Plant Physiol. (1999) 120: 257-274

Two SNF1-Related Protein Kinases from Spinach Leaf Phosphorylate and Inactivate 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase, Nitrate Reductase, and Sucrose Phosphate Synthase in Vitro¹

Christopher Sugden², Paul G. Donaghy^{2, 3}, Nigel G. Halford, and D. Grahame Hardie^*

Biochemistry Department, Dundee University, Medical Sciences Institute/Wellcome Trust Building Complex, Dow Street, Dundee DD1 5EH, Scotland, United Kingdom (C.S., P.G.D., D.G.H.); and IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol BS41 9AF, United Kingdom (N.G.H.)

	ABSTRACT

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We resolved from spinach (Spinacia oleracea) leaf extracts four Ca²⁺-independent protein kinase activities that phosphorylate the AMARAASAAALARRR (AMARA) and HMRSAMSGLHLVKRR (SAMS) peptides, originally designed as specific substrates for mammalian AMP-activated protein kinase and its yeast homolog, SNF1. The two major activities, HRK-A and HRK-C (3-hydroxy-3-methylglutaryl-coenzyme A reductase kinase A and C) were extensively purified and shown to be members of the plant SnRK1 (SNF1-related protein kinase 1) family using the following criteria: (a) They contain 58-kD polypeptides that cross-react with an antibody against a peptide sequence characteristic of the SnRK1 family; (b) they have similar native molecular masses and specificity for peptide substrates to mammalian AMP-activated protein kinase and the cauliflower homolog; (c) they are inactivated by homogeneous protein phosphatases and can be reactivated using the mammalian upstream kinase; and (d) they phosphorylate 3-hydroxy-3-methylglutaryl-coenzyme A reductase from Arabidopsis at the inactivating site, serine (Ser)-577. We propose that HRK-A and HRK-C represent either distinct SnRK1 isoforms or the same catalytic subunit complexed with different regulatory subunits. Both kinases also rapidly phosphorylate nitrate reductase purified from spinach, which is associated with inactivation of the enzyme that is observed only in the presence of 14-3-3 protein, a characteristic of phosphorylation at Ser-543. Both kinases also inactivate spinach sucrose phosphate synthase via phosphorylation at Ser-158. The SNF1-related kinases therefore potentially regulate several major biosynthetic pathways in plants: isoprenoid synthesis, sucrose synthesis, and nitrogen assimilation for the synthesis of amino acids and nucleotides.

	INTRODUCTION

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Recent studies have defined a subfamily of plant protein kinases that are related to mammalian AMPK and the SNF1 protein kinase from the yeast Saccharomyces cerevisiae (for review, see Hardie and Carling, 1997; Halford and Hardie, 1998; Hardie et al., 1998). Mammalian AMPK switches off ATP-consuming anabolic pathways and switches on ATP-producing catabolic pathways by phosphorylating key regulatory enzymes such as HMG-CoA reductase (Corton et al., 1995). AMPK is activated by increased AMP and decreased ATP via a complex mechanism involving allosteric regulation (Corton et al., 1995), promotion of phosphorylation by an upstream protein kinase (AMPKK) (Hawley et al., 1995), and inhibition of dephosphorylation (Davies et al., 1995). Since AMP is elevated under conditions in which ATP is depleted because of the action of adenylate kinase, the kinase cascade is activated in a sensitive manner in response to cellular stresses that cause ATP depletion. We propose that AMPK acts as a "fuel gauge," protecting cells against the effects of environmental or nutritional stresses that deplete ATP (Hardie and Carling, 1997; Hardie et al., 1998).

A 1992 study (MacKintosh et al., 1992) reported that extracts of several plant species contained protein kinase(s) that phosphorylated the HMRSAMSGLHLVKRR (SAMS) peptide, a synthetic peptide designed as a specific substrate for mammalian AMPK (Davies et al., 1989). One of these protein kinases was purified from cauliflower (MacKintosh et al., 1992) and was shown to have properties very similar to those of mammalian AMPK in terms of both specificity for peptide substrates (Weekes et al., 1993; Dale et al., 1995b) and regulation by phosphorylation (MacKintosh et al., 1992). It also phosphorylated and inactivated HMG1, an HMG-CoA reductase from Arabidopsis, at a site (Ser-577) equivalent to that at which AMPK phosphorylated mammalian HMG-CoA reductase (Dale et al., 1995a). The one feature that was different between the plant and animal kinases was that the former was not activated by AMP, so that we could not adopt the name AMPK. Since HMG-CoA reductase was likely to be a physiological substrate, we tentatively named it HRK (HMG-CoA reductase kinase). It was subsequently termed HRK-A to distinguish it from a distinct form, HRK-B, that was purified from cauliflower and had a similar substrate specificity but a lower native molecular mass (Ball et al., 1994).

The Saccharomyces cerevisiae SNF1 gene (also known as CAT1 or CCR1) was identified via mutants that would not grow on carbon sources other than Glc, such as Suc or Gal. Genes required for growth on these carbon sources are repressed by Glc, and a functional SNF1 gene is required for derepression (Gancedo, 1998). SNF1 encodes a protein kinase (Snf1p) (Celenza and Carlson, 1986) that is closely related to the catalytic subunit of AMPK (Carling et al., 1994; Mitchelhill et al., 1994), and the two accessory subunits associated with these catalytic subunits in mammals and yeast are also closely related (Hardie et al., 1998). The yeast SNF1 kinase complex is rapidly and dramatically activated by phosphorylation in response to Glc deprivation, and this is associated with large increases in the cellular AMP:ATP ratio (Wilson et al., 1996). Therefore, there are obvious analogies between the roles of the AMPK and SNF1 systems, although, like cauliflower HRK-A, the SNF1 complex is not directly activated by AMP.

In 1991 a DNA encoding an Snf1 homolog was cloned from rye (Alderson et al., 1991), and homologs were subsequently cloned from barley (Halford et al., 1992; Hannappel et al., 1995), Arabidopsis (Le Guen et al., 1992), tobacco (Muranaka et al., 1994), and potato (Man et al., 1997). These higher plant kinases are now termed the SnRK1 (SNF1-related protein kinase 1) subfamily to distinguish them from other plant kinases somewhat more distantly related to Snf1 (Halford and Hardie, 1998). DNAs encoding rye (Alderson et al., 1991) or tobacco (Muranaka et al., 1994) SnRK1 complemented snf1 mutations in S. cerevisiae. Plant SnRK1 DNAs predict protein products of approximately 58 kD, and it was shown that the catalytic subunit of cauliflower HRK-A was a 58-kD polypeptide that cross-reacted with antibodies raised against a sequence, the PFDDDNIPNLFKKIK (NIP) peptide, which is conserved in the SnRK1 family (Ball et al., 1994, 1995). Evidence was also obtained that the barley SnRK1 gene (BKIN12) encoded a protein kinase with a similar specificity to cauliflower HRK-A (Barker et al., 1996). Recently, expression of potato SnRK1 DNA in the antisense orientation has been shown to dramatically decrease expression of Suc synthase mRNA in tubers and to abolish the induction of Suc synthase mRNA by Suc in leaves (Purcell et al., 1998). Since Suc synthase catalyzes a primary step in Suc catabolism in these tissues, this reinforces the idea that one function of SnRK1 kinases is to promote catabolic pathways, in this case via changes in gene expression.

Our biochemical characterization of the SnRK1 kinases in plants was originally performed using cauliflower inflorescences (MacKintosh et al., 1992; Ball et al., 1994, 1995; Dale et al., 1995a, 1995b), an abundant source for kinase purification. We have now switched our attention to spinach (Spinacia oleracea) leaves, partly because this system is more amenable to physiological studies, but also because there have been more extensive biochemical and metabolic studies using this system. We were particularly interested in the possibility that Ca²⁺-independent protein kinases resolved from spinach leaf extracts by anion-exchange chromatography, which regulate SPS (McMichael et al., 1995a) or NR (Douglas et al., 1997), might be members of the SnRK1 family. This seemed possible because the sequences around the regulatory sites on both SPS (McMichael et al., 1993) and NR (Douglas et al., 1995; Bachmann et al., 1996b; Su et al., 1996) matched the recognition motif that we had previously established for cauliflower HRK-A (Dale et al., 1995b).

McMichael et al. (1995a) resolved three kinase activities, of which the third (peak III) was Ca²⁺ independent. Peak III inactivated SPS and had a native molecular mass (150 kD) similar to HRK-A but was not purified beyond the initial column and remained poorly characterized. Douglas et al. (1997) also resolved three kinase peaks of which the third (PK_III) was Ca²⁺ independent. PK_III appeared similar to HRK-A in that it phosphorylated the SAMS peptide and Arabidopsis HMG-CoA reductase, was inactivated by protein phosphatases, had a similar specificity with synthetic peptides, and had a similar native molecular mass (140 kD). Fractions containing PK_III also contained an activity that inactivated NR in an ATP- and 14-3-3 protein-dependent manner and a 58-kD polypeptide that cross-reacted with an antibody raised against a fusion between maltose-binding protein and a rye SnRK1 (MBP-RKIN1). However, the latter result was not conclusive proof that PK_III was an snRK1 kinase, because the 58-kD polypeptide did not comigrate exactly with the peptide kinase activity.

Although these results suggested that PK_III (Douglas et al., 1997) and/or peak III (McMichael et al., 1995a) might be members of the SnRK1 family, several important questions remained: Was peak III (McMichael et al., 1995a) the same entity as PK_III (Douglas et al., 1997)? Although there were certain similarities, peak III was reported not to regulate NR, whereas PK_III had not been tested on SPS. Were peak III and/or PK_III members of the SnRK1 kinase family? Were the NR kinase and the peptide kinase in the PK_III fractions the same molecular entity?

In this paper we have re-examined these questions. We report that spinach leaf contains at least four Ca²⁺-independent protein kinases that phosphorylate synthetic peptides designed as substrates for AMPK/SNF1-related protein kinases, that at least two of these (HRK-A and -C) are indeed members of the SnRK1 family, and that HRK-A and HRK-C can phosphorylate and inactivate HMG-CoA reductase, NR, and SPS in vitro. HRK-C may correspond to peak III (McMichael et al., 1995a) and PK_III (Douglas et al., 1997), but it has now been more extensively purified and characterized. HRK-B, HRK-C, and HRK-D appear to represent previously undescribed entities.

	MATERIALS AND METHODS

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Materials

We purified commercial samples of Glc-6-P (supplied as disodium salts) by dissolving them in water and passing them through a column containing Chelex 100 (2.5 mL, Na⁺ form) layered on top of the ion-exchange resin AG50 (1 mL, H⁺ form). Fractions were collected in preweighed tubes and freeze-dried. The weighed Glc-6-P (now in the free acid form) was dissolved in water and neutralized with NaOH to make a 500 mM stock solution.

Peptides, Proteins, and Antibodies

Enzyme Assays

Purification of Peptide Kinases from Spinach Leaf

The supernatant was stirred with an equal volume of DEAE-Sepharose slurry equilibrated in buffer A, and the slurry was packed into a column. The column was washed with buffer A until the A₂₈₀ of the eluate was <0.15, and kinases were eluted using 300 mL of buffer A plus 0.25 M NaCl. The eluate was made 12% (w/v) with respect to PEG 6000 and stirred for 20 min, and the precipitated protein was collected by centrifugation at 22,000g for 20 min. The pellet was resuspended in 30 mL of buffer B (buffer A at pH 7.5) and clarified by centrifugation at 10,000g for 5 min. The supernatant was loaded at 3 mL/min onto a 20-mL Q-Sepharose HiLoad 16/10 column (equilibrated in buffer B) at 3 mL/min. The column was washed with buffer B until the A₂₈₀ had returned to baseline, and then kinases were eluted using a 180-mL gradient from 0 to 0.4 M NaCl in buffer B.

Two peaks of AMARA kinase activity were resolved: These were pooled and mixed together, and protein was precipitated by adding (NH₄)₂SO₄ to 50% saturation, stirring for 20 min, and centrifuging at 22,000g for 20 min. The pellet was resuspended in 5 mL of buffer C (50 mM Na Hepes, pH 8.0, 50 mM NaF, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM benzamidine, 0.1 mM PMSF, 0.02% [w/v] Brij-35, and 10% [v/v] glycerol) and was dialyzed overnight against three changes of buffer C. The volume was made up to 20 mL, and the suspension was clarified by centrifugation at 10,000g for 5 min. The supernatant was applied to a 1-mL Mono-Q HR 5/5 column equilibrated in buffer C at 1 mL/min. The column was eluted using a 33-mL gradient from 0 to 0.4 M NaCl in buffer C (Fig. 1).

View larger version (35K): [in this window] [in a new window]   Figure 1. Separation of AMARA peptide kinases and immunoreactive 58-kD polypeptide(s) from spinach leaf by Mono-Q chromatography. The column was eluted with a gradient of increasing NaCl. Upper panel, Fractions were analyzed for peptide kinase activity (). Peaks I, II, and III are indicated. Protein content in the eluate (continuous line) was monitored by A280, and the NaCl concentration (dashed line) was monitered by conductivity. U, Units. Lower panels, Individual numbered fractions from the column analyzed by western blotting.

Three peaks of AMARA kinase activity (I, II, and III) were pooled separately, concentrated to approximately 500 µL in a Vivaspin-30 (Vivascience, Lincoln, UK), frozen in liquid N₂, and stored at 80°C. For further purification, peaks I, II, and III were diluted to 1 mL with buffer D (buffer C at pH 7.4 and without NaF) and applied individually to 1-mL Mono-Q HR 5/5 columns equilibrated in buffer D. The columns were eluted with gradients from 0 to 100 mM (peaks I and II) or 0 to 200 mM (peak III) MgCl₂ in buffer D (Fig. 2). Peaks of AMARA kinase activity (HRK-A, HRK-B, HRK-C, and HRK-D) were concentrated in a Vivaspin-30, frozen in liquid N₂, and stored at 80°C.

View larger version (35K): [in this window] [in a new window]   Figure 2. Rechromatography of fractions I, II, and III on Mono-Q, with elution using a gradient of increasing MgCl2 (dashed line). Other symbols are as for Figure 1.

For some studies HRK-A and HRK-C were purified by a modification of the above method, referred to in the text as "protocol 2." Homogenization was identical except that the leaves (600 g) were homogenized in 1.2 L of homogenization buffer containing 8 g of PVPP. The homogenate was centrifuged at 10,500g for 15 min, and the supernatant was squeezed through two layers of cheesecloth and then one layer of Miracloth. Solid (NH₄)₂SO₄ was added with stirring to give 50% saturation. The suspension was centrifuged at 28,000g for 20 min, and the pellet was resuspended in buffer A and dialyzed against two changes of buffer A. The dialyzed sample was applied to DEAE-Sepharose equilibrated in 160 mL of buffer A, the column was washed extensively with buffer A, and the kinases were eluted with a 500-mL linear gradient from 0 to 0.5 M NaCl in buffer A. Fractions containing AMARA peptide kinase activity were pooled and (NH₄)₂SO₄ was added to 50% saturation.

The suspension was centrifuged (24,000g; 20 min), and the pellet was resuspended in buffer E (buffer C at pH 7.0), dialyzed against two changes of buffer E, and applied at 1 mL/min to a reactive Blue-Sepharose 30-mL column. After the sample was washed extensively with buffer E, the kinases were eluted with buffer E containing 0.5 M NaCl. Fractions containing AMARA peptide kinase activity were pooled and dialyzed against two changes of buffer C. The suspension was centrifuged at 24,000g for 10 min and applied to a Mono-Q column equilibrated with buffer C at 1 mL/min. After the sample was washed extensively with buffer C, the kinases were eluted with a gradient from 0 to 50 mM NaCl (1 mL), 50 to 400 mM NaCl (20 mL), and then 0.4 to 1.0 M NaCl (1 mL), all in buffer C.

Four peaks of AMARA peptide kinase activity were obtained, eluting at approximately 130, 200, 250, and 350 mM NaCl. The profile (not shown) was similar to that in Figure 1, except that HRK-D appeared as a discrete peak eluting between HRK-A and HRK-C. The four peaks were pooled separately, desalted by repeated dilution with buffer D and concentration in a Vivaspin-30, and applied to a Mono-Q column equilibrated in buffer D. After the sample was washed with buffer D, HRK-A, HRK-B, and HRK-D were eluted using gradients in buffer D from 0 to 30 mM MgCl₂ (2 mL), 30 to 70 mM MgCl₂ (13 mL), and then 70 to 100 mM MgCl₂ (5 mL) or 0 to 30 mM MgCl₂ (2 mL); then HRK-C using a gradient from 30 to 100 mM MgCl₂ (23 mL). HRK-A, HRK-B, HRK-C, and HRK-D eluted at approximately 50, 35, 90, and 70 mM MgCl₂ respectively. This step removed substantial contamination of HRK-B and HRK-D by HRK-A and a small contamination of HRK-C by HRK-D. Kinase peaks were pooled, concentrated in a Vivaspin-30, made 50% (v/v) with respect to glycerol, and stored at 20°C.

Further Purification of Peptide Kinases on ATP--Sepharose

Native Molecular Mass Determination

Phosphorylation of HMG-CoA Reductase and Analysis of Phosphorylation Site

Partial Purification of NR

Partial Purification of Spinach SPS

After the sample was loaded, the Blue-Sepharose column was washed with 30 mL of buffer K to wash any remaining unbound proteins onto the Q-Sepharose and then disconnected. The Q-Sepharose column was washed extensively with buffer K containing 0.2 M NaCl until the A₂₈₀ fell below 0.15, and then the protein was eluted with buffer K containing 0.5 M NaCl. SPS activity was measured in the 4-mL fractions collected, and active fractions were pooled and concentrated to 2.5 mL in a Vivaspin-30 before they were applied to a PD10 desalting column. The desalted sample was further concentrated to approximately 1 mL, diluted 3-fold with buffer K, and added to 4 mL of AMP-agarose resin. The suspension was stirred and allowed to settle for 15 min in a column, and this procedure was repeated twice. The column was washed with 20 mL of buffer K and then eluted with the same volume of buffer K containing 0.5 M NaCl. Fractions (1.4 mL) were collected throughout the wash and elution steps and were assayed for SPS and AMARA peptide kinase activity. Fractions containing SPS activity in the initial wash (SPS1) and in 0.5 M NaCl eluate (SPS2) were pooled separately, and both were concentrated to approximately 0.3 mL in a Vivaspin-30. Glycerol was added to a final concentration of 20% (v/v), and the samples were snap-frozen in small aliquots and stored at 80°C.

Pretreatment of SPS with Protein Phosphatase

Phosphorylation and Inactivation of SPS

To study phosphorylation of SPS, incubations were identical except that [-³²P]ATP (500-1000 cpm/pmol) was used. The reaction was terminated by the addition of SDS sample buffer, and the sample was boiled and subjected to SDS-PAGE in 8% gels. After Coomassie Blue staining the gel was dried and subjected to autoradiography to determine the incorporation of ³²P into the 130-kD SPS polypeptide. To estimate the stoichiometry of phosphorylation, the amount of the 130-kD polypeptide was estimated by densitometry of stained gels by comparison with BSA standards run on the same gel. The 130-kD polypeptide was then excised from the gel and its radioactivity was determined by Cerenkov counting.

Peptide Analysis and Sequencing

PP2A-treated SPS1 was phosphorylated and analyzed by SDS-PAGE as in the previous section. The gel was dried, and the ³²P-labeled SPS polypeptide was detected by autoradiography and excised from the gel. The gel slice was then rehydrated in water and redried on a centrifugal vacuum evaporator. It was then rehydrated in buffer N (50 mM Tris-HCl, pH 8.0, and 0.05% [v/v] Zwittergent 3-16) at 37°C for 60 min and then digested with trypsin (1:10, w/w) at 37°C for 60 min. The supernatant was removed and the gel slice was washed twice with buffer N. The combined supernatants were taken to near dryness on a centrifugal vacuum evaporator and diluted in 0.1% (v/v) TFA.

³²P-labeled peptides were analyzed on a C₁₈ protein and peptide HPLC column (0.25 × 45 cm; Vydac, Hesperia, CA) on an HPLC system (Gilson, Middleton, WI) run in 0.1% (v/v) TFA. Peptides were eluted in the gradient from water to acetonitrile, as indicated in Figure 12, and detected by Cerenkov counting using an analytical on-line monitor (Reeve Analytical, Glasgow, UK). To determine which residue was phosphorylated, peptides were subjected to solid-phase sequencing using a procedure based on that of Bodwell et al. (1991).

View larger version (25K): [in this window] [in a new window]   Figure 12. Reversed-phase HPLC analysis of 32P-labeled tryptic peptides derived from synthetic GRM peptide phosphorylated by HRK-A (A); synthetic GRM peptide phosphorylated by HRK-C (B); purified SPS phosphorylated by HRK-A (C); and purified SPS phosphorylated by HRK-C (D). See ``Materials and Methods'' for experimental details. The continuous line shows the radioactivity determined by Cerenkov counting; the dashed line shows the percentage of acetonitrile in the eluant.

PAGE, Western Blotting, and Other Analytical Procedures

	RESULTS

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Resolution of AMARA Kinases from Spinach Leaf Extracts by Anion-Exchange Chromatography

Protein kinases in spinach leaf extracts were extracted in buffers containing 50 mM NaF to inhibit protein phosphatases. They were initially purified by PEG precipitation and chromatography on DEAE-Sepharose, the latter using a step (rather than gradient) elution. No significant resolution of multiple forms of peptide kinase occurred in this step. Chromatography on Q-Sepharose with elution using an NaCl gradient resulted in two broad peaks of AMARA kinase activity (not shown). These were recombined and then separated on a Mono-Q column eluted with an NaCl gradient (Fig. 1), which resulted in the resolution of three peaks of peptide kinase activity designated I, II, and III. Peaks I and II eluted from Mono-Q at NaCl concentrations similar to those of HRK-B and HRK-A previously purified from cauliflower (Ball et al., 1994). Fraction II indeed appears to be very similar to cauliflower HRK-A by several criteria (see below) and will henceforth be referred to as HRK-A. However, fraction I turned out to be a mixture of two kinases. The kinase(s) corresponding to fraction III was not observed in the previous study of cauliflower (Ball et al., 1994).

If the fractions from the Mono-Q column were assayed with the SAMS peptide (used to purify the cauliflower kinases by Ball et al. [1994]) or the SP peptide (based on the phosphorylation site on SPS; see ``Materials and Methods''), identical profiles were obtained, but the specific activities were approximately one-half those obtained with the AMARA peptide (not shown). None of the three AMARA kinase peaks was inhibited by EGTA or stimulated by Ca²⁺; 1 mM Ca²⁺ caused a slight inhibition of all activities (not shown).

If fraction I was rechromatographed using the same gradient, only 56% of the recovered activity eluted in the fraction I position, whereas 44% eluted in the same position as fraction II/HRK-A (not shown). Fraction I from the first column therefore contained at least two protein kinase activities. Fractions I, II, and III from the first column were further purified by chromatography on Mono-Q with elution using a gradient of increasing MgCl₂ rather than NaCl (Fig. 2). This resulted in the resolution of the peptide kinase activities from a considerable amount of contaminating protein, particularly for fractions I and II. When fraction I was analyzed in this way, approximately 80% of the activity eluted in exactly the same position as fraction II/HRK-A (although we suspect it to be identical to HRK-A, this fraction will be designated HRK-A), with 20% eluting as a discrete earlier peak designated HRK-B.

When fraction III was analyzed using the MgCl₂ gradient, approximately 90% of the activity (designated HRK-C) eluted right at the end of the gradient, with about 10% (designated HRK-D) eluting at an intermediate position between HRK-A and HRK-C. At this stage the specific activities of HRK-A, HRK-B, HRK-C, and HRK-D were increased 95-, 46-, 120-, and 100-fold, respectively, compared with the initial extract (2.5 units/mg). However, these figures are underestimates of the purification achieved, because the activity of the initial extract represents the sum of the activity of all four kinases.

Analysis of AMARA Kinases by Gel Filtration

View larger version (19K): [in this window] [in a new window]   Figure 3. Analysis of fractions II (A), I (B), and III (C) by gel filtration on Superdex 200. Fractions were analyzed for peptide kinase activity (). Elution volumes of marker proteins are indicated by arrows, and molecular masses in kilodaltons are shown in B.

Further Purification of HRK-A on ATP--Sepharose

View larger version (36K): [in this window] [in a new window]   Figure 4. Purification of HRK-A (derived from a separation similar to Fig. 2) on ATP--Sepharose. Top, Protein/activity profile. The bulk of proteins did not bind to the column, as shown by the protein peak () in fractions 1 to 3. After extensive washing, the column was eluted with buffer containing ATP. Fractions were analyzed for peptide kinase activity (). Middle, SDS-PAGE analysis followed by silver staining. Lane L, Load; lane B, breakthrough (fractions 1-3); lane 1, fractions 21 to 25; lane 2, fractions 26 to 30; lane 3, fractions 31 to 35; and lane 4, fractions 36 to 40. Bottom, Western-blot analysis of fractions using anti-NIP antibody. Arrows at right indicate that the 58-kD polypeptide detected with the antibody aligns with the central band of the silver-stained triplet.

Analysis of Peptide Kinase Peaks by Western Blotting and Sequence Analysis

Specificity of HRK-A and HRK-C with Synthetic Peptides

View larger version (65K): [in this window] [in a new window]   Figure 5. Rate of phosphorylation of variants of the AMARA peptide (40 µM) by HRK-A and HRK-C. The sequence of the peptide is shown at left, with the amino acids changed from the parent AMARA peptide highlighted in bold and underlined. Results are expressed relative to the initial velocity obtained with the parent AMARA peptide.

Phosphorylation of HMG-CoA Reductase

Phosphorylation of NR

View larger version (52K): [in this window] [in a new window]   Figure 6. Coomassie Blue-stained gel (left) and corresponding autoradiogram (right) after incubation of partially purified NR with [-32P]ATP and HRK-A or HRK-C (both 1 unit/mL, 25 min). The presence or absence of NR or kinases in individual lanes is indicated at the bottom. Migrations of marker proteins (in kilodaltons) are indicated on the left. Identification of NR polypeptides (arrows at right) is based on their molecular masses and previous studies using a similar purification protocol (Douglas et al., 1995). NR (0.2 unit/mL) was incubated with [-32P]ATP (200 µM, approximately 1000 cpm/pmol) in a total volume of 20 µL of 50 mM Hepes-NaOH, pH 7.4, 10 mM MgCl2, 10 µM FAD, and 1 mM DTT in the presence or absence of HRK-A or HRK-C (1 unit/mL). After 10 min at 30°C the reactions were terminated by the addition of SDS-PAGE sample buffer, and the samples were boiled and loaded onto an 8% gel. After Coomassie Blue staining, the dried gels were subjected to autoradiography using Hyperfilm-MP.

View larger version (23K): [in this window] [in a new window]   Figure 7. Effect of incubation with MgATP with or without HRK-A (top) or HRK-C (bottom) with or without 14-3-3 protein, on the activity of partially purified NR. NR was incubated with MgATP alone (), MgATP plus kinase (), MgATP plus 14-3-3 protein (), or MgATP plus kinase plus 14-3-3 protein (). NR (4 milliunits/mL) was incubated at 30°C in a total volume of 850 µL of 50 mM Na-Hepes, pH 7.4, 10 mM MgCl2, 10 µM FAD, 1 mM DTT, and 1 mg/mL BSA. When added, MgATP was ATP (200 µM) plus MgCl2 (5 mM), HRK-A or HRK-C were at 1 unit/mL, and 14-3-3 protein (yeast Bmh2 protein) was at 0.02 mg/mL. Aliquots (200 µL) were removed at various times and assayed for NR activity in the presence of 5 nM staurosporine, which inhibits HRK-A and HRK-C and blocks further phosphorylation. Results are expressed as the percentages of initial activity, which was 4 milliunits/mL in both cases.

Phosphorylation of SPS

View larger version (19K): [in this window] [in a new window]   Figure 8. A, Partial resolution of SPS activity and AMARA peptide kinase activity on AMP-agarose. An SPS fraction purified to the Q-Sepharose step as described in ``Materials and Methods'' was applied to a column of AMP-agarose, which was washed with starting buffer. At the point shown by the arrow, bound proteins were eluted with buffer plus 0.5 M NaCl. Fractions were assayed for SPS () and AMARA peptide kinase activity (). B, SPS1 fraction was analyzed by SDS-PAGE in 8% gels. Gels were either stained with Coomassie Blue (left) or the proteins were electrophoretically transferred to nitrocellulose and the membranes probed with an anti-SPS antibody (right). The migration of marker proteins are indicated on the left.

The SPS specific activity of the SPS1 fraction was 0.4 µmol min¹ mg¹. When analyzed by SDS-PAGE, the preparation contained a number of polypeptides, but there was a prominent one with an apparent molecular mass of 130 kD. Although this is slightly higher than the mass predicted from the DNA sequence of spinach SPS (122 kD), this polypeptide cross-reacted with anti-SPS antibody (Fig. 8B). When the amount of protein in the 130-kD polypeptide was estimated by densitometry of Coomassie Blue-stained gels using BSA as a standard, specific activities of 150 ± 50 µmol min¹ mg¹ (means ± SE) were calculated for three different SPS1 preparations. This is identical to the value quoted by Huber and Huber (1996) for the specific activity of pure SPS. The SPS1 fraction was still contaminated with trace amounts of peptide kinase and SPS-inactivating activities. However, the residual peptide kinase activity could be inactivated in a time- and dose-dependent manner by treatment with the homogeneous catalytic subunit of PP2A; this treatment also almost completely abolished the ATP-dependent SPS inactivation (not shown). The PP2A-treated SPS1 fraction represents an essentially kinase-free SPS preparation that was used for further studies, PP2A being inhibited before subsequent studies by the addition of okadaic acid.

We assessed the effect of phosphorylation on SPS activity by measuring the ratio of activities under limiting conditions (3 mM Fru-6-P, 12 mM Glc-6-P, and 10 mM Pi) and nonlimiting conditions (10 mM Fru-6-P and 40 mM Glc-6-P), a protocol established by Huber et al. (1989). Incubation of PP2A-treated SPS1 with MgATP alone did not give a significant decrease in the activity ratio, but further addition of HRK-A, HRK-B, HRK-C, or HRK-D (equal amounts of peptide kinase activity) led to a decrease in activity ratio in every case from 0.4 to 0.20 to 0.25 (Fig. 9). This was associated with phosphorylation of the 130-kD SPS polypeptide (Fig. 9). At least some of this phosphorylation occurred at Ser-158, because after incubation with MgATP and added kinases, but not with MgATP alone, the signal obtained in western blots using an antibody that only recognizes the dephospho form of the Ser-158 sequence (Weiner, 1995) was significantly diminished (Fig. 9).

View larger version (33K): [in this window] [in a new window]   Figure 9. Inactivation and phosphorylation of SPS by HRK-A, HRK-B, HRK-C, and HRK-D. SPS1 was pretreated with PP2A to inactivate the endogenous kinase. Incubations (30 min at 30°C) contained SPS, MgATP, and HRK-A, HRK-B, HRK-C, or HRK-D (0.34 unit/mL) as indicated. Top, Aliquots were assayed for SPS activity. Results (means ± SE for triplicate assays) are expressed as the ratio of activities under limiting conditions (3 mM Fru-6-P, 12 mM Glc-6-P, and 10 mM Pi) and nonlimiting conditions (10 mM Fru-6-P and 40 mM Glc-6-P). Middle, Incubations utilized [-32P]ATP and were analyzed by SDS-PAGE and autoradiography. The 130-kD SPS polypeptide is indicated by the arrow on the left. Bottom, Incubations were analyzed by western blotting using an anti-peptide antibody that only recognizes SPS when it is not phosphorylated at Ser-158.

Because HRK-B and HRK-D are less well characterized than HRK-A and HRK-C and because HRK-C was available in larger amounts than HRK-A, more detailed studies of the inactivation of SPS were conducted with HRK-C. No inactivation of SPS by HRK-C was observed if MgATP was omitted, and inactivation was dependent on the dose of HRK-C added (not shown). Both the decrease in the activity ratio and the phosphorylation of the 130-kD polypeptide of SPS produced by HRK-C were time dependent (Fig. 10). With the particular SPS1 preparation used in Figure 10, there was some decrease in the activity ratio and an increase in the phosphorylation of SPS, even in the absence of added kinase. However, the addition of HRK-C greatly stimulated both effects. At later times the phosphorylation produced by HRK-C reached a stoichiometry of about 0.3 mol phosphate mol¹ 130-kD subunit. This represents an approximate estimate only, because the SPS protein content was estimated by densitometric comparison of the Coomassie Blue-stained 130-kD polypeptide using known amounts of BSA as standard. Inactivation of SPS could be at least partially reversed by subsequent incubation with homogeneous mammalian PP2A or PP2C (Fig. 11). This experiment does not distinguish whether the reactivation was due to reversal of phosphorylation by the endogenous kinase or by HRK-C. However, since both phosphorylate Ser-158 exclusively (see below), this does not affect the interpretation.

View larger version (34K): [in this window] [in a new window]   Figure 10. Time course of inactivation of SPS by HRK-C. The conditions were the same as in Figure 9, except that a different SPS1 preparation was used and HRK-C was at 0.4 unit/mL. Top, Aliquots were taken at various times for SPS assay. The results (means ± SE for triplicate assays) are expressed as the ratios of activities under limiting and nonlimiting conditions. Bottom, Parallel experiment performed using [-32P]ATP. At various times aliquots were analyzed by SDS-PAGE. Results of Coomassie Blue staining (top) and autoradiography (bottom) of the 130-kD SPS polypeptide are shown. There were other prominent polypeptides on the stained gel but, as in Figure 9, the 130-kD polypeptide was the only one that was significantly labeled with 32P.

View larger version (20K): [in this window] [in a new window]   Figure 11. Inactivation of SPS by phosphorylation can be partially reversed by dephosphorylation. Inactivation was as for Figure 5 except that the SPS1 was not pretreated with PP2A (this explains the slightly lower activity ratio in the control, since the sample contained higher endogenous kinase activity). The SPS was inactivated for 35 min at 30°C with HRK-C and 1 mM ATP. At the point shown by the arrow, 10 nM staurosporine was added to inhibit the kinase, followed by 4 milliunits/mL PP2A, 4 milliunits/mL PP2C, or 200 nM okadaic acid. Separate experiments showed that staurosporine inhibited HRK-C with an IC50 of 1 nM. Staurosporine (10 nM) did not affect SPS activity, and was also added to the SPS assays.

Since SPS is phosphorylated at multiple sites, not all of which are regulatory (Huber and Huber, 1996), it was important to examine the site(s) of phosphorylation. The experiment presented in Figure 9 using the dephospho-specific antibody suggested that HRK-A, HRK-B, HRK-C, and HRK-D all phosphorylated SPS on Ser-158 but did not reveal whether all of the phosphate incorporated into the 130-kD polypeptide was located at this site. To address this, we synthesized the GRM peptide, which corresponds to residues 151 to 170 on spinach SPS. This peptide was an excellent substrate for both HRK-A and HRK-C (K_m[HRK-A] = 6.4 ± 0.8 µM; K_m[HRK-C] = 10.9 ± 1.2 µM). Solid-phase sequence analysis (not shown) demonstrated that all of the phosphate was released at cycle 8, as would be expected if the Ser residue corresponding to Ser-158 in the full-length sequence was phosphorylated. The phosphorylated peptide was digested with trypsin (which would cleave after Arg-5, yielding the limit tryptic peptide ISS(p)VEMMDNWANTFK) and purified by reversed-phase HPLC (Fig. 12, A and B). The major radioactive peptide retained by the column eluted at 66 min for HRK-A and HRK-C. Solid-phase sequencing of these peptides indicated that almost all of the radioactivity was now released at cycle 3 as expected (not shown).

The PP2A-treated SPS1 preparation was phosphorylated using [-³²P]ATP and HRK-A or HRK-C, and proteins were separated by SDS-PAGE. A gel slice containing the 130-kD SPS polypeptide was dried and digested with trypsin, leading to release of 70% to 80% of the radioactivity into solution. On analysis by reversed-phase HPLC, the radioactivity retained by the column was recovered as a major peptide eluting at 66 min (exactly comigrating with the tryptic peptide obtained after phosphorylation of the GRM peptide), plus a minor peptide eluting just ahead of it at 64 min (Fig. 12, C and D). This minor form was also present in the analyses of the synthetic peptides (Fig. 12, A and B), although it was much less prominent. Solid-phase sequencing of the peptides eluting at 64 or 66 min revealed that the radioactivity was released at cycle 3 in every case (not shown). Although the peptide eluting at 64 min was not conclusively identified, we suspect that it is a form of the ISS(p)VEMMDNWANTFK peptide in which one or both of the Met residues had been oxidized during preparation.

Regulation of HRK-A and HRK-C by Phosphorylation

View larger version (23K): [in this window] [in a new window]   Figure 13. Inactivation of HRK-A (open symbols) and HRK-C (closed symbols) by PP2A (top) or PP2C (bottom). Incubations were performed with kinase alone (circles), kinase plus PP2A, or PP2C plus Mg2+ (squares), and kinase plus PP2A and okadaic acid, or PP2C minus Mg2+ (triangles). HRK-A and -C were purified as far as the Mono-Q column (Mg2+ gradient) but omitting NaF from the final purification buffer. The kinases were incubated at 30°C in 50 mM Na-Hepes, pH 7.0, 0.02% Brij-35 with PP2A. When added, PP2A or PP2C were at 10 units/mL, MgCl2 at 10 mM, and okadaic acid at 100 nM. Samples were removed at various times for assay of peptide kinase activity. Results are expressed as the percentage of the initial activity.

Lack of Effect of Glc-6-P on HRK-A and HRK-C

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

The key findings of this paper are that two Ca²⁺-independent protein kinases from spinach leaf, currently termed HRK-A and HRK-C, phosphorylate and inactivate not only HMG-CoA reductase but also SPS and NR. Our results also provide compelling evidence that these kinases represent members of the SNF1-related SnRK1 family. Our results pull together several previous studies of the regulation of HMG-CoA reductase, SPS, and NR via phosphorylation by Ca²⁺-independent protein kinases (Dale et al., 1995a; McMichael et al., 1995a; Douglas et al., 1997). However, in these previous studies the protein kinases were poorly characterized in molecular terms, and it was not clear that single protein kinases could regulate all three metabolic enzymes. Although further work is required to prove that these enzymes are targets for the SnRK1 kinases in vivo, this work shows that the SnRK1 kinases could potentially regulate both isoprenoid and Suc synthesis and nitrogen assimilation for amino acid and nucleotide biosynthesis. These are three major biosynthetic pathways in the plant, and it is interesting to note that the mammalian SNF1 homolog, AMPK, also regulates multiple biosynthetic pathways in animals (Hardie and Carling, 1997; Hardie et al., 1998).

Using peptides designed as substrates for the AMPK/SNF1 protein kinase subfamily, this study also shows that the situation in spinach leaves is more complex than that in cauliflower inflorescences (Ball et al., 1994). In the latter case only two Ca²⁺-independent peptide kinases were defined, HRK-A and HRK-B. HRK-A had a catalytic subunit of 58 kD, which cross-reacted with an antibody raised against a peptide conserved in the plant SnRK1 subfamily (Ball et al., 1995) and had a native molecular mass of 160 to 200 kD (Ball et al., 1994). In these and several other respects, such as elution positions on Mono-Q in NaCl and MgCl₂ gradients, peptide specificity, inactivation by protein phosphatases, and reactivation by mammalian kinase kinase (MacKintosh et al., 1992; Ball et al., 1994; Dale et al., 1995b), cauliflower and spinach HRK-A are very similar. Spinach HRK-B did not cross-react with the plant SnRK1 antibody and had a native molecular mass of 45 kD: In this respect it appears to be similar to the previously defined cauliflower HRK-B (Ball et al., 1994).

The analysis of spinach HRK-A