Molecular Basis for Ser/Thr Specificity in PKA Signaling
cAMP-dependent protein kinase (PKA) is the major receptor of the second messenger cAMP and a prototype for Ser/Thr-specific protein kinases. Although PKA strongly prefers serine over threonine substrates, little is known about the molecular basis of this substrate specificity. We employ classical en...
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2020-06-01
|
| Series: | Cells |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2073-4409/9/6/1548 |
| _version_ | 1827714269913808896 |
|---|---|
| author | Matthias J. Knape Maximilian Wallbott Nicole C. G. Burghardt Daniela Bertinetti Jan Hornung Sven H. Schmidt Robin Lorenz Friedrich W. Herberg |
| author_facet | Matthias J. Knape Maximilian Wallbott Nicole C. G. Burghardt Daniela Bertinetti Jan Hornung Sven H. Schmidt Robin Lorenz Friedrich W. Herberg |
| author_sort | Matthias J. Knape |
| collection | DOAJ |
| description | cAMP-dependent protein kinase (PKA) is the major receptor of the second messenger cAMP and a prototype for Ser/Thr-specific protein kinases. Although PKA strongly prefers serine over threonine substrates, little is known about the molecular basis of this substrate specificity. We employ classical enzyme kinetics and a surface plasmon resonance (SPR)-based method to analyze each step of the kinase reaction. In the absence of divalent metal ions and nucleotides, PKA binds serine (PKS) and threonine (PKT) substrates, derived from the heat-stable protein kinase inhibitor (PKI), with similar affinities. However, in the presence of metal ions and adenine nucleotides, the Michaelis complex for PKT is unstable. PKA phosphorylates PKT with a higher turnover due to a faster dissociation of the product complex. Thus, threonine substrates are not necessarily poor substrates of PKA. Mutation of the DFG+1 phenylalanine to β-branched amino acids increases the catalytic efficiency of PKA for a threonine peptide substrate up to 200-fold. The PKA Cα mutant F187V forms a stable Michaelis complex with PKT and shows no preference for serine versus threonine substrates. Disease-associated mutations of the DFG+1 position in other protein kinases underline the importance of substrate specificity for keeping signaling pathways segregated and precisely regulated. |
| first_indexed | 2024-03-10T18:53:21Z |
| format | Article |
| id | doaj.art-805e36a159e441b8a7fbffef26b07b35 |
| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-10T18:53:21Z |
| publishDate | 2020-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cells |
| spelling | doaj.art-805e36a159e441b8a7fbffef26b07b352023-11-20T04:58:15ZengMDPI AGCells2073-44092020-06-0196154810.3390/cells9061548Molecular Basis for Ser/Thr Specificity in PKA SignalingMatthias J. Knape0Maximilian Wallbott1Nicole C. G. Burghardt2Daniela Bertinetti3Jan Hornung4Sven H. Schmidt5Robin Lorenz6Friedrich W. Herberg7Department of Biochemistry, University of Kassel, 34132 Kassel, GermanyDepartment of Biochemistry, University of Kassel, 34132 Kassel, GermanyDepartment of Biochemistry, University of Kassel, 34132 Kassel, GermanyDepartment of Biochemistry, University of Kassel, 34132 Kassel, GermanyDepartment of Biochemistry, University of Kassel, 34132 Kassel, GermanyDepartment of Biochemistry, University of Kassel, 34132 Kassel, GermanyDepartment of Biochemistry, University of Kassel, 34132 Kassel, GermanyDepartment of Biochemistry, University of Kassel, 34132 Kassel, GermanycAMP-dependent protein kinase (PKA) is the major receptor of the second messenger cAMP and a prototype for Ser/Thr-specific protein kinases. Although PKA strongly prefers serine over threonine substrates, little is known about the molecular basis of this substrate specificity. We employ classical enzyme kinetics and a surface plasmon resonance (SPR)-based method to analyze each step of the kinase reaction. In the absence of divalent metal ions and nucleotides, PKA binds serine (PKS) and threonine (PKT) substrates, derived from the heat-stable protein kinase inhibitor (PKI), with similar affinities. However, in the presence of metal ions and adenine nucleotides, the Michaelis complex for PKT is unstable. PKA phosphorylates PKT with a higher turnover due to a faster dissociation of the product complex. Thus, threonine substrates are not necessarily poor substrates of PKA. Mutation of the DFG+1 phenylalanine to β-branched amino acids increases the catalytic efficiency of PKA for a threonine peptide substrate up to 200-fold. The PKA Cα mutant F187V forms a stable Michaelis complex with PKT and shows no preference for serine versus threonine substrates. Disease-associated mutations of the DFG+1 position in other protein kinases underline the importance of substrate specificity for keeping signaling pathways segregated and precisely regulated.https://www.mdpi.com/2073-4409/9/6/1548cAMP-dependent protein kinasePKAcAMP signalingprotein kinaseskinase functionphosphorylation |
| spellingShingle | Matthias J. Knape Maximilian Wallbott Nicole C. G. Burghardt Daniela Bertinetti Jan Hornung Sven H. Schmidt Robin Lorenz Friedrich W. Herberg Molecular Basis for Ser/Thr Specificity in PKA Signaling Cells cAMP-dependent protein kinase PKA cAMP signaling protein kinases kinase function phosphorylation |
| title | Molecular Basis for Ser/Thr Specificity in PKA Signaling |
| title_full | Molecular Basis for Ser/Thr Specificity in PKA Signaling |
| title_fullStr | Molecular Basis for Ser/Thr Specificity in PKA Signaling |
| title_full_unstemmed | Molecular Basis for Ser/Thr Specificity in PKA Signaling |
| title_short | Molecular Basis for Ser/Thr Specificity in PKA Signaling |
| title_sort | molecular basis for ser thr specificity in pka signaling |
| topic | cAMP-dependent protein kinase PKA cAMP signaling protein kinases kinase function phosphorylation |
| url | https://www.mdpi.com/2073-4409/9/6/1548 |
| work_keys_str_mv | AT matthiasjknape molecularbasisforserthrspecificityinpkasignaling AT maximilianwallbott molecularbasisforserthrspecificityinpkasignaling AT nicolecgburghardt molecularbasisforserthrspecificityinpkasignaling AT danielabertinetti molecularbasisforserthrspecificityinpkasignaling AT janhornung molecularbasisforserthrspecificityinpkasignaling AT svenhschmidt molecularbasisforserthrspecificityinpkasignaling AT robinlorenz molecularbasisforserthrspecificityinpkasignaling AT friedrichwherberg molecularbasisforserthrspecificityinpkasignaling |