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

Serine phosphorylation of STAT3 is regulated by oxidation of an adjacent methionine in Ras-driven lung adenocarcinoma (#521)

Dilanka Fernando 1 2 , Daniel Garama 1 2 , Shuai Nie 3 , Daniel Gough 1 2
  1. Department of Molecular and Translational Science, Monash University, 27-31 Wright Street, Clayton, VIC 3168, Australia
  2. Centre for Cancer Research, Hudson Institute of Medical Research, 27-31 Wright Street, Clayton, VIC 3168, Australia
  3. The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, VIC, Australia

Signal Transducer and Activator of Transcription (STAT) 3 is a critical signalling molecule that relays signals in response to cytokines and growth factors to regulate immune responses, metabolism and cell death. Deregulation of STAT3 activity as a transcription factor results in many diseases including cancers. Y705-phosphorylation of STAT3 is dominant for its transcriptional output which is augmented by S727-phosphorylation. Additionally we have discovered a new, non-nuclear function dependent on S727. This S727 site located within a CDK/MAPK consensus motif ie, PMSP can undergo phosphorylation by numerous serine kinases, most prominently via the MEK-ERK pathway. Mass spectrometric analyses of STAT3 post-translational modifications in A549 (Ras-driven lung cancer) cell line revealed that S727-phosphorylation was concurrent with adjacent methionine (M726) oxidation. To determine whether M726 oxidation altered S727 phosphorylation, we undertook a series of experiments. STAT3 mutants were generated that could not undergo oxidation (M726L), or mimicked oxidation (M7276Q) and stably expressed in A549 cells. We observed that the M727L blocked S727-phosphorylation whilst M727Q augmented S727-phosphorylation. Moreover we show that loss of oxidation (M727L) reduced the capacity for anchorage independent growth – indicative of reduced tumour forming potential. We next used a cell free system to directly assess the ability of Erk to phosphorylate STAT3 on S727. In these experiments we combined active, recombinant Erk2 with chemically synthesized peptides that consist of Erk2 recognition motifs in control peptides or from STAT3 and observed the effect of hydrogen peroxide-induced methionine oxidation on phosphorylation of the adjacent serine using LCMS. Our results suggest that oxidation of amino acids adjacent to Erk-catalysed phosphor-sites alters the efficacy of the kinase reaction. This has implications in cancer.  Ras-driven tumours comprise ~25% of all cancers and have elevated levels of reactive oxygen species. Our data suggests that a consequence of this altered redox environment is more permissive signal transduction.