Abstract
We have isolated a full-length cDNA clone (CpCDPK1) encoding a calcium-dependent protein kinase (CDPK) gene from zucchini (Cucurbita pepo L.). The predicted amino acid sequence of the cDNA shows a remarkably high degree of similarity to members of the CDPK gene family from Arabidopsis thaliana, especially AtCPK1 and AtCPK2.
Northern analysis of steady-state mRNA levels for CpCPK1 in etiolated and light-grown zucchini seedlings shows that the transcript is most abundant in etiolated hypocotyls and overall expression is suppressed by light.
As described for other members of the CDPK gene family from different species, the CpCPK1 clone has a putative N- terminal myristoylation sequence. In this study, site-directed mutagenesis and an in vitro coupled transcription/translation system were used to demonstrate that the protein encoded by this cDNA is specifically myristoylated by a plant N-myristoyl transferase. This is the first demonstration of myristoylation of a CDPK protein which may contribute to the mechanism by which this protein is localized to the plasma membrane.
Similar content being viewed by others
References
Abo-el Saad M, Wu R: A rice calcium-dependent protein kinase is induced by gibberellin. Plant Physiol 108: 77–793 (1995).
Allan AC, Fricker MD, Ward J, Beale M, Trewavas AJ: Caged ABA-induced calcium transients in guard cells of Commelina communis are dependent on previous growth temperature. Plant Cell 6: 1319–1328 (1994).
Botella JR, Arteca JM, Somadevilla M, Arteca RN: Calcium-dependent protein kinase gene expression in response to physical and chemical stimuli in mungbean (Vigna radiata). Plant Mol Biol 30: 1129–1137 (1996).
Breviario D, Morello L, Giani S: Molecular cloning of two novel rice cDNA sequences encoding putative calcium-dependent protein kinases. Plant Mol Biol 27: 953–967 (1995).
Bush DS: Calcium regulation in plant cells and its role in signaling. Annu Rev Plant Physiol Plant Mol Biol 46: 95–122 (1995).
Bush DS: Regulation of cytosolic calcium in plants. Plant Physiol 103: 7–13 (1993).
Casey PJ: Protein lipidation in cell signaling. Science 268: 221–225 (1995).
Chomczynski P, Sacci N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159 (1987).
Church GM, Gilbert W: Genomic sequencing. Proc Natl Acad Sci USA 81: 1991–1995 (1984).
Dizhoor AM, Ericsson LH, Johnson RS, Kumar S, Olshevskaya E, Zozulya S, Neubert TA, Stryer L, Hurley JB, Walsh KA: The NH 2 terminus of retinal recoverin is acylated by a small family of fatty acids. J Biol Chem 267: 16033–16036 (1992).
Dizhoor AM, Ray S, Kumar S, Niemi G, Spencer M, Brolley D, Walsh KA, Philipov PP, Hurley JB, Stryer L: Recoverin: a calcium sensitive activator of retinal rod guanylate cyclase. Science 251: 915–918 (1991).
Estruch JJ, Kadwell W, Merlin E, Crossland L: Cloning and characterization of a maize pollen-specific calcium-dependent calmodulin-independent protein kinase. Proc Natl Acad Sci USA 91: 8837–8841 (1994).
Franklin-Tong VE, Ryde JP, Read ND, Trewavas AJ, Franklin C: The self incompatibility response in Papaver rhoeas is mediated by free cytosolic calcium. Plant J 4: 163–177 (1993).
Furumoto T, Ogawa N, Hata S, Izui K: Plant calcium-dependent protein kinase-related kinases (CRKs) do not require calcium for their activities. FEBS Lett 396: 147–151 (1996).
Gilroy S, Bethke PC, Jones RL: Calcium homeostasis in plants. J Cell Sci 106: 453–462 (1993).
Grabski S, Xie XG, Holland JF, Schindler M: Lipids trigger changes in the elasticity of the cytoskeleton in plant cells: a cell optical displacement assay for live cell measurements. J Cell Biol 126: 713–726 (1994).
Hanks SK: Homology probing: identification of cDNA clones encoding members of the protein-serine kinase family. Proc Natl Acad Sci USA 84: 388–392 (1987).
Hanks SK, Quinn AM, Hunter T: The protein kinases family: conserved features and deduced phylogeny of the catalytic domains. Science 24: 42–52 (1988).
Harmon AC, McCurdy DW: Calcium-dependent protein kinase and its possible role in regulation of the cytoskeleton. Curr Top Plant Biochem Physiol 9: 119–128 (1990).
Harper JF, Huang J-F, Lloyd SJ: Genetic identification of an autoinhibitor in CDPK, a protein kinase with a calmodulin-like domain. Biochemistry 33: 7267–7277 (1994).
Harper JF, Binder BM, Sussman MR: Calcium and lipid regulation of an Arabidopsis protein kinase expressed in Escherichia coli. Biochemistry 32: 3282–3290 (1993).
Harper JF, Sussman MR, Schaller GE, Putnam-Evans C, Charbonneau H, Harmon AC: A calcium-dependent protein kinase with a regulatory domain similar to calmodulin. Science 252: 951–954 (1991).
Heuckeroth RO, Towler DA, Adams SP, Glaser L, Gordon JI: 11-(Ethylthio)undecanoic acid. A myristic acid analogue of altered hydrophobicity which is functional for peptid N-myristoylation with wheat germ and yeast acyltransferase. J Biol Chem 263: 2127–2133 (1988).
Hong Y, Takano M, Liu CM, Gasch A, Chye ML, Chua N-H: Expression of three members of the calcium-dependent protein kinase gene family in Arabidopsis thaliana. PlantMol Biol 30: 1259–1275 (1996).
Hrabak EM, Dickmann LJ, Satterlee JS, Sussman MR: Characterization of eight new members of the calmodulin-like domain protein kinase gene family from Arabidopsis thaliana. Plant Mol Biol 31: 405–412 (1996).
Huang J-F, Teyton L, Harper JF: Activation of a Ca2+-dependent protein kinase involves intramolecular binding of a calmodulin-like regulatory domain. Biochemistry 35: 13222–13230 (1996).
Johnson DR, Bhatnager RS, Knoll LJ, Gordon JI: Genetic and biochemical studies of protein N-myristoylation. Annu Rev Biochem 63: 869–914 (1994).
Kawasaki T, Hayashida N, Shinozaki K, Shimada H: The gene encoding a calcium-dependent protein kinase located near the sbe1 gene encoding starch branching enzyme I is specifically expressed in developing rice seeds. Gene 129: 183–189 (1993).
Kluais E, Polya GM: Localization, solubilization, and characterization of plant membrane-associated protein kinases. Plant Physiol 88: 164–171 (1988).
Lindzen E, Choi JH: A carrot cDNA encoding an atypical protein kinase homologous to plant calcium-dependent protein kinases. Plant Mol Biol 28: 785–797 (1995).
Malho R, Read ND, Pais MS, Trewavas AJ: Role of cytosolic free calcium in the reorientation of pollen tube growth. Plant J 5: 331–341 (1994).
Mochly-Rosen D: Localization of protein kinases by anchoring proteins: a theme in signal transduction. Science 268: 247–251 (1995).
Putnam-Evans CL, Harmon AC, Cormier MJ: Purification and characterization of a novel calcium-dependent protein kinase from soybean. Biochemistry 29: 2488–2495 (1990).
Putnam-Evans CL, Harmon AC, Palevitz BA, Fechheimer M, Cormier MJ: Calcium-dependent protein kinase is localized with F-actin in plant cells. Cell Motil Cytoskel 12: 12–22 (1989).
Risinger MA, Korsgren C, Cohen CM: Role of N-myristylation in targeting of band 4.2 (pallidin) in nonery-throid cells. Exp Cell Res 229: 421–431 (1996).
Roberts DM, Harmon AC: Calcium-modulated proteins: targets of intracellular calcium signals in higher plants. Annu Rev Plant Physiol Plant Mo] Biol 43: 375–414 (1992).
Rogers SO, Bendich AJ: Extraction of DNA from milligram amounts of fresh, herbarium, and mummified plant tissues. Plant Mol Biol 5: 69–76 (1985).
Rommens CMT, Salmeron JM, Baulcombe DC, Staskawicz BJ: Use of a gene expression system based in Potato Virus X to rapidly identify and characterize a tomato pto homolog that controls fenthion sensitivity. Plant Cell 7: 249–257 (1995).
Sambrook J, Maniatis E, Fritsch T: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988).
Schaller GE, Sussman MR: Phosphorylation of the plasma-membrane H +-ATPase of oat roots by calcium-stimulated protein kinases. Planta 173: 509–518 (1988).
Schultz AM, Oroszlan S: In vivo modification of retroviral gag gene-encoded polyproteins by myristic acid. J Virol 46: 355–361 (1993).
Shacklock P, Read ND, Trewavas AJ: Cytosolic free calcium mediates red light-induced photomorphogenesis. Nature 358: 753–755 (1992).
Stephenson M, Ryals PE, Thompson Jr GA: Fatty acid acylated proteins of the halotolerant alga, Dunaliella salina. Plant Physiol 90: 549–552 (1989).
Stone JM, Walker JC: Plant protein kinase families and signal transduction. Plant Physiol 108: 451–457 (1995).
Suen K-L, Choi JH: Isolation and sequence analysis of a cDNA clone for a carrot calcium-dependent protein kinase: homology to calcium/calmodulin-dependent protein kinases and to calmodulin. Plant Mol Biol 17: 581–590 (1991).
Tanaka T, Ames JB, Harvey TS, Stryer L, Ikura M: Sequestration of the membrane-targeting myristoyl group of recoverin in the calcium-free state. Nature 376: 444–447 (1995).
Towler DA, Adams SP, Eubanks SR, Towery DS, Jackson-Machelski E, Glaser L, Gordon JI: Myristoyl CoA:Protein N-myristoyltransferase activities from rat liver and yeast possess overlapping yet distinct peptide substrate specificities. J Biol Chem 262: 1784–1790 (1988).
Towler DA, Adams SP, Eubanks SR, Towery DS, Jackson-Machelski E, Glaser L, Gordon JI: Purification and characterization of yeast myristoyl CoA:Protein N-myristoyltransferase. Proc Natl Acad Sci USA 84: 2708–2712 (1987).
Trewavas A, Knight M: Mechanical signaling, calcium and plant form. Plant Mol Biol 26: 1329–1341 (1994).
Urao T, Katagiri T, Mizoguchi T, Yamaguchi-Shinozaki K, Hayashida N, Shinozaki K: Two genes that encode Ca+2-dependent protein kinases are induced by drought and high-salt stresses in Arabidopsis thaliana. Mol Gen Genet 244: 331–340 (1994).
Walker F, deBlaquiere J, Burgess AW: Translocation of pp60 c-src from the plasma membrane to the cytosol after stimulation by platelet-derived growth factor. J Biol Chem 268: 19552–19558 (1993).
Weinberg RA, McWherter CA, Freeman SK, Wood DC, Gordon JI, Lee SC: Genetic studies reveal that myristoyl-CoA: Protein N-myristoyltransferase is an essential enzyme in Candida albicans. Mol Microbiol 16: 241–250 (1995).
Verhey SD, Gaiser JC, Lomax TL: Protein kinases in zucchini: characterization of calcium-requiring plasma membrane kinases. Plant Physiol 103: 413–419 (1993).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Ellard-Ivey, M., Hopkins, R.B., White, T. et al. Cloning, expression and N-terminal myristoylation of CpCPK1, a calcium-dependent protein kinase from zucchini (Cucurbita pepo L.). Plant Mol Biol 39, 199–208 (1999). https://doi.org/10.1023/A:1006125918023
Issue Date:
DOI: https://doi.org/10.1023/A:1006125918023