Aufsatz
Molecular Basis for Ser/Thr Specificity in PKA Signaling
Abstract
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.
Sponsorship
Gefördert durch den Publikationsfonds der Universität KasselCitation
@article{doi:10.17170/kobra-202007011392,
author={Knape, Matthias Joseph and Wallbott, Maximilian and Burghardt, Nicole C. G. and Bertinetti, Daniela and Hornung, Jan and Schmidt, Sven H. and Lorenz, Robin and Herberg, Friedrich Wilhelm},
title={Molecular Basis for Ser/Thr Specificity in PKA Signaling},
journal={Cells},
year={2020}
}
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3000 Knape, Matthias Joseph
3010 Wallbott, Maximilian
3010 Burghardt, Nicole C. G.
3010 Bertinetti, Daniela
3010 Hornung, Jan
3010 Schmidt, Sven H.
3010 Lorenz, Robin
3010 Herberg, Friedrich Wilhelm
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<resource xsi:schemaLocation="http://datacite.org/schema/kernel-2.2 http://schema.datacite.org/meta/kernel-2.2/metadata.xsd"> 2020-07-02T09:41:53Z 2020-07-02T09:41:53Z 2020-06-25 doi:10.17170/kobra-202007011392 http://hdl.handle.net/123456789/11620 Gefördert durch den Publikationsfonds der Universität Kassel eng Namensnennung 4.0 International http://creativecommons.org/licenses/by/4.0/ cAMP-dependent protein kinase PKA cAMP signaling protein kinases kinase function phosphorylation substrate specificity Ser/Thr specificity surface plasmon resonance 540 Molecular Basis for Ser/Thr Specificity in PKA Signaling Aufsatz 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. open access Knape, Matthias Joseph Wallbott, Maximilian Burghardt, Nicole C. G. Bertinetti, Daniela Hornung, Jan Schmidt, Sven H. Lorenz, Robin Herberg, Friedrich Wilhelm doi:10.3390/cells9061548 Proteinkinase A Proteinkinasen Phosphorylierung publishedVersion EISSN 2073-4409 Issue 6 Cells 1548 Volume 9 false </resource>
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