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CDPKs are dual-specificity protein kinases and tyrosine autophosphorylation attenuates kinase activity
FEBS Letters 586 (2012) 4070–4075
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CDPKs are dual-specificity protein kinases and tyrosine autophosphorylation attenuates kinase activity Man-Ho Oh a, Xia Wu a,1, Hyoung Seok Kim a, Jeffrey F. Harper b, Raymond E. Zielinski a, Steven D. Clouse c, Steven C. Huber a,d,⇑ a
Department of Plant Biology, University of Illinois, Urbana, IL 61801, USA Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV 89557, USA Department of Horticultural Science, NC State University, Raleigh, NC 27695, USA d U.S. Department of Agriculture, Agricultural Research Service, Urbana, IL 61801, USA b c
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Article history: Received 25 September 2012 Accepted 28 September 2012 Available online 16 October 2012 Edited by Daniela Ruffell Keywords: Calcium-dependent protein kinase Tyrosine autophosphorylation Site-directed mutagenesis Calcium signaling Peptide kinase activity
a b s t r a c t Although calcium-dependent protein kinases (CDPKs or CPKs) are classified as serine/threonine protein kinases, autophosphorylation on tyrosine residues was observed for soybean CDPKb and several Arabidopsis isoforms (AtCPK4 and AtCPK34). We identified Ser-8, Thr-17, Tyr-24 (in the kinase domain), Ser-304, and Ser-358 as autophosphorylation sites of His6-GmCDPKb. Overall autophosphorylation increased kinase activity with synthetic peptides, but autophosphorylation of Tyr-24 appears to attenuate kinase activity based on studies with the Y24F directed mutant. While much remains to be done, it is clear that several CDPKs are dual-specificity kinases, which raises the possibility that phosphotyrosine signaling may play a role in Ca2+/CDPK-mediated processes. Structured summary of protein interactions: GmCDPKb phosphorylates GmCDPKb by protein kinase assay (View interaction) Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
1. Introduction Calcium signaling is an essential second messenger controlling key aspects of plant growth and development and many specific mechanisms are involved in transducing Ca2+ signals [1]. One important mechanism involves the regulation of protein phosphorylation by Ca2+ and this is accomplished in part by the family of calcium-dependent protein kinases (CDPKs or CPKs) that are found in terrestrial plants, green algae, and certain protists (ciliates and apicomplexans) [2]. The CDPKs are classified as serine/threonine protein kinases that are activated by micromolar or submicromolar concentrations of free Ca2+ and do not require exogenous calmodulin. This can occur because the plant CDPKs have a calmodulinlike domain (CLD) that contains four Ca2+-binding EF-hands. The CLD is located C-terminal to the kinase domain and between the kinase domain and CLD is an autoinhibitory junction domain that Abbreviations: CDPK or CPK, calcium-dependent protein kinase; CLD, calmodulin-like domain; PTP1B, protein tyrosine phosphatase 1B ⇑ Corresponding author at: 1201 W. Gregory Drive, 197 ERML, University of Illinois, Urbana, IL 61801-3838, USA. Fax: +1 217 244 4419. E-mail address: [email protected] (S.C. Huber). 1 Present address: Department of Genome Sciences, University of Washington, Seattle, WA 98109, USA.
inhibits the kinase domain in the absence of Ca2+ [3–5]. In response to an increase in Ca2+, binding to the CLD occurs and results in an interaction of the Ca2+-CLD with the junction domain that alleviates the inhibition of kinase activity. Calcium activation of the CDPKs is certainly the hallmark of this family of protein kinases but regulation may also involve autophosphorylation. However, it is clear that regulation of CDPKs by autophosphorylation is not as simple as it is for some RD-type kinases that are directly activated by autophosphorylation of residue(s) within the activation segment. The RD-type kinases contain the RD (arginine-aspartate)-dipeptide sequence in subdomain VIb, which is typically associated with kinases that require autophosphorylation within the activation loop for activity [6]. The CDPKs are RD-kinases but have an acidic residue at the position where autophosphorylation is often required (9 residues Nterminal to the APE motif) thereby removing the requirement for autophosphorylation [7]. However, numerous studies have demonstrated that CDPKs autophosphorylate on serine and threonine residues in a Ca2+-dependent manner [3,8–11]. In the most extensive survey of recombinant Arabidopsis CDPKs undertaken to date [10], 35 specific sites of serine and threonine autophosphorylation were identified on 8 CDPK isoforms and 2 CDPK-related kinases (CRKs). What is not completely clear is how kinase activity is affected by
0014-5793/$36.00 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. http://dx.doi.org/10.1016/j.febslet.2012.09.040
M.-H. Oh et al. / FEBS Letters 586 (2012) 4070–4075
autophosphorylation, although the balance of evidence is suggestive that autophosphorylation promotes activity [12,13]. Here we report the autophosphorylation of soybean CDPKb, AtCPK4 and AtCPK34 on serine, threonine and (unexpectedly) tyrosine residue(s), which suggests that at least some CDPKs are dual specificity kinases rather than serine/threonine kinases as currently classified. Several sites of autophosphorylation are identified including Tyr-24, which is the first residue of the kinase domain and is strictly conserved in Arabidopsis CDPKs. We show that overall autophosphorylation of GmCDPKb increases kinase activity and that substitution of Tyr-24 with phenylalanine results in increased kinase activity suggesting that tyrosine autophosphorylation inhibits activity while serine and threonine autophosphorylation enhances activity.
2. Materials and methods 2.1. Recombinant protein production and immunoblotting The site-directed mutant of GmCDPKb where Tyr-24 was substituted with phenylalanine, to generate the Y24F directed mutant, was performed using the QuikChange XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). After sequencing to confirm mutant regions, His6-GmCDPKb (Y24F) mutant or the wild type protein was expressed in BL21(DE3)pLysS cells (Novagen). In most experiments, Escherichia coli cells were cultured in LB media supplemented with 1 mM CaCl2 or 1 mM EGTA, as indicated. Purification of His6-tagged CDPKb proteins was performed as described using Ni2+-NTA beads [14]. Autophosphorylation of the recombinant CDPKs was analyzed by immunoblotting after transfer to PVDF membranes using anti-phosphotyrosine or anti-phosphothreonine antibodies, or staining with Pro-Q Diamond phosphoprotein stain (Invitrogen, Carlsbad, CA, USA). The recombinant protein was detected by immunoblotting with anti-His6 antibodies (GenScript, Piscataway, NJ, USA). Detection of immune-complexes using fluorescent secondary antibodies (Invitrogen) was as described by [15]. Immunoblotting was done as described [16], and densitometry was performed using an Odyssey Infrared Imaging System (Li-COR Bioscience, Lincoln, NE, USA) to quantify immunoblot results. The primers used for site-directed mutagenesis were: Forward 50 -GCG AGG CTA AGG GAC CAC TTC GTT CTG GGG AAG AAG CTG, and Reverse 50 -CAG CTT CTT CCC CAG AAC GAA GTG GTC CCT TAG CCT CGC. In general, all experiments were repeated at least twice and representative results are presented.
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ium containing 1 mM EGTA. Purified protein (1.5 lg) was incubated in 10 mM MOPS-KOH, pH 7.0, containing 2 mM EGTA, 100 mM KCl, 20 lM ATP and 10 mM MgCl2 with the different concentrations of CaCl2 to generate the free [Ca2+] ranging from 95 to 750 nM. Reactions were run for 1 h at room temperature before analysis by immunoblotting. Values reported are means ± S.E.M. from three determinations and are representative of at least two independent experiments. 3. Results 3.1. CDPKs autophosphorylate on serine, threonine and tyrosine residues A common experimental protocol is to express an epitopetagged recombinant CDPK in E. coli, affinity purify the protein, and autophosphorylate in vitro by incubation with ATP prior to analysis of phosphoamino acids [10,12,18]. However, protein kinases can autophosphorylate during production in bacterial cells [19], and we used this system to examine autophosphorylation of CDPKs. Fig. 1 documents the autophosphorylation of three CDPKs as a function of time during IPTG induction in E. coli. At each time point, the His6-tagged proteins were affinity purified and equal amounts of protein were analyzed by immunoblotting with
2.2. LC–MS/MS analysis The E. coli proteins were extracted with 6 M urea in 50 mM ammonium bicarbonate (pH 7.8). Protein extracts were digested with trypsin (Promega, Madison, WI, USA) and tryptic peptides were collected with C18 SPE column (Alltech, Deerfield, IL, USA). The phosphopeptides were enriched either by Pierce TiO2 spin column (Thermo-Fisher Scientific, Rockford, IL, USA) or by immobilized metal affinity chromatography (IMAC; PHOS-Select Iron Affinity gel, Sigma–Aldrich, St. Louis, MO, USA). The enriched peptides were purified with Pierce graphite spin columns (Thermo Scientific, Rockford, IL, USA) for the LC–MS/MS analysis (see Supplementary data). 2.3. Peptide kinase assays For routine assays, peptide kinase activity was measured with the indicated synthetic peptide substrate as previously described with 1 mM free Ca2+ [14,17]. In the experiment of Fig. 4, His6GmCDPKb protein was purified from E. coli cells grown in LB med-
Fig. 1. Autophosphorylation of recombinant CDPKs in E. coli. (A) His6-GmCDPKb, (B) His6-AtCPK4, and (C) His6-AtCPK34 proteins were induced by addition of IPTG at time zero and at the times indicated, recombinant proteins were affinity purified and analyzed for autophosphorylation status by staining with ProQ Diamond stain or cross reaction with anti-phosphothreonine (anti-pThr) or anti-phosphotyrosine (anti-pY) antibodies.
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M.-H. Oh et al. / FEBS Letters 586 (2012) 4070–4075
generic phosphoamino acid antibodies that detect phosphothreonine (anti-pThr) or phosphotyrosine (anti-pY). The protein present at time zero reflects the CDPK protein expressed as a result of bleed-through of the T7 promoter and was weakly autophosphorylated on threonine, but not tyrosine, residues. As shown, autophosphorylation on threonine residues increased with time of induction for all three CDPKs tested. The surprising result, however, was that all three CDPKs also autophosphorylated on tyrosine residues (Fig. 1). With GmCDPKb and AtCPK4, threonine autophosphorylation preceded tyrosine autophosphorylation, whereas with AtCPK34 autophosphorylation on both residues followed similar time courses but were clearly delayed relative to the other isoforms tested. The surprising and most important result was that CDPKs, which are classified as serine/threonine protein kinases, also have tyrosine kinase activity and thus are dual-specificity kinases. In the follow-up studies, we focused primarily on GmCDPKb as we attempted to extend this result. We identifyied specific sites of autophosphorylation of GmCDPKb by LC–MS/MS analysis, including three serine residues (Ser-8, Ser-304 and Ser-358), one threonine residue (Thr-17) and importantly, one tyrosine residue (Tyr-24) (Fig. 2A). The location of these residues is shown schematically in Fig. 2B. GmCDPKb is an ortholog of AtCPK4, and previous studies [10] identified Ser360 (homologous to GmCDPKb Ser-358) as a site of autophosphorylation. None of the other sites have been identified before, and in particular, no sites of tyrosine autophosphorylation have been reported or identified. Tyr-24 is the first residue of the kinase domain of GmCDPKb and is strictly conserved among members of the Arabidopsis CPK family, suggesting that other CDPKs may also autophosphorylate at this position. It is also worth noting that Ser-358 corresponds to the ‘site II’ locus where autophosphorylation of several other Arabidopsis CDPKs was identified [12]. Finally, it is also interesting that 3 of the 5 autophosphorylation sites identified in the present study were clustered in, or close to, the N-terminal domain. This clustering was noted before in a broader survey study [10] and is particularly interesting given that in some cases the CDPK N-terminus might function in substrate specificity [20].
The stoichiometry of tyrosine phosphorylation on recombinant GmCDPKb was quite high, because >80% of the GmCDPKb protein could be immunoprecipitated with immobilized antiphosphotyrosine antibodies (Fig. 3). Thus, the failure to detect phosphotyrosine by [32P]-phosphoamino acid analysis may be because the recombinant proteins used were already highly autophosphorylated on tyrosine residues as isolated from E. coli, or that autophosphorylation in vitro is not as effective as in situ autophosphorylation, at least with respect to tyrosine. Regardless, the important point to note here is that the stoichiometry of tyrosine autophosphorylation of GmCDPKb was quite high and thus could be functionally important. 3.2. Tyrosine autophosphorylation in vitro is Ca2+dependent In our studies with GmCDPKb, tyrosine autophosphorylation in vitro could be readily monitored. For these experiments, recombinant protein was purified after a 10 h induction period, when tyrosine autophosphorylation of the protein as purified from E. coli was low (see Fig. 1A). After purification, the protein was allowed autophosphorylation for 1 h at room temperature in reaction mixtures containing all necessary components and variable concentrations of free Ca2+. In the absence of Ca2+, phosphotyrosine remained low but increased in the presence of even low concentrations of free Ca2+ to reach a maximum effect at submicromolar concentrations of Ca2+ (Fig. 4A). In these experiments, the CDPK as isolated contained some phosphothreonine, which increased in parallel with phosphotyrosine (Fig. 4A and B). The important result is that tyrosine autophosphorylation can indeed be observed to occur in vitro and does so in a strictly Ca2+-dependent manner, as would be expected. This indicates that tyrosine autophosphorylation is not an artifact of overexpression of the kinase in bacteria or somehow influenced by bacterial proteins. 3.3. Phosphorylation of Tyr-24 attenuates CDPK peptide kinase activity To investigate the role of Tyr-24 of GmCDPKb, we produced a site-directed mutant that is unable to autophosphorylate at this
Fig. 2. Identification of autophosphorylation sites on GmCDPKb. (A) Summary of LC–MS/MS results identifying GmCDPKb autophosphorylation sites obtained by enrichment of phosphopeptides using TiO2 or Fe3+- IMAC as indicated. ‘P-bodies’ refers to analysis of the insoluble ‘protein body’ fraction that was digested and enriched with TiO2. For each enrichment, spectral counts obtained are indicated. M, the number of missed cleavages; Score, Mascot score, which is a probability based implementation of the Mowse algorithm. The total score is the absolute probability that the observed match is a random event and is calculated as 10LOG10(P), where P is the absolute probability. Expect, expectation value, which is directly equivalent to the E-value in a Blast search result. The lower the expectation value, the more significant the score. Spectra for each of these phosphosites are provided as Supplemental Figures. (B) Schematic representation of GmCDPKb showing the location of the phosphosites listed in (A). KD, kinase domain; J, autoinhibitory junction domain; CLD, calmodulin-like domain with four EF hands (open ovals).
M.-H. Oh et al. / FEBS Letters 586 (2012) 4070–4075
Fig. 3. High stoichiometry of tyrosine phosphorylation of recombinant His6GmCDPKb. An aliquot of GmCDPKb protein was directly immunoprecipitated with anti-His6 antibodies (lane 1). A second equivalent aliquot of GmCDPKb protein was subjected to 5 rounds of sequential immunoprecipitation with immobilized anti-pY antibodies (lanes 2–6) followed by immunoprecipitation with anti-His6 antibodies of the remaining supernatant (lane 7). In each case, washed immunoprecipitates were analyzed by immunoblotting with antiphosphotyrosine (anti-pY), antiphosphothreonine (anti-pT), or anti-His6 antibodies as indicated. As is readily apparent, the anti-pY antibodies effectively removed GmCDPKb protein from solution, and densitometry suggested that >80% of the GmCDPKb protein was phosphorylated on tyrosine residues.
Fig. 4. Tyrosine autophosphorylation is Ca2+-dependent in vitro (A) Immunoblots showing phosphotyrosine (pY) and phosphothreonine (pThr) content after autophosphorylation in vitro with the indicated concentration of free Ca2+. (B) Densitometry of the immunoblots shown in (A). There was no change in phosphotyrosine or phosphothreonine content during the in vitro autophosphorylation in the absence of Ca2+. His6-GmCDPKb protein was extracted from E. coli cells grown in LB medium containing 1 mM EGTA. Purified protein (1.5 lg) was incubated in reaction mixtures containing10 mM MOPS–KOH, pH 7, 2 mM EGTA, 100 mM KCl, 20 lM ATP and 10 mM MgCl2 with the indicated concentration of free Ca2+ for 1 h at room temperature.
site by substituting tyrosine with phenylalanine. The corresponding directed mutant protein, Y24F, was compared with GmCDPKb in terms of autophosphorylation and ability to catalyze transphosphorylation reactions using a variety of synthetic peptides as substrates. As expected, tyrosine autophosphorylation of wild type CDPKb was apparent in preparations purified from E. coli that had been induced with IPTG for 16 h. Interestingly, tyrosine autophosphorylation of the Y24F directed mutant was almost completely eliminated whereas there was little impact on
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autophosphorylation on threonine residues (Fig. 5A). These results suggest that Tyr-24 is not essential for kinase activity and that this residue may be a major site of tyrosine autophosphorylation. In order to assess the role of autophosphorylation on CDPK transphosphorylation activity, we purified wild type CDPK and the Y24F directed mutant from E. coli cultures immediately after addition of IPTG (time zero) or after 16 h of induction. As shown in Fig. 5A, the time zero protein samples had low autophosphorylation on threonine (and presumably serine) residues and were devoid of phosphotyrosine. The 16 h preparations had increased autophosphorylation on threonine (and presumably serine) but differed dramatically in phosphotyrosine content, specifically at the Tyr-24 position. The synthetic peptide substrates (sequences shown in Fig. 5B) correspond to the different motifs that are recognized and phosphorylated by CDPKs. These motifs are distinguished on the basis of positioning of both basic and hydrophobic residues. The NR6, SP42 and UGP1 peptides all conform to the classical motif with a basic residue (an arginine in each case) at the 3 position and hydrophobic residues at 5 and +4 [21,22]. All three synthetic peptides were phosphorylated but the SP42 peptide had the lowest activity. However, in all cases, activity was highest with the kinase prepared from 16-h cultures and the Y24F directed mutant had higher activity than the wild type enzyme (Fig. 5C). Generally similar results were obtained with peptide substrates conforming to the non-canonical motifs. For example, the ACA2–7 peptide, which conforms to the non-canonical ‘ACA2’ motif that is defined by basic residues at 6 and beyond (but not 3) and at +2 [17], was the best substrate tested and corresponding differences in activities among the preparations was very pronounced. Similarly, the other non-canonical motif tested was originally described for the Ser-460 regulatory phosphorylation site of tomato ACS (1-amino-cyclopropane-1-carboxylate synthase) 2, and as is distinguished by lack of both the basic residue at P-3 and the hydrophobic residue at P-5 that are hallmarks of the classic motif. Rather, with the ‘ACS’ motif, basic residues at 2 and +3 and +4 are key positive recognition elements. The ACSM +1 peptide was a good substrate for GmCDPKb and interestingly showed the largest relative effects of kinase autophosphorylation on activity. With wild type GmCDPKb, phosphorylation of the ACSM +1 peptide increased three-fold comparing the 0 and 16 h enzyme preparations, and the increase was even more dramatic (9.4-fold) for the Y24F directed mutant. Comparing the 16-h enzyme preparations with the ACSM +1 peptide, the Y24F directed mutant had almost twice the activity of wild type. Collectively, these results suggest that in general, autophosphorylation enhances CDPK peptide kinase activity as evidenced by increased activity with the 16-h hyperphosphorylated enzyme compared to the 0-h hypophosphorylated enzyme. However, at least some autophosphorylation sites are inhibitory to enzyme activity, and Tyr-24 may be one such site. Accordingly, when autophosphorylation at this position is prevented as with the Y24F directed mutant, the increase in activity with autophosphorylation is even greater.
4. Discussion The results of the present study document for the first time that CDPKs, which are classified as serine/threonine protein kinases, also autophosphorylate on tyrosine residues and thus are dualspecificity kinases. While much remains to be done, the results strongly suggest that autophosphorylation on tyrosine residues has functional significance. At least with GmCDPKb, autophosphorylation of Tyr-24 attenuates kinase activity, as demonstrated with synthetic peptide substrates in vitro (Fig. 5). It will be important in future studies to determine how dual specificity impacts the cellular functions of CDPKs in vivo, but these observations uncover the
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Fig. 5. Tyrosine-24 is a major site of GmCDPKb autophosphorylation and attenuates kinase activity (A) The native sequence or Y24F directed mutant of GmCDPKb were expressed in E. coli and after 0, 6 or 16 h of induction, recombinant proteins were purified and analyzed by immunoblotting or staining with CBB as indicated. Equivalent amounts of protein (1.5 lg per lane) were loaded in each lane. The anti-pThr immunoblot and CBB-stained blot were obtained with the kinase preparations used for activity measurements in panel C. The anti-Tyr immunoblot was from another preparation but all of the results are representative of several independent experiments. (B) Peptides used to monitor kinase activity in vitro. (C) Peptide kinase activity of GmCDPKb and the Y24F directed mutant (0.5 lg protein) prepared at time zero (hypophosphorylated) or after 16 h of induction (hyperphosphorylated). Values are the means ± S.E.M. from three determinations and are representative of several independent experiments.
potential for a new component of phosphotyrosine signaling in a large family of protein kinases that function to transduce Ca2+ signals in the regulation of growth and development and plant response to biotic and abiotic stresses. While Tyr-24 appears to be a major site of autophosphorylation of GmCDPKb, it is possible that other tyrosine residues are autophosphorylated as well. However, it is clear that prevention of phosphorylation of Tyr-24 plays a role in regulation of activity based on the results with the Y24F directed mutant (Fig. 5C). Tyrosine autophosphorylation can also be a high stoichiometry modification, based on the ability to remove CDPK protein from solution by immunoprecipitation with anti-phosphotyrosine antibodies (Fig. 3). Because a tyrosine residue at the start of the kinase domain (corresponding to Tyr-24 in GmCDPKb) is conserved among Arabidopsis CPK isoforms, the potential for similar regulation of other members of the family is likely and will be interesting to explore. Our studies also established that autophosphorylation on serine and threonine residues stimulate CDPK transphosphorylation activity. This is based on the observation that increased autophosphorylation of GmCDPKb that occurred as the protein was produced in E. coli was associated with increased peptide kinase activity (Fig. 5) and is consistent with earlier reports [12,13]. Our results suggest that autophosphorylation on serine and/or threonine residues stimulates while tyrosine autophosphorylation inhibits peptide kinase activity. It will be important in future studies to determine which specific phosphosites are activating kinase activity. Interestingly, the magnitude of the effect of autophosphorylation on kinase activity varied with the synthetic peptide used as substrate (Fig. 5C), suggesting that autophosphorylation may also influence target specificity. Given that the N-terminus plays an important role in substrate specificity [20] and sites of serine/ threonine autophosphorylation tend to cluster in the N-terminus ([10] and this study), an effect of autophosphorylation on CDPK activity and selectivity can be readily understood. In summary, an emerging concept is that several protein families classified as serine/threonine protein kinases are actually dual-specificity kinases capable of phosphorylation on serine, threonine and tyrosine residues. This is true for many, but likely not all,
members of the large receptor-like kinase family in Arabidopsis [15,19,23–26], and a similar situation is emerging with the CDPK family. Clearly, we do not have a complete understanding of the kinase domain motifs that confer dual specificity. Much remains to be done with both the receptor kinases and CDPKs in terms of tyrosine phosphorylation, but recognizing that it occurs is certainly the essential first step. With the CDPKs, we have established that tyrosine autophosphorylation has a functional role in vitro that may oppose the activation caused by autophosphorylation on serine/ threonine residues. Establishing which specific sites are of regulatory significance and whether they function in this manner in vivo are important objectives for future studies. Acknowledgements This work was supported in part by the National Science Foundation (IOS-1022177, MCB-0740211, MCB-1021363 and MCB-0920624) and the US Department of Agriculture (USDA)Agricultural Research Service (ARS). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2012. 09.040. References [1] Hepler, P.K. (2005) Calcium: a central regulator of plant growth and development. Plant Cell 17, 2142–2155. [2] Hrabak, E.M. et al. (2003) The Arabidopsis CDPK-SnRK superfamily of protein kinases. Plant Physiol. 132, 666–680. [3] Harmon, A.C., Putnam-Evans, C. and Cormier, M.J. (1987) A calcium-dependent but calmodulin-independent protein kinase from soybean. Plant Physiol. 83, 830–837. [4] Harmon, A.C., Gribskov, M. and Harper, J.F. (2000) CDPKs—a kinase for every Ca2+ signal? Trends Plant Sci. 5, 154. [5] Harmon, A.C., Gribskov, M., Gubrium, E. and Harper, J.F. (2001) The CDPK superfamily of protein kinases. New Phytol. 151, 175. [6] Johnson, L.N., Noble, M.E. and Owen, D.J. (1996) Active and inactive protein kinases: structural basis for regulation. Cell 85, 149–158.
M.-H. Oh et al. / FEBS Letters 586 (2012) 4070–4075 [7] Harper, J.F., Breton, G. and Harmon, A. (2004) Decoding Ca2+ signals through plant protein kinases. Annu. Rev. Plant Biol. 55, 263–288. [8] Mee Yoon, G., Sun Cho, H., Jung Ha, H., Ryol Liu, J. and Pai Lee, H.-S. (1999) Characterization of NtCDPK1, a calcium-dependent protein kinase gene in Nicotiana tabacum, and the activity of its encoded protein. Plant Mol. Biol. 39, 991–1001. [9] Anil, V.S., Harmon, A.C. and Rao, K.S. (2000) Spatio-temporal accumulation and activity of calcium-dependent protein kinases during embryogenesis, seed development, and germination in sandalwood. Plant Physiol. 122, 1035–1044. [10] Hegeman, A.D. et al. (2006) A phyloproteomic characterization of in vitro autophosphorylation in calcium-dependent protein kinases. Proteomics 6, 3649–3664. [11] Nemoto, K., Seto, T., Takahashi, H., Nozawa, A., Seki, M., Shinozaki, K., Endo, Y. and Sawasaki, T. (2011) Autophosphorylation profiling of Arabidopsis protein kinases using the cell-free system. Phytochemistry 72, 1136–1144. [12] Chehab, E.W., Patharkar, O.R., Hegeman, A.D., Taybi, T. and Cushman, J.C. (2004) Autophosphorylation and subcellular localization dynamics of a saltand water deficit-induced calcium-dependent protein kinase from ice plant. Plant Physiol. 135, 1430–1446. [13] Chaudhuri, S., Seal, A. and DasGupta, M. (1999) Autophosphorylationdependent activation of a calcium-dependent protein kinase from groundnut. Plant Physiol. 120, 859. [14] Huang, J.Z. and Huber, S.C. (2001) Phosphorylation of synthetic peptides by a CDPK and plant SNF1-related protein kinase. Plant Cell Physiol. 42, 1079. [15] Oh, M.H., Wang, X., Kota, U., Goshe, M.B., Clouse, S.D. and Huber, S.C. (2009) Tyrosine phosphorylation of the BRI1 receptor kinase emerges as a component of brassinosteroid signaling in Arabidopsis. Proc. Natl. Acad. Sci. USA 106, 658–663. [16] Duncan, K.A., Hardin, S.C. and Huber, S.C. (2006) The three maize sucrose synthase isoforms differ in distribution, localization, and phosphorylation. Plant Cell Physiol 47, 959–971.
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[17] Huang, J.Z., Hardin, S.C. and Huber, S.C. (2001) Identification of a novel phosphorylation motif for CDPKs: phosphorylation of synthetic peptides lacking basic residues at P-3/P-4. Arch. Biochem. Biophys. 393, 61. [18] Rutschmann, F., Stalder, U., Piotrowski, M., Oecking, C. and Schaller, A. (2002) LeCPK1, a calcium-dependent protein kinase from tomato. Plasma membrane targeting and biochemical characterization. Plant Physiol. 129, 156–168. [19] Oh, M.-H., Wang, X., Wu, X., Zhao, Y., Clouse, S.D. and Huber, S.C. (2010) Autophosphorylation of Tyr-610 in the receptor kinase BAK1 plays a role in brassinosteroid signaling and basal defense gene expression. Proc. Natl. Acad. Sci. USA 107, 17827–17832. [20] Ito, T., Nakata, M., Fukazawa, J., Ishida, S. and Takahashi, Y. (2010) Alteration of substrate specificity: the variable n-terminal domain of tobacco Ca2+-dependent protein kinase is important for substrate recognition. Plant Cell 22, 1592–1604. [21] Bachmann, M., Shiraishi, N., Campbell, W.H., Yoo, B.C., Harmon, A.C. and Huber, S.C. (1996) Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase. Plant cell 8, 505–517. [22] McMichael, R.W., Klein, R.R., Salvucci, M.E. and Huber, S.C. (1993) Identification of the major regulatory phosphorylation site in sucrosephosphate synthase. Arch. Biochem. Biophys. 307, 248–252. [23] Oh, M.H., Wu, X., Clouse, S.D. and Huber, S.C. (2011) Functional importance of BAK1 tyrosine phosphorylation in vivo. Plant Signal. Behav. 6, 400–405. [24] Oh, M.H., Kim, H.S., Wu, X., Clouse, S.D., Zielinski, R.E. and Huber, S.C. (2012) Calcium/calmodulin inhibition of the Arabidopsis BRI1 receptor kinase provides a possible link between calcium- and brassinosteroid-signaling. Biochem. J. 443, 515–523. [25] Oh, M.-H., Clouse, S.D. and Huber, S.C. (2012) Tyrosine phosphorylation of the BRI1 receptor kinase occurs via a posttranslational modification and is activated by the juxtamembrane domain. Front. Plant Sci. 3, 175, http://dx/ doi.org/10.3389/fpls.2012.00175. [26] Oh, M.H., Clouse, S.D. and Huber, S.C. (2009) Tyrosine phosphorylation in brassinosteroid signaling. Plant Signal. Behav. 4, 1–4.
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CDPKs are dual-specificity protein kinases and tyrosine autophosphorylation attenuates kinase activity Man-Ho Oh a, Xia Wu a,1, Hyoung Seok Kim a, Jeffrey F. Harper b, Raymond E. Zielinski a, Steven D. Clouse c, Steven C. Huber a,d,⇑ a
Department of Plant Biology, University of Illinois, Urbana, IL 61801, USA Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV 89557, USA Department of Horticultural Science, NC State University, Raleigh, NC 27695, USA d U.S. Department of Agriculture, Agricultural Research Service, Urbana, IL 61801, USA b c
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Article history: Received 25 September 2012 Accepted 28 September 2012 Available online 16 October 2012 Edited by Daniela Ruffell Keywords: Calcium-dependent protein kinase Tyrosine autophosphorylation Site-directed mutagenesis Calcium signaling Peptide kinase activity
a b s t r a c t Although calcium-dependent protein kinases (CDPKs or CPKs) are classified as serine/threonine protein kinases, autophosphorylation on tyrosine residues was observed for soybean CDPKb and several Arabidopsis isoforms (AtCPK4 and AtCPK34). We identified Ser-8, Thr-17, Tyr-24 (in the kinase domain), Ser-304, and Ser-358 as autophosphorylation sites of His6-GmCDPKb. Overall autophosphorylation increased kinase activity with synthetic peptides, but autophosphorylation of Tyr-24 appears to attenuate kinase activity based on studies with the Y24F directed mutant. While much remains to be done, it is clear that several CDPKs are dual-specificity kinases, which raises the possibility that phosphotyrosine signaling may play a role in Ca2+/CDPK-mediated processes. Structured summary of protein interactions: GmCDPKb phosphorylates GmCDPKb by protein kinase assay (View interaction) Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
1. Introduction Calcium signaling is an essential second messenger controlling key aspects of plant growth and development and many specific mechanisms are involved in transducing Ca2+ signals [1]. One important mechanism involves the regulation of protein phosphorylation by Ca2+ and this is accomplished in part by the family of calcium-dependent protein kinases (CDPKs or CPKs) that are found in terrestrial plants, green algae, and certain protists (ciliates and apicomplexans) [2]. The CDPKs are classified as serine/threonine protein kinases that are activated by micromolar or submicromolar concentrations of free Ca2+ and do not require exogenous calmodulin. This can occur because the plant CDPKs have a calmodulinlike domain (CLD) that contains four Ca2+-binding EF-hands. The CLD is located C-terminal to the kinase domain and between the kinase domain and CLD is an autoinhibitory junction domain that Abbreviations: CDPK or CPK, calcium-dependent protein kinase; CLD, calmodulin-like domain; PTP1B, protein tyrosine phosphatase 1B ⇑ Corresponding author at: 1201 W. Gregory Drive, 197 ERML, University of Illinois, Urbana, IL 61801-3838, USA. Fax: +1 217 244 4419. E-mail address: [email protected] (S.C. Huber). 1 Present address: Department of Genome Sciences, University of Washington, Seattle, WA 98109, USA.
inhibits the kinase domain in the absence of Ca2+ [3–5]. In response to an increase in Ca2+, binding to the CLD occurs and results in an interaction of the Ca2+-CLD with the junction domain that alleviates the inhibition of kinase activity. Calcium activation of the CDPKs is certainly the hallmark of this family of protein kinases but regulation may also involve autophosphorylation. However, it is clear that regulation of CDPKs by autophosphorylation is not as simple as it is for some RD-type kinases that are directly activated by autophosphorylation of residue(s) within the activation segment. The RD-type kinases contain the RD (arginine-aspartate)-dipeptide sequence in subdomain VIb, which is typically associated with kinases that require autophosphorylation within the activation loop for activity [6]. The CDPKs are RD-kinases but have an acidic residue at the position where autophosphorylation is often required (9 residues Nterminal to the APE motif) thereby removing the requirement for autophosphorylation [7]. However, numerous studies have demonstrated that CDPKs autophosphorylate on serine and threonine residues in a Ca2+-dependent manner [3,8–11]. In the most extensive survey of recombinant Arabidopsis CDPKs undertaken to date [10], 35 specific sites of serine and threonine autophosphorylation were identified on 8 CDPK isoforms and 2 CDPK-related kinases (CRKs). What is not completely clear is how kinase activity is affected by
0014-5793/$36.00 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. http://dx.doi.org/10.1016/j.febslet.2012.09.040
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autophosphorylation, although the balance of evidence is suggestive that autophosphorylation promotes activity [12,13]. Here we report the autophosphorylation of soybean CDPKb, AtCPK4 and AtCPK34 on serine, threonine and (unexpectedly) tyrosine residue(s), which suggests that at least some CDPKs are dual specificity kinases rather than serine/threonine kinases as currently classified. Several sites of autophosphorylation are identified including Tyr-24, which is the first residue of the kinase domain and is strictly conserved in Arabidopsis CDPKs. We show that overall autophosphorylation of GmCDPKb increases kinase activity and that substitution of Tyr-24 with phenylalanine results in increased kinase activity suggesting that tyrosine autophosphorylation inhibits activity while serine and threonine autophosphorylation enhances activity.
2. Materials and methods 2.1. Recombinant protein production and immunoblotting The site-directed mutant of GmCDPKb where Tyr-24 was substituted with phenylalanine, to generate the Y24F directed mutant, was performed using the QuikChange XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). After sequencing to confirm mutant regions, His6-GmCDPKb (Y24F) mutant or the wild type protein was expressed in BL21(DE3)pLysS cells (Novagen). In most experiments, Escherichia coli cells were cultured in LB media supplemented with 1 mM CaCl2 or 1 mM EGTA, as indicated. Purification of His6-tagged CDPKb proteins was performed as described using Ni2+-NTA beads [14]. Autophosphorylation of the recombinant CDPKs was analyzed by immunoblotting after transfer to PVDF membranes using anti-phosphotyrosine or anti-phosphothreonine antibodies, or staining with Pro-Q Diamond phosphoprotein stain (Invitrogen, Carlsbad, CA, USA). The recombinant protein was detected by immunoblotting with anti-His6 antibodies (GenScript, Piscataway, NJ, USA). Detection of immune-complexes using fluorescent secondary antibodies (Invitrogen) was as described by [15]. Immunoblotting was done as described [16], and densitometry was performed using an Odyssey Infrared Imaging System (Li-COR Bioscience, Lincoln, NE, USA) to quantify immunoblot results. The primers used for site-directed mutagenesis were: Forward 50 -GCG AGG CTA AGG GAC CAC TTC GTT CTG GGG AAG AAG CTG, and Reverse 50 -CAG CTT CTT CCC CAG AAC GAA GTG GTC CCT TAG CCT CGC. In general, all experiments were repeated at least twice and representative results are presented.
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ium containing 1 mM EGTA. Purified protein (1.5 lg) was incubated in 10 mM MOPS-KOH, pH 7.0, containing 2 mM EGTA, 100 mM KCl, 20 lM ATP and 10 mM MgCl2 with the different concentrations of CaCl2 to generate the free [Ca2+] ranging from 95 to 750 nM. Reactions were run for 1 h at room temperature before analysis by immunoblotting. Values reported are means ± S.E.M. from three determinations and are representative of at least two independent experiments. 3. Results 3.1. CDPKs autophosphorylate on serine, threonine and tyrosine residues A common experimental protocol is to express an epitopetagged recombinant CDPK in E. coli, affinity purify the protein, and autophosphorylate in vitro by incubation with ATP prior to analysis of phosphoamino acids [10,12,18]. However, protein kinases can autophosphorylate during production in bacterial cells [19], and we used this system to examine autophosphorylation of CDPKs. Fig. 1 documents the autophosphorylation of three CDPKs as a function of time during IPTG induction in E. coli. At each time point, the His6-tagged proteins were affinity purified and equal amounts of protein were analyzed by immunoblotting with
2.2. LC–MS/MS analysis The E. coli proteins were extracted with 6 M urea in 50 mM ammonium bicarbonate (pH 7.8). Protein extracts were digested with trypsin (Promega, Madison, WI, USA) and tryptic peptides were collected with C18 SPE column (Alltech, Deerfield, IL, USA). The phosphopeptides were enriched either by Pierce TiO2 spin column (Thermo-Fisher Scientific, Rockford, IL, USA) or by immobilized metal affinity chromatography (IMAC; PHOS-Select Iron Affinity gel, Sigma–Aldrich, St. Louis, MO, USA). The enriched peptides were purified with Pierce graphite spin columns (Thermo Scientific, Rockford, IL, USA) for the LC–MS/MS analysis (see Supplementary data). 2.3. Peptide kinase assays For routine assays, peptide kinase activity was measured with the indicated synthetic peptide substrate as previously described with 1 mM free Ca2+ [14,17]. In the experiment of Fig. 4, His6GmCDPKb protein was purified from E. coli cells grown in LB med-
Fig. 1. Autophosphorylation of recombinant CDPKs in E. coli. (A) His6-GmCDPKb, (B) His6-AtCPK4, and (C) His6-AtCPK34 proteins were induced by addition of IPTG at time zero and at the times indicated, recombinant proteins were affinity purified and analyzed for autophosphorylation status by staining with ProQ Diamond stain or cross reaction with anti-phosphothreonine (anti-pThr) or anti-phosphotyrosine (anti-pY) antibodies.
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generic phosphoamino acid antibodies that detect phosphothreonine (anti-pThr) or phosphotyrosine (anti-pY). The protein present at time zero reflects the CDPK protein expressed as a result of bleed-through of the T7 promoter and was weakly autophosphorylated on threonine, but not tyrosine, residues. As shown, autophosphorylation on threonine residues increased with time of induction for all three CDPKs tested. The surprising result, however, was that all three CDPKs also autophosphorylated on tyrosine residues (Fig. 1). With GmCDPKb and AtCPK4, threonine autophosphorylation preceded tyrosine autophosphorylation, whereas with AtCPK34 autophosphorylation on both residues followed similar time courses but were clearly delayed relative to the other isoforms tested. The surprising and most important result was that CDPKs, which are classified as serine/threonine protein kinases, also have tyrosine kinase activity and thus are dual-specificity kinases. In the follow-up studies, we focused primarily on GmCDPKb as we attempted to extend this result. We identifyied specific sites of autophosphorylation of GmCDPKb by LC–MS/MS analysis, including three serine residues (Ser-8, Ser-304 and Ser-358), one threonine residue (Thr-17) and importantly, one tyrosine residue (Tyr-24) (Fig. 2A). The location of these residues is shown schematically in Fig. 2B. GmCDPKb is an ortholog of AtCPK4, and previous studies [10] identified Ser360 (homologous to GmCDPKb Ser-358) as a site of autophosphorylation. None of the other sites have been identified before, and in particular, no sites of tyrosine autophosphorylation have been reported or identified. Tyr-24 is the first residue of the kinase domain of GmCDPKb and is strictly conserved among members of the Arabidopsis CPK family, suggesting that other CDPKs may also autophosphorylate at this position. It is also worth noting that Ser-358 corresponds to the ‘site II’ locus where autophosphorylation of several other Arabidopsis CDPKs was identified [12]. Finally, it is also interesting that 3 of the 5 autophosphorylation sites identified in the present study were clustered in, or close to, the N-terminal domain. This clustering was noted before in a broader survey study [10] and is particularly interesting given that in some cases the CDPK N-terminus might function in substrate specificity [20].
The stoichiometry of tyrosine phosphorylation on recombinant GmCDPKb was quite high, because >80% of the GmCDPKb protein could be immunoprecipitated with immobilized antiphosphotyrosine antibodies (Fig. 3). Thus, the failure to detect phosphotyrosine by [32P]-phosphoamino acid analysis may be because the recombinant proteins used were already highly autophosphorylated on tyrosine residues as isolated from E. coli, or that autophosphorylation in vitro is not as effective as in situ autophosphorylation, at least with respect to tyrosine. Regardless, the important point to note here is that the stoichiometry of tyrosine autophosphorylation of GmCDPKb was quite high and thus could be functionally important. 3.2. Tyrosine autophosphorylation in vitro is Ca2+dependent In our studies with GmCDPKb, tyrosine autophosphorylation in vitro could be readily monitored. For these experiments, recombinant protein was purified after a 10 h induction period, when tyrosine autophosphorylation of the protein as purified from E. coli was low (see Fig. 1A). After purification, the protein was allowed autophosphorylation for 1 h at room temperature in reaction mixtures containing all necessary components and variable concentrations of free Ca2+. In the absence of Ca2+, phosphotyrosine remained low but increased in the presence of even low concentrations of free Ca2+ to reach a maximum effect at submicromolar concentrations of Ca2+ (Fig. 4A). In these experiments, the CDPK as isolated contained some phosphothreonine, which increased in parallel with phosphotyrosine (Fig. 4A and B). The important result is that tyrosine autophosphorylation can indeed be observed to occur in vitro and does so in a strictly Ca2+-dependent manner, as would be expected. This indicates that tyrosine autophosphorylation is not an artifact of overexpression of the kinase in bacteria or somehow influenced by bacterial proteins. 3.3. Phosphorylation of Tyr-24 attenuates CDPK peptide kinase activity To investigate the role of Tyr-24 of GmCDPKb, we produced a site-directed mutant that is unable to autophosphorylate at this
Fig. 2. Identification of autophosphorylation sites on GmCDPKb. (A) Summary of LC–MS/MS results identifying GmCDPKb autophosphorylation sites obtained by enrichment of phosphopeptides using TiO2 or Fe3+- IMAC as indicated. ‘P-bodies’ refers to analysis of the insoluble ‘protein body’ fraction that was digested and enriched with TiO2. For each enrichment, spectral counts obtained are indicated. M, the number of missed cleavages; Score, Mascot score, which is a probability based implementation of the Mowse algorithm. The total score is the absolute probability that the observed match is a random event and is calculated as 10LOG10(P), where P is the absolute probability. Expect, expectation value, which is directly equivalent to the E-value in a Blast search result. The lower the expectation value, the more significant the score. Spectra for each of these phosphosites are provided as Supplemental Figures. (B) Schematic representation of GmCDPKb showing the location of the phosphosites listed in (A). KD, kinase domain; J, autoinhibitory junction domain; CLD, calmodulin-like domain with four EF hands (open ovals).
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Fig. 3. High stoichiometry of tyrosine phosphorylation of recombinant His6GmCDPKb. An aliquot of GmCDPKb protein was directly immunoprecipitated with anti-His6 antibodies (lane 1). A second equivalent aliquot of GmCDPKb protein was subjected to 5 rounds of sequential immunoprecipitation with immobilized anti-pY antibodies (lanes 2–6) followed by immunoprecipitation with anti-His6 antibodies of the remaining supernatant (lane 7). In each case, washed immunoprecipitates were analyzed by immunoblotting with antiphosphotyrosine (anti-pY), antiphosphothreonine (anti-pT), or anti-His6 antibodies as indicated. As is readily apparent, the anti-pY antibodies effectively removed GmCDPKb protein from solution, and densitometry suggested that >80% of the GmCDPKb protein was phosphorylated on tyrosine residues.
Fig. 4. Tyrosine autophosphorylation is Ca2+-dependent in vitro (A) Immunoblots showing phosphotyrosine (pY) and phosphothreonine (pThr) content after autophosphorylation in vitro with the indicated concentration of free Ca2+. (B) Densitometry of the immunoblots shown in (A). There was no change in phosphotyrosine or phosphothreonine content during the in vitro autophosphorylation in the absence of Ca2+. His6-GmCDPKb protein was extracted from E. coli cells grown in LB medium containing 1 mM EGTA. Purified protein (1.5 lg) was incubated in reaction mixtures containing10 mM MOPS–KOH, pH 7, 2 mM EGTA, 100 mM KCl, 20 lM ATP and 10 mM MgCl2 with the indicated concentration of free Ca2+ for 1 h at room temperature.
site by substituting tyrosine with phenylalanine. The corresponding directed mutant protein, Y24F, was compared with GmCDPKb in terms of autophosphorylation and ability to catalyze transphosphorylation reactions using a variety of synthetic peptides as substrates. As expected, tyrosine autophosphorylation of wild type CDPKb was apparent in preparations purified from E. coli that had been induced with IPTG for 16 h. Interestingly, tyrosine autophosphorylation of the Y24F directed mutant was almost completely eliminated whereas there was little impact on
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autophosphorylation on threonine residues (Fig. 5A). These results suggest that Tyr-24 is not essential for kinase activity and that this residue may be a major site of tyrosine autophosphorylation. In order to assess the role of autophosphorylation on CDPK transphosphorylation activity, we purified wild type CDPK and the Y24F directed mutant from E. coli cultures immediately after addition of IPTG (time zero) or after 16 h of induction. As shown in Fig. 5A, the time zero protein samples had low autophosphorylation on threonine (and presumably serine) residues and were devoid of phosphotyrosine. The 16 h preparations had increased autophosphorylation on threonine (and presumably serine) but differed dramatically in phosphotyrosine content, specifically at the Tyr-24 position. The synthetic peptide substrates (sequences shown in Fig. 5B) correspond to the different motifs that are recognized and phosphorylated by CDPKs. These motifs are distinguished on the basis of positioning of both basic and hydrophobic residues. The NR6, SP42 and UGP1 peptides all conform to the classical motif with a basic residue (an arginine in each case) at the 3 position and hydrophobic residues at 5 and +4 [21,22]. All three synthetic peptides were phosphorylated but the SP42 peptide had the lowest activity. However, in all cases, activity was highest with the kinase prepared from 16-h cultures and the Y24F directed mutant had higher activity than the wild type enzyme (Fig. 5C). Generally similar results were obtained with peptide substrates conforming to the non-canonical motifs. For example, the ACA2–7 peptide, which conforms to the non-canonical ‘ACA2’ motif that is defined by basic residues at 6 and beyond (but not 3) and at +2 [17], was the best substrate tested and corresponding differences in activities among the preparations was very pronounced. Similarly, the other non-canonical motif tested was originally described for the Ser-460 regulatory phosphorylation site of tomato ACS (1-amino-cyclopropane-1-carboxylate synthase) 2, and as is distinguished by lack of both the basic residue at P-3 and the hydrophobic residue at P-5 that are hallmarks of the classic motif. Rather, with the ‘ACS’ motif, basic residues at 2 and +3 and +4 are key positive recognition elements. The ACSM +1 peptide was a good substrate for GmCDPKb and interestingly showed the largest relative effects of kinase autophosphorylation on activity. With wild type GmCDPKb, phosphorylation of the ACSM +1 peptide increased three-fold comparing the 0 and 16 h enzyme preparations, and the increase was even more dramatic (9.4-fold) for the Y24F directed mutant. Comparing the 16-h enzyme preparations with the ACSM +1 peptide, the Y24F directed mutant had almost twice the activity of wild type. Collectively, these results suggest that in general, autophosphorylation enhances CDPK peptide kinase activity as evidenced by increased activity with the 16-h hyperphosphorylated enzyme compared to the 0-h hypophosphorylated enzyme. However, at least some autophosphorylation sites are inhibitory to enzyme activity, and Tyr-24 may be one such site. Accordingly, when autophosphorylation at this position is prevented as with the Y24F directed mutant, the increase in activity with autophosphorylation is even greater.
4. Discussion The results of the present study document for the first time that CDPKs, which are classified as serine/threonine protein kinases, also autophosphorylate on tyrosine residues and thus are dualspecificity kinases. While much remains to be done, the results strongly suggest that autophosphorylation on tyrosine residues has functional significance. At least with GmCDPKb, autophosphorylation of Tyr-24 attenuates kinase activity, as demonstrated with synthetic peptide substrates in vitro (Fig. 5). It will be important in future studies to determine how dual specificity impacts the cellular functions of CDPKs in vivo, but these observations uncover the
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Fig. 5. Tyrosine-24 is a major site of GmCDPKb autophosphorylation and attenuates kinase activity (A) The native sequence or Y24F directed mutant of GmCDPKb were expressed in E. coli and after 0, 6 or 16 h of induction, recombinant proteins were purified and analyzed by immunoblotting or staining with CBB as indicated. Equivalent amounts of protein (1.5 lg per lane) were loaded in each lane. The anti-pThr immunoblot and CBB-stained blot were obtained with the kinase preparations used for activity measurements in panel C. The anti-Tyr immunoblot was from another preparation but all of the results are representative of several independent experiments. (B) Peptides used to monitor kinase activity in vitro. (C) Peptide kinase activity of GmCDPKb and the Y24F directed mutant (0.5 lg protein) prepared at time zero (hypophosphorylated) or after 16 h of induction (hyperphosphorylated). Values are the means ± S.E.M. from three determinations and are representative of several independent experiments.
potential for a new component of phosphotyrosine signaling in a large family of protein kinases that function to transduce Ca2+ signals in the regulation of growth and development and plant response to biotic and abiotic stresses. While Tyr-24 appears to be a major site of autophosphorylation of GmCDPKb, it is possible that other tyrosine residues are autophosphorylated as well. However, it is clear that prevention of phosphorylation of Tyr-24 plays a role in regulation of activity based on the results with the Y24F directed mutant (Fig. 5C). Tyrosine autophosphorylation can also be a high stoichiometry modification, based on the ability to remove CDPK protein from solution by immunoprecipitation with anti-phosphotyrosine antibodies (Fig. 3). Because a tyrosine residue at the start of the kinase domain (corresponding to Tyr-24 in GmCDPKb) is conserved among Arabidopsis CPK isoforms, the potential for similar regulation of other members of the family is likely and will be interesting to explore. Our studies also established that autophosphorylation on serine and threonine residues stimulate CDPK transphosphorylation activity. This is based on the observation that increased autophosphorylation of GmCDPKb that occurred as the protein was produced in E. coli was associated with increased peptide kinase activity (Fig. 5) and is consistent with earlier reports [12,13]. Our results suggest that autophosphorylation on serine and/or threonine residues stimulates while tyrosine autophosphorylation inhibits peptide kinase activity. It will be important in future studies to determine which specific phosphosites are activating kinase activity. Interestingly, the magnitude of the effect of autophosphorylation on kinase activity varied with the synthetic peptide used as substrate (Fig. 5C), suggesting that autophosphorylation may also influence target specificity. Given that the N-terminus plays an important role in substrate specificity [20] and sites of serine/ threonine autophosphorylation tend to cluster in the N-terminus ([10] and this study), an effect of autophosphorylation on CDPK activity and selectivity can be readily understood. In summary, an emerging concept is that several protein families classified as serine/threonine protein kinases are actually dual-specificity kinases capable of phosphorylation on serine, threonine and tyrosine residues. This is true for many, but likely not all,
members of the large receptor-like kinase family in Arabidopsis [15,19,23–26], and a similar situation is emerging with the CDPK family. Clearly, we do not have a complete understanding of the kinase domain motifs that confer dual specificity. Much remains to be done with both the receptor kinases and CDPKs in terms of tyrosine phosphorylation, but recognizing that it occurs is certainly the essential first step. With the CDPKs, we have established that tyrosine autophosphorylation has a functional role in vitro that may oppose the activation caused by autophosphorylation on serine/ threonine residues. Establishing which specific sites are of regulatory significance and whether they function in this manner in vivo are important objectives for future studies. Acknowledgements This work was supported in part by the National Science Foundation (IOS-1022177, MCB-0740211, MCB-1021363 and MCB-0920624) and the US Department of Agriculture (USDA)Agricultural Research Service (ARS). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2012. 09.040. References [1] Hepler, P.K. (2005) Calcium: a central regulator of plant growth and development. Plant Cell 17, 2142–2155. [2] Hrabak, E.M. et al. (2003) The Arabidopsis CDPK-SnRK superfamily of protein kinases. Plant Physiol. 132, 666–680. [3] Harmon, A.C., Putnam-Evans, C. and Cormier, M.J. (1987) A calcium-dependent but calmodulin-independent protein kinase from soybean. Plant Physiol. 83, 830–837. [4] Harmon, A.C., Gribskov, M. and Harper, J.F. (2000) CDPKs—a kinase for every Ca2+ signal? Trends Plant Sci. 5, 154. [5] Harmon, A.C., Gribskov, M., Gubrium, E. and Harper, J.F. (2001) The CDPK superfamily of protein kinases. New Phytol. 151, 175. [6] Johnson, L.N., Noble, M.E. and Owen, D.J. (1996) Active and inactive protein kinases: structural basis for regulation. Cell 85, 149–158.
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