Label-free Kinase Profiling Using Phosphate Affinity Polyacrylamide Gel Electrophoresis

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Herein we describe three applications of label-free kinase profiling using a novel type of phosphate affinity polyacrylamide gel electrophoresis. The phosphate affinity site is a polyacrylamide-bound dinuclear Mn2+ complex that enables the mobility shift detection of phosphorylated proteins from their nonphosphorylated counterpart. The first application is in vitro kinase activity profiling for the analysis of varied phosphoprotein isotypes in phosphorylation status. The activity profiles of six kinds of kinases, glycogen synthase kinase-3β, cyclin-dependent kinase 5/p35, protein kinase A, mitogen-activated protein kinase (MAPK), casein kinase II, and calmodulin-dependent protein kinase II, were determined using a substrate protein, Tau, which has a number of phosphorylation sites. Each kinase demonstrated characteristic multiple electrophoresis migration bands up-shifted from the nonphosphorylated Tau due to differences in the phosphorylation sites and stoichiometry. The second application is in vivo kinase activity profiling for the analysis of protein phosphorylation involved in intracellular signal transduction. The time course changes in the epidermal growth factor-induced phosphorylation levels of Shc and MAPK in A431 cells were visualized as highly up-shifted migration bands by subsequent immunoblotting with anti-Shc and anti-MAPK antibodies. The third application is in vitro kinase inhibition profiling for the quantitative screening of kinase-specific inhibitors. The inhibition profile of a tyrosine kinase, Abl (a histidine-tagged recombinant mouse Abl kinase), was determined using the substrate Abltide-GST (a fusion protein consisting of a specific substrate peptide for Abl and glutathione S-transferase) and the approved drug Glivec (an ATP competitor). In the kinase assay, the slower migration band, monophosphorylated Abltide-GST, increased time-dependently, whereas the faster migration band, nonphosphorylated Abltide-GST, decreased. The dose-dependent inhibition of Glivec was determined by a change in the ratio of the faster and slower migration bands, which showed an IC50 value of 1.6 μm in the presence of 0.10 mm ATP. Herein we describe three applications of label-free kinase profiling using a novel type of phosphate affinity polyacrylamide gel electrophoresis. The phosphate affinity site is a polyacrylamide-bound dinuclear Mn2+ complex that enables the mobility shift detection of phosphorylated proteins from their nonphosphorylated counterpart. The first application is in vitro kinase activity profiling for the analysis of varied phosphoprotein isotypes in phosphorylation status. The activity profiles of six kinds of kinases, glycogen synthase kinase-3β, cyclin-dependent kinase 5/p35, protein kinase A, mitogen-activated protein kinase (MAPK), casein kinase II, and calmodulin-dependent protein kinase II, were determined using a substrate protein, Tau, which has a number of phosphorylation sites. Each kinase demonstrated characteristic multiple electrophoresis migration bands up-shifted from the nonphosphorylated Tau due to differences in the phosphorylation sites and stoichiometry. The second application is in vivo kinase activity profiling for the analysis of protein phosphorylation involved in intracellular signal transduction. The time course changes in the epidermal growth factor-induced phosphorylation levels of Shc and MAPK in A431 cells were visualized as highly up-shifted migration bands by subsequent immunoblotting with anti-Shc and anti-MAPK antibodies. The third application is in vitro kinase inhibition profiling for the quantitative screening of kinase-specific inhibitors. The inhibition profile of a tyrosine kinase, Abl (a histidine-tagged recombinant mouse Abl kinase), was determined using the substrate Abltide-GST (a fusion protein consisting of a specific substrate peptide for Abl and glutathione S-transferase) and the approved drug Glivec (an ATP competitor). In the kinase assay, the slower migration band, monophosphorylated Abltide-GST, increased time-dependently, whereas the faster migration band, nonphosphorylated Abltide-GST, decreased. The dose-dependent inhibition of Glivec was determined by a change in the ratio of the faster and slower migration bands, which showed an IC50 value of 1.6 μm in the presence of 0.10 mm ATP. Protein phosphorylation is essential for the regulatory events of biological processes, such as signal transduction, apoptosis, proliferation, differentiation, and metabolism, in all living cells (1Hunter T. Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling..Cell. 1995; 80: 225-236Abstract Full Text PDF PubMed Scopus (2577) Google Scholar, 2Hunter T. Signaling—2000 and beyond..Cell. 2000; 100: 113-127Abstract Full Text Full Text PDF PubMed Scopus (2244) Google Scholar). It occurs on several amino acid residues, including histidine, aspartic acid, glutamic acid, lysine, arginine, and cysteine on which it is very labile and difficult to detect, whereas more stable and well studied phosphorylation takes place on the three specific residues serine, threonine, and tyrosine (3Mann M. Ong S.E. Grønborg M. Steen H. Jensen O.N. Pandey A. Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome..Trends Biotechnol. 2002; 20: 261-268Abstract Full Text Full Text PDF PubMed Scopus (789) Google Scholar). The balance of the kinase and phosphatase reactions controls the phosphorylation status of a certain protein. Perturbation of the balance triggers severe pathologies, such as cancer and inflammation. Many of the genetic changes that play a causal role in the cancer phenotype involve mutations of protein kinases and phosphatases (4Hunter T. Tyrosine phosphorylation in cell signaling and disease..Keio J. Med. 2002; 51: 61-71Crossref PubMed Scopus (32) Google Scholar). There has been considerable progress in the development of selective inhibitors for the protein kinase and phosphatase involved in disease (5Cohen P. Protein kinases—the major drug targets of the twenty-first century?.Nat. Rev. Drug Discov. 2002; 1: 309-315Crossref PubMed Scopus (1827) Google Scholar). Some of these inhibitors have been recently approved for use in humans for the treatment of cancer. Furthermore the activities of several protein kinases are dysregulated, leading to a hyperphosphorylation state of the microtubule-associated protein Tau, which is a classical hallmark of Alzheimer disease, a neurodegenerative disorder (6Morishima-Kawashima M. Hasegawa M. Takio K. Suzuki M. Yoshida H. Watanabe A. Titani K. Ihara Y. Hyperphosphorylation of Tau in PHF..Neurobiol. Aging. 1995; 16: 365-380Crossref PubMed Scopus (193) Google Scholar, 7Lee V.M.-Y. Goedert M. Trojanowski J.Q. Neurodegenerative tauopathies..Annu. Rev. Neurosci. 2001; 24: 1121-1159Crossref PubMed Scopus (2098) Google Scholar). The phosphorylation site and stoichiometry of the Tau protein are correlated with the pathological characteristics of the disease.Methods for the determination of the phosphorylation status of a protein are thus very important with respect to the evaluation of the basis for understanding the molecular origins of diseases and for drug design. A conventionally used method for defining a particular phosphorylation event is the incorporation of a radioactive label, i.e. a 32P or 33P isotope, in a phosphorylated protein, which is followed by PAGE and autoradiography. The phosphorylation state of the target protein is detected and quantified as radioactivity. A newer, non-radioactive method using poly- and monoclonal antibodies has been well established for the detection of site-specific phosphorylation. The anti-phosphoprotein antibody used in such as the assay, and a for defining a number of phosphorylation events were using a peptide followed by J. M. kinase activities by using a peptide and mass PubMed Scopus Google and K. M. Y. Y. T. T. Y. and of phosphorylated by using a phosphate PubMed Scopus Google Scholar). of the phosphate has been used for site in peptide mass and subsequent analysis the Biotechnol. 2001; PubMed Scopus Google Scholar, H. A to the analysis of protein Biotechnol. 2001; PubMed Scopus Google Scholar, Y. T. analysis of phosphorylated proteins as a for the Biotechnol. 2001; PubMed Scopus Google Scholar, for sites of protein Biotechnol. PubMed Scopus Google we that a dinuclear complex of as a used glycogen synthase cyclin-dependent kinase protein kinase mitogen-activated protein casein kinase calmodulin-dependent protein kinase epidermal growth ratio of the 32P signal to the of used glycogen synthase cyclin-dependent kinase protein kinase mitogen-activated protein casein kinase calmodulin-dependent protein kinase epidermal growth ratio of the 32P signal to the of in an H. A. M. A. T. mass of phosphorylated using a novel phosphate PubMed Scopus Google Scholar, M. H. M. T. of phosphate by an dinuclear PubMed Scopus Google Scholar, A. H. T. affinity for and phosphorylated PubMed Scopus Google Scholar, K. T. a to phosphorylated Full Text Full Text PDF PubMed Scopus Google Scholar, A. M. T. of phosphorylated proteins from cell using a novel PubMed Scopus Google Scholar). The has a on that is for a as a A such as and the of in has to the development of phosphate affinity electrophoresis for the mobility shift detection of from their nonphosphorylated K. T. a to phosphorylated Full Text Full Text PDF PubMed Scopus Google Scholar). an as a novel i.e. a of the gel in The the and are The time course quantitative ratio of the phosphorylated and nonphosphorylated proteins The phosphate is of the amino acid A such as is The is to that of the we describe three novel applications of The first is in vitro kinase activity profiling for the analysis of the phosphoprotein isotypes from kinase The activity profiles of six kinds of kinases, glycogen synthase cyclin-dependent kinase protein kinase A mitogen-activated protein kinase (MAPK), casein kinase and calmodulin-dependent protein kinase were determined using the substrate Tau protein. The second application is in vivo kinase activity profiling for the analysis of protein phosphorylation. The of epidermal growth phosphorylation levels of Shc and were demonstrated using the of A431 The third application is in vitro kinase inhibition profiling for the quantitative analysis of a kinase-specific The profile of a tyrosine kinase, Abl (a histidine-tagged recombinant mouse Abl kinase), was demonstrated using the substrate Abltide-GST (a fusion protein consisting of a specific substrate peptide for Abl and glutathione S-transferase) and the approved drug Glivec (a used for the treatment of J. H. T. M. of the Abl kinase in vitro and in vivo by a Google Scholar, M. J. a tyrosine kinase the growth of cells and fusion PubMed Google Scholar, T. P. J. for inhibition of tyrosine 2000; PubMed Scopus Google Scholar, has tyrosine kinase activity that is by of the and by regulatory 2000; Full Text Full Text PDF PubMed Scopus Google we have three kinds of protein profiling using or The method is on the mobility shift of phosphorylated proteins from the nonphosphorylated a kinase the of phosphorylated and nonphosphorylated proteins determined using or protein phosphorylation occurs of a target protein, the monophosphorylated and nonphosphorylated proteins are as migration bands on In the of the phosphorylated as multiple bands, on the phosphorylation such as the number and of the phosphate first application is in vitro kinase activity profiling for the analysis of varied phosphoprotein isotypes in a phosphorylation status. determined the activity profiles of six kinds of Alzheimer kinases, and using a substrate protein, Tau, which has a number of phosphorylation sites. Each kinase the kinase-specific gel from nonphosphorylated Tau due to differences in the phosphorylation sites and stoichiometry. the the phosphorylation status and the biological antibodies or have been these are for the analysis of varied phosphoprotein isotypes in a phosphorylation status. established method the detection of the isotypes by kinase activities in a polyacrylamide The immunoblotting using the site-specific Tau antibodies the of phosphorylation of Tau and that progress in by application and such as mass A peptide analysis of the phosphorylated Tau by is in kinase activity profiling on the kinase novel on the protein phosphorylation and the biological second application is in vivo kinase activity profiling for the analysis of protein phosphorylation involved in intracellular signal transduction. The time course changes of phosphorylation levels of Shc and in A431 cells were visualized as highly up-shifted migration bands by subsequent immunoblotting with anti-Shc and antibodies. demonstrated the of to phosphorylated in vivo in a from a nonphosphorylated in the presence of method an in profiling the phosphorylation state of in a complex biological it is to the phosphorylation events by in has been to a protein is shift and proteins that are to phosphorylated gel in In up-shifted of phosphorylated MAPK was of In was to gel of varied phosphoprotein of the method is of for of the phosphorylation state of third application is in vitro kinase inhibition profiling for the quantitative screening of kinase-specific inhibitors. demonstrated the inhibition profile of the tyrosine kinase Abl using the substrate Abltide-GST and the specific The dose-dependent inhibition of Glivec was determined by an in the ratio of the monophosphorylated substrate slower migration and the nonphosphorylated faster in an The inhibition showed an IC50 value of 1.6 in the presence of 0.10 mm ATP. that application enables quantitative analysis of kinase activities to the inhibition IC50 of Glivec with Abl in vitro have been to μm J. H. T. M. of the Abl kinase in vitro and in vivo by a Google μm J. M. kinase activities by using a peptide and mass PubMed Scopus Google and μm has tyrosine kinase activity that is by of the and by regulatory 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). in the differences in Abl ATP or kinase inhibition profiling in for A to phosphatase inhibition profiling using an phosphorylated which is of the important the of Many isotypes by phosphorylation site and stoichiometry a number of biological A. Hasegawa M. Suzuki M. Takio K. M. Titani K. T. Ihara Y. In vivo phosphorylation sites in and Full Text PDF PubMed Google Scholar, kinase an of as the phosphate Full Text PDF PubMed Google Scholar, M. A. and of by PubMed Scopus Google Scholar, T. K. phosphorylation of by protein kinase A and of by protein phosphatase and 2002; PubMed Scopus Google Scholar, K. J. to A. PubMed Scopus Google Scholar, H. K. of and phosphorylation by Neurosci. PubMed Scopus Google Scholar, M. K. H. T. Y. H. T. T. of of phosphorylation in PubMed Scopus Google Scholar, Y. Y. H. A regulatory phosphorylation for of the complex J. PubMed Scopus Google Scholar). Hyperphosphorylation of a certain protein cells or and It has been difficult to the role of isotypes such the complex the for the of the isotypes of are very important in in biological and by using phosphate affinity i.e. the of the detection of protein phosphorylation and the method in phosphorylation events and a in the of the Protein phosphorylation is essential for the regulatory events of biological processes, such as signal transduction, apoptosis, proliferation, differentiation, and metabolism, in all living cells (1Hunter T. Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling..Cell. 1995; 80: 225-236Abstract Full Text PDF PubMed Scopus (2577) Google Scholar, 2Hunter T. Signaling—2000 and beyond..Cell. 2000; 100: 113-127Abstract Full Text Full Text PDF PubMed Scopus (2244) Google Scholar). It occurs on several amino acid residues, including histidine, aspartic acid, glutamic acid, lysine, arginine, and cysteine on which it is very labile and difficult to detect, whereas more stable and well studied phosphorylation takes place on the three specific residues serine, threonine, and tyrosine (3Mann M. Ong S.E. Grønborg M. Steen H. Jensen O.N. Pandey A. Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome..Trends Biotechnol. 2002; 20: 261-268Abstract Full Text Full Text PDF PubMed Scopus (789) Google Scholar). The balance of the kinase and phosphatase reactions controls the phosphorylation status of a certain protein. Perturbation of the balance triggers severe pathologies, such as cancer and inflammation. Many of the genetic changes that play a causal role in the cancer phenotype involve mutations of protein kinases and phosphatases (4Hunter T. Tyrosine phosphorylation in cell signaling and disease..Keio J. Med. 2002; 51: 61-71Crossref PubMed Scopus (32) Google Scholar). There has been considerable progress in the development of selective inhibitors for the protein kinase and phosphatase involved in disease (5Cohen P. Protein kinases—the major drug targets of the twenty-first century?.Nat. Rev. Drug Discov. 2002; 1: 309-315Crossref PubMed Scopus (1827) Google Scholar). Some of these inhibitors have been recently approved for use in humans for the treatment of cancer. Furthermore the activities of several protein kinases are dysregulated, leading to a hyperphosphorylation state of the microtubule-associated protein Tau, which is a classical hallmark of Alzheimer disease, a neurodegenerative disorder (6Morishima-Kawashima M. Hasegawa M. Takio K. Suzuki M. Yoshida H. Watanabe A. Titani K. Ihara Y. Hyperphosphorylation of Tau in PHF..Neurobiol. Aging. 1995; 16: 365-380Crossref PubMed Scopus (193) Google Scholar, 7Lee V.M.-Y. Goedert M. Trojanowski J.Q. Neurodegenerative tauopathies..Annu. Rev. Neurosci. 2001; 24: 1121-1159Crossref PubMed Scopus (2098) Google Scholar). The phosphorylation site and stoichiometry of the Tau protein are correlated with the pathological characteristics of the for the determination of the phosphorylation status of a protein are thus very important with respect to the evaluation of the basis for understanding the molecular origins of diseases and for drug design. A conventionally used method for defining a particular phosphorylation event is the incorporation of a radioactive label, i.e. a 32P or 33P isotope, in a phosphorylated protein, which is followed by PAGE and autoradiography. The phosphorylation state of the target protein is detected and quantified as radioactivity. A newer, non-radioactive method using poly- and monoclonal antibodies has been well established for the detection of site-specific phosphorylation. The anti-phosphoprotein antibody used in such as the assay, and a for defining a number of phosphorylation events were using a peptide followed by J. M. kinase activities by using a peptide and mass PubMed Scopus Google and K. M. Y. Y. T. T. Y. and of phosphorylated by using a phosphate PubMed Scopus Google Scholar). of the phosphate has been used for site in peptide mass and subsequent analysis the Biotechnol. 2001; PubMed Scopus Google Scholar, H. A to the analysis of protein Biotechnol. 2001; PubMed Scopus Google Scholar, Y. T. analysis of phosphorylated proteins as a for the Biotechnol. 2001; PubMed Scopus Google Scholar, for sites of protein Biotechnol. PubMed Scopus Google Scholar). we that a dinuclear complex of as a used glycogen synthase cyclin-dependent kinase protein kinase mitogen-activated protein casein kinase calmodulin-dependent protein kinase epidermal growth ratio of the 32P signal to the of used glycogen synthase cyclin-dependent kinase protein kinase mitogen-activated protein casein kinase calmodulin-dependent protein kinase epidermal growth ratio of the 32P signal to the of in an H. A. M. A. T. mass of phosphorylated using a novel phosphate PubMed Scopus Google Scholar, M. H. M. T. of phosphate by an dinuclear PubMed Scopus Google Scholar, A. H. T. affinity for and phosphorylated PubMed Scopus Google Scholar, K. T. a to phosphorylated Full Text Full Text PDF PubMed Scopus Google Scholar, A. M. T. of phosphorylated proteins from cell using a novel PubMed Scopus Google Scholar). The has a on that is for a as a A such as and the of in has to the development of phosphate affinity electrophoresis for the mobility shift detection of from their nonphosphorylated K. T. a to phosphorylated Full Text Full Text PDF PubMed Scopus Google Scholar). an as a novel i.e. a of the gel in The the and are The time course quantitative ratio of the phosphorylated and nonphosphorylated proteins The phosphate is of the amino acid A such as is The is to that of the Herein we describe three novel applications of The first is in vitro kinase activity profiling for the analysis of the phosphoprotein isotypes from kinase The activity profiles of six kinds of kinases, glycogen synthase cyclin-dependent kinase protein kinase A mitogen-activated protein kinase (MAPK), casein kinase and calmodulin-dependent protein kinase were determined using the substrate Tau protein. The second application is in vivo kinase activity profiling for the analysis of protein phosphorylation. The of epidermal growth phosphorylation levels of Shc and were demonstrated using the of A431 The third application is in vitro kinase inhibition profiling for the quantitative analysis of a kinase-specific The profile of a tyrosine kinase, Abl (a histidine-tagged recombinant mouse Abl kinase), was demonstrated using the substrate Abltide-GST (a fusion protein consisting of a specific substrate peptide for Abl and glutathione S-transferase) and the approved drug Glivec (a used for the treatment of J. H. T. M. of the Abl kinase in vitro and in vivo by a Google Scholar, M. J. a tyrosine kinase the growth of cells and fusion PubMed Google Scholar, T. P. J. for inhibition of tyrosine 2000; PubMed Scopus Google Scholar, has tyrosine kinase activity that is by of the and by regulatory 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). we have three kinds of protein profiling using or The method is on the mobility shift of phosphorylated proteins from the nonphosphorylated a kinase the of phosphorylated and nonphosphorylated proteins determined using or protein phosphorylation occurs of a target protein, the monophosphorylated and nonphosphorylated proteins are as migration bands on In the of the phosphorylated as multiple bands, on the phosphorylation such as the number and of the phosphate first application is in vitro kinase activity profiling for the analysis of varied phosphoprotein isotypes in a phosphorylation status. determined the activity profiles of six kinds of Alzheimer kinases, and using a substrate protein, Tau, which has a number of phosphorylation sites. Each kinase the kinase-specific gel from nonphosphorylated Tau due to differences in the phosphorylation sites and stoichiometry. the the phosphorylation status and the biological antibodies or have been these are for the analysis of varied phosphoprotein isotypes in a phosphorylation status. established method the detection of the isotypes by kinase activities in a polyacrylamide The immunoblotting using the site-specific Tau antibodies the of phosphorylation of Tau and that progress in by application and such as mass A peptide analysis of the phosphorylated Tau by is in kinase activity profiling on the kinase novel on the protein phosphorylation and the biological second application is in vivo kinase activity profiling for the analysis of protein phosphorylation involved in intracellular signal transduction. The time course changes of phosphorylation levels of Shc and in A431 cells were visualized as highly up-shifted migration bands by subsequent immunoblotting with anti-Shc and antibodies. demonstrated the of to phosphorylated in vivo in a from a nonphosphorylated in the presence of method an in profiling the phosphorylation state of in a complex biological it is to the phosphorylation events by in has been to a protein is shift and proteins that are to phosphorylated gel in In up-shifted of phosphorylated MAPK was of In was to gel of varied phosphoprotein of the method is of for of the phosphorylation state of third application is in vitro kinase inhibition profiling for the quantitative screening of kinase-specific inhibitors. demonstrated the inhibition profile of the tyrosine kinase Abl using the substrate Abltide-GST and the specific The dose-dependent inhibition of Glivec was determined by an in the ratio of the monophosphorylated substrate slower migration and the nonphosphorylated faster in an The inhibition showed an IC50 value of 1.6 in the presence of 0.10 mm ATP. that application enables quantitative analysis of kinase activities to the inhibition IC50 of Glivec with Abl in vitro have been to μm J. H. T. M. of the Abl kinase in vitro and in vivo by a Google μm J. M. kinase activities by using a peptide and mass PubMed Scopus Google and μm has tyrosine kinase activity that is by of the and by regulatory 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). in the differences in Abl ATP or kinase inhibition profiling in for A to phosphatase inhibition profiling using an phosphorylated which is of the important the of Many isotypes by phosphorylation site and stoichiometry a number of biological A. Hasegawa M. Suzuki M. Takio K. M. Titani K. T. Ihara Y. In vivo phosphorylation sites in and Full Text PDF PubMed Google Scholar, kinase an of as the phosphate Full Text PDF PubMed Google Scholar, M. A. and of by PubMed Scopus Google Scholar, T. K. phosphorylation of by protein kinase A and of by protein phosphatase and 2002; PubMed Scopus Google Scholar, K. J. to A. PubMed Scopus Google Scholar, H. K. of and phosphorylation by Neurosci. PubMed Scopus Google Scholar, M. K. H. T. Y. H. T. T. of of phosphorylation in PubMed Scopus Google Scholar, Y. Y. H. A regulatory phosphorylation for of the complex J. PubMed Scopus Google Scholar). Hyperphosphorylation of a certain protein cells or and It has been difficult to the role of isotypes such the complex the for the of the isotypes of are very important in in biological and by using phosphate affinity i.e. the of the detection of protein phosphorylation and the method in phosphorylation events and a in the of the In we have three kinds of protein profiling using or The method is on the mobility shift of phosphorylated proteins from the nonphosphorylated a kinase the of phosphorylated and nonphosphorylated proteins determined using or protein phosphorylation occurs of a target protein, the monophosphorylated and nonphosphorylated proteins are as migration bands on In the of the phosphorylated as multiple bands, on the phosphorylation such as the number and of the phosphate The first application is in vitro kinase activity profiling for the analysis of varied phosphoprotein isotypes in a phosphorylation status. determined the activity profiles of six kinds of Alzheimer kinases, and using a substrate protein, Tau, which has a number of phosphorylation sites. Each kinase the kinase-specific gel from nonphosphorylated Tau due to differences in the phosphorylation sites and stoichiometry. the the phosphorylation status and the biological antibodies or have been these are for the analysis of varied phosphoprotein isotypes in a phosphorylation status. established method the detection of the isotypes by kinase activities in a polyacrylamide The immunoblotting using the site-specific Tau antibodies the of phosphorylation of Tau and that progress in by application and such as mass A peptide analysis of the phosphorylated Tau by is in kinase activity profiling on the kinase novel on the protein phosphorylation and the biological The second application is in vivo kinase activity profiling for the analysis of protein phosphorylation involved in intracellular signal transduction. The time course changes of phosphorylation levels of Shc and in A431 cells were visualized as highly up-shifted migration bands by subsequent immunoblotting with anti-Shc and antibodies. demonstrated the of to phosphorylated in vivo in a from a nonphosphorylated in the presence of method an in profiling the phosphorylation state of in a complex biological it is to the phosphorylation events by in has been to a protein is shift and proteins that are to phosphorylated gel in In up-shifted of phosphorylated MAPK was of In was to gel of varied phosphoprotein of the method is of for of the phosphorylation state of The third application is in vitro kinase inhibition profiling for the quantitative screening of kinase-specific inhibitors. demonstrated the inhibition profile of the tyrosine kinase Abl using the substrate Abltide-GST and the specific The dose-dependent inhibition of Glivec was determined by an in the ratio of the monophosphorylated substrate slower migration and the nonphosphorylated faster in an The inhibition showed an IC50 value of 1.6 in the presence of 0.10 mm ATP. that application enables quantitative analysis of kinase activities to the inhibition IC50 of Glivec with Abl in vitro have been to μm J. H. T. M. of the Abl kinase in vitro and in vivo by a Google μm J. M. kinase activities by using a peptide and mass PubMed Scopus Google and μm has tyrosine kinase activity that is by of the and by regulatory 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). in the differences in Abl ATP or kinase inhibition profiling in for A to phosphatase inhibition profiling using an phosphorylated protein. Protein which is of the important the of Many isotypes by phosphorylation site and stoichiometry a number of biological A. Hasegawa M. Suzuki M. Takio K. M. Titani K. T. Ihara Y. In vivo phosphorylation sites in and Full Text PDF PubMed Google Scholar, kinase an of as the phosphate Full Text PDF PubMed Google Scholar, M. A. and of by PubMed Scopus Google Scholar, T. K. phosphorylation of by protein kinase A and of by protein phosphatase and 2002; PubMed Scopus Google Scholar, K. J. to A. PubMed Scopus Google Scholar, H. K. of and phosphorylation by Neurosci. PubMed Scopus Google Scholar, M. K. H. T. Y. H. T. T. of of phosphorylation in PubMed Scopus Google Scholar, Y. Y. H. A regulatory phosphorylation for of the complex J. PubMed Scopus Google Scholar). Hyperphosphorylation of a certain protein cells or and It has been difficult to the role of isotypes such the complex the for the of the isotypes of are very important in in biological and by using phosphate affinity i.e. the of the detection of protein phosphorylation and the method in phosphorylation events and a in the of the