A systematic MS-based approach for identifying in vitro substrates of PKA and PKG in rat uteri

Sheng Yu Huang, Mei-Ling Tsai, Guan Yuan Chen, Chin Jen Wu, Shu-Hui Chen

研究成果: Article

45 引文 斯高帕斯(Scopus)

摘要

Protein phosphorylation is an important modulator of many cellular processes, and identification of kinase substrates provides critical insights for signal transduction. However, this identification process is often difficult and many kinase substrates remain unexplored. Herein, a systematic proteomics approach solely depending on MS detection is reported for identifying substrates of PKA and PKG, which are suspected to have similar specificity determinants, in pregnant rat uteri. Instead of radioisotopes that are commonly used to couple with MS for substrate identification, this study developed an efficient in vitro kinase assay on depleted tissue homogenates to reveal substrate candidates directly by MS. To facilitate MS detection, exogenous phosphatases were added to remove intrinsic phosphorylation followed by a heating step to inactivate all enzymes. No observable interference caused by endogenous kinases or background phosphorylation was detected in the control experiment in which no kinase was externally added. A total of 61 and 12 substrate candidates were identified in vitro for PKA and PKG, respectively, and most of these identified sites contain consensus motifs of each kinase with only a few sites overlapped, indicating a good specificity. Moreover, differential phosphoproteomics analysis using stable isotope dimethyl labeling and MS was performed to detect the change of protein phosphorylation upon kinase stimulation in vivo. Four identified in vitro PKA substrates including three reported sites on HSP27 or filamin A were significantly phosphorylated in vivo, giving them high confidences as physiological substrates in pregnant rat uteri. Moreover, telokin, a known PKG substrate on S1880, and actin-binding proteins such as Arp 3, titin, and desmuslin were also identified to be in vitro PKG substrates in pregnant rat uteri. These proteins are all expected to be involved in the regulation of actin-mediated cytoskeletal remodeling.

原文English
頁(從 - 到)2674-2684
頁數11
期刊Journal of Proteome Research
6
發行號7
DOIs
出版狀態Published - 2007 七月 1

    指紋

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Chemistry(all)

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