-length His₆-SnRK2.4(K33A) (lane 3) in the presence of [γ-³²P]ATP. The reactions were resolved by SDS-PAGE and transferred to nitrocellulose membranes. The top panels show ³²P-labeled proteins visualized by autoradiography. The middle panels show total GST-GRIK proteins detected by immunoblotting with anti-GST antibodies, while the bottom panels correspond to total His₆-SnRKs detected with an anti-His₆ antibody.

Figure 2.

The GRIKs phosphorylate a conserved Thr residue in the SnRK1 activation loop. A, Amino acid sequence alignment of the activation loops of yeast SNF1, human AMPKα, and Arabidopsis SnRK1.1, SnRK1.2, SnRK2.4, and SnRK3.11. The box encloses the SNF1 homologs, and the asterisks mark identical amino acids in the included sequences. The arrow indicates the conserved Thr residue. The highlighted residues were mutated to Ala residues to generate activation loop mutants. B and C, Protein phosphorylation assays containing GST-GRIK1 (B) or GST-GRIK2 (C) were performed using [γ-³²P]ATP (top panels). The lanes contained His₆-tagged wild-type (wt) activation loop SnRKs, including inactive kinase domains of SnRK1.1(KD, K48A) (lane 1), SnRK1.2(KD, K49A) (lane 3), SnRK3.11(KD, K40A) (lane 8), or full-length SnRK2.4(KD, K33A) (lane 5) or activation loop mutant (m) forms of SnRKs, including SnRK1.1(KD, T175A) (lane 2), SnRK1.2(KD, T176A) (lane 4), SnRK2.4(K33A, S158A) (lane 6), SnRK2.4(K33A, T157A) (lane 7), and SnRK3.11(KD, K40A, T168A, T169A) (lane 9). D, Immunoblot kinase assays using antibodies against a human AMPKα phospho-T172 peptide with GST-GRIK1 (wt, lanes 1–4), kinase-inactive GST-GRIK1(K147A) (m, lanes 5 and 6), GST-GRIK2 (wt, lanes 7–10), or kinase-inactive GST-GRIK2(K136A) (m, lanes 11 and 12). The different GRIKs are enclosed by boxes. The substrates are inactive kinase domains with wild-type activation loop sequences (wt) of SnRK1.1(KD, K48A) (lanes 1, 5, 7, and 11) or SnRK1.2(KD, K49A) (lanes 3, 6, 9, and 12) or kinase domains with mutant activation loop sequences (m) of SnRK1.1(KD, T175A) (lanes 2 and 8) or SnRK1.2(KD, T176A) (lanes 4 and 10), as indicated above the top panel. In B to D, total GST-GRIK and His₆-SnRK were visualized using anti-GST (middle panels) and anti-His₆ antibodies (bottom panels).

Figure 3.

The GRIKs interact with SnRK1.1. Equal concentrations (150 nm) of His₆-tagged SnRK1.1 kinase domain (lanes 1–5) or full-length SnRK2.4 (lanes 6–10) were incubated with GST (lanes 1 and 6), GST-tagged GRIK1 (lanes 2 and 7), GST-tagged kinase-inactive GRIK1(K137A) (mGRIK1; lanes 3 and 8), GST-tagged GRIK2 (lanes 4 and 9), or GST-tagged kinase-inactive GRIK2(K136A) (mGRIK2; lanes 5 and 10). The protein mixtures were incubated with glutathione-Sepharose, and the bound fractions were resolved by SDS-PAGE and visualized by immunoblotting with antibodies to the His₆ (top panel) or GST (bottom panel) tags. Aliquots equivalent to one-fourth of the input for His₆-tagged SnRK1.1 (lane 11) and SnRK2.4 (lane 12) were included on the gels.

Figure 4.

The GRIKs activate SnRK1 kinase activity. A and B, Protein phosphorylation assays with wild-type (lanes 3–5) or kinase-inactive forms (lane 7) of GST-tagged GRIK1 (A) or GRIK2 (B) were performed using [γ-³²P]ATP (top panels). The lanes contained the His₆-tagged SnRK1.1 kinase domain (lanes 5–7) or the corresponding kinase-inactive mutant (K48A; lanes 1 and 4) or activation loop mutant (T175A; lanes 2 and 3). Total GST-GRIK and His₆-SnRK1.1 were visualized using anti-GST (middle panels) and anti-His₆ antibodies (bottom panels). C, His₆-tagged wild-type (wt) SnRK1.1 kinase domain, its kinase-inactive form (m), or activation loop mutant (m) was incubated alone or in the presence of GST-tagged wild-type (wt) or kinase-inactive (m) GRIK1 or GRIK2. SnRK1 kinase activity was monitored by ³²P-labeling of a peptide substrate derived from Suc-P synthase. The mean activities and sds for three experiments using different SnRK preparations are shown.

Figure 5.

Influence of cations, 5′-AMP, and STO-609 on GRIK activity. Phosphorylation assays containing GST-tagged GRIK1 (top) or GRIK2 (bottom) were performed in the presence of unlabeled ATP and detected by immunoblotting with antibodies against a human AMPKα phospho-T172 peptide. A, The reactions included 5 mm Mg²⁺ (lanes 1, 3, 5, and 7), 5 mm Mn²⁺ (lanes 2, 4, 6, and 8), 1 mm EGTA (lanes 1, 2, 5, and 6), or 1 mm Ca²⁺ (lanes 3, 4, 7, and 8). Lanes 1 to 4 contained His₆-SnRK1.1(KD, K48A), while lanes 5 to 8 contained His₆-SnRK1.2(KD, K49A). B, The reactions contained no AMP or GMP (lanes 1 and 4), 0.5 mm AMP (lanes 2 and 5), or 0.5 mm GMP (lanes 3 and 6). Lanes 1 to 3 contained SnRK1.1, while lanes 4 to 6 contained SnRK1.2. C, The reactions were performed in the absence of STO-609 (lanes 1 and 3) or in the presence of 20 μm STO-609 (lanes 2 and 4). Lanes 1 and 2 contained SnRK1.1, while lanes 3 and 4 contained SnRK1.2.

Figure 6.

GRIK protein expression and SnRK1 phosphorylation overlap in planta. A, Total protein extracts from Arabidopsis SAM and young leaves (≤0.5 cm, lane 1), expanding leaves (0.5–2 cm, lane 2), fully expanded leaves (2–3 cm, lane 3), and senescing leaves (older than the fully expanded, lane 4) were resolved by SDS-PAGE and analyzed by immunoblotting. B, Total protein extracts from mock (lane 1) and CaLCuV-infected leaves (lane 2) were analyzed. Antibodies against the GRIK1 protein (top panels), a human AMPKα phospho-T172 peptide (middle panels), or SnRK1.1 (bottom panels) were used in A and B.