Redox Modification of PKA-Cα Differentially Affects Its Substrate Selection.
Redox Modification of PKA-Cα Differentially Affects Its Substrate Selection.
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DOI：
10.3390/life13091811
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发表时间：
2023-08-26
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影响因子：
--
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环腺苷酸依赖性蛋白激酶（cyclicAMP-dependent protein kinase，PKA）在许多重要的细胞过程的调节中起重要作用，并且在一些广泛性疾病中失调，包括糖尿病、心血管疾病和各种神经退行性疾病。以往的研究表明，PKA催化亚基的α亚型（PKA-Cα）在体外和原位都在C199上被氧化。然而，这些修饰对PKA-Cα底物选择的分子后果在很大程度上尚未探索。C199位于PKA-Cα活性位点的P + 1环上，表明氧化还原修饰可能影响其激酶活性。考虑到C199与底物结合口袋的接近性，我们假设氧化可以不同地改变PKA-Cα对其底物的活性。为此，我们使用生物化学（即，转磷酸化测定和稳态动力学分析）和生物物理学（即，表面等离子体共振和荧光偏振测定）策略。这些研究表明，PKA-Cα的氧化还原修饰对不同底物的活性有不同的影响。例如，我们发现二酰胺介导的氧化导致PKA-Cα对某些底物的活性显著降低（例如，Kemptide和CREBtide），而对其他药物（例如，Crosstide）。相反，在相对较低的H2 O2浓度下，PKA-Cα的H2 O2依赖性氧化导致其对每种底物的活性增加，在较高的过氧化物浓度下具有不同的作用。总之，这些研究为PKA介导的氧化还原和磷酸化依赖性信号通路之间的串扰提供了新的见解。同样，由于C199在AGC激酶家族成员中高度保守，它们也为将来旨在阐明生理和病理状态下激酶底物选择的氧化还原依赖性修饰的作用的研究奠定了基础。
The cyclic AMP-dependent protein kinase (PKA) plays an essential role in the regulation of many important cellular processes and is dysregulated in several pervasive diseases, including diabetes, cardiovascular disease, and various neurodegenerative disorders. Previous studies suggest that the alpha isoform of the catalytic subunit of PKA (PKA-Cα) is oxidized on C199, both in vitro and in situ. However, the molecular consequences of these modifications on PKA-Cα’s substrate selection remain largely unexplored. C199 is located on the P + 1 loop within PKA-Cα’s active site, suggesting that redox modification may affect its kinase activity. Given the proximity of C199 to the substrate binding pocket, we hypothesized that oxidation could differentially alter PKA-Cα’s activity toward its substrates. To this end, we examined the effects of diamide- and H2O2-dependent oxidation on PKA-Cα’s activity toward select peptide and protein substrates using a combination of biochemical (i.e., trans-phosphorylation assays and steady-state kinetics analysis) and biophysical (i.e., surface plasmon resonance and fluorescence polarization assays) strategies. These studies suggest that redox modification of PKA-Cα differentially affects its activity toward different substrates. For instance, we found that diamide-mediated oxidation caused a marked decrease in PKA-Cα’s activity toward some substrates (e.g., Kemptide and CREBtide) while having little effect on others (e.g., Crosstide). In contrast, H2O2-dependent oxidation of PKA-Cα led to an increase in its activity toward each of the substrates at relatively low H2O2 concentrations, with differential effects at higher peroxide concentrations. Together, these studies offer novel insights into crosstalk between redox- and phosphorylation-dependent signaling pathways mediated by PKA. Likewise, since C199 is highly conserved among AGC kinase family members, they also lay the foundation for future studies designed to elucidate the role of redox-dependent modification of kinase substrate selection in physiological and pathological states.
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影响因子：
4.8
作者：
Humphries, Kenneth M.;Pennypacker, Juniper K.;Taylor, Susan S.
通讯作者：
Taylor, Susan S.
影响因子：
5.7
作者：
Cao, Baohua;Lu, Tsan-Wen;Zhang, Ping
通讯作者：
Zhang, Ping
影响因子：
4.8
作者：
Brennan, Jonathan P.;Bardswell, Sonya C.;Eaton, Philip
通讯作者：
Eaton, Philip
DOI：
10.3390/antiox10050663
发表时间：
2021-04-24
期刊：
Antioxidants (Basel, Switzerland)
影响因子：
--
作者：
Cuello F;Herberg FW;Stathopoulou K;Henning P;Diering S
通讯作者：
Diering S
影响因子：
13.8
作者：
Omar MH;Scott JD
通讯作者：
Scott JD