New Candidate Targets of AMP-Activated Protein Kinase in Murine Brain Revealed by a Novel Multidimensional Substrate-Screen for Protein Kinases
AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine kinase that is involved in the maintenance of energy homeostasis and recovery from metabolic stresses both at the cellular and whole body level. AMPK is found in all tissues examined so far, and a number of downstream targets h...
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2020
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| _version_ | 1851600839037157376 |
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| author | Roland D. Tuerk (8660466) |
| author2 | Ramon F. Thali (8660469) Yolanda Auchli (247893) Helene Rechsteiner (8660472) René A. Brunisholz (8660475) Uwe Schlattner (18191) Theo Wallimann (18196) Dietbert Neumann (311099) |
| author2_role | author author author author author author author |
| author_facet | Roland D. Tuerk (8660466) Ramon F. Thali (8660469) Yolanda Auchli (247893) Helene Rechsteiner (8660472) René A. Brunisholz (8660475) Uwe Schlattner (18191) Theo Wallimann (18196) Dietbert Neumann (311099) |
| author_role | author |
| dc.creator.none.fl_str_mv | Roland D. Tuerk (8660466) Ramon F. Thali (8660469) Yolanda Auchli (247893) Helene Rechsteiner (8660472) René A. Brunisholz (8660475) Uwe Schlattner (18191) Theo Wallimann (18196) Dietbert Neumann (311099) |
| dc.date.none.fl_str_mv | 2020-04-02T16:33:45Z |
| dc.identifier.none.fl_str_mv | 10.1021/pr070160a.s002 |
| dc.relation.none.fl_str_mv | https://figshare.com/articles/dataset/New_Candidate_Targets_of_AMP-Activated_Protein_Kinase_in_Murine_Brain_Revealed_by_a_Novel_Multidimensional_Substrate-Screen_for_Protein_Kinases/12067209 |
| dc.rights.none.fl_str_mv | CC BY-NC 4.0 info:eu-repo/semantics/openAccess |
| dc.subject.none.fl_str_mv | Biophysics Biochemistry Cell Biology Genetics Molecular Biology Physiology Pharmacology Biotechnology Immunology Developmental Biology Inorganic Chemistry Infectious Diseases Chemical Sciences not elsewhere classified Physical Sciences not elsewhere classified appetite control AMP-Activated Protein Kinase murine brain lysates column method mass spectrometry Novel Multidimensional Substrate-Screen radiolabeled ATP candidate substrate proteins Recent work phosphorylation events energy homeostasis pilot screen New Candidate Kinase assays AMPK substrates body level Protein Kinases AMP-activated protein kinase brain-specific substrates kinase assays radiolabeled proteins SDS-PAGE Murine Brain Revealed AMPK targets MALDI 53 AMPK-specific phospho-spots |
| dc.title.none.fl_str_mv | New Candidate Targets of AMP-Activated Protein Kinase in Murine Brain Revealed by a Novel Multidimensional Substrate-Screen for Protein Kinases |
| dc.type.none.fl_str_mv | Dataset info:eu-repo/semantics/publishedVersion dataset |
| description | AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine kinase that is involved in the maintenance of energy homeostasis and recovery from metabolic stresses both at the cellular and whole body level. AMPK is found in all tissues examined so far, and a number of downstream targets have been identified. Recent work suggests that AMPK has specialized functions in the brain, such as involvement in appetite control. Nevertheless, brain-specific substrates of AMPK are unknown. Here, we performed a proteomic <i>in vitro</i> screen to identify new putative AMPK targets in brain. Prefractionation of murine brain lysates by liquid chromatography, utilizing four different, serially connected columns with different chemistries was found to be superior to a single column method. A pilot screen involving incubation of small volumes of individual fractions with radiolabeled ATP in the presence or absence of active AMPK, followed by one-dimensional SDS-PAGE and autoradiography, revealed the presence of potential AMPK substrates in a number of different fractions. On the basis of these results, several kinase assays were repeated with selected fractions on a preparative scale. Following separation of the radiolabeled proteins by two-dimensional electrophoresis and comparison of samples with or without added AMPK by differential autoradiography, 53 AMPK-specific phospho-spots were detected and excised. Thereof, 26 unique proteins were identified by mass spectrometry and were considered as new potential downstream targets of AMPK. Kinase assays with 14 highly purified candidate substrate proteins confirmed that at least 12 were direct targets of AMPK <i>in vitro</i>. Although the physiological consequences of these phosphorylation events remain to be established, hypotheses concerning the most intriguing potential targets of AMPK that have been identified by this search are discussed herein. Our data suggests that signaling by AMPK in brain is likely to be involved in the regulation of pathways that have not yet been linked to this kinase. Keywords: AMP-activated protein kinase • phosphorylation • multidimensional separation • liquid chromatography • two-dimensional electrophoresis • phosphoproteomics • MALDI • mass spectrometry • multidimensional substrate-screen for protein kinases • MudSeek |
| eu_rights_str_mv | openAccess |
| id | Manara_1cfbf46756b51f0f276d42cf971ea10c |
| identifier_str_mv | 10.1021/pr070160a.s002 |
| network_acronym_str | Manara |
| network_name_str | ManaraRepo |
| oai_identifier_str | oai:figshare.com:article/12067209 |
| publishDate | 2020 |
| repository.mail.fl_str_mv | |
| repository.name.fl_str_mv | |
| repository_id_str | |
| rights_invalid_str_mv | CC BY-NC 4.0 |
| spelling | New Candidate Targets of AMP-Activated Protein Kinase in Murine Brain Revealed by a Novel Multidimensional Substrate-Screen for Protein KinasesRoland D. Tuerk (8660466)Ramon F. Thali (8660469)Yolanda Auchli (247893)Helene Rechsteiner (8660472)René A. Brunisholz (8660475)Uwe Schlattner (18191)Theo Wallimann (18196)Dietbert Neumann (311099)BiophysicsBiochemistryCell BiologyGeneticsMolecular BiologyPhysiologyPharmacologyBiotechnologyImmunologyDevelopmental BiologyInorganic ChemistryInfectious DiseasesChemical Sciences not elsewhere classifiedPhysical Sciences not elsewhere classifiedappetite controlAMP-Activated Protein Kinasemurine brain lysatescolumn methodmass spectrometryNovel Multidimensional Substrate-Screenradiolabeled ATPcandidate substrate proteinsRecent workphosphorylation eventsenergy homeostasispilot screenNew CandidateKinase assaysAMPK substratesbody levelProtein Kinases AMP-activated protein kinasebrain-specific substrateskinase assaysradiolabeled proteinsSDS-PAGEMurine Brain RevealedAMPK targetsMALDI53 AMPK-specific phospho-spotsAMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine kinase that is involved in the maintenance of energy homeostasis and recovery from metabolic stresses both at the cellular and whole body level. AMPK is found in all tissues examined so far, and a number of downstream targets have been identified. Recent work suggests that AMPK has specialized functions in the brain, such as involvement in appetite control. Nevertheless, brain-specific substrates of AMPK are unknown. Here, we performed a proteomic <i>in vitro</i> screen to identify new putative AMPK targets in brain. Prefractionation of murine brain lysates by liquid chromatography, utilizing four different, serially connected columns with different chemistries was found to be superior to a single column method. A pilot screen involving incubation of small volumes of individual fractions with radiolabeled ATP in the presence or absence of active AMPK, followed by one-dimensional SDS-PAGE and autoradiography, revealed the presence of potential AMPK substrates in a number of different fractions. On the basis of these results, several kinase assays were repeated with selected fractions on a preparative scale. Following separation of the radiolabeled proteins by two-dimensional electrophoresis and comparison of samples with or without added AMPK by differential autoradiography, 53 AMPK-specific phospho-spots were detected and excised. Thereof, 26 unique proteins were identified by mass spectrometry and were considered as new potential downstream targets of AMPK. Kinase assays with 14 highly purified candidate substrate proteins confirmed that at least 12 were direct targets of AMPK <i>in vitro</i>. Although the physiological consequences of these phosphorylation events remain to be established, hypotheses concerning the most intriguing potential targets of AMPK that have been identified by this search are discussed herein. Our data suggests that signaling by AMPK in brain is likely to be involved in the regulation of pathways that have not yet been linked to this kinase. Keywords: AMP-activated protein kinase • phosphorylation • multidimensional separation • liquid chromatography • two-dimensional electrophoresis • phosphoproteomics • MALDI • mass spectrometry • multidimensional substrate-screen for protein kinases • MudSeek2020-04-02T16:33:45ZDatasetinfo:eu-repo/semantics/publishedVersiondataset10.1021/pr070160a.s002https://figshare.com/articles/dataset/New_Candidate_Targets_of_AMP-Activated_Protein_Kinase_in_Murine_Brain_Revealed_by_a_Novel_Multidimensional_Substrate-Screen_for_Protein_Kinases/12067209CC BY-NC 4.0info:eu-repo/semantics/openAccessoai:figshare.com:article/120672092020-04-02T16:33:45Z |
| spellingShingle | New Candidate Targets of AMP-Activated Protein Kinase in Murine Brain Revealed by a Novel Multidimensional Substrate-Screen for Protein Kinases Roland D. Tuerk (8660466) Biophysics Biochemistry Cell Biology Genetics Molecular Biology Physiology Pharmacology Biotechnology Immunology Developmental Biology Inorganic Chemistry Infectious Diseases Chemical Sciences not elsewhere classified Physical Sciences not elsewhere classified appetite control AMP-Activated Protein Kinase murine brain lysates column method mass spectrometry Novel Multidimensional Substrate-Screen radiolabeled ATP candidate substrate proteins Recent work phosphorylation events energy homeostasis pilot screen New Candidate Kinase assays AMPK substrates body level Protein Kinases AMP-activated protein kinase brain-specific substrates kinase assays radiolabeled proteins SDS-PAGE Murine Brain Revealed AMPK targets MALDI 53 AMPK-specific phospho-spots |
| status_str | publishedVersion |
| title | New Candidate Targets of AMP-Activated Protein Kinase in Murine Brain Revealed by a Novel Multidimensional Substrate-Screen for Protein Kinases |
| title_full | New Candidate Targets of AMP-Activated Protein Kinase in Murine Brain Revealed by a Novel Multidimensional Substrate-Screen for Protein Kinases |
| title_fullStr | New Candidate Targets of AMP-Activated Protein Kinase in Murine Brain Revealed by a Novel Multidimensional Substrate-Screen for Protein Kinases |
| title_full_unstemmed | New Candidate Targets of AMP-Activated Protein Kinase in Murine Brain Revealed by a Novel Multidimensional Substrate-Screen for Protein Kinases |
| title_short | New Candidate Targets of AMP-Activated Protein Kinase in Murine Brain Revealed by a Novel Multidimensional Substrate-Screen for Protein Kinases |
| title_sort | New Candidate Targets of AMP-Activated Protein Kinase in Murine Brain Revealed by a Novel Multidimensional Substrate-Screen for Protein Kinases |
| topic | Biophysics Biochemistry Cell Biology Genetics Molecular Biology Physiology Pharmacology Biotechnology Immunology Developmental Biology Inorganic Chemistry Infectious Diseases Chemical Sciences not elsewhere classified Physical Sciences not elsewhere classified appetite control AMP-Activated Protein Kinase murine brain lysates column method mass spectrometry Novel Multidimensional Substrate-Screen radiolabeled ATP candidate substrate proteins Recent work phosphorylation events energy homeostasis pilot screen New Candidate Kinase assays AMPK substrates body level Protein Kinases AMP-activated protein kinase brain-specific substrates kinase assays radiolabeled proteins SDS-PAGE Murine Brain Revealed AMPK targets MALDI 53 AMPK-specific phospho-spots |