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Purification and characterization of a calcium-dependent protein kinase from beetroot plasma membranes

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Abstract

Several calcium-dependent protein kinases (CDPKs) are located in plant plasma membranes where they phosphorylate enzymes and transporters, like the H+-ATPase and water channels, thereby regulating their activities. In order to determine which kinases phosphorylate the H+-ATPase, a calcium-dependent kinase was purified from beetroot (Beta vulgaris L.) plasma membranes by anion-exchange chromatography, centrifugation in glycerol gradients and hydrophobic interaction chromatography. The kinetic parameters of this kinase were determined (V max: 3.5 μmol mg−1 min−1, K m for ATP: 67 μM, K m for syntide 2: 15 μM). The kinase showed an optimum pH of 6.8 and a marked dependence on low-micromolar Ca2+ concentrations (K d : 0.77 μM). During the purification procedure, a 63-kDa protein with an isoelectric point of 4.7 was enriched. However, this protein was shown not to be a kinase by mass spectrometry. Kinase activity gels showed that a 50-kDa protein could be responsible for most of the activity in purified kinase preparations. This protein was confirmed to be a CDPK by mass spectrometry, possibly the red beet ortholog of rice CDPK2 and Arabidopsis thaliana CPK9, both found associated with membranes. This kinase was able to phosphorylate purified H+-ATPase in a Ca2+-dependent manner.

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Abbreviations

CDPK:

Calcium-dependent protein kinase

CID:

Collision-induced dissociation

ESI:

Electrospray ionization

GFP:

Green fluorescent protein

HEDTA:

N-hydroxyethylethylenediamine-triacetic acid

K d :

Dissociation constant

MALDI-ToF:

Matrix-assisted laser desorption ionization-time of flight

MBP:

Myelin basic protein

MS:

Mass spectrometry

PDI:

Protein disulfide isomerase

PMF:

Peptide-mass fingerprinting

PMSF:

Phenylmethylsulfonyl fluoride

Q-ToF:

Quadrupole-time of flight

References

  • Alexandersson E, Saalbach G, Larsson C, Kjellbom P (2004) Arabidopsis plasma membrane proteomics identifies components of transport, signal transduction and membrane trafficking. Plant Cell Physiol 45:1543–1556

    Article  PubMed  CAS  Google Scholar 

  • Anthon GE, Spanswick RM (1986) Purification and properties of the H+-translocating ATPase from the plasma membrane of tomato roots. Plant Physiol 81:1080–1085

    Article  PubMed  CAS  Google Scholar 

  • Asano T, Tanaka N, Yang G, Hayashi N, Komatsu S (2005) Genome-wide identification of the rice calcium-dependent protein kinase and its closely related kinase gene families: Comprehensive analysis of the CDPKs gene family in rice. Plant Cell Physiol 46:356–366

    Article  PubMed  CAS  Google Scholar 

  • Baizabal-Aguirre VM, González de la Vara LE (1994) Characterization of the protein kinase activity in beet root plasma membranes. Physiol Plant 91:147–154

    Article  CAS  Google Scholar 

  • Baizabal-Aguirre VM, González de la Vara LE (1997) Purification and characterization of a calcium-regulated protein kinase from beet root (Beta vulgaris) plasma membranes. Physiol Plant 99:135–143

    Article  CAS  Google Scholar 

  • Buchanan BB, Balmer Y (2005) Redox regulation: A broadening horizon. Annu Rev Plant Biol 56:187–220

    Article  PubMed  CAS  Google Scholar 

  • Camoni L, Fullone MR, Marra M, Aducci P (1998) The plasma membrane H+-ATPase from maize roots is phosphorylated in the C-terminal domain by a calcium-dependent protein kinase. Physiol Plant 104:549–555

    Article  CAS  Google Scholar 

  • Chehab EW, Patharkar OR, Hegeman AD, Taybi T, Cushman JC (2004) Autophosphorylation and subcellular localization dynamics of a salt- and water deficit-induced calcium-dependent protein kinase from ice plant. Plant Physiol 135:1430–1446

    Article  PubMed  CAS  Google Scholar 

  • Dammann C, Ichida A, Hong B, Romanowski SM, Hrabak EM, Harmon AC, Pickard BG, Harper JF (2003) Subcellular targeting of nine calcium-dependent kinase isoforms from Arabidopsis. Plant Physiol 132:1840–1848

    Article  PubMed  CAS  Google Scholar 

  • Desbrosses G, Stelling J, Renaudin JP (1998) Dephosphorylation activates the purified plant plasma membrane H+-ATPase-possible function of phosphothreonine residues in a mechanism not involving the regulatory C-terminal domain of the enzyme. Eur J Biochem 251:496–503

    Article  PubMed  CAS  Google Scholar 

  • Douglas P, Moorhead G, Hong Y, Morrice N, MacKintosh C (1998) Purification of a nitrate reductase kinase from Spinacea oleracea leaves, and its identification as a calmodulin-domain protein kinase. Planta 206:435–442

    Article  PubMed  CAS  Google Scholar 

  • Frattini M, Morello L, Breviario D (1999) Rice calcium-dependent protein kinase isoforms OsCDPK2 and OsCDPK11 show different responses to light and different expression patterns during seed development. Plant Mol Biol 41:753–764

    Article  PubMed  CAS  Google Scholar 

  • Fuglsang AT, Visconti S, Drumm K, Jahn T, Stensballe A, Mattei B, Jensen ON, Aducci P, Palmgren MG (1999) Binding of 14-3-3 protein to the plasma membrane H+-ATPase AHA2 involves the three C-terminal residues Tyr946-Thr-Val and requires phosphorylation of Thr947. J Biol Chem 274:36774–36780

    Article  PubMed  CAS  Google Scholar 

  • Harmon AC, Gribskov M, Harper JF (2000) CDPKs - a kinase for every Ca2+ signal? Trends Plant Sci 5:154–159

    Article  PubMed  CAS  Google Scholar 

  • Harper JE, Breton G, Harmon AC (2004) Decoding Ca2+ signals through plant protein kinases. Annu Rev Plant Biol 55:263–288

    Article  PubMed  CAS  Google Scholar 

  • Henkeshoven J, Dernick R (1985) Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism for silver staining. Electrophoresis 6:103-112

    Article  Google Scholar 

  • Hrabak EM (2000) Calcium-dependent protein kinases and their relatives. Adv Bot Res 32:185–223

    CAS  Google Scholar 

  • Hrabak EM, Chan CWM, Gribskov M, Harper JF, Choi JH, Halford N, Kudla J, Luan S, Nimmo HG, Sussman MR, Thomas M, Walker-Simmons K, Zhu J-K, Harmon AC (2003) The Arabidopsis CDPK-SnRK superfamily of protein kinases. Plant Physiol 132:666–680

    Article  PubMed  CAS  Google Scholar 

  • Iwata Y, Kuriyama M, Nakakita M, Kojima H, Ohto M-A, Nakamura K (1998) Characterization of a calcium-dependent protein kinase of tobacco leaves that is associated with the plasma membrane and is inducible by sucrose. Plant Cell Physiol 39:1176–1183

    PubMed  CAS  Google Scholar 

  • Johansson I, Larsson C, Ek B, Kjellbom P (1996) The major integral proteins of spinach leaf plasma membranes are putative aquaporins and are phosphorylated in response to Ca2+ and apoplastic water potential. Plant Cell 8:1181–1191

    Article  PubMed  CAS  Google Scholar 

  • Kinoshita T, Shimazaki K (1999) Blue light activates the plasma membrane H+-ATPase by phosphorylation of the C-terminus in stomatal guard cells. EMBO J 18:5548–5558

    Article  PubMed  CAS  Google Scholar 

  • Kinoshita T, Shimazaki K (2001) Analysis of the phosphorylation level in guard-cell plasma membrane H+-ATPase in response to fusicoccin. Plant Cell Physiol 42:424–432

    Article  PubMed  CAS  Google Scholar 

  • Lee J-Y, Yoo B-C, Harmon AC (1998) Kinetic and calcium-binding properties of three calcium-dependent protein kinase isoenzymes from soybean. Biochemistry 37:6801–6809

    Article  PubMed  CAS  Google Scholar 

  • Levitan A, Trebitsh T, Kiss V, Pereg Y, Dangoor I, Danon A (2005) Dual targeting of the protein disulfide isomerase RB60 to the chloroplast and the endoplasmic reticulum. Proc Natl Acad Sci USA 102:6225–6230

    Article  PubMed  CAS  Google Scholar 

  • Lino B, Baizabal-Aguirre VM, González de la Vara LE (1998) The plasma-membrane H+-ATPase from beet root is inhibited by a calcium-dependent phosphorylation. Planta 204:352-359

    Article  PubMed  CAS  Google Scholar 

  • Ludwig A, Romeis T, Jones JDG (2004) CDPK-mediated signalling pathways:specificity and cross-talk. J Exp Bot 55:181–188

    Article  PubMed  CAS  Google Scholar 

  • Marmagne A, Rouet M-A, Ferro M, Rolland N, Alcon C, Joyard J, Garin J, Barbier-Brygoo H, Ephritikhine G (2004) Identification of new intrinsic proteins in Arabidopsis plasma membrane proteome. Mol Cell Proteomics 3:675–691

    Article  PubMed  CAS  Google Scholar 

  • Martín ML, Busconi L (2000) Membrane localization of a rice calcium-dependent protein kinase (CDPK) is mediated by myristoylation and palmitoylation. Plant J 24:429–435

    Article  PubMed  Google Scholar 

  • Nühse TS, Stensballe A, Jensen ON, Peck SC (2003) Large-scale analysis of in vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry. Mol Cell Proteomics 2:1234–1243

    Article  PubMed  CAS  Google Scholar 

  • Palmer T (1981) Understanding enzymes. Ellis Horwood ltd. Publishers, Chichester, p 172

    Google Scholar 

  • Palmgren MG (2001) Plant plasma membrane H+-ATPases:Powerhouses for nutrient uptake. Ann Rev Plant Physiol Plant Mol Biol 52:817–845

    Article  CAS  Google Scholar 

  • Putnam-Evans CL, Harmon AC, Cormier MJ (1990) Purification and characterization of a novel calcium-dependent protein kinase from soybean. Biochemistry 29:2488–2495

    Article  PubMed  CAS  Google Scholar 

  • Rutschmann F, Stalder U, Piotrowski M, Oecking C, Schaller A (2002) LeCPK1, a calcium-dependent protein kinase from tomato, plasma membrane targeting and biochemical characterization. Plant Physiol 129:156–168

    Article  PubMed  CAS  Google Scholar 

  • Satterlee JS, Sussman MR (1998) Unusual membrane-associated protein kinases in higher plants. J Membr Biol 164:205–213

    Article  PubMed  CAS  Google Scholar 

  • Schägger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368-379

    Article  PubMed  Google Scholar 

  • Schaller GE, Sussman MR (1988) Phosphorylation of the plasma membrane H+-ATPase of oat roots by a calcium stimulated protein kinase. Planta 173:509–518

    Article  CAS  Google Scholar 

  • Schaller GE, Harmon AC, Sussman MR (1992) Characterization of a calcium- and lipid-dependent protein kinase associated with the plasma membrane of oat. Biochemistry 31:1721–1727

    Article  PubMed  CAS  Google Scholar 

  • Schoenmakers TJM, Visser GJ, Flik G, Theuvenet APR (1992) CHELATOR: An improved method for computing metal ion concentrations in physiological solutions. Biotechniques 12:870–879

    PubMed  CAS  Google Scholar 

  • Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 68:850–858

    Article  PubMed  CAS  Google Scholar 

  • Shevchenko A, Sunyaev S, Loboda A, Shevchenko A, Bork P, Ens W, Standing KG (2001) Charting the proteomes of organisms with unsequenced genomes by MALDI-quadrupole time-of-flight mass spectrometry and BLAST homology searching. Anal Chem 73:1917–1926

    Article  PubMed  CAS  Google Scholar 

  • Stoschek CM (1990) Increased uniformity in the response of the coomassie blue G protein assay to different proteins. Anal Biochem 184:111–116

    Article  Google Scholar 

  • Szczegielniak J, Klimecka M, Liwosz A, Ciesielski A, Kaczanowski S, Dobrowolska G, Harmon AC, Muszynska G (2005) A wound-responsive and phospholipid-regulated maize calcium-dependent protein kinase. Plant Physiol 139:1970–1983

    Article  PubMed  CAS  Google Scholar 

  • Vera-Estrella R, Higgins VJ, Blumwald E (1994) Plant defense response to fungal pathogens. (Activation of host-plasma membrane H+-ATPase by elicitor-induced enzyme dephosphorylation). Plant Physiol 104:209–215

    PubMed  CAS  Google Scholar 

  • Wooten MW (2002) In-gel kinase assay as a method to identify kinase substrates. Science’s STKE http://www.stke.org/cgi/content/full/sigtrans;2002/153/pl15

Download references

Acknowledgments

We thank Dr. Gregory W. Kilby from Agilent Technologies and Dr. Tonya Pekar and Dr. Larry Burchfield from Thermo Electron Corporation for performing the MS analyses of the active kinases. We also thank Dr. June Simpson for kindly reviewing this manuscript. This work was supported in part by Conacyt, Mexico (grant no. 31802N). Conacyt also granted a postdoctoral fellowship to MTCR.

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Authors and Affiliations

  1. Departamento de Biotecnología y Bioquímica, Unidad Irapuato, Centro de Investigación y de Estudios Avanzados del IPN, Apartado postal 629, 36500, Irapuato Gto, México

    Bárbara Lino & Luis E. González de la Vara

  2. Departamento de Ingeniería genética, Unidad Irapuato, Centro de Investigación y de Estudios Avanzados del IPN, Apartado postal 629, 36500, Irapuato Gto, México

    M. Teresa Carrillo-Rayas

  3. Unidad Irapuato, Centro de Investigación y de Estudios Avanzados del IPN, Apartado postal 629, 36500, Irapuato Gto, México

    Alicia Chagolla

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  1. Bárbara Lino
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  2. M. Teresa Carrillo-Rayas
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  3. Alicia Chagolla
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  4. Luis E. González de la Vara
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Correspondence to Luis E. González de la Vara.

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Lino, B., Carrillo-Rayas, M.T., Chagolla, A. et al. Purification and characterization of a calcium-dependent protein kinase from beetroot plasma membranes. Planta 225, 255–268 (2006). https://doi.org/10.1007/s00425-006-0343-8

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  • DOI: https://doi.org/10.1007/s00425-006-0343-8

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