Oral Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

In Vitro Profiling of Ser/Thr/Tyr Selectivity of Human Protein Kinome (#167)

Naoyuki Sugiyama 1 , Kazutaka Yachigo 1 , Masato Tsuyuguchi 2 , Takayoshi Kinoshita 2 , Yasushi Ishihama 1
  1. Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, KYOTO, Japan
  2. Graduate School of Science, Osaka Prefecture University, Osaka

Protein phosphorylation mediated by protein kinases play significant roles for cellular signal transduction networks. Human genome sequence analysis has revealed at least 518 genes encode protein kinases. These protein kinases have a phosphoacceptor preference, namely that serine/threonine kinases (STKs) and tyrosine kinases (TKs) specifically phosphorylate Ser/Thr and Tyr, respectively, and most STKs show Ser preference rather than Thr preference. There are some reports describing correlations between phosphoacceptor preference and amino acid sequence in a kinase activation loop1,2, however it still remains unclear how the Ser/Thr/Tyr (S/T/Y) preferences of kinases arise. Here we investigated the phosphoacceptor preferences of human protein kinome by using in vitro kinase assay and phosphoproteomic approach, and extracted rules to determine S/T/Y preference from the kinome sequences.

HeLa cell lysate was dephosphorylated with alkaline phosphatase, and reacted individually with a recombinant human protein kinase. Tryptic digest was isotopically labeled with formaldehyde to distinguish substrates and endogenous phosphosites. Phosphopeptides were enriched with hydroxy acid-modified titanium dioxide chromatography and then analyzed with nanoLC/MS/MS.

We profiled 354 kinds of wild-type human protein kinases by using the in vitro kinase assay. Most STKs were strongly directed to Ser as expected, but MAPK, CDK families and some kinases had a relatively low Ser preference. Furthermore, most of STE group and a portion of the kinases belonging to TKL and others groups preferentially phosphorylated Thr. As reported by Chen et al1, the amino acid residues following the common motif “DFG” in the kinase activation loop were highly correlated with Ser/Thr preference. We also found other amino acids governing S/T/Y preference in the kinase activation loop and experimentally validated them by in vitro assay with mutant kinases and synthetic peptides.

  1. Chen, C. et al. Mol Cell 53, 140-147 (2014)
  2. van de Kooij B et al. Elife 8: e44635 (2019)