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• W2033582712 abstract "The Arabidopsis SOS2 (Salt Overly Sensitive 2)-like protein kinases (PKS) are novel protein kinases that contain an SNF1-like catalytic domain with a putative activation loop and a regulatory domain with an FISL motif that binds calcium sensors. Very little biochemical and functional information is currently available on this family of kinases. Here we report on the expression of the PKS11 gene, activation and characterization of the gene product, and transgenic evaluation of its function in plants.PKS11 transcript was preferentially expressed in roots ofArabidopsis plants. Recombinant glutathioneS-transferase fusion protein of PKS11 was inactive in substrate phosphorylation. However, the kinase can be highly activated by a threonine 161 to aspartate substitution (designated PKS11T161D) in the putative activation loop. Interestingly, PKS11 can also be activated by substitution of either a serine or tyrosine with aspartate within the activation loop. Deletion of the FISL motif also resulted in a slight activation of PKS11. PKS11T161D displayed an uncommon preference for Mn2+ over Mg2+ for substrate phosphorylation and autophosphorylation. The optimal pH and temperature values of PKS11T161D were determined to be 7.5 and 30 °C, respectively. The activated kinase showed substrate specificity, high affinity, and catalytic efficiency for a peptide substrate p3 and for ATP. AMP or ADP at concentrations from 10 μm to 1 mm did not activate PKS11T161D. TransgenicArabidopsis plants expressing PKS11T161D were more resistant to high concentrations of glucose, suggesting the involvement of this protein kinase in sugar signaling in plants. These results provide insights into the function as well as regulation and biochemical properties of the PKS protein kinase. The Arabidopsis SOS2 (Salt Overly Sensitive 2)-like protein kinases (PKS) are novel protein kinases that contain an SNF1-like catalytic domain with a putative activation loop and a regulatory domain with an FISL motif that binds calcium sensors. Very little biochemical and functional information is currently available on this family of kinases. Here we report on the expression of the PKS11 gene, activation and characterization of the gene product, and transgenic evaluation of its function in plants.PKS11 transcript was preferentially expressed in roots ofArabidopsis plants. Recombinant glutathioneS-transferase fusion protein of PKS11 was inactive in substrate phosphorylation. However, the kinase can be highly activated by a threonine 161 to aspartate substitution (designated PKS11T161D) in the putative activation loop. Interestingly, PKS11 can also be activated by substitution of either a serine or tyrosine with aspartate within the activation loop. Deletion of the FISL motif also resulted in a slight activation of PKS11. PKS11T161D displayed an uncommon preference for Mn2+ over Mg2+ for substrate phosphorylation and autophosphorylation. The optimal pH and temperature values of PKS11T161D were determined to be 7.5 and 30 °C, respectively. The activated kinase showed substrate specificity, high affinity, and catalytic efficiency for a peptide substrate p3 and for ATP. AMP or ADP at concentrations from 10 μm to 1 mm did not activate PKS11T161D. TransgenicArabidopsis plants expressing PKS11T161D were more resistant to high concentrations of glucose, suggesting the involvement of this protein kinase in sugar signaling in plants. These results provide insights into the function as well as regulation and biochemical properties of the PKS protein kinase. Salt Overly Sensitive 2 abscisic acid AMP-activated protein kinase [bis(2-hydroxyethy)amino]-2-(hydroxymethyl)propane-1,3-diol calcium-dependent/calmodulin-like domain protein kinase glutathione S-transferase Murashige and Skoog protein kinase C protein kinase D phenylmethanesulfonyl fluoride sucrose-non-fermenting protein kinase SOS3-like calcium-binding proteins SOS2-like protein kinases Protein phosphorylation plays crucial roles in cellular functions, including cell division, metabolism, and response to hormonal, developmental, and environmental signals. The Arabidopsisgenome encodes a large number of protein kinases (1Arabidopsis Genome Initiative Nature. 2000; 408: 796-815Crossref PubMed Scopus (7004) Google Scholar). The calcium-dependent protein kinase or calmodulin-like domain protein kinase family is responsive to calcium, because they contain a kinase catalytic domain fused with a calmodulin-like regulatory domain (2Roberts D.M. Curr. Opin. Cell Biol. 1993; 5: 242-246Crossref PubMed Scopus (58) Google Scholar). Recent studies suggest that the family of Salt Overly Sensitive 2 (SOS2)1-like protein kinases (i.e. PKSes) in plants is also responsive to calcium through interaction with the SOS3 (Salt Overly Sensitive 3) family of calcium-binding proteins, and thus may be functionally analogous to animal calcium/calmodulin-dependent protein kinases (3Guo Y. Halfer U. Ishitani M. Zhu J.-K. Plant Cell. 2001; 13: 1383-1399Crossref PubMed Scopus (372) Google Scholar). The Arabidopsis SOS2 and SOS3 genes are required for sodium and potassium ion homeostasis and salt tolerance (4Liu J. Ishitani M. Halfter U. Kim C.-S. Zhu J.-K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3730-3734Crossref PubMed Scopus (597) Google Scholar, 5Liu J. Zhu J.-K. Science. 1998; 280: 1943-1945Crossref PubMed Scopus (661) Google Scholar). SOS3 encodes a myristoylated EF-hand calcium-binding protein (5Liu J. Zhu J.-K. Science. 1998; 280: 1943-1945Crossref PubMed Scopus (661) Google Scholar, 6Ishitani M. Liu J. Halfter U. Kim C.-S. Wei M. Zhu J.-K. Plant Cell. 2000; 12: 1667-1677Crossref PubMed Scopus (371) Google Scholar) that may sense the calcium signal elicited by salt stress. SOS2 encodes a serine/threonine protein kinase with an N-terminal kinase catalytic domain similar to SNF1/AMPK (7Hardie D.G. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 97-131Crossref PubMed Scopus (246) Google Scholar) and a novel C-terminal regulatory domain (4Liu J. Ishitani M. Halfter U. Kim C.-S. Zhu J.-K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3730-3734Crossref PubMed Scopus (597) Google Scholar). SOS3 physically interacts with SOS2 in the yeast two-hybrid system as well as in vitro (8Halfter U. Ishitani M. Zhu J.-K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3735-3740Crossref PubMed Scopus (578) Google Scholar). A 21-amino acid sequence in the regulatory domain of SOS2, the FISL motif, has been determined to be necessary and sufficient to bind SOS3 (3Guo Y. Halfer U. Ishitani M. Zhu J.-K. Plant Cell. 2001; 13: 1383-1399Crossref PubMed Scopus (372) Google Scholar). In the presence of calcium, SOS3 activates the substrate phosphorylation of SOS2 (8Halfter U. Ishitani M. Zhu J.-K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3735-3740Crossref PubMed Scopus (578) Google Scholar). Salt stress up-regulation of the SOS1 (Salt Overly Sensitive 1) gene encoding a putative Na+/H+ antiporter is partially under control of the SOS3-SOS2 regulatory pathway (9Shi H. Ishitani M. Kim C.-S. Zhu J.-K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6896-6901Crossref PubMed Scopus (1182) Google Scholar). Arabidopsis contains 23 PKS genes, several of which have been cloned and their transcript expression analyzed (3Guo Y. Halfer U. Ishitani M. Zhu J.-K. Plant Cell. 2001; 13: 1383-1399Crossref PubMed Scopus (372) Google Scholar). However, neither the biochemical properties nor the physiological functions of the PKS gene products are known. By analogy to SOS2, these PKSes do not seem to have substrate phosphorylation activity in the absence of specific interacting proteins,i.e. the SOS3-like calcium-binding proteins, 2D. Gong, Z. Gong, Y. Guo, X. Chen, and J.-K. Zhu, unpublished observations. 2D. Gong, Z. Gong, Y. Guo, X. Chen, and J.-K. Zhu, unpublished observations. and thus further characterization is difficult to carry out. Therefore, it is of crucial importance to make these inactive PKSes active in order to characterize their biochemical properties. In addition to being excellent materials for biochemical characterization, active forms of PKSes may be expressed in plants to probe their in vivo functions. In this current work, we cloned the cDNA and analyzed the tissue-specific expression of a PKS gene, PKS11. We found that PKS11 was preferentially expressed in roots ofArabidopsis plants. A highly active PKS11 mutant form was constructed by substituting a threonine residue with aspartate (designated PKS11T161D) within the putative activation loop (10Johnson L.N. Noble M.E.M. Owen D.J. Cell. 1996; 85: 149-158Abstract Full Text Full Text PDF PubMed Scopus (1156) Google Scholar). This observation strongly suggests that activation loop phosphorylation may be an important determinant of the kinase activity in vivo. We then further characterized the activated PKS11 in terms of cofactor preference, substrate specificity, kinetic properties, effect of ADP and AMP, and pH and temperature dependence. We expressed the constitutively active PKS11 kinase mutant in transgenicArabidopsis, and we found that the transgenic plants were more resistant to high levels of glucose. Our results provide the first detailed biochemical characterization of the PKS and suggest that PKS11 is involved in sugar signaling in plants. A cDNA containing the complete open reading frame of PKS11was obtained by reverse transcriptase (Invitrogen)-PCR. Template mRNA was isolated from 2-week-old wild-type Arabidopsis(Columbia ecotype) plants. PKS11-specific primer pairs containing KpnI and EcoRI sites at the termini are as follows: 5′-GCGGTACCATGGTGGTAAGGAAGGTGGGCATATG-3′ (forward) and 5′-CGGAATTCAACGTCTTTTACTCTTGGCCTTGGTGAC-3′ (reverse) (MWG Biotec, High Point, NC). The PCR products were gel-purified, digested, and cloned into a modified pGEX-2T-CMS vector and completely sequenced. Arabidopsis wild-type seedlings were grown on Murashige and Skoog (MS) nutrient agar plates under continuous light (11Wu S. Ding L. Zhu J.-K. Plant Cell. 1996; 8: 617-627Crossref PubMed Google Scholar), and 10-day-old seedlings were treated with NaCl, abscisic acid (ABA), cold, and drought as described previously (9Shi H. Ishitani M. Kim C.-S. Zhu J.-K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6896-6901Crossref PubMed Scopus (1182) Google Scholar, 12Shi H. Xiong L. Stevenson B., Lu, T. Zhu J.-K. Plant Cell. 2002; 14: 465-477Crossref PubMed Scopus (925) Google Scholar). For the collection of different tissues, wild-type plants were grown in Turface soil to facilitate root harvesting. Roots and leaves were collected from 3-week-old-seedlings, and stems, flowers, and siliques were harvested from mature plants. Total RNA isolation and Northern blot analysis were performed as described previously (13Zhu J.-K. Liu J. Xiong L. Plant Cell. 1998; 8: 1181-1191Crossref Scopus (531) Google Scholar). For analysis of transgene expression, total RNA was isolated from 10-day-old seedlings grown on MS agar plates containing 3% glucose. Thirty micrograms of total RNA was loaded in each lane, size-fractionated by electrophoresis, and blotted onto a nylon membrane. The blot was hybridized with a gene-specific DNA probe for PKS11. A 1207-bp promoter region of the PKS11 gene was amplified by PCR from genomic DNA with the following primer pair introducing a BamHI site at the 5′ end and an SmaI site at the 3′ end to facilitate cloning: 5′-CGGGATCCATTATTTAGGAGAC-3′ (BamHI site underlined) and 5′-TCCCGGGCATTTCTTCAAGTCTAG-3′ (SmaI site underlined). The fragment was cloned intoBamHI- and SmaI-digested pBI101 vector to obtain a transcriptional fusion of the PKS11 promoter and the β-glucuronidase coding sequence. Transgenic plants harboring this construct were generated as described previously (12Shi H. Xiong L. Stevenson B., Lu, T. Zhu J.-K. Plant Cell. 2002; 14: 465-477Crossref PubMed Scopus (925) Google Scholar). For β-glucuronidase assay, materials were stained at 37 °C overnight in 100 mm sodium phosphate buffer, pH 7.0, containing 1 mg/ml 5-bromo-4-chloro-3-indoxyl-β-D glucuronic acid, 5 mm potassium ferricyanide, 5 mm potassium ferrocyanide, and 0.03% (v/v) Triton X-100. Both the T/S/Y to D change within the activation loop and the FISL motif deletion mutation of the PKS11 were introduced using oligonucleotide-directed in vitromutagenesis. The mutagenic primers for T/S/Y to D changed mutation are as follows: pPKS11T161D-forward, 5′-CAAGGAGTTACCATCCTAAAGGACACATGTGGAACTCCC-3′; pPKS11T161D-reverse, 5′-AATTGGGAGTTCCACATGTGTCCTTTAGGATGGTAACTC-3′; pPKS11S154D-forward, 5′-ATATCTGATTTTGCCTCGACGCATTACCTGAACAAGGAG-3′; pPKS11S154D-reverse, 5′-TCCTTGTTCAGGTAATGCGTCGAGGCCAAAATCAGATATC-3′; pPKS11Y173D-forward, 5′-ACATGTGGAACTCCCAATGACGTTGCTCCTGAGGTTCTCAG-3′; and pPKS11Y173D-reverse, 5′-GAGAACCTCAGGAGCAACGTCATTGGGAGTTCCACATGTTG-3′. The mutagenic primers for deletion mutation are as follows: pPKS11Δ F-forward, 5′-TCCACTAACTGGAAAGGACTCCATGAAGCACCAGACAAGG-3′; and pPKS11ΔF-reverse, 5′-AGTCCTTTCC- AGTTAGTGGACCTGTGTCTCTTGTTCCATC-3′. Mutagenesis reactions were carried out on the double-stranded plasmid DNA using an enzyme mix of LA Tag (Takara Shuzo Ltd., Kyoto) and Pfu Turbo DNA polymerase (1:1) (Stratagene, La Jolla, CA) with the following PCR cycle: 95 °C for 30 s, followed by 16 cycles of 95 °C for 30 s, 58 °C for 1 min, and 72 °C for 7 min. The PCR products were gel-purified and treated with DpnI to digest the parental supercoiled double-stranded DNA. The digested PCR products were transformed into DH5α-competent cells. The sequences of mutation as well as the fidelity of the rest of the DNA in all constructs were confirmed by direct DNA sequencing. GlutathioneS-transferase (GST)-PKS11 was obtained as described previously (8Halfter U. Ishitani M. Zhu J.-K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3735-3740Crossref PubMed Scopus (578) Google Scholar). The PKS11 open reading frame was cloned into pGEX-2T-CMS vector and expressed in bacteria as a C-terminal fusion protein with the bacterial GST under control of the isopropyl β-d-thiogalactopyranoside-inducible tacpromoter. All GST fusion constructs were transformed intoEscherichia coli BL21 codon plus cells (Stratagene). A 10-ml overnight Luria-Bertani (LB) culture was transferred to a fresh 1000 ml of LB and further cultured at 37 °C until theA 600 reached about 0.8. Recombinant protein expression was induced by 0.6 mm isopropyl β-d-thiogalactopyranoside for 4 h. The cells were harvested by centrifugation, and the pellets were resuspended in ice-cold lysis buffer, pH 7.5, containing 140 mm NaCl, 2.7 mm KCl, 10.1 mmNa2HPO4, 1.8 mmKH2PO4, 10% (v/v) glycerol, 5 mmdithiothreitol, 2 μg/ml aprotinin, 2 μg/ml leupeptin, and 2 mm phenylmethanesulfonyl fluoride. Lysozyme (1 mg/ml) and Triton X-100 (1%, v/v) were added to the suspension and incubated on ice with gentle shaking for 1 h before sonication. The sonicate was then clarified by centrifugation, and the recombinant proteins were affinity-purified by glutathione-Sepharose 4B (Amersham Biosciences). SDS-PAGE (10%, w/v) analysis was used to evaluate the protein composition of each preparation. Gels were stained with Coomassie Brilliant Blue R-250 (Bio-Rad). In vitro phosphorylation assays using a synthetic peptide p3 (ALARAASAAALARRR, Research Genetics, Huntsville, AL) as substrate were performed as described previously (8Halfter U. Ishitani M. Zhu J.-K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3735-3740Crossref PubMed Scopus (578) Google Scholar) with modification. Peptide phosphorylation was measured as the incorporation of radioactivity from [γ-32P]ATP (PerkinElmer Life Sciences) into the peptide substrate. Reactions without the peptide p3 or kinase proteins were used as controls. The kinase assay buffer contained 20 mm Tris-HCl, pH 7.2, 2.5 mm MnCl2 or 5 mm MgCl2, 0.5 mm CaCl2, 10 μm ATP, and 2 mm dithiothreitol. Kinase reactions (in a total volume of 40 μl) were started by adding 150 μm p3 and 5 μCi of [γ-32P]ATP (specific activity of 600 cpm/pmol), and reaction mixtures were immediately transferred to 30 °C for 30 min. All reactions contained ∼400–500 ng of purified proteins. Protein concentration was determined by the Bradford method using a dye binding assay (Bio-Rad) with bovine serum albumin as a standard. The stained bands on SDS-PAGE gels were also compared with a bovine serum albumin dilution series to adjust for the potential presence of other minor proteins that may copurify with the kinases. Enzyme activities were linear with respect to incubation time and amount of enzyme assayed. Reactions were terminated by adding 1 μl of 0.5 m EDTA, and the GST fusion proteins bound to glutathione-Sepharose beads were pelleted. Fifteen microliters of the supernatant was spotted onto P-81 phosphocellulose paper (Whatman) for peptide phosphorylation analysis. The P-81 paper was then washed 4 times in cold 1% (v/v) phosphoric acid (10 min per wash) and dried, and the phosphorylated peptide was quantified by phosphorimaging using a STORM 860 PhosphorImager (Amersham Biosciences) with the ImageQuant software. To the remaining 25 μl of reaction mixture, 5 μl of 6× SDS-PAGE sample buffer was added and denatured by boiling for 3 min, the samples were then separated by a 10% SDS-PAGE gel. The gel was dried and exposed to x-ray film (Eastman Kodak) to detect kinase autophosphorylation. For the analysis of a cofactor requirement, peptide phosphorylation and autophosphorylation assays were performed in the kinase assay buffer with 0–20 mm MnCl2 or MgCl2, whereas the concentrations of p3 (150 μm) and ATP (10 μM) were fixed. For substrate specificity assays, peptides p1 (LRRASLG) and p2 (VRKRTLRRL) (Sigma) were used in addition to the p3. Individual kinetic parameters were determined by varying the concentrations of p3 (0–300 μm) while holding ATP constant (10 μm). Alternatively, ATP concentrations were varied (0–20 μm) while keeping p3 constant (150 μm). The amount of recombinant proteins added to individual assays and the time of incubation were varied to maintain substrate conversion within a linear range. Optimal concentration of MnCl2 was used in the activity assays for the determination of kinetic parameters. Kinase assay buffers containing 10 μm to 1 mm ADP or AMP were used to test the effect of ADP or AMP on substrate phosphorylation. For the determination of optimal temperature of substrate phosphorylation, reaction mixtures were incubated at 15–42 °C instead of 30 °C. The effect of pH on substrate phosphorylation activity was determined using 20 mm BisTris titrated to the desired pH with either HCl or KOH in place of 20 mm Tris-HCl buffer. To generate the expression construct of PKS11T161D, PCR was carried out using two restriction sitesXbaI/SstI containing primers (5′-GCTCTAGAATGGTGGTAAGGAAGGTGGGCAAGTG-3′, forward,XbaI site underlined, and 5′-CGAGCTCTCAACGTCTTTTACTCTTGGCCTTGGTG-3′, reverse,SstI site underlined) on the PKS11T161D cDNA template. The PCR products were purified from agarose gel, digested, and cloned into the binary vector, pBIB, under control of the super promoter (14Narasimhulu S.B. Deng X. Sarria R. Gelvin S.B. Plant Cell. 1996; 8: 873-886PubMed Google Scholar). This promoter is located upstream of thePKS11T161D coding region and causes expression in all tissues constitutively. The construct was introduced intoAgrobacterium tumefaciens strain GV3101 by electroporation, and Arabidopsis transformation was performed according to the method described previously (15Clough S.J. Bent A.F. Plant J. 1998; 16: 735-743Crossref PubMed Google Scholar). The wild-type PKS11coding sequence was similarly overexpressed in Arabidopsisunder control of the super promoter. After harvesting, the seeds were planted on MS agar medium containing 40 mg/liter hygromycin and 500 mg/liter vancomycin, and the transgenic lines were selected out. The transformed seedlings were transferred into soil to set seed under routine conditions. Seeds of wild-type and transgenic plants were surface-sterilized in 100% bleach for 10 min, followed by 5 washes in sterile distilled water. The seeds were embedded on MS agar plates and germinated and grown on the vertical plates at 22 °C, 300 PAR, 16-h light and 8-h dark photoperiod. Seed germination and seedling growth of the wild-type control plants, T2and T3 generation transgenic plants expressingPKS11T161D or PKS11 were tested for responses to various concentrations of ABA, salt, mannitol, and glucose treatments. To observe the effect of glucose on seed germination and seedling growth, transgenic and control lines were germinated and grown on MS plates containing 1–5% glucose, 0.1 to 0.5 mm2-deoxyglucose, or 1–5% 3-O-methylglucose. Seedlings were grown in the dark for 6 days, and hypocotyl length was measured from 20 seedlings at each glucose concentration in the control and transgenic plants. The values of apparent K m and maximal velocityV max for p3 and ATP were determined by at least triplicate measurements of initial velocity for different concentrations of p3 and ATP. Eadie-Hofstee regression was used to fit the data in a defined concentration range to a straight line, andK m and V max values were determined from the regression equation. In order to determine experimentally the open reading frame of PKS11gene, the cDNA was cloned by reverse transcription-PCR. The deduced amino acid sequence of PKS11 was found to be identical to that in the database, which was obtained from computer-based annotation. As a first step toward functional analysis, the steady-state transcript level ofPKS11 gene in different tissues of mature plants as well as under various stresses was determined. Blots of total RNA from different tissues or from stress-treated young seedlings were hybridized to a specific DNA probe for PKS11. PKS11 was expressed in all tissues examined, but the expression level in roots was substantially higher than that in leaves, stems, flowers, or siliques of mature Arabidopsis plants (Fig. 1 A). Because of our interest in plant stress responses, potential regulation of thePKS gene by salt, cold, drought, and ABA was examined in young Arabidopsis seedlings (Fig. 1 B). No significant induction or repression of PKS11 was observed under any of the treatments. A promoter-glucuronidase reporter fusion was used to investigate further the tissue distribution of PKS11 expression. InArabidopsis seedlings, promoter-glucuronidase staining was readily detected in roots, but the staining in other tissues was very weak or below the detection limit (Fig.1 C). Like SOS2, the founding member of the PKS family, PKS11 also contains an N-terminal SNF1-like kinase catalytic domain and a C-terminal regulatory domain (Fig.2 A). An alignment of the deduced amino acid sequence of PKS11 with SOS2 showed that these kinases are highly conserved throughout the entire length (Fig.2 B). In the superfamily of protein kinases, the PKSes belong to SNF1/AMPK family (16Hanks S.K. Hunter T. The Protein Kinase Facts Book. Academic Press, London1995: 7-47Google Scholar). Like many other protein kinases including SOS2, PKS11 contains a putative “activation loop” or “activation segment” in the kinase catalytic domain, located between the conserved DFG and APE sequences (Fig. 2 B). The kinase also contains a conserved FISL motif, a stretch of 21 amino acid residues, located near the kinase domain (Fig. 2 B). The FISL motif in SOS2 has been identified recently as the SOS3-interacting sequence and is autoinhibitory to substrate phosphorylation (3Guo Y. Halfer U. Ishitani M. Zhu J.-K. Plant Cell. 2001; 13: 1383-1399Crossref PubMed Scopus (372) Google Scholar). PKS11 contains an open reading frame of 1338 bp and is predicted to encode a protein of 446 amino acid residues with an estimated molecular mass of 50.4 kDa.PKS11 is located on chromosome 4, based on information in the Arabidopsis genomic sequence database (www.arabidopsis.org). We expressed PKS11 and a number of other PKS proteins in bacteria, and we found that none had any kinase activity against commonly used protein or peptide substrates (data not shown). In order to biochemically characterize the enzyme, we attempted to construct active forms of the kinase. Previously, we have found that aspartate substitution of Thr168 in the putative activation loop of SOS2 could activate the kinase (3Guo Y. Halfer U. Ishitani M. Zhu J.-K. Plant Cell. 2001; 13: 1383-1399Crossref PubMed Scopus (372) Google Scholar). A comparison of the putative activation loop of PKS11 and a number of other PKSes (data not shown) with that of SOS2 showed that the threonine residue is conserved (Fig.2 B). This suggests that the threonine residue in PKS11, Thr161, could be a target site for phosphorylation by a putative upstream activating kinase(s). To produce active PKS11 protein, we substituted the threonine residue with aspartate to partially mimic phosphorylation by an upstream kinase(s) using site-directed mutagenesis on the PKS11 cDNA. The resulting mutant, designated PKS11T161D, was produced by changing Thr161 to Asp (Fig. 2 B). In addition, the FISL motif in PKS11 may be autoinhibitory to the kinase activity. Therefore, a FISL motif deletion mutant, designated PKS11ΔF, was constructed by deleting the FISL motif between Lys304 and Leu325 (Fig.2 B) using site-directed mutagenesis. PKS11 wild-type and mutant proteins were expressed in E. coli BL21 cells as GST fusion proteins and purified by affinity chromatography on glutathione-Sepharose (Fig.3 A). The expression level of the recombinant PKS11 mutant proteins was similar to that of the wild-type counterpart, as shown by SDS-PAGE analysis. These purified GST-PKS11 fusion proteins showed the expected apparent molecular mass of about 80 kDa, with GST-PKS11ΔF slightly smaller. We have shown previously (8Halfter U. Ishitani M. Zhu J.-K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3735-3740Crossref PubMed Scopus (578) Google Scholar) that SOS2 can phosphorylate a peptide p3 in the presence of SOS3. We measured substrate phosphorylation of the peptide and autophosphorylation in vitro for the mutant and wild-type kinases in the presence of 5 mm Mg2+ as a cofactor. The FISL motif deletion mutant, PKS11ΔF, displayed a 3-fold increase in substrate phosphorylation compared with the wild-type kinase (designated PKS11WT) (Fig. 3 B). In contrast, the activation loop mutant, PKS11T161D, was extremely active, with 130-fold higher activity in p3 phosphorylation than PKS11WT (Fig.3 B). Both PKS11 mutants also had higher autophosphorylation activity than PKS11WT (Fig. 3 C). In addition to the threonine residue, a serine and a tyrosine residue within the activation loop are also completely conserved in all PKSes (Fig. 2 B and data not shown). We wanted to investigate whether changing the conserved serine or tyrosine to aspartate could also make the kinase constitutively active. We thus constructed PKS11S154D and PKS11Y173D, respectively, by mutating Ser154to Asp and Tyr173 to Asp, respectively, via site-directed mutagenesis. Kinase assays showed that PKS11S154D and PKS11Y173D exhibited 16- and 15-fold higher activity, respectively, in p3 phosphorylation than the wild-type kinase (Fig. 3 D). The most active mutant, PKS11T161D, was therefore used for subsequent biochemical and functional analysis. To determine the cofactor preference for divalent cations in vitro of PKS11T161D, we measured substrate phosphorylation activity in the presence of various concentrations of two divalent cations, Mg2+ and Mn2+. Divalent cations were absolutely required for substrate phosphorylation of p3 as well as autophosphorylation of the kinase, as shown by the lack of activity in the absence of the cations (Fig. 4 A). Substrate phosphorylation increased as the concentrations of Mn2+ or Mg2+ in the range of 0–2.5 mm(Mn2+) or 0–5.0 mm (Mg2+) increased. Interestingly, Mn2+ appeared to be a much more effective cofactor than Mg2+ for PKS11T161D. As low as 0.25 mm Mn2+ could activate substrate phosphorylation of PKS11T161D. Optimal activation was observed at around 2.5 mm Mn2+, and higher concentrations (>5 mm Mn2+) became inhibitory. In contrast, Mg2+ did not activate PKS11T161D at concentrations of less than 1 mm. Optimal activation was achieved at 5 mm or higher concentrations of Mg2+ (Fig.4 A). The intracellular concentration of Mn2+ is in the micromolar range, whereas that of Mg2+ is in the millimolar range (17White M.F. Kahn C.R. Enzymes. 1986; 17: 248-361Google Scholar). Nevertheless, these results suggest that Mn2+ could play a role in activity regulation of the PKS under physiological conditions. We tested whether PKS11T161D also preferred Mn2+ over Mg2+ as a cofactor for autophosphorylation. Autophosphorylation was assayed in the presence of various concentrations of the two divalent cations. Mn2+ also strongly activated autophosphorylation of PKS11T161D even in the micromolar range (Fig. 4 B). In contrast, Mg2+only weakly activated the autophosphorylation, and the activation required millimolar concentrations of Mg2+. These results suggest that PKS11 is a novel protein kinase with an uncommon cofactor preference. With 2.5 mm Mn2+ as a cofactor in the kinase assay, PKS11T161D displayed even higher peptide phosphorylation (Fig. 4 C) as well as autophosphorylation activity (data not shown). The PKS family of proteins tested thus far does not show any kinase activity against commonly used protein substrates, such as myelin basic protein, histone H1, and casein. However, three synthetic peptide substrates (p1, p2, and p3), derived from the recognition sequences of protein kinase C or SNF1/AMPK, are known to be phosphorylated by SOS2 (8Halfter U. Ishitani M. Zhu J.-K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3735-3740Crossref PubMed Scopus (578) Google Scholar). These peptides were thus chosen to analyze the substrate specificity of PKS11T161D in the present study. The above results show that PKS11T161D can phosphorylate the peptide substrate p3. To determine the subs" @default.
• W2033582712 created "2016-06-24" @default.
• W2033582712 creator A5040216938 @default.
• W2033582712 creator A5041543923 @default.
• W2033582712 creator A5048412219 @default.
• W2033582712 creator A5048925227 @default.
• W2033582712 creator A5049293054 @default.
• W2033582712 date "2002-08-01" @default.
• W2033582712 modified "2023-09-28" @default.
• W2033582712 title "Biochemical and Functional Characterization of PKS11, a Novel Arabidopsis Protein Kinase" @default.
• W2033582712 cites W1258742671 @default.
• W2033582712 cites W1480905987 @default.
• W2033582712 cites W1494075612 @default.
• W2033582712 cites W1504436115 @default.
• W2033582712 cites W1526381231 @default.
• W2033582712 cites W1578035536 @default.
• W2033582712 cites W1580304949 @default.
• W2033582712 cites W1755773572 @default.
• W2033582712 cites W1906849814 @default.
• W2033582712 cites W1973813211 @default.
• W2033582712 cites W1976254673 @default.
• W2033582712 cites W1979498518 @default.
• W2033582712 cites W1988811776 @default.
• W2033582712 cites W2001206569 @default.
• W2033582712 cites W2005165257 @default.
• W2033582712 cites W2010978356 @default.
• W2033582712 cites W2013463985 @default.
• W2033582712 cites W2019097955 @default.
• W2033582712 cites W2019982745 @default.
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• W2033582712 cites W2047345705 @default.
• W2033582712 cites W2051933489 @default.
• W2033582712 cites W2057069307 @default.
• W2033582712 cites W2059707517 @default.
• W2033582712 cites W2061840340 @default.
• W2033582712 cites W2081964845 @default.
• W2033582712 cites W2084086228 @default.
• W2033582712 cites W2097404112 @default.
• W2033582712 cites W2110627230 @default.
• W2033582712 cites W2116122104 @default.
• W2033582712 cites W2117544585 @default.
• W2033582712 cites W2121745326 @default.
• W2033582712 cites W2124150183 @default.
• W2033582712 cites W2135018494 @default.
• W2033582712 cites W2139287985 @default.
• W2033582712 cites W2144338881 @default.
• W2033582712 cites W2147145620 @default.
• W2033582712 cites W2152479669 @default.
• W2033582712 cites W2153216467 @default.
• W2033582712 cites W2164331942 @default.
• W2033582712 cites W4231058641 @default.
• W2033582712 cites W4254769864 @default.
• W2033582712 cites W4256394625 @default.
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