C02 Identification of novel kinase targets up- and downstream of mTOR by Kinase Correlation Profiling

26

27

  • B01
    • Walz
    • Kammerer
  • B02
    • Köttgen
  • B04
    • Bergmann
  • B06
    • Lausch
    • Schmidts
  • B07
    • Lienkamp
    • Arnold

28

29

  • Z01
    • Nitschke
    • Eimer
  • Z02
    • Brox
    • Ronneberger
Jörn Dengjel

Jörn Dengjel

Principal investigator of

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease and the fourth most single cause of end-stage renal disease. Major treatment strategies aim to (a) reduce cAMP levels, (b) inhibit mTOR and cell proliferation, and (c) reduce fluid secretion. Several drugs that are currently being tested in preclinical or clinical trials target kinases that are known to be important in ADPKD pathology. However, knowledge about up- and downstream targets of respective kinases (e.g. mTOR) and their functional role in ADPKD is rather limited. Importantly, signaling pathways being deregulated in ADPKD, such as mTOR signaling and the regulation of autophagy, have also been identified to play a general role in the maintenance of specialized kidney epithelia including tubular cells and glomerular podocytes. We plan to employ mass spectrometry (MS)-based phosphoproteomics to comprehensively identify targets downstream of kinases promoting ADPKD and kidney disease progression. The overall goal is to identify direct targets of kinases upstream of mTOR, including AMP-activated protein kinase (AMPK), B-Raf, and epidermal growth factor receptor (EGFR), and downstream targets of ULK1, a kinase phosphorylated by mTOR involved in energy sensing and autophagosome formation. Therefore, we will utilize Kinase Correlation Profiling, which combines kinase activity modulation by pharmacological modulators, shRNA-based knockdown, or overexpression with high-resolution, high-accuracy MS-based quantitative phosphoproteomics. To identify direct in vivo substrates stimulus specific and time-resolved quantitative phosphoproteomics data will be acquired. Based on the quantitative information attenuation or accentuation profiles of phosphorylation sites can be obtained and direct targets of the inhibited/activated kinase will be found. Phosphorylation profiles of known substrates can be used for correlation between established and putatively novel substrates that will also be analyzed by in vitro kinase assays. Functional studies will test for the relevance of newly identified phosphosites and target proteins addressing cell proliferation, renal epithelial maintenance and ADPKD progression.