Comparative receptor tyrosine kinase profiling identifies a novel role for AXL in human stem cell pluripotency

Hum Mol Genet. 2014 Apr 1;23(7):1802-16. doi: 10.1093/hmg/ddt571. Epub 2013 Nov 11.

Abstract

The extensive molecular characterization of human pluripotent stem cells (hPSCs), human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) is required before they can be applied in the future for personalized medicine and drug discovery. Despite the efforts that have been made with kinome analyses, we still lack in-depth insights into the molecular signatures of receptor tyrosine kinases (RTKs) that are related to pluripotency. Here, we present the first detailed and distinct repertoire of RTK characteristic for hPSC pluripotency by determining both the expression and phosphorylation profiles of RTKs in hESCs and hiPSCs using reverse transcriptase-polymerase chain reaction with degenerate primers that target conserved tyrosine kinase domains and phospho-RTK array, respectively. Among the RTKs tested, the up-regulation of EPHA1, ERBB2, FGFR4 and VEGFR2 and the down-regulation of AXL, EPHA4, PDGFRB and TYRO3 in terms of both their expression and phosphorylation levels were predominantly related to the maintenance of hPSC pluripotency. Notably, the specific inhibition of AXL was significantly advantageous in maintaining undifferentiated hESCs and hiPSCs and for the overall efficiency and kinetics of hiPSC generation. Additionally, a global phosphoproteomic analysis showed that ∼30% of the proteins (293 of 970 phosphoproteins) showed differential phosphorylation upon AXL inhibition in undifferentiated hPSCs, revealing the potential contribution of AXL-mediated phosphorylation dynamics to pluripotency-related signaling networks. Our findings provide a novel molecular signature of AXL in pluripotency control that will complement existing pluripotency-kinome networks.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Axl Receptor Tyrosine Kinase
  • Cell Cycle / genetics
  • Cell Differentiation / genetics*
  • Cells, Cultured
  • Chromatography, Liquid
  • Embryonic Stem Cells / cytology*
  • Enzyme Activation
  • Gene Expression Profiling
  • Homeodomain Proteins / genetics
  • Humans
  • Induced Pluripotent Stem Cells / cytology*
  • Molecular Sequence Data
  • Nanog Homeobox Protein
  • Octamer Transcription Factor-3 / genetics
  • Phosphorylation
  • Proto-Oncogene Proteins / antagonists & inhibitors
  • Proto-Oncogene Proteins / genetics*
  • Proto-Oncogene Proteins / metabolism
  • Receptor Protein-Tyrosine Kinases / antagonists & inhibitors
  • Receptor Protein-Tyrosine Kinases / genetics*
  • Receptor Protein-Tyrosine Kinases / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • SOXB1 Transcription Factors / genetics
  • Signal Transduction / genetics
  • Tandem Mass Spectrometry

Substances

  • Homeodomain Proteins
  • NANOG protein, human
  • Nanog Homeobox Protein
  • Octamer Transcription Factor-3
  • POU5F1 protein, human
  • Proto-Oncogene Proteins
  • SOX2 protein, human
  • SOXB1 Transcription Factors
  • Receptor Protein-Tyrosine Kinases
  • Axl Receptor Tyrosine Kinase
  • AXL protein, human