scholarly journals Calculation of centralities in protein kinase A

2022 ◽  
Author(s):  
Alexandr P Kornev ◽  
Phillip Aoto ◽  
Susan Taylor
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Mutual Information ◽  
Protein Kinase A ◽  
Topological Analysis ◽  
Correlation Coefficients ◽  
Eigenvector Centrality ◽  
Simple Fact ◽  
Centrality Metrics ◽  
Kinase A

Topological analysis of amino acid networks is a common method that can help to understand the roles of individual residues. The most popular approach for network construction is to create a connection between residues if they interact. These interactions are usually weighted by absolute values of correlation coefficients or mutual information. Here we argue that connections in such networks have to reflect levels of cohesion within the protein instead of a simple fact of interaction between residues. If this is correct, an indiscriminate combination of correlation and anti-correlation, as well as the all-inclusive nature of the mutual information metrics, should be detrimental for the analysis. To test our hypothesis, we studied amino acid networks of the protein kinase A created by Local Spatial Pattern alignment, a method that can detect conserved patterns formed by Cα-Cβ vectors. Our results showed that, in comparison with the traditional methods, this approach is more efficient in detecting functionally important residues. Out of four studied centrality metrics, Closeness centrality was the least efficient measure of residue importance. Eigenvector centrality proved to be ineffective as the spectral gap values of the networks were very low due to the bilobal structure of the kinase. We recommend using joint graphs of Betweenness centrality and Degree centrality to visualize different aspects of amino acid roles.

Mutants of protein kinase A that mimic the ATP-binding site of Aurora kinase

2011 ◽  
Vol 440 (1) ◽  
pp. 85-93 ◽  
Author(s):  
Alexander Pflug ◽  
Taianá Maia de Oliveira ◽  
Dirk Bossemeyer ◽  
Richard A. Engh
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Binding Site ◽  
Protein Kinase A ◽  
Aurora Kinase ◽  
Atp Binding ◽  
Atp Binding Site ◽  
High Affinity ◽  
Specific Inhibitors ◽  
Kinase A

We describe in the present paper mutations of the catalytic subunit α of PKA (protein kinase A) that introduce amino acid side chains into the ATP-binding site and progressively transform the pocket to mimic that of Aurora protein kinases. The resultant PKA variants are enzymatically active and exhibit high affinity for ATP site inhibitors that are specific for Aurora kinases. These features make the Aurora-chimaeric PKA a valuable tool for structure-based drug discovery tasks. Analysis of crystal structures of the chimaera reveal the roles for individual amino acid residues in the binding of a variety of inhibitors, offering key insights into selectivity mechanisms. Furthermore, the high affinity for Aurora kinase-specific inhibitors, combined with the favourable crystallizability properties of PKA, allow rapid determination of inhibitor complex structures at an atomic resolution. We demonstrate the utility of the Aurora-chimaeric PKA by measuring binding kinetics for three Aurora kinase-specific inhibitors, and present the X-ray structures of the chimaeric enzyme in complex with VX-680 (MK-0457) and JNJ-7706621 [Aurora kinase/CDK (cyclin-dependent kinase) inhibitor].

scholarly journals Nβ-methylation changes the recognition pattern of aza-β3-amino acid containing peptidomimetic substrates by protein kinase A

2011 ◽  
Vol 1 (1) ◽  
pp. 16 ◽  
Author(s):  
Ksenija Kisseljova ◽  
Michèle Baudy-Floc'h ◽  
Aleksei Kuznetsov ◽  
Jaak Järv
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Recognition Pattern ◽  
Kinase A

scholarly journals Identifying protein kinase target preferences using mass spectrometry

2012 ◽  
Vol 303 (7) ◽  
pp. C715-C727 ◽  
Author(s):  
Jacqueline Douglass ◽  
Ruwan Gunaratne ◽  
Davis Bradford ◽  
Fahad Saeed ◽  
Jason D. Hoffert ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Amino Acid ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Protein Kinases ◽  
Phosphorylation Site ◽  
General Question ◽  
Computational Tool ◽  
Kinase A

A general question in molecular physiology is how to identify candidate protein kinases corresponding to a known or hypothetical phosphorylation site in a protein of interest. It is generally recognized that the amino acid sequence surrounding the phosphorylation site provides information that is relevant to identification of the cognate protein kinase. Here, we present a mass spectrometry-based method for profiling the target specificity of a given protein kinase as well as a computational tool for the calculation and visualization of the target preferences. The mass spectrometry-based method identifies sites phosphorylated in response to in vitro incubation of protein mixtures with active recombinant protein kinases followed by standard phosphoproteomic methodologies. The computational tool, called “PhosphoLogo,” uses an information-theoretic algorithm to calculate position-specific amino acid preferences and anti-preferences from the mass-spectrometry data ( http://helixweb.nih.gov/PhosphoLogo/ ). The method was tested using protein kinase A (catalytic subunit α), revealing the well-known preference for basic amino acids in positions −2 and −3 relative to the phosphorylated amino acid. It also provides evidence for a preference for amino acids with a branched aliphatic side chain in position +1, a finding compatible with known crystal structures of protein kinase A. The method was also employed to profile target preferences and anti-preferences for 15 additional protein kinases with potential roles in regulation of epithelial transport: CK2, p38, AKT1, SGK1, PKCδ, CaMK2δ, DAPK1, MAPKAPK2, PKD3, PIM1, OSR1, STK39/SPAK, GSK3β, Wnk1, and Wnk4.

scholarly journals High-affinity AKAP7δ–protein kinase A interaction yields novel protein kinase A-anchoring disruptor peptides

2006 ◽  
Vol 396 (2) ◽  
pp. 297-306 ◽  
Author(s):  
Christian Hundsrucker ◽  
Gerd Krause ◽  
Michael Beyermann ◽  
Anke Prinz ◽  
Bastian Zimmermann ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Binding Domain ◽  
Full Length ◽  
Amino Acid Residues ◽  
High Affinity ◽  
Binding Domains ◽  
Kinase A

PKA (protein kinase A) is tethered to subcellular compartments by direct interaction of its regulatory subunits (RI or RII) with AKAPs (A kinase-anchoring proteins). AKAPs preferentially bind RII subunits via their RII-binding domains. RII-binding domains form structurally conserved amphipathic helices with unrelated sequences. Their binding affinities for RII subunits differ greatly within the AKAP family. Amongst the AKAPs that bind RIIα subunits with high affinity is AKAP7δ [AKAP18δ; Kd (equilibrium dissociation constant) value of 31 nM]. An N-terminally truncated AKAP7δ mutant binds RIIα subunits with higher affinity than the full-length protein presumably due to loss of an inhibitory region [Henn, Edemir, Stefan, Wiesner, Lorenz, Theilig, Schmidtt, Vossebein, Tamma, Beyermann et al. (2004) J. Biol. Chem. 279, 26654–26665]. In the present study, we demonstrate that peptides (25 amino acid residues) derived from the RII-binding domain of AKAP7δ bind RIIα subunits with higher affinity (Kd=0.4±0.3 nM) than either full-length or N-terminally truncated AKAP7δ, or peptides derived from other RII binding domains. The AKAP7δ-derived peptides and stearate-coupled membrane-permeable mutants effectively disrupt AKAP–RII subunit interactions in vitro and in cell-based assays. Thus they are valuable novel tools for studying anchored PKA signalling. Molecular modelling indicated that the high affinity binding of the amphipathic helix, which forms the RII-binding domain of AKAP7δ, with RII subunits involves both the hydrophobic and the hydrophilic faces of the helix. Alanine scanning (25 amino acid peptides, SPOT technology, combined with RII overlay assays) of the RII binding domain revealed that hydrophobic amino acid residues form the backbone of the interaction and that hydrogen bond- and salt-bridge-forming amino acid residues increase the affinity of the interaction.

Regulation of spontaneous and induced resumption of meiosis in mouse oocytes by different intracellular pathways

Reproduction ◽  
2000 ◽  
pp. 377-383 ◽  
Author(s):  
L Leonardsen ◽  
A Wiersma ◽  
M Baltsen ◽  
AG Byskov ◽  
CY Andersen
Keyword(s):  
Signal Transduction ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Mitogen Activated Protein Kinase ◽  
Meiotic Maturation ◽  
Mouse Oocytes ◽  
Mitogen Activated Protein ◽  
Dependent Signal ◽  
Kinase Pathway ◽  
Kinase A

The mitogen-activated protein kinase-dependent and the cAMP-protein kinase A-dependent signal transduction pathways were studied in cultured mouse oocytes during induced and spontaneous meiotic maturation. The role of the mitogen-activated protein kinase pathway was assessed using PD98059, which specifically inhibits mitogen-activated protein kinase 1 and 2 (that is, MEK1 and MEK2), which activates mitogen-activated protein kinase. The cAMP-dependent protein kinase was studied by treating oocytes with the protein kinase A inhibitor rp-cAMP. Inhibition of the mitogen-activated protein kinase pathway by PD98059 (25 micromol l(-1)) selectively inhibited the stimulatory effect on meiotic maturation by FSH and meiosis-activating sterol (that is, 4,4-dimethyl-5alpha-cholest-8,14, 24-triene-3beta-ol) in the presence of 4 mmol hypoxanthine l(-1), whereas spontaneous maturation in the absence of hypoxanthine was unaffected. This finding indicates that different signal transduction mechanisms are involved in induced and spontaneous maturation. The protein kinase A inhibitor rp-cAMP induced meiotic maturation in the presence of 4 mmol hypoxanthine l(-1), an effect that was additive to the maturation-promoting effect of FSH and meiosis-activating sterol, indicating that induced maturation also uses the cAMP-protein kinase A-dependent signal transduction pathway. In conclusion, induced and spontaneous maturation of mouse oocytes appear to use different signal transduction pathways.

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