Molecular basis for isoform-specific autoregulation of protein kinase A

详细信息	查看全文 | 推荐本文 |

• 作者：M ; y Diskar ; Hans-Michael Zenn ; Alex ; ra Kaupisch ; Anke Prinz ; Friedrich W. Herberg
• 关键词：cAMP ; PKA ; PrKX ; Autoinhibitory site ; Bioluminescence Resonance Energy Transfer ; Surface Plasmon Resonance
• 刊名：Cellular Signalling
• 出版年：2007
• 期刊代码：42_08986568
• 类别：bio
• 出版时间：October 2007
• 卷：19
• 期：10
• 页码：2024-2034
• 文件大小：1253 K

摘要

Protein kinase A (PKA) isozymes are distinguishable by the inhibitory pattern of their regulatory (R) subunits with RI subunits containing a pseudophosphorylation P⁰-site and RII subunits being a substrate. Under physiological conditions, RII does not inhibit PrKX, the human X chromosome encoded PKA catalytic (C) subunit. Using a live cell Bioluminescence Resonance Energy Transfer (BRET) assay, Surface Plasmon Resonance (SPR) and kinase activity assays, we identified the P⁰-position of the R subunits as the determinant of PrKX autoinhibition. Holoenzyme formation only takes place with an alanine at position P⁰, whereas RI subunits containing serine, phosphoserine or aspartate do not bind PrKX. Surprisingly, PrKX reversibly associates with RII when changing P⁰ from serine to alanine. In contrast, PKA-Cα forms holoenzyme complexes with all wildtype and mutant R subunits; however, holoenzyme re-activation by cAMP is severely affected. Only PKA type II or mutant PKA type I holoenzymes (P⁰: Ser or Asp) are able to dissociate fully upon maximally elevated intracellular cAMP. The data are of particular significance for understanding PKA isoform-specific activation patterns in living cells.