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Characterizing activities of eukaryotic-like protein kinases with atypical catalytic loop motifs from Myxococcus xanthus
Journal of Bioscience and Bioengineering VOL. 119 No. 5, 511e514, 2015 www.elsevier.com/locate/jbiosc
NOTE
Characterizing activities of eukaryotic-like protein kinases with atypical catalytic loop motifs from Myxococcus xanthus Yoshio Kimura,* Maho Urata, and Reiko Okamoto Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795, Japan Received 5 August 2014; accepted 3 October 2014 Available online 1 November 2014
Myxococcus xanthus has eukaryotic-like protein kinases (EPKs) with different atypical catalytic loop motifs. Seven out of 14 recombinant M. xanthus EPKs containing atypical motifs in the catalytic loop showed protein kinase activity against myelin basic protein and four autophosphorylated EPKs were detected using anti-phosphotyrosine antibody by western blotting. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Myxococcus xanthus; Eukaryotic-like protein kinase; Dual-specificity kinase; MBP kinase activity; Atypical catalytic loop]
Protein kinases play a central role in several signal transduction cascades by phosphorylating the target protein at specific amino acid residues. The phosphorylation of amino acids can induce conformational changes in the target protein that alters their function (1). Eukaryotic protein kinases share a catalytic domain of 250e300 amino acids that is divided into 12 subdomains with highly conserved motifs and amino acid residues (2). In eukaryotes, most protein kinases (Ser/Thr protein kinases) can phosphorylate on serine and threonine, and a distinct group (Tyr protein kinases) phosphorylates on tyrosine. These eukaryotic protein kinases can be further divided into arginine-aspartate (RD) kinases and non-RD kinases depending on the conservation of arginine immediately preceding the catalytic aspartate in the consensus motif of the catalytic loop (3). Ser/Thr protein kinases contain consensus motif RDxKxxN in the catalytic loop (4). This segment contains many of the key residues that direct the g-phosphate of ATP to the protein substrate. Aspartate residue is presumed to act as a catalytic base to accept the proton from the hydroxyl nucleophile, and lysine residue is thought to assist the phosphotransfer by neutralizing the local negative charge of the g-phosphate of the MgATP. Furthermore, asparagine residue assists in the stabilization of the catalytic loop and chelates Mg2þ (5). On the other hand, Tyr protein kinases contain consensus motif HRDx(A/R)A(A/R)N in the catalytic loop. Myxococcus xanthus is a soil bacterium, which moves in coordinated swarms (6,7). In response to nutritional stress, M. xanthus initiates a complex developmental program, and hundreds of thousands of vegetative cells aggregate to form fruiting bodies. Analysis of the complete M. xanthus genomic sequence has revealed that this strain contains w100 EPKs, and approximately 50% of M. xanthus EPKs have various atypical motifs in the catalytic loop;
* Corresponding author. Tel.: þ81 87 891 3118; fax: þ81 87 891 3021. E-mail address: [email protected] (Y. Kimura).
SxxN (26 kinases), TxxN (4 kinases), AxxN (3 kinase), DxxH (4 kinases), NxxN (1 kinase), and RxxN (1 kinase) sequences are found in the catalytic loops of M. xanthus EPKs. To determine whether these M. xanthus EPKs have protein kinase activity, we selected 20 that have atypical catalytic loop in subdomain VIb. These EPKs have all the 12 subdomains necessary for the kinase activity. These EPK encoding genes were amplified by PCR using primers. The products were inserted into a cold shock expression vector, pCold or pCold-TF vector (Takara Bio) and transformed in competent Escherichia coli cells. Fourteen out of 20 these EPKs were expressed in the soluble fraction, while the remaining 6 (MXAN_1234, 2911, 3693, 5517, 5976, and 6545) did not. Recombinant kinases were purified by affinity chromatography on a Talon CellThru column (Clontech). When recombinant kinases were expressed as a His-tagged trigger-factor (TF) fusion protein using pCold-TF, the TF was cleaved by human rhinovirus (HRV) 3C protease at 4 C for 16 h. Alignment analysis of 14 expressed EPKs are shown in Fig. 1. MXAN_0396, 0552, 2059, 2980, 5116, and 6669 (designated DspA) contain SPxN sequence; MXAN_0018, 0023, and 4479 contain AxxN sequence; and MXAN_1297 and 6312 contain a NPAN sequence and a TPDN sequence, respectively. MXAN_2177, 3183, and 4841 contain DVxH sequences, which have a His residue instead of Asn residue. These EPKs contain all essential amino acid residues for catalytic activity. Purified recombinant EPKs were tested for autophosphorylation and protein kinase activity in the presence of either MnCl2 or MgCl2. The assay was performed at 37 C, because the optimum temperature for growth of M. xanthus is between 34 C and 36 C (8). Also, 2.5 mM MnCl2 or 2.5 mM MgCl2 was used for the assay, because some M. xanthus eukaryotic-like Ser/Thr kinase activities were inhibited by 5 or 10 mM MnCl2 or MgCl2 (9, unpublished data). The autophosphorylation assay was performed in volume of 20 mL in buffer containing 100 mM TriseHCl buffer (pH 7.5), 2.5 mM MnCl2 or MgCl2, 0.2 mM ATP, 0.15 MBq [g-32P] ATP and enzyme at
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.10.002
512
KIMURA ET AL.
J. BIOSCI. BIOENG.,
FIG. 1. Amino acid sequence alignment of kinase region of 14 EPKs from M. xanthus. Highly conserved residues common to eukaryotic protein kinases are shown in bold. Identical residues are denoted by an asterisk, and the amino acids of catalytic loops are boxed.
37 C for 60 min. Following SDS-PAGE analysis, the bands corresponding to EPK proteins were sliced from the gel and subjected to liquid scintillation counting. MXAN_2980 possessed strong autophosphorylation activity (Table 1), while MXAN_0552, 6669, 0018, 0023, and 1297 showed weak activities. These kinases showed higher activity in the presence of Mn2þ than Mg2þ. The remaining 8 EPKs did not show autophosphorylation activity. We next examined protein kinase activity using a myelin basic protein (MBP), which is commonly used as the substrate for in vitro protein kinase assay. The assay was performed in a total volume of 20 mL containing 50 mM TriseHCl (pH 7.5), 2.5 mM MnCl2 or MgCl2,
0.2 mM ATP, 0.15 MBq [g-32P] ATP and enzyme at 37 C for 60 min. Similar to the test mentioned above, phosphorylated protein such as MBP was sliced from the SDS-PAGE gel and subjected to liquid scintillation counting. In these recombinant M. xanthus EPKs, MXAN_0018 showed the highest kinase activity against MBP (Table 1). The specific activity of MXAN_0018 for MBP in the presence of Mn2þ was 13.1 0.4 nmol/min/mg, which was lower by w18 fold in comparison to rabbit cAMP-dependent protein kinase (PKA) and by w107 fold than rat protein kinase C (PKC), respectively (10). MXAN_6669 (DspA) also showed high kinase activity (Table 1); however, it was unstable and inactivated when stocked
VOL. 119, 2015
NOTE
TABLE 1. Specific activities of autophosphorylation and MBP kinases of M. xanthus EPKs in the presence of Mg2þ and Mn2þ. Enzyme
MXAN_0396 MXAN_0552 MXAN_2059 MXAN_2980 MXAN_5116 MXAN_6669 MXAN_0018 MXAN_0023 MXAN_4479 MXAN_2177 MXAN_3183 MXAN_4841 MXAN_1297 MXAN_6312
Catalytic loop
RDLSPAN RDVSPQN RDVSPSN CDVSPSN RDVSPEN RDLSPHN RDVAQKN RDVAEKN RDVAPRN RAVDVEH RAVDVEH RAIDVAH RDVNPAN RDFTPDN
Autophosphorylation activity (pmol/min/mg)
MBP kinase activity (pmol/min/mg)
Mg2þ
Mn2þ
Mg2þ
Mn2þ
ND 20 ND 17 5 ND 30 41 20 ND ND ND ND 10 ND
ND 62 ND 80 8 ND 81 10 2 31 ND ND ND ND 20 ND
10 20 ND 18 3 ND 480 40 5060 250 31 ND ND ND ND 50 ND
20 40 ND 50 5 ND 1110 110 13100 380 82 ND ND ND ND 12 2 ND
ND, not detected. These assays were carried out in the presence of 2.5 mM MgCl2 or 2.5 mM MnCl2. Specific activity is pmol of phosphate transferred per min per mg of protein. The data represent the mean standard error of three independent experiments.
at 25 C. MXAN_0396, 0552, 2980, 0023, and 1297 displayed weak protein kinase activities against MBP, but these activities of EPKs were significantly lower than their eukaryotic counterparts. All tested EPKs showed higher activity in the presence of Mn2þ compared with Mg2þ. Among eukaryotic protein kinases known to utilize manganese, Mn2þ-dependent protein kinases have been reported in bacteria and plants (11,12). MXAN_2980 showed high autophosphorylation activity but low kinase activity. Most RD kinases are activated by phosphorylation of Thr, Ser or Tyr residues in the activation loop between the conserved DFG motif in the Mg-binding loop and the PE motif in the P þ 1 loop. The arginine residue in RD motif interacts with phosphoamino acid(s) in the activation loop and stabilizes a conformation favorable for catalysis. The catalytic loop of MXAN_2980, which has a CDVSPSN sequence, lacks the conserved RD motif. Similarly, non-RD kinases FLS2 and EFR of Arabidopsis, which contain a CDLKPAN and a CDIKPSN sequence in the catalytic loop,
513
respectively, display low MBP kinase activity in contrast to the RD kinase of Arabidopsis (13). All canonical eukaryotic protein kinases contain a conserved amino acid consensus motif GxGxxG in the glycine-rich G loop, which controls ATP binding and phosphate transfer in protein kinases. In PKA, two mutations (G50S and G52S in the G50TG52SFG55 sequence) reduced the affinity for ATP by approximately 10-fold, and three mutations (G50S, G52S and G55S) lowered the rate of phosphoryl transfer by 7e300-fold (14). Barring MXAN_0396, remaining M. xanthus EPKs lack a canonical GxGxxG sequence in subdomain I. Instead, they contain GxG, GxxG or GxxxxG sequences (Fig. 1). MXAN_6669 (DspA) contains a GxG and GxxG sequence in the subdomain I. The enzyme showed Km of 250 mM for ATP (9), which is 12.5-fold higher but the specific protein kinase activity w450-fold lower than that of PKA. Presumably, M. xanthus EPKs lacking GxGxxG motif may attribute to their low protein kinase activity. MXAN_2177, 3183, and 4841 containing RAxDVxH sequence in the catalytic loop had neither autophosphorylation nor MBP kinase activities. Asparagine (Asn) residue in the catalytic loop coordinates with ATP-bound Mg2þ; however, these EPKs have a histidine (His) in place of the Asn in the loop. A sugar kinase from Pseudomonas aeruginosa, WaaP, which phosphorylates heptose I in lipopolysaccharide shares sequence homology with eukaryotic protein kinases. This enzyme has a His residue at this position in its catalytic loop (RDCYICH), but it catalyzes Tyr self-phosphorylation and phosphorylation of poly (Glu, Tyr) (15). Furthermore, these EPKs lack many crucial residues that are conserved especially the aspartate (Asp) residue in the RD motif. The Asp mutation to Ala in the RD motif in yeast PKA led to a near complete loss of activity (16). We previously reported that M. xanthus DspA is a dual-specificity kinase that autophosphorylates on two Tyr residues (Y35 and Y111) (9). To determine if these EPKs phosphorylate Tyr residue(s) on self or MBP, western blot analysis was performed using an anti-phosphotyrosine monoclonal antibody PY20. Recombinant EPKs were incubated for 60 min at 37 C with 5 mM ATP and 2.5 mM MnCl2 in 100 mM TriseHCl buffer (pH 7.5). As shown in Fig. 2, autophosphorylated MXAN_0552, 6669 (DspA), 0018, and 0023 were detected by treating the western blot membranes with antibody PY20. These EPKs displayed no detectable Tyr phosphorylation of MBP (data not shown). In addition, these EPKs showed no Tyr kinase
FIG. 2. Western blotting analysis of autophosphorylated 14 EPKs. MXAN_0396 (48 kDa), 0552 (43 kDa), 2980 (38 kDa), 6669 (41 kDa), 3183 (43 kDa), 1297 (34 kDa), 2059 (31 kDa), 2177 (36 kDa) and 6312 (33 kDa) were expressed in E. coli cells by pCold-TF vector, and MXAN_4841 (43 kDa), 4479 (45 kDa), 0018 (42 kDa), 0023 (40 kDa) and 5116 (47 kDa) were expressed using pCold vector. Autophosphorylated recombinant EPKs were subjected to SDS-PAGE, transferred to PVDF membrane and analyzed with a horseradish peroxidaseconjugated anti-phosphotyrosine antibody PY20. Protein size is indicated by molecular mass markers in kD.
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activity against histone 2B, bovine serum albumin and casein, when studied by western blot analysis using PY20 (data not shown). These four EPKs have a G(K/R)YE(L/V)(L/V) sequence (the Tyr residue in the sequence is autophosphorylated by DspA) in the subdomain I, suggesting that these sequences may be important for autophosphorylation on Tyr residue. Taken together, seven out of 14 M. xanthus recombinant EPKs containing atypical catalytic loops showed protein kinase activities, and four EPKs were dual-specificity kinases; however, these kinase activities, except for two EPKs, were significantly lower than those of Ser/Thr/Tyr protein kinases of eukaryotes. This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (25440087).
References 1. Weiwad, M., Kullertz, G., Schutkowski, M., and Fischer, G.: Evidence that the substrate backbone conformation is critical to phosphorylation by p42 MAP kinase, FEBS Lett., 478, 39e42 (2000). 2. Hanks, S. K.: Genomic analysis of the eukaryotic protein kinase superfamily: a perspective, Genome Biol., 4, 111 (2003). 3. Dardick, C. and Ronald, P.: Plant and animal pathogen recognition receptors signal through non-RD kinases, PLoS Pathog., 2, e2 (2006). 4. Hanks, S. K., Quinn, A. M., and Hunter, T.: The protein kinase family: conserved features and deduced phylogeny of the catalytic domains, Science, 241, 42e52 (1988). 5. Zhou, J. and Adams, J. A.: Is there a catalytic base in the active site of cAMP-dependent protein kinase? Biochemistry, 36, 2977e2984 (1997).
J. BIOSCI. BIOENG., 6. Jelsbak, L. and Søgaard-Andersen, L.: Cell behavior and cell-cell communication during fruiting body morphogenesis in Myxococcus xanthus, J. Microbiol. Methods, 55, 829e839 (2003). 7. Kaiser, D.: Signaling in myxobacteria, Annu. Rev. Microbiol., 58, 75e98 (2004). 8. Janssen, G. R., Wireman, J. W., and Dworkin, M.: Effect of temperature on the growth of Myxococcus xanthus, J. Bacteriol., 130, 561e562 (1977). 9. Okamoto, R., Takegawa, K., and Kimura, Y.: Regulation of eukaryotic-like protein kinase activity of DspA from Myxococcus xanthus by autophosphorylation, J. Biochem., 155, 99e106 (2014). 10. Kishimoto, A., Nishiyama, K., Nakanishi, H., Uratsuji, Y., Nomura, H., Takeyama, Y., and Nishizuka, Y.: Studies on the phosphorylation of myelin basic protein by protein kinase C and adenosine 30 :50 -monophosphatedependent protein kinase, J. Biol. Chem., 260, 12492e12499 (1985). 11. Schwessinger, B., Roux, M., Kadota, Y., Ntoukakis, V., Sklenar, J., Jones, A., and Zipfel, C.: Phosphorylation-dependent differential regulation of plant growth, cell death, and innate immunity by the regulatory receptor-like kinase BAK1, PLoS Genet., 7, e1002046 (2011). 12. Pérez, J., Castañeda-García, A., Jenke-Kodama, H., Müller, R., and MuñozDorado, J.: Eukaryotic-like protein kinases in the prokaryotes and the myxobacterial kinome, Proc. Natl. Acad. Sci. USA, 105, 15950e15955 (2008). 13. Reddy, M. M. and Rajasekharan, R.: Role of threonine residues in the regulation of manganese-dependent arabidopsis serine/threonine/tyrosine protein kinase activity, Arch. Biochem. Biophys., 455, 99e109 (2006). 14. Grant, B. D., Hemmer, W., Tsigelny, I., Adams, J. A., and Taylor, S. S.: Kinetic analyses of mutations in the glycine-rich loop of cAMP-dependent protein kinase, Biochemistry, 37, 7708e7715 (1998). 15. Zhao, X. and Lam, J. S.: WaaP of Pseudomonas aeruginosa is a novel eukaryotic type protein-tyrosine kinase as well as a sugar kinase essential for the biosynthesis of core lipopolysaccharide, J. Biol. Chem., 277, 4722e4730 (2002). 16. Gibbs, C. S. and Zoller, M. J.: Rational scanning mutagenesis of a protein kinase identifies functional regions involved in catalysis and substrate interactions, J. Biol. Chem., 266, 8923e8931 (1991).
NOTE
Characterizing activities of eukaryotic-like protein kinases with atypical catalytic loop motifs from Myxococcus xanthus Yoshio Kimura,* Maho Urata, and Reiko Okamoto Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795, Japan Received 5 August 2014; accepted 3 October 2014 Available online 1 November 2014
Myxococcus xanthus has eukaryotic-like protein kinases (EPKs) with different atypical catalytic loop motifs. Seven out of 14 recombinant M. xanthus EPKs containing atypical motifs in the catalytic loop showed protein kinase activity against myelin basic protein and four autophosphorylated EPKs were detected using anti-phosphotyrosine antibody by western blotting. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Myxococcus xanthus; Eukaryotic-like protein kinase; Dual-specificity kinase; MBP kinase activity; Atypical catalytic loop]
Protein kinases play a central role in several signal transduction cascades by phosphorylating the target protein at specific amino acid residues. The phosphorylation of amino acids can induce conformational changes in the target protein that alters their function (1). Eukaryotic protein kinases share a catalytic domain of 250e300 amino acids that is divided into 12 subdomains with highly conserved motifs and amino acid residues (2). In eukaryotes, most protein kinases (Ser/Thr protein kinases) can phosphorylate on serine and threonine, and a distinct group (Tyr protein kinases) phosphorylates on tyrosine. These eukaryotic protein kinases can be further divided into arginine-aspartate (RD) kinases and non-RD kinases depending on the conservation of arginine immediately preceding the catalytic aspartate in the consensus motif of the catalytic loop (3). Ser/Thr protein kinases contain consensus motif RDxKxxN in the catalytic loop (4). This segment contains many of the key residues that direct the g-phosphate of ATP to the protein substrate. Aspartate residue is presumed to act as a catalytic base to accept the proton from the hydroxyl nucleophile, and lysine residue is thought to assist the phosphotransfer by neutralizing the local negative charge of the g-phosphate of the MgATP. Furthermore, asparagine residue assists in the stabilization of the catalytic loop and chelates Mg2þ (5). On the other hand, Tyr protein kinases contain consensus motif HRDx(A/R)A(A/R)N in the catalytic loop. Myxococcus xanthus is a soil bacterium, which moves in coordinated swarms (6,7). In response to nutritional stress, M. xanthus initiates a complex developmental program, and hundreds of thousands of vegetative cells aggregate to form fruiting bodies. Analysis of the complete M. xanthus genomic sequence has revealed that this strain contains w100 EPKs, and approximately 50% of M. xanthus EPKs have various atypical motifs in the catalytic loop;
* Corresponding author. Tel.: þ81 87 891 3118; fax: þ81 87 891 3021. E-mail address: [email protected] (Y. Kimura).
SxxN (26 kinases), TxxN (4 kinases), AxxN (3 kinase), DxxH (4 kinases), NxxN (1 kinase), and RxxN (1 kinase) sequences are found in the catalytic loops of M. xanthus EPKs. To determine whether these M. xanthus EPKs have protein kinase activity, we selected 20 that have atypical catalytic loop in subdomain VIb. These EPKs have all the 12 subdomains necessary for the kinase activity. These EPK encoding genes were amplified by PCR using primers. The products were inserted into a cold shock expression vector, pCold or pCold-TF vector (Takara Bio) and transformed in competent Escherichia coli cells. Fourteen out of 20 these EPKs were expressed in the soluble fraction, while the remaining 6 (MXAN_1234, 2911, 3693, 5517, 5976, and 6545) did not. Recombinant kinases were purified by affinity chromatography on a Talon CellThru column (Clontech). When recombinant kinases were expressed as a His-tagged trigger-factor (TF) fusion protein using pCold-TF, the TF was cleaved by human rhinovirus (HRV) 3C protease at 4 C for 16 h. Alignment analysis of 14 expressed EPKs are shown in Fig. 1. MXAN_0396, 0552, 2059, 2980, 5116, and 6669 (designated DspA) contain SPxN sequence; MXAN_0018, 0023, and 4479 contain AxxN sequence; and MXAN_1297 and 6312 contain a NPAN sequence and a TPDN sequence, respectively. MXAN_2177, 3183, and 4841 contain DVxH sequences, which have a His residue instead of Asn residue. These EPKs contain all essential amino acid residues for catalytic activity. Purified recombinant EPKs were tested for autophosphorylation and protein kinase activity in the presence of either MnCl2 or MgCl2. The assay was performed at 37 C, because the optimum temperature for growth of M. xanthus is between 34 C and 36 C (8). Also, 2.5 mM MnCl2 or 2.5 mM MgCl2 was used for the assay, because some M. xanthus eukaryotic-like Ser/Thr kinase activities were inhibited by 5 or 10 mM MnCl2 or MgCl2 (9, unpublished data). The autophosphorylation assay was performed in volume of 20 mL in buffer containing 100 mM TriseHCl buffer (pH 7.5), 2.5 mM MnCl2 or MgCl2, 0.2 mM ATP, 0.15 MBq [g-32P] ATP and enzyme at
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.10.002
512
KIMURA ET AL.
J. BIOSCI. BIOENG.,
FIG. 1. Amino acid sequence alignment of kinase region of 14 EPKs from M. xanthus. Highly conserved residues common to eukaryotic protein kinases are shown in bold. Identical residues are denoted by an asterisk, and the amino acids of catalytic loops are boxed.
37 C for 60 min. Following SDS-PAGE analysis, the bands corresponding to EPK proteins were sliced from the gel and subjected to liquid scintillation counting. MXAN_2980 possessed strong autophosphorylation activity (Table 1), while MXAN_0552, 6669, 0018, 0023, and 1297 showed weak activities. These kinases showed higher activity in the presence of Mn2þ than Mg2þ. The remaining 8 EPKs did not show autophosphorylation activity. We next examined protein kinase activity using a myelin basic protein (MBP), which is commonly used as the substrate for in vitro protein kinase assay. The assay was performed in a total volume of 20 mL containing 50 mM TriseHCl (pH 7.5), 2.5 mM MnCl2 or MgCl2,
0.2 mM ATP, 0.15 MBq [g-32P] ATP and enzyme at 37 C for 60 min. Similar to the test mentioned above, phosphorylated protein such as MBP was sliced from the SDS-PAGE gel and subjected to liquid scintillation counting. In these recombinant M. xanthus EPKs, MXAN_0018 showed the highest kinase activity against MBP (Table 1). The specific activity of MXAN_0018 for MBP in the presence of Mn2þ was 13.1 0.4 nmol/min/mg, which was lower by w18 fold in comparison to rabbit cAMP-dependent protein kinase (PKA) and by w107 fold than rat protein kinase C (PKC), respectively (10). MXAN_6669 (DspA) also showed high kinase activity (Table 1); however, it was unstable and inactivated when stocked
VOL. 119, 2015
NOTE
TABLE 1. Specific activities of autophosphorylation and MBP kinases of M. xanthus EPKs in the presence of Mg2þ and Mn2þ. Enzyme
MXAN_0396 MXAN_0552 MXAN_2059 MXAN_2980 MXAN_5116 MXAN_6669 MXAN_0018 MXAN_0023 MXAN_4479 MXAN_2177 MXAN_3183 MXAN_4841 MXAN_1297 MXAN_6312
Catalytic loop
RDLSPAN RDVSPQN RDVSPSN CDVSPSN RDVSPEN RDLSPHN RDVAQKN RDVAEKN RDVAPRN RAVDVEH RAVDVEH RAIDVAH RDVNPAN RDFTPDN
Autophosphorylation activity (pmol/min/mg)
MBP kinase activity (pmol/min/mg)
Mg2þ
Mn2þ
Mg2þ
Mn2þ
ND 20 ND 17 5 ND 30 41 20 ND ND ND ND 10 ND
ND 62 ND 80 8 ND 81 10 2 31 ND ND ND ND 20 ND
10 20 ND 18 3 ND 480 40 5060 250 31 ND ND ND ND 50 ND
20 40 ND 50 5 ND 1110 110 13100 380 82 ND ND ND ND 12 2 ND
ND, not detected. These assays were carried out in the presence of 2.5 mM MgCl2 or 2.5 mM MnCl2. Specific activity is pmol of phosphate transferred per min per mg of protein. The data represent the mean standard error of three independent experiments.
at 25 C. MXAN_0396, 0552, 2980, 0023, and 1297 displayed weak protein kinase activities against MBP, but these activities of EPKs were significantly lower than their eukaryotic counterparts. All tested EPKs showed higher activity in the presence of Mn2þ compared with Mg2þ. Among eukaryotic protein kinases known to utilize manganese, Mn2þ-dependent protein kinases have been reported in bacteria and plants (11,12). MXAN_2980 showed high autophosphorylation activity but low kinase activity. Most RD kinases are activated by phosphorylation of Thr, Ser or Tyr residues in the activation loop between the conserved DFG motif in the Mg-binding loop and the PE motif in the P þ 1 loop. The arginine residue in RD motif interacts with phosphoamino acid(s) in the activation loop and stabilizes a conformation favorable for catalysis. The catalytic loop of MXAN_2980, which has a CDVSPSN sequence, lacks the conserved RD motif. Similarly, non-RD kinases FLS2 and EFR of Arabidopsis, which contain a CDLKPAN and a CDIKPSN sequence in the catalytic loop,
513
respectively, display low MBP kinase activity in contrast to the RD kinase of Arabidopsis (13). All canonical eukaryotic protein kinases contain a conserved amino acid consensus motif GxGxxG in the glycine-rich G loop, which controls ATP binding and phosphate transfer in protein kinases. In PKA, two mutations (G50S and G52S in the G50TG52SFG55 sequence) reduced the affinity for ATP by approximately 10-fold, and three mutations (G50S, G52S and G55S) lowered the rate of phosphoryl transfer by 7e300-fold (14). Barring MXAN_0396, remaining M. xanthus EPKs lack a canonical GxGxxG sequence in subdomain I. Instead, they contain GxG, GxxG or GxxxxG sequences (Fig. 1). MXAN_6669 (DspA) contains a GxG and GxxG sequence in the subdomain I. The enzyme showed Km of 250 mM for ATP (9), which is 12.5-fold higher but the specific protein kinase activity w450-fold lower than that of PKA. Presumably, M. xanthus EPKs lacking GxGxxG motif may attribute to their low protein kinase activity. MXAN_2177, 3183, and 4841 containing RAxDVxH sequence in the catalytic loop had neither autophosphorylation nor MBP kinase activities. Asparagine (Asn) residue in the catalytic loop coordinates with ATP-bound Mg2þ; however, these EPKs have a histidine (His) in place of the Asn in the loop. A sugar kinase from Pseudomonas aeruginosa, WaaP, which phosphorylates heptose I in lipopolysaccharide shares sequence homology with eukaryotic protein kinases. This enzyme has a His residue at this position in its catalytic loop (RDCYICH), but it catalyzes Tyr self-phosphorylation and phosphorylation of poly (Glu, Tyr) (15). Furthermore, these EPKs lack many crucial residues that are conserved especially the aspartate (Asp) residue in the RD motif. The Asp mutation to Ala in the RD motif in yeast PKA led to a near complete loss of activity (16). We previously reported that M. xanthus DspA is a dual-specificity kinase that autophosphorylates on two Tyr residues (Y35 and Y111) (9). To determine if these EPKs phosphorylate Tyr residue(s) on self or MBP, western blot analysis was performed using an anti-phosphotyrosine monoclonal antibody PY20. Recombinant EPKs were incubated for 60 min at 37 C with 5 mM ATP and 2.5 mM MnCl2 in 100 mM TriseHCl buffer (pH 7.5). As shown in Fig. 2, autophosphorylated MXAN_0552, 6669 (DspA), 0018, and 0023 were detected by treating the western blot membranes with antibody PY20. These EPKs displayed no detectable Tyr phosphorylation of MBP (data not shown). In addition, these EPKs showed no Tyr kinase
FIG. 2. Western blotting analysis of autophosphorylated 14 EPKs. MXAN_0396 (48 kDa), 0552 (43 kDa), 2980 (38 kDa), 6669 (41 kDa), 3183 (43 kDa), 1297 (34 kDa), 2059 (31 kDa), 2177 (36 kDa) and 6312 (33 kDa) were expressed in E. coli cells by pCold-TF vector, and MXAN_4841 (43 kDa), 4479 (45 kDa), 0018 (42 kDa), 0023 (40 kDa) and 5116 (47 kDa) were expressed using pCold vector. Autophosphorylated recombinant EPKs were subjected to SDS-PAGE, transferred to PVDF membrane and analyzed with a horseradish peroxidaseconjugated anti-phosphotyrosine antibody PY20. Protein size is indicated by molecular mass markers in kD.
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activity against histone 2B, bovine serum albumin and casein, when studied by western blot analysis using PY20 (data not shown). These four EPKs have a G(K/R)YE(L/V)(L/V) sequence (the Tyr residue in the sequence is autophosphorylated by DspA) in the subdomain I, suggesting that these sequences may be important for autophosphorylation on Tyr residue. Taken together, seven out of 14 M. xanthus recombinant EPKs containing atypical catalytic loops showed protein kinase activities, and four EPKs were dual-specificity kinases; however, these kinase activities, except for two EPKs, were significantly lower than those of Ser/Thr/Tyr protein kinases of eukaryotes. This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (25440087).
References 1. Weiwad, M., Kullertz, G., Schutkowski, M., and Fischer, G.: Evidence that the substrate backbone conformation is critical to phosphorylation by p42 MAP kinase, FEBS Lett., 478, 39e42 (2000). 2. Hanks, S. K.: Genomic analysis of the eukaryotic protein kinase superfamily: a perspective, Genome Biol., 4, 111 (2003). 3. Dardick, C. and Ronald, P.: Plant and animal pathogen recognition receptors signal through non-RD kinases, PLoS Pathog., 2, e2 (2006). 4. Hanks, S. K., Quinn, A. M., and Hunter, T.: The protein kinase family: conserved features and deduced phylogeny of the catalytic domains, Science, 241, 42e52 (1988). 5. Zhou, J. and Adams, J. A.: Is there a catalytic base in the active site of cAMP-dependent protein kinase? Biochemistry, 36, 2977e2984 (1997).
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