Ahnak1 interaction is affected by phosphorylation of Ser-296 on Cav尾₂

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• 作者：Ines Pankonien^a ; ^b ; ^{i_pankonien@hotmail.com} ; Albrecht Otto^a ; ^{alot@mdc-berlin.de} ; Nathan Dascal^c ; ^{dascaln@post.tau.ac.il} ; Ingo Morano^a ; ^b ; ^{imorano@mdc-berlin.de} ; Hannelore Haase^a ; ^{haase@mdc-berlin.de}
• 关键词：Ahnak ; Cav尾2 ; L-type calcium channel ; PKA phosphorylation ; Protein&ndash ; protein interaction
• 刊名：Biochemical and Biophysical Research Communications
• 出版年：2012
• 期刊代码：159_0006291x
• 类别：bio
• 出版时间：4 May, 2012
• 卷：421
• 期：2
• 页码：184-189
• 文件大小：727 K

摘要

Ahnak1 has been implicated in protein kinase A (PKA)-mediated control of cardiac L-type Ca²⁺ channels (Cav1.2) through its interaction with the Cav尾₂ regulatory channel subunit. Here we corroborate this functional linkage by immunocytochemistry on isolated cardiomyocytes showing co-localization of ahnak1 and Cav尾₂ in the T-tubule system. In previous studies Cav尾₂ attachment sites which impacted the channel鈥檚 PKA regulation have been located to ahnak1鈥檚 proximal C-terminus (ahnak1^4889-5535, ahnak1^5462-5535). In this study, we mapped the ahnak1-interacting regions in Cav尾₂ and investigated whether Cav尾₂ phosphorylation affects its binding behavior. In vitro binding assays with Cav尾₂ truncation mutants and ahnak1^4889-5535 revealed that the core region of Cav尾₂ consisting of Src-homology 3 (SH3), HOOK, and guanylate kinase (GK) domains was important for ahnak1 interaction while the C- and N-terminal regions were dispensable. Furthermore, Ser-296 in the GK domain of Cav尾₂ was identified as novel PKA phosphorylation site by mass spectrometry. Surface plasmon resonance (SPR) binding analysis showed that Ser-296 phosphorylation did not affect the high affinity interaction (K_D 鈮?#xA0;35 nM) between Cav尾₂ and the 伪_1C I-II linker, but affected ahnak1 interaction in a complex manner. SPR experiments with ahnak1^5462-5535 revealed that PKA phosphorylation of Cav尾₂ significantly increased the binding affinity and, in parallel, it reduced the binding capacity. Intriguingly, the phosphorylation mimic substitution Glu-296 fully reproduced both effects, increased the affinity by 鈮?.4-fold and reduced the capacity by 鈮?0%. Our results are indicative for the release of a population of low affinity interaction sites following Cav尾₂ phosphorylation on Ser-296. We propose that this phosphorylation event is one mechanism underlying ahnak1麓s modulator function on Cav1.2 channel activity.