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Signaling through dynamic linkers as revealed by PKA.

ABSTRACT: Protein kinase A (PKA) is a prototype of multidomain signaling proteins functioning as allosteric conformational switches. Allosteric transitions have been the subject of extensive structural and dynamic investigations focusing mainly on folded domains. However, the current understanding of the allosteric role of partially unstructured linkers flanking globular domains is limited. Here, we show that a dynamic linker in the regulatory subunit (R) of PKA serves not only as a passive covalent thread, but also as an active allosteric element that controls activation of the kinase subunit (C) by tuning the inhibitory preequilibrium of a minimally populated intermediate (apo R). Apo R samples both C-binding competent (inactive) and incompetent (active) conformations within a nearly degenerate free-energy landscape and such degeneracy maximally amplifies the response to weak (?2RT), but conformation-selective interactions elicited by the linker. Specifically, the R linker that in the R:C complex docks in the active site of C in apo R preferentially interacts with the C-binding incompetent state of the adjacent cAMP-binding domain (CBD). These unanticipated findings imply that the formation of the intermolecular R:C inhibitory interface occurs at the expense of destabilizing the intramolecular linker/CBD interactions in R. A direct implication of this model, which was not predictable solely based on protein structure, is that the disruption of a linker/CBD salt bridge in the R:C complex unexpectedly leads to increased affinity of R for C. The linker includes therefore sites of R:C complex frustration and frustration-relieving mutations enhance the kinase inhibitory potency of R without compromising its specificity.

SUBMITTER: Akimoto M

PROVIDER: S-EPMC3761587 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Signaling through dynamic linkers as revealed by PKA.

Akimoto Madoka M Selvaratnam Rajeevan R McNicholl E Tyler ET Verma Geeta G Taylor Susan S SS Melacini Giuseppe G

Proceedings of the National Academy of Sciences of the United States of America 20130814 35

Protein kinase A (PKA) is a prototype of multidomain signaling proteins functioning as allosteric conformational switches. Allosteric transitions have been the subject of extensive structural and dynamic investigations focusing mainly on folded domains. However, the current understanding of the allosteric role of partially unstructured linkers flanking globular domains is limited. Here, we show that a dynamic linker in the regulatory subunit (R) of PKA serves not only as a passive covalent threa ...[more]

PMID: 23946424

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Project description:BackgroundExcitotoxicity plays a key role in nervous system disease and can trigger a critical cascade of reaction which affects cell viability and promotes neuronal death. Tetramethylpyrazine (TMP) reveals its effect in the treatment of neurovascular diseases by antiapoptosis. Recently, there were several studies that demonstrated that the PKA/CREB signaling pathway played a role in neural disease because of excitotoxicity, such as stroke, AD, and Parkinson's disease. In this study, we wanted to focus on the protective effect of tetramethylpyrazine against excitotoxicity through the PKA/CREB signaling pathway.MethodsIn order to verify whether tetramethylpyrazine can attenuate excitotoxicity through the PKA/CREB signaling pathway, we first used molecular docking technology to predict the combinational strength and mode of tetramethylpyrazine with the proteins in the PKA/CREB signaling pathway. Then, we determined the optimal concentration and time according to the model effect of glutamate (Glu) with different concentration gradients and action times in PC12 cells. After the determination of concentration and time of glutamate in the previous step as the model way, tetramethylpyrazine was added to determine its influence on the cell viability under different doses and times. The TUNEL assay and flow cytometry were used to detect apoptosis. RT-PCR was used to detect the expression of Bcl-2, Bax, PKA, and 5CREB genes, and Western blot was used to detect the expression of these factors.ResultTetramethylpyrazine had a good docking score (-5.312) with PKA and had a moderately docking score (-3.838) with CREB. The CCK-8 cell activity assay showed that the activity of PC12 cells decreased gradually with the increase in glutamate concentration and time, and PC12 cells were treated with 10 mM/L glutamate (the half of the inhibitory concentration (IC50)) for 12 hours. Then, the cell viability increased gradually following the increased concentration of tetramethylpyrazine. When PC12 cells were treated with 0.1 mM/L tetramethylpyrazine, the cell viability was increased significantly compared with the control group (P < 0.05). The TUNEL assay and flow cytometry also showed that tetramethylpyrazine could decrease the apoptosis induced by glutamate. In the result of RT-PCR, the transcriptional levels of Bcl-2, PKA, and CREB were increased and Bax was decreased. Meanwhile, Western blot showed that expression levels of Bcl-2, PKA, CREB, and p-CREB were increased and Bax was decreased.ConclusionsThis study provided evidence that tetramethylpyrazine can protect against apoptosis caused by neuroexcitotoxicity and the protective mechanism is closely related to the activation of the PKA/CREB signaling pathway.

Project description:Introduction:This study aimed to investigate the role of ?2 adrenergic receptor (?2AR) in insulin signaling transduction in H9C2 cardiomyoblast cells to understand the formation of the ?2AR-insulin receptor (IR) protein complex and its role in insulin-induced Glut4 expression. Methods:H9C2 cells were treated with various protein inhibitors (CGP, ?1AR inhibitor CGP20712; ICI, ?2AR inhibitor ICI 118,551; PKI, PKA inhibitor myristoylated PKI; PD 0325901, MEK inhibitor; SP600125, JNK inhibitor) with or without insulin or isoproterenol (ISO) before RNA-sequencing (RNA-Seq) and quantitative-PCR (Q-PCR). Yeast two-hybrid, co-immunoprecipitation and His-tag pull-down assay were carried out to investigate the formation of the ?2AR-IR protein complex. The intracellular concentrations of cAMP in H9C2 cells were tested by high performance liquid chromatography (HPLC) and the phosphorylation of JNK was tested by Western blot. Results:Gene Ontology (GO) analysis revealed that the most significantly enriched processes in the domain of molecular function (MF) were catalytic activity and binding, whereas in the domain of biological processes (BP) were metabolic process and cellular process. Furthermore, the enriched processes in the domain of cellular components (CC) were cell and cell parts. The Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that the most significant pathways that have been altered included the PI3K-Akt and MAPK signaling pathways. Q-PCR, which was performed to verify the gene expression levels exhibited consistent results. In evaluating the signaling pathways, the sustained stimulation of ?2AR by ISO inhibited insulin signalling, and the effect was primarily through the cAMP-PKA-JNK pathway and MEK/JNK signaling pathway. Yeast two-hybrid, co-immunoprecipitation and His-tag pull-down assay revealed that ?2AR, IR, insulin receptor substrate 1 (IRS1), Grb2-associated binding protein 1 (GAB1) and Grb2 existed in the same protein complex. Conclusion:The sustained stimulation of ?2AR might inhibit insulin signaling transduction through the cAMP-PKA-JNK and MEK/JNK pathways in H9C2 cells.

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Signaling through dynamic linkers as revealed by PKA.

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Signaling through dynamic linkers as revealed by PKA.

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