Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
권호 표지
DOI QR코드

DOI QR Code

Phosphorylation Properties of Recombinant OsCPK11, a Calcium-dependent Protein Kinase from Rice

벼의 칼슘-의존적 단백질 카이네즈인 재조합 OsCPK11의 인산화 특성

  • Received : 2016.09.22
  • Accepted : 2017.11.13
  • Published : 2017.12.30
Download PDF
⟨ Previous Next ⟩

Abstract

In plants, calcium ($Ca^{2+}$)-dependent protein kinases (CDPKs) are important sensors of $Ca^{2+}$ signals. Previous research demonstrated the expression of the OsCPK11 gene in various tissues at the transcription level, but its developmental and biochemical functions at the protein level were not determined. This study was aimed to identify biochemical characteristics of OsCPK11. GST- OsCPK11 was expressed in E. coli and used for an in vitro kinase assay. Biochemical analyses identified OsCPK11 as a CDPK. OsCPK11 autophosphorylated itself and transphosphorylated histone III-s and MBP as substrates in the presence of $Ca^{2+}$. The activity of the recombinant OsCPK11 was influenced by $Mg^{2+}$, with optimum activity detected at pH 7.0-7.5. OsCPK11 activity was not affected by $Mg^{2+}$, $Mn^{2+}$, or $Na^+$ in the presence of a high level of $Ca^{2+}$. Autophosphorylation of OsCPK11 decreased $Ca^{2+}$ sensitivity of OsCPK11. An anti-OsCPK11 rabbit antibody recognized 95.5 kD of GST-OsCPK11, as shown by an immunoblot analysis. These results shed light on the function of OsCPK11 in $Ca^{2+}$-mediated signaling in rice.

식물에서, 칼슘-의존적 단백질 카이네즈(CDPKs)는 $Ca^{2+}$ 신호전달에서 중요한 $Ca^{2+}$ 수용체이다. 벼(Oryza sativa L.)의 CDPKs인 3개의 OsCPKs는 생물정보에 대한 분석이 이루어졌으나, OsCPK11 유전자는 연구가 완전히 수행되지 않았다. 다양한 조직에서 OsCPK11 유전자가 전사수준에서 발현한다는 것은 알려져 있으나, 단백질 수준에서 발현과 생화학적인 특징은 잘 알려져 있지 않다. 이 연구는 OsCPK11의 몇 가지 생화학적 특징을 알아보기 위해 이루어졌다. 먼저 in vitro에서 E. coli를 이용하여 GST-OsCPK11를 발현시키고, 카이네즈 활성 측정과 칼슘-의존적 단백질 카이네즈로서 OsCPK11의 생화학적 분석도 수행하였다. OsCPK11은 스스로 자가인산화하며, $Ca^{2+}$의 존재 하에서 기질로서 histone III-s와 MBP로 인산기 전달 작용을 수행한다. 재조합 OsCPK11의 활성은 $Mg^{2+}$에 의해 영향을 받으며, pH 7.0-7.5에서 최적의 활성을 보인다. 또한 OsCPK11의 활성은 높은 수준의 $Ca^{2+}$가 존재하는 조건에서는 $Mg^{2+}$, $Mn^{2+}$, $Na^+$의 영향을 받지 않는다. 또한 OsCPK11의 자가인산화는 OsCPK11의 $Ca^{2+}$ 민감도를 감소시키는 것으로 밝혀졌다. 마지막으로, OsCPK11의 N-말단 다양화 지역으로 토끼 항체를 만들었고, immunoblot을 기초로 polyclonal antibody는 95.5 kD의 GST-OsCPK11를 인식하는 것으로 나타났다. 이 결과는 벼의 $Ca^{2+}$ 매개 신호전달에서 OsCPK11의 기능을 더 잘 이해하는데 도움을 줄 것이며, 심화 연구를 위해 다양한 OsCPKs의 단백질 정보를 결정하는 것이 필요할 것이다.

Keywords

References

  1. Anil, V. S. and Rao, K. S. 2000. Calcium-mediated signaling during sandalwood somatic embryogenesis. Role for exogenous calcium as second messenger. Plant Physiol. 123, 1301-1311. https://doi.org/10.1104/pp.123.4.1301
  2. Anil, V. S. and Rao, K. S. 2001. Purification and characterization of a $Ca^{2+}$-dependent protein kinase from sandalwood (Santalum album L.): evidence for $Ca^{2+}$-induced conformational changes. Phytochemistry 58, 203-212. https://doi.org/10.1016/S0031-9422(01)00231-X
  3. Arabidopsis Genome Initiative 2000. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408, 796-815. https://doi.org/10.1038/35048692
  4. Asano, T., Kunieda, N., Omura, Y., Ibe, H., Kawasaki, T., Takano, M., Sato, M., Furuhashi, H., Mujin, T., Takaiwa, F., Wu, C., Tada, Y., Satozawa, T., Sakamoto, M. and Shimada, H. 2002. Rice SPK, a calmodulin-like domain protein kinase, is required for storage product accumulation during seed development: phosphorylation of sucrose synthase is a possible factor. Plant Cell 14, 619-628. https://doi.org/10.1105/tpc.010454
  5. Asano, T., Tanaka, N., Yang, G., Hayashi, N. and Komatsu, S. 2005. Genome-wide identification of the rice calciumdependent protein kinase and its closely related kinase gene families: Comprehensive analysis of the CDPKs gene family in rice. Plant Cell Physiol. 46, 356-366. https://doi.org/10.1093/pcp/pci035
  6. Chaudhuri, S., Seal, A. and Dasgupta, M. 1999. Autophosphorylation-dependent activation of a calcium-dependent protein kinase from ground nut. Plant Physiol. 120, 859-866. https://doi.org/10.1104/pp.120.3.859
  7. Cheng, S. H., Willmann, M. R., Chen, H. C. and Sheen, J. 2002. Calcium signaling through protein kinases. The Arabidopsis calcium dependent protein kinase gene family. Plant Physiol. 129, 469-485. https://doi.org/10.1104/pp.005645
  8. Cho, I. S. 2010 Biochemical characterization of the recombinant OsCPK11, a calcium-dependent protein kinase from rice. Thesis for master's degree. Korea National University of Education, Chungbuk, Korea.
  9. Dasgupta, M. 1994. Characterization of a calcium-dependent protein kinase from Arachis hypogea (groundnut) seeds. Plant Physiol. 104, 961-969. https://doi.org/10.1104/pp.104.3.961
  10. Dixit, A. K. and Jayabaskaran, C. 2012. Molecular cloning, soluble expression and characterization of autophosphorylation in recombinant calcium dependent protein kinase 1(CaCDPK1) from Cicer arietinum. Appl. Microbiol. Biotechnol. 97, 3429-3439.
  11. Evans, N H., McAinsh, M. R. and Hetherington, A. M. 2001. Calcium oscillations in higher plants. Curr. Opin. Plant Biol. 4, 415-420. https://doi.org/10.1016/S1369-5266(00)00194-1
  12. Ganguly, S. and Singh, M. 1999. Purification and characterization of a protein phosphatase from winged bean. Phytochemistry 52, 239-246. https://doi.org/10.1016/S0031-9422(98)00473-7
  13. Guo, Y. L. and Roux, S. J. 1990. Partial purification and characterization of a $Ca^{2+}$-dependent protein kinase from the green alga, Dunaliella salina. Plant Physiol. 94, 143-150. https://doi.org/10.1104/pp.94.1.143
  14. Hanks, S. K. and Hunter, T. 1995. The eukaryotic protein kinase super family: kinase (catalytic) domain structure and classification. FASEB J. 9, 546-596.
  15. Harmon, A. C., Yoo, B. C. and McCaffery, C. 1994. Pseudosubstrate inhibition of CDPK, a protein kinase with a calmodulin-like domain. Biochemistry 33, 7278-7287. https://doi.org/10.1021/bi00189a032
  16. Harmon, A. C., Gribskov, M. and Harper, J. F. 2000. CDPKs: a kinase for every $Ca^{2+}$ signal? Trends Plant Sci. 5, 154-159. https://doi.org/10.1016/S1360-1385(00)01577-6
  17. Harper, J. F., Sussman, M. R., Schaller, G. E., Putnam-Evans, C., Charbonneau, H. and Harmon, A. C. 1991. A calciumdependent protein kinase with a regulatory domain similar to calmodulin. Science 252, 951-954. https://doi.org/10.1126/science.1852075
  18. Harper, J. F., Breton, G. and Harmon, A. 2004. Decoding $Ca^{2+}$ signals through plant protein kinases. Annu. Rev. Plant Physiol. Plant Mol. Biol. 55, 263-288. https://doi.org/10.1146/annurev.arplant.55.031903.141627
  19. Hong, Y., Takano, M., Liu, C. M., Gasch, A., Chye, M. L. and Chua, N. H. 1996. Expression of three members of the calcium-dependent protein kinase gene family in Arabidopsis thaliana. Plant Mol. Biol. 30, 1259-1275. https://doi.org/10.1007/BF00019557
  20. Hrabak, E. M., Chan, C. W., Gribskov, M., Harper, J. F., Choi, J. H., Halford, N., Kudla, J., Luan, S., Nimmo, H. G., Sussman, M. R., Thomas, M., Walker-Simmons, K., Zhu, J. K. and Harmon, A. C. 2003. The Arabidopsis CDPK-SnRK superfamily of protein kinases. Plant Physiol. 132, 666-680. https://doi.org/10.1104/pp.102.011999
  21. Huang, Q. S., Wang, H. Y., Gao, P., Wang, G. Y. and Xia, G. X. 2008. Cloning and characterization of a calcium dependent protein kinase gene associated with cotton fiber development. Plant Cell Rep. 27, 1869-875. https://doi.org/10.1007/s00299-008-0603-0
  22. Johnson, D. R., Bhatnagar, R. S., Knoll, L. J. and Gordon, J. I. 1994. Genetic and biochemical studies of protein Nmyristoylation. Annu. Rev. Biochem. 63, 869-914. https://doi.org/10.1146/annurev.bi.63.070194.004253
  23. Klimecka, M. and Muszynska, G. 2007. Structure and functions of plant calcium-dependent protein kinases. Acta Biochim. Pol. 54, 219-233.
  24. Komatsu, S., Yang, G., Khan, M., Onodera, H., Toki, S. and Yamaguchi, M. 2007. Over-expression of calcium-dependent protein kinase 13 and calreticulin interacting protein 1 confers cold tolerance on rice plants. Mol. Genet. Genomics 277, 713-723. https://doi.org/10.1007/s00438-007-0220-6
  25. Lachaud, C., Prigent, E., Thuleau, P., Grat, S., Da Silva, D., Brière, C., Mazars, C. and Cotelle, V. 2013. 14-3-3-regulated $Ca^{2+}$-dependent protein kinase CPK3 is required for sphingolipid-induced cell death in Arabidopsis. Cell Death Differ. 20, 209-217. https://doi.org/10.1038/cdd.2012.114
  26. Lee, J. Y., Yoo, B. C. and Harmon, A. C. 1998. Kinetic and calcium-binding properties of three calcium-dependent protein kinase isoenzymes from soybean. Biochemistry 37, 6801-6809. https://doi.org/10.1021/bi980062q
  27. Lee, S. H. 2009. Functional characterization of OsCPK11, a calcium-dependent protein kinase gene from rice and its cDNA cloning. Thesis for master's degree. Korea National University of Education, Chungbuk, Korea.
  28. Liese, A and Romeis, T. 2013. Biochemical regulation of in vivo function of plant calcium-dependent protein kinases (CDPK). Biochim. Biophys. Acta. 1833, 1582-1589. https://doi.org/10.1016/j.bbamcr.2012.10.024
  29. Martin, M. L. and Busconi, L. 2000. Membrane localization of a rice calcium-dependent protein kinase (CDPK) is mediated by myristoylation and palmitoylation. Plant J. 24, 429-435. https://doi.org/10.1046/j.1365-313x.2000.00889.x
  30. Millaway, R. and Wiersholm, L. 1979. Calcium and metabolic disorders. Commun. Soil Sci. Plant Anal. 10, 1-28. https://doi.org/10.1080/00103627909366875
  31. Reddy, V. S. and Reddy, A. S. 2004. Proteomics of calcium-signaling components in plants. Phytochemistry 65, 1745-1776. https://doi.org/10.1016/j.phytochem.2004.04.033
  32. Ritchie, S. and Gilroy, S. 1998. Calcium-dependent protein phosphorylation may mediate the gibberellic acid response in barley aleurone. Plant Physiol. 116, 765-776. https://doi.org/10.1104/pp.116.2.765
  33. Roberts, D. M. and Harmon, A. C. 1992. Calcium-modulated proteins: targets of intracellular calcium signals in higher plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 43, 375-414. https://doi.org/10.1146/annurev.pp.43.060192.002111
  34. Romeis, T., Piedras, P. and Jones, J. D. 2000. Resistance gene-dependent activation of a calcium-dependent protein kinase in the plant defense response. Plant Cell 12, 803-815. https://doi.org/10.1105/tpc.12.5.803
  35. Romeis, T., Ludwig, A. A., Martin, R. and Jones, J. D. 2001. Calcium-dependent protein kinases play an essential role in a plant defense response. EMBO J. 20, 5556-5567. https://doi.org/10.1093/emboj/20.20.5556
  36. Rudd, J. J. and Franklin-Tong, V. E. 2001. Unravelling response-specificity in $Ca^{2+}$ signalling pathways in plant cells. New Phytol. 151,7-33. https://doi.org/10.1046/j.1469-8137.2001.00173.x
  37. Saha, P. and Singh, M. 1995. Characterization of a winged bean (Psophocarpus tetragonolobus) protein-kinase with calmodulin-like domain regulation by autophosphorylation. Biochem. J. 305, 205-210. https://doi.org/10.1042/bj3050205
  38. Saijo, Y., Hata, S., Kyozuka, J., Shimamoto, K. and Izui, K. 2000. Over-expression of a single $Ca^{2+}$-dependent protein kinase confers both cold and salt/drought tolerance on rice plants. Plant J. 23, 319-327. https://doi.org/10.1046/j.1365-313x.2000.00787.x
  39. Sambrook, J. and Russell, D. W. 2001. Molecular cloning: a laboratory manual (3rd ed.). NY: CSHL press.
  40. Sanders, D., Brownlee, C., and Harper, J. F. 1999. Communicating with calcium. Plant Cell 11, 691-706. https://doi.org/10.1105/tpc.11.4.691
  41. Sanders, D., Pelloux, J., Brownlee, C. and Harper, J. F. 2002. Calcium at the cross roads of signaling. Plant Cell 14, S401-S417. https://doi.org/10.1105/tpc.002899
  42. See, Y. and Jackowski, G. 1989. Protein structure: A practical approach (Creighton, T. E. ed.). Oxford: IRL Press.
  43. Szczegielniak, J., Klimecka, M., Liwosz, A., Ciesielski, A., Kaczanowski, S., Dobrowolska, G., Hormon, A. C. and Muszynka, G. 2005. A wound-responsive and phospholipid-regulated maize calcium-dependent protein kinase. Plant Physiol. 139, 970-983.
  44. Urao, T., Katagiri, T., Mizoguchi, T., Yamaguchi-Shinozaki, K., Hayashida, N. and Shinozaki, K. 1994. 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.
  45. Wernimont, A. K., Amani, M., Qiu, W., Pizarro, J. C., Artz, J. D., Lin, Y. H., Lew, J., Hutchinson, A. and Hui, R. 2011. Structures of parasitic CDPK domains point to a common mechanism of activation. Proteins 79, 803-820. https://doi.org/10.1002/prot.22919
  46. Wernimont, A. K., Artz, J. D., Finerty, P. Jr., Lin, Y. H., Amani, M., Allali-Hassani, A., Senisterra, G., Vedadi, M., Tempel, W., Mackenzie, F., Chau, I., Lourido, S., Sibley, L. D. and Hui, R. 2010. Structures of apicomplexan calciumdependent protein kinases reveal mechanism of activation by calcium. Nat. Struct. Mol. Biol. 17, 596-601. https://doi.org/10.1038/nsmb.1795
  47. Ye, S., Wang, L., Xie, W., Wan, B., Li, X. and Lin, Y. 2009. Expression profile of calcium-dependent protein kinase (CDPKs) genes during the whole life span and under phyto hormone treatment conditions in rice (Oryza sativa L. ssp. indica). Plant Mol. Biol. 70, 311-325. https://doi.org/10.1007/s11103-009-9475-0
  48. Yoon, G. M., Cho, H. S., Ha, H. J., Liu, J. R. and 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. https://doi.org/10.1023/A:1006170512542
  49. Zhang, M. and Yuan, T. 1998. Molecular mechanisms of calmodulin's functional versatility. Biochem. Cell Biol. 76, 313-323. https://doi.org/10.1139/o98-027
  50. Zhang, M., Liang, S. and Lu, Y. T. 2005. Cloning and functional characterization of NtCPK4, a new tobacco calciumdependent protein kinase. Biochim. Biophys. Acta 1729, 174-185. https://doi.org/10.1016/j.bbaexp.2005.04.006
  51. Zhao, Y., Pokutta, S., Maurer, P., Lindt, M., Franklin, R. M. and Kappes, B. 1994. Calcium-binding properties of a calcium-dependent protein kinases from Plasmodium falciparum and the significance of individual calcium-binding sites for kinase activation. Biochemistry 33, 3714-3721. https://doi.org/10.1021/bi00178a031