Partial Purification and Characterization of the Rat Parotid Gland Protein Kinase Catalyzing Phosphorylation of Matured Destrin at Ser-2
Author(s)
Eriko Osumi,
Chihiro Kondo,
Mitsumasa Mizuno,
Takahiro Suzuki,
Mamoru Matsubara,
Kazuo Shimozato,
Takao Kanamori
Affiliation(s)
Department of Maxillofacial Surgery, School of Dentistry, Aichi-Gakuin University, Nagoya, Japan. Department of Biochemistry, School of Dentistry, Aichi-Gakuin University, Nagoya, Japan. Department of Bioscience and Biotechnology, Faculty of Bioenvironmental Science, Kyotogakuen University, Kyoto, Japan.
Department of Maxillofacial Surgery, School of Dentistry, Aichi-Gakuin University, Nagoya, Japan. Department of Biochemistry, School of Dentistry, Aichi-Gakuin University, Nagoya, Japan. Department of Bioscience and Biotechnology, Faculty of Bioenvironmental Science, Kyotogakuen University, Kyoto, Japan.
ABSTRACT
Destrin, also called actin-depolymerizing factor (ADF), exists in resting parotid tissue as phosphorylated (inactive) and dephosphorylated (active) forms, and β-adrenergic stimulation of this tissue induces dephosphorylation of destrin. It is suggested that destrin dephosphorylation is involved in cortical F-actin disruption observed in parallel with β-agonist-induced amylase secretion. At present, the phosphorylation/dephosphorylation mechanism of destrin in parotid tissue is not known. We previously detected, in a crude rat parotid extract, a constitutively active protein kinase catalyzing phosphorylation of destrin; however, its identification has been hampered by difficulty in its enrichment. The purpose of this study was to explore a simple purification method(s) for this enzyme. To this end, we first developed a high-throughput dot-blot assay for the kinase with an anti-phosphodestrin antibody and then studied its purification by column chromatography on several media. We found that the kinase could be partially purified by sequential chromatography on DEAE-cellulose, phenyl-Sepharose, and hydroxyapatite columns. In each chromatography, however, the kinase could be eluted, at the cost of resolution, only by sharp increases in the elution power of the eluent; gradual increases in the elution power resulted in unacceptably poor recovery. We confirmed that enzymatic properties of the kinase were not basically altered during the purification. Further purification of the kinase was achieved by native polyacrylamide gel electrophoresis (PAGE), which resolved the kinase activity into two bands and separated the activity from most proteins (the kinase activity after PAGE was detected with destrin-coated polyvinylidene difluoride membranes and the anti-phosphodestrin antibody). The two bands seem to constitute the major destrin-phosphorylating activity in the resting rat parotid gland. We here report its partial purification and characterization together with the detection methods.
Destrin, also called actin-depolymerizing factor (ADF), exists in resting parotid tissue as phosphorylated (inactive) and dephosphorylated (active) forms, and β-adrenergic stimulation of this tissue induces dephosphorylation of destrin. It is suggested that destrin dephosphorylation is involved in cortical F-actin disruption observed in parallel with β-agonist-induced amylase secretion. At present, the phosphorylation/dephosphorylation mechanism of destrin in parotid tissue is not known. We previously detected, in a crude rat parotid extract, a constitutively active protein kinase catalyzing phosphorylation of destrin; however, its identification has been hampered by difficulty in its enrichment. The purpose of this study was to explore a simple purification method(s) for this enzyme. To this end, we first developed a high-throughput dot-blot assay for the kinase with an anti-phosphodestrin antibody and then studied its purification by column chromatography on several media. We found that the kinase could be partially purified by sequential chromatography on DEAE-cellulose, phenyl-Sepharose, and hydroxyapatite columns. In each chromatography, however, the kinase could be eluted, at the cost of resolution, only by sharp increases in the elution power of the eluent; gradual increases in the elution power resulted in unacceptably poor recovery. We confirmed that enzymatic properties of the kinase were not basically altered during the purification. Further purification of the kinase was achieved by native polyacrylamide gel electrophoresis (PAGE), which resolved the kinase activity into two bands and separated the activity from most proteins (the kinase activity after PAGE was detected with destrin-coated polyvinylidene difluoride membranes and the anti-phosphodestrin antibody). The two bands seem to constitute the major destrin-phosphorylating activity in the resting rat parotid gland. We here report its partial purification and characterization together with the detection methods.
KEYWORDS
Protein Kinase Purification, Protein Kinase Assay, Destrin Kinase, Actin-Depolymerizing Factor, Rat Parotid Gland
Protein Kinase Purification, Protein Kinase Assay, Destrin Kinase, Actin-Depolymerizing Factor, Rat Parotid Gland
Cite this paper
Osumi, E. , Kondo, C. , Mizuno, M. , Suzuki, T. , Matsubara, M. , Shimozato, K. and Kanamori, T. (2014) Partial Purification and Characterization of the Rat Parotid Gland Protein Kinase Catalyzing Phosphorylation of Matured Destrin at Ser-2. Advances in Enzyme Research, 2, 100-112. doi: 10.4236/aer.2014.22011.
Osumi, E. , Kondo, C. , Mizuno, M. , Suzuki, T. , Matsubara, M. , Shimozato, K. and Kanamori, T. (2014) Partial Purification and Characterization of the Rat Parotid Gland Protein Kinase Catalyzing Phosphorylation of Matured Destrin at Ser-2. Advances in Enzyme Research, 2, 100-112. doi: 10.4236/aer.2014.22011.
References
[1] Spearman, T.N. and Butcher, F.R. (1989) Cellular Regulation of Amylase Secretion by the Parotid Gland. In: Forte, J.G., Ed., Handbook of Physiology, Section 6, The Gastrointestinal System, American Physiological Society, Bethesda, 63-77. [2] Takuma, T. and Ichida, T. (1994) Catalytic Subunit of Protein Kinase A Induces Amylase Release from Streptolysin O-Permeabilized Parotid Acini. The Journal of Biological Chemistry, 269, 22124-22128. [3] Kanamori, T., Hayakawa, T., Suzuki, M. and Titani, K. (1995) Identification of Two 17-kDa Rat Parotid Gland Phosphoproteins, Subjects for Dephosphorylation upon β-Adrenergic Stimulation, as Destrin- and Cofilin-Like Proteins. The Journal of Biological Chemistry, 270, 8061-8067.
http://dx.doi.org/10.1074/jbc.270.14.8061 [4] Kanamori, T., Suzuki, M. and Titani, K. (1998) Complete Amino Acid Sequences and Phosphorylation Sites, Determined by Edman Degradation and Mass Spectrometry, of Rat Parotid Destrin- and Cofilin-Like Proteins. Archives of Oral Biology, 43, 955-967. http://dx.doi.org/10.1016/S0003-9969(98)00083-1 [5] Moon, A. and Drubin, D.G. (1995) The ADF/Cofilin Proteins: Stimulus-Responsive Modulators of Actin Dynamics. Molecular Biology of the Cell, 6, 1423-1431. http://dx.doi.org/10.1091/mbc.6.11.1423 [6] Perrin, D., Moller, K., Hanke, K. and Soling, H.D. (1992) cAMP and Ca2+-Mediated Secretion in Parotid Acinar Cells Is Associated with Reversible Changes in the Organization of the Cytoskeleton. The Journal of Cell Biology, 116, 127-134. http://dx.doi.org/10.1083/jcb.116.1.127 [7] Nakano, S., Kanamori, T., Suzuki, M. and Titani, K. (2003) Detection and Characterization of a Rat Parotid Gland Protein Kinase That Catalyzes Phosphorylation of Matured Destrin at Ser-2. Archives of Oral Biology, 48, 649-661. http://dx.doi.org/10.1016/S0003-9969(03)00129-8 [8] Kondo, C., Nakano, S., Suzuki, T. and Kanamori, T. (2007) An Easily Constructed, Inexpensive Device for Dot Blotting. Analytical Biochemistry, 370, 115-117. http://dx.doi.org/10.1016/j.ab.2007.05.015 [9] Armstrong, I.L. and Tate, W.P. (1980) A Simple Device for the Dialysis of Small Volumes. Analytical Biochemistry, 106, 469-470. http://dx.doi.org/10.1016/0003-2697(80)90549-7 [10] Fling, S.P. and Gregerson, D.S. (1986) Peptide and Protein Molecular Weight Determination by Electrophoresis Using a High-Molarity Tris Buffer System without Urea. Analytical Biochemistry, 155, 83-88. http://dx.doi.org/10.1016/0003-2697(86)90228-9 [11] Hartree, E.F. (1972) Determination of Protein: A Modification of the Lowry Method That Gives a Linear Photometric Response. Analytical Biochemistry, 48, 422-427.
http://dx.doi.org/10.1016/0003-2697(72)90094-2 [12] Ruzzene, M. and Pinna, L.A. (1999) Assay of Protein Kinases and Phosphatases Using Specific Peptide Substrates. In: Hardie, D.G., Ed., Protein Phosphorylation, 2nd Edition, Oxford University Press, New York, 221-253. [13] Marcu, M.G., Chadli, A., Bouhouche, I., Catelli, M. and Neckers, L.M. (2000) The Heat Shock Protein 90 Antagonist Novobiocin Interacts with a Previously Unrecognized ATP-Binding Domain in the Carboxyl Terminus of the Chaperone. The Journal of Biological Chemistry, 275, 37181-37186.
http://dx.doi.org/10.1074/jbc.M003701200 [14] Hathaway, G.M., Lubben, T.H. and Traugh, J.A. (1980) Inhibition of Casein Kinase II by Heparin. The Journal of Biological Chemistry, 255, 8038-8041. [15] Bain, J., McLauchlan, H., Elliott, M. and Cohen, P. (2003) The Specificities of Protein Kinase Inhibitors: An Update. Biochemical Journal, 371, 199-204. http://dx.doi.org/10.1042/BJ20021535 [16] Bain, J., Plater, L., Elliott, M., Shpiro, N., Hastie, C.J., McLauchlan, H., Klevernic, I., Arthur, J.S., Alessi, D.R. and Cohen, P. (2007) The Selectivity of Protein Kinase Inhibitors: A Further Update. Biochemical Journal, 408, 297-315. http://dx.doi.org/10.1042/BJ20070797 [17] Davies, S.P., Reddy, H., Caivano, M. and Cohen, P. (2000) Specificity and Mechanism of Action of Some Commonly Used Protein Kinase Inhibitors. Biochemical Journal, 351, 95-105.
http://dx.doi.org/10.1042/0264-6021:3510095 [18] Morgan, T.E., Lockerbie, R.O., Minamide, L.S., Browning, M.D. and Bamburg, J.R. (1993) Isolation and Characterization of a Regulated Form of Actin Depolymerizing Factor. The Journal of Cell Biology, 122, 623-633. http://dx.doi.org/10.1083/jcb.122.3.623 [19] Hidaka, H. and Tanaka, T. (1983) Naphthalenesulfonamides as Calmodulin Antagonists. Methods in Enzymology, 102, 185-194. http://dx.doi.org/10.1016/S0076-6879(83)02019-4 [20] Caplan, A.J., Mandal, A.K. and Theodoraki, M.A. (2007) Molecular Chaperones and Protein Kinase Quality Control. Trends in Cell Biology, 17, 87-92. http://dx.doi.org/10.1016/j.tcb.2006.12.002 [21] D’Arcangelo, J.G., Stahmer, K.R. and Miller, E.A. (2013) Vesicle-Mediated Export from the ER: COPII Coat Function and Regulation. Biochimica et Biophysica Acta, 1833, 2464-2472.
http://dx.doi.org/10.1016/j.bbamcr.2013.02.003 [22] Wei, N. and Deng, X.W. (2003) The COP9 Signalosome. Annual Review of Cell and Developmental Biology, 19, 261- 286. http://dx.doi.org/10.1146/annurev.cellbio.19.111301.112449 [23] Shugrue, C.A., Kolen, E.R., Peters, H., Czernik, A., Kaiser, C., Matovcik, L., Hubbard, A.L. and Gorelick, F. (1999) Identification of the Putative Mammalian Orthologue of Sec31P, a Component of the COPII Coat. Journal of Cell Science, 112, 4547-4556. [24] Lazarides, E. and Lindberg, U. (1974) Actin Is the Naturally Occurring Inhibitor of Deoxyribonuclease I. Proceedings of the National Academy of Sciences of the United States of America, 71, 4742-4746. http://dx.doi.org/10.1073/pnas.71.12.4742 [25] Morrissey, J.H. (1981) Silver Stain for Proteins in Polyacrylamide Gels: A Modified Procedure with Enhanced Uniform Sensitivity. Analytical Biochemistry, 117, 307-310.
http://dx.doi.org/10.1016/0003-2697(81)90783-1 [26] Shevchenko, A., Tomas, H., Havlis, J., Olsen, J.V. and Mann, M. (2006) In-Gel Digestion for Mass Spectrometric Characterization of Proteins and Proteomes. Nature Protocols, 1, 2856-2860.
http://dx.doi.org/10.1038/nprot.2006.468 [27] Kanamori, T., Suzuki, M. and Titani, K. (1998) Complete Amino Acid Sequences and Phosphorylation Sites, Determined by Edman Degradation and Mass Spectrometry, of Rat Parotid Destrin- and Cofilin-Like Proteins. Archives of Oral Biology, 43, 955-967.
http://dx.doi.org/10.1016/S0003-9969(98)00083-1 [28] Wu, J., Gage, D.A. and Watson, J.T. (1996) A Strategy to Locate Cysteine Residues in Proteins by Specific Chemical Cleavage Followed by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. Analytical Biochemistry, 235, 161-174.
http://dx.doi.org/10.1006/abio.1996.0108 [29] Brune, D.C. (1992) Alkylation of Cysteine with Acrylamide for Protein Sequence Analysis. Analytical Biochemistry, 207, 285-290.
http://dx.doi.org/10.1016/0003-2697(92)90013-W [30] Cavins, J.F. and Friedman, M. (1968) Specific Modification of Protein Sulfhydryl Groups with α, β-Unsaturated Compounds. The Journal of Biological Chemistry, 243, 3357-3360. [31] Wei, N. and Deng, X.W. (2003) The COP9 Signalosome. Annual Review of Cell and Developmental Biology, 19, 261- 286.
http://dx.doi.org/10.1146/annurev.cellbio.19.111301.112449
[1] Spearman, T.N. and Butcher, F.R. (1989) Cellular Regulation of Amylase Secretion by the Parotid Gland. In: Forte, J.G., Ed., Handbook of Physiology, Section 6, The Gastrointestinal System, American Physiological Society, Bethesda, 63-77. [2] Takuma, T. and Ichida, T. (1994) Catalytic Subunit of Protein Kinase A Induces Amylase Release from Streptolysin O-Permeabilized Parotid Acini. The Journal of Biological Chemistry, 269, 22124-22128. [3] Kanamori, T., Hayakawa, T., Suzuki, M. and Titani, K. (1995) Identification of Two 17-kDa Rat Parotid Gland Phosphoproteins, Subjects for Dephosphorylation upon β-Adrenergic Stimulation, as Destrin- and Cofilin-Like Proteins. The Journal of Biological Chemistry, 270, 8061-8067.
http://dx.doi.org/10.1074/jbc.270.14.8061 [4] Kanamori, T., Suzuki, M. and Titani, K. (1998) Complete Amino Acid Sequences and Phosphorylation Sites, Determined by Edman Degradation and Mass Spectrometry, of Rat Parotid Destrin- and Cofilin-Like Proteins. Archives of Oral Biology, 43, 955-967. http://dx.doi.org/10.1016/S0003-9969(98)00083-1 [5] Moon, A. and Drubin, D.G. (1995) The ADF/Cofilin Proteins: Stimulus-Responsive Modulators of Actin Dynamics. Molecular Biology of the Cell, 6, 1423-1431. http://dx.doi.org/10.1091/mbc.6.11.1423 [6] Perrin, D., Moller, K., Hanke, K. and Soling, H.D. (1992) cAMP and Ca2+-Mediated Secretion in Parotid Acinar Cells Is Associated with Reversible Changes in the Organization of the Cytoskeleton. The Journal of Cell Biology, 116, 127-134. http://dx.doi.org/10.1083/jcb.116.1.127 [7] Nakano, S., Kanamori, T., Suzuki, M. and Titani, K. (2003) Detection and Characterization of a Rat Parotid Gland Protein Kinase That Catalyzes Phosphorylation of Matured Destrin at Ser-2. Archives of Oral Biology, 48, 649-661. http://dx.doi.org/10.1016/S0003-9969(03)00129-8 [8] Kondo, C., Nakano, S., Suzuki, T. and Kanamori, T. (2007) An Easily Constructed, Inexpensive Device for Dot Blotting. Analytical Biochemistry, 370, 115-117. http://dx.doi.org/10.1016/j.ab.2007.05.015 [9] Armstrong, I.L. and Tate, W.P. (1980) A Simple Device for the Dialysis of Small Volumes. Analytical Biochemistry, 106, 469-470. http://dx.doi.org/10.1016/0003-2697(80)90549-7 [10] Fling, S.P. and Gregerson, D.S. (1986) Peptide and Protein Molecular Weight Determination by Electrophoresis Using a High-Molarity Tris Buffer System without Urea. Analytical Biochemistry, 155, 83-88. http://dx.doi.org/10.1016/0003-2697(86)90228-9 [11] Hartree, E.F. (1972) Determination of Protein: A Modification of the Lowry Method That Gives a Linear Photometric Response. Analytical Biochemistry, 48, 422-427.
http://dx.doi.org/10.1016/0003-2697(72)90094-2 [12] Ruzzene, M. and Pinna, L.A. (1999) Assay of Protein Kinases and Phosphatases Using Specific Peptide Substrates. In: Hardie, D.G., Ed., Protein Phosphorylation, 2nd Edition, Oxford University Press, New York, 221-253. [13] Marcu, M.G., Chadli, A., Bouhouche, I., Catelli, M. and Neckers, L.M. (2000) The Heat Shock Protein 90 Antagonist Novobiocin Interacts with a Previously Unrecognized ATP-Binding Domain in the Carboxyl Terminus of the Chaperone. The Journal of Biological Chemistry, 275, 37181-37186.
http://dx.doi.org/10.1074/jbc.M003701200 [14] Hathaway, G.M., Lubben, T.H. and Traugh, J.A. (1980) Inhibition of Casein Kinase II by Heparin. The Journal of Biological Chemistry, 255, 8038-8041. [15] Bain, J., McLauchlan, H., Elliott, M. and Cohen, P. (2003) The Specificities of Protein Kinase Inhibitors: An Update. Biochemical Journal, 371, 199-204. http://dx.doi.org/10.1042/BJ20021535 [16] Bain, J., Plater, L., Elliott, M., Shpiro, N., Hastie, C.J., McLauchlan, H., Klevernic, I., Arthur, J.S., Alessi, D.R. and Cohen, P. (2007) The Selectivity of Protein Kinase Inhibitors: A Further Update. Biochemical Journal, 408, 297-315. http://dx.doi.org/10.1042/BJ20070797 [17] Davies, S.P., Reddy, H., Caivano, M. and Cohen, P. (2000) Specificity and Mechanism of Action of Some Commonly Used Protein Kinase Inhibitors. Biochemical Journal, 351, 95-105.
http://dx.doi.org/10.1042/0264-6021:3510095 [18] Morgan, T.E., Lockerbie, R.O., Minamide, L.S., Browning, M.D. and Bamburg, J.R. (1993) Isolation and Characterization of a Regulated Form of Actin Depolymerizing Factor. The Journal of Cell Biology, 122, 623-633. http://dx.doi.org/10.1083/jcb.122.3.623 [19] Hidaka, H. and Tanaka, T. (1983) Naphthalenesulfonamides as Calmodulin Antagonists. Methods in Enzymology, 102, 185-194. http://dx.doi.org/10.1016/S0076-6879(83)02019-4 [20] Caplan, A.J., Mandal, A.K. and Theodoraki, M.A. (2007) Molecular Chaperones and Protein Kinase Quality Control. Trends in Cell Biology, 17, 87-92. http://dx.doi.org/10.1016/j.tcb.2006.12.002 [21] D’Arcangelo, J.G., Stahmer, K.R. and Miller, E.A. (2013) Vesicle-Mediated Export from the ER: COPII Coat Function and Regulation. Biochimica et Biophysica Acta, 1833, 2464-2472.
http://dx.doi.org/10.1016/j.bbamcr.2013.02.003 [22] Wei, N. and Deng, X.W. (2003) The COP9 Signalosome. Annual Review of Cell and Developmental Biology, 19, 261- 286. http://dx.doi.org/10.1146/annurev.cellbio.19.111301.112449 [23] Shugrue, C.A., Kolen, E.R., Peters, H., Czernik, A., Kaiser, C., Matovcik, L., Hubbard, A.L. and Gorelick, F. (1999) Identification of the Putative Mammalian Orthologue of Sec31P, a Component of the COPII Coat. Journal of Cell Science, 112, 4547-4556. [24] Lazarides, E. and Lindberg, U. (1974) Actin Is the Naturally Occurring Inhibitor of Deoxyribonuclease I. Proceedings of the National Academy of Sciences of the United States of America, 71, 4742-4746. http://dx.doi.org/10.1073/pnas.71.12.4742 [25] Morrissey, J.H. (1981) Silver Stain for Proteins in Polyacrylamide Gels: A Modified Procedure with Enhanced Uniform Sensitivity. Analytical Biochemistry, 117, 307-310.
http://dx.doi.org/10.1016/0003-2697(81)90783-1 [26] Shevchenko, A., Tomas, H., Havlis, J., Olsen, J.V. and Mann, M. (2006) In-Gel Digestion for Mass Spectrometric Characterization of Proteins and Proteomes. Nature Protocols, 1, 2856-2860.
http://dx.doi.org/10.1038/nprot.2006.468 [27] Kanamori, T., Suzuki, M. and Titani, K. (1998) Complete Amino Acid Sequences and Phosphorylation Sites, Determined by Edman Degradation and Mass Spectrometry, of Rat Parotid Destrin- and Cofilin-Like Proteins. Archives of Oral Biology, 43, 955-967.
http://dx.doi.org/10.1016/S0003-9969(98)00083-1 [28] Wu, J., Gage, D.A. and Watson, J.T. (1996) A Strategy to Locate Cysteine Residues in Proteins by Specific Chemical Cleavage Followed by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. Analytical Biochemistry, 235, 161-174.
http://dx.doi.org/10.1006/abio.1996.0108 [29] Brune, D.C. (1992) Alkylation of Cysteine with Acrylamide for Protein Sequence Analysis. Analytical Biochemistry, 207, 285-290.
http://dx.doi.org/10.1016/0003-2697(92)90013-W [30] Cavins, J.F. and Friedman, M. (1968) Specific Modification of Protein Sulfhydryl Groups with α, β-Unsaturated Compounds. The Journal of Biological Chemistry, 243, 3357-3360. [31] Wei, N. and Deng, X.W. (2003) The COP9 Signalosome. Annual Review of Cell and Developmental Biology, 19, 261- 286.
http://dx.doi.org/10.1146/annurev.cellbio.19.111301.112449