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.
Highlights
Signaling via the second messenger 30,50 -cyclic adenosine monophosphate is a common concept of eukaryotic signal transduction. cAMP signaling regulates a myriad of physiological conditions such as the metabolism of glycogen and lipids, long-term potentiation, and endocrine function [1,2]
Using a spectrophotometric kinase assay, we recapitulated that protein kinase (PKA) Cα phosphorylates the peptide substrate S-Kemptide with lower KM and higher kcat compared to T-Kemptide (Table 1, Figure S1) [20,29]
Discussion cAMP-dependent protein kinase is the main receptor of the second messenger cAMP and plays cAMP-dependent protein kinase is the main receptor of the second messenger cAMP and plays a critical role in various signaling pathways controlling energy metabolism, neurophysiology, and a critical role in various signaling pathways controlling energy metabolism, neurophysiology, and endocrine response
Summary
Signaling via the second messenger 30 ,50 -cyclic adenosine monophosphate (cAMP) is a common concept of eukaryotic signal transduction. cAMP signaling regulates a myriad of physiological conditions such as the metabolism of glycogen and lipids, long-term potentiation, and endocrine function [1,2]. Signaling via the second messenger 30 ,50 -cyclic adenosine monophosphate (cAMP) is a common concept of eukaryotic signal transduction. CAMP signaling regulates a myriad of physiological conditions such as the metabolism of glycogen and lipids, long-term potentiation, and endocrine function [1,2]. Several human diseases have recently been linked to abnormalities of the cAMP signaling pathway [3]. Binding of a first messenger such as epinephrine to its respective seven-transmembrane G protein-coupled receptor leads to the dissociation of a Gsα protein, which in turn activates adenylate cyclases to produce cAMP out of ATP. CAMP binds to and activates effector proteins. The major receptor of cAMP is the cAMP-dependent protein kinase (PKA) [4,5]. PKA forms a heterotetramer consisting of a regulatory (R)
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