Signaling by
growth factor receptor tyrosine kinases is manifest through networks of
proteins that are substrates and/or bind to the activated receptors. FGF receptor-3 (FGFR3) is a drug target in a subset of human
multiple myelomas (MM) and is mutationally activated in some cervical and colon and many
bladder cancers and in certain skeletal dysplasias. To define the FGFR3 network in
multiple myeloma, mass spectrometry was used to identify and quantify
phosphotyrosine (pY) sites modulated by FGFR3 activation and inhibition in myeloma-derived KMS11 cells. Label-free quantification of
peptide ion currents indicated the activation of FGFR3 by phosphorylation of tandem tyrosines in the
kinase domain activation loop when cellular pY
phosphatases were inhibited by
pervanadate. Among the 175
proteins that accumulated pY in response to
pervanadate was a subset of 52 including FGFR3 that contained a total of 61 pY sites that were sensitive to inhibition by the FGFR3 inhibitor
PD173074. The FGFR3
isoform containing the tandem pY motif in its activation loop was targeted by
PD173074. Forty of the drug-sensitive pY sites, including two located within the 35-residue cytoplasmic domain of the transmembrane
growth factor binding
proteoglycan (and
multiple myeloma biomarker)
Syndecan-1/CD138, were also stimulated in cells treated with the
ligand FGF1, providing additional validation of their link to FGFR3. The identification of these overlapping sets of co-modulated
tyrosine phosphorylations presents an outline of an FGFR3 network in the MM model and demonstrates the potential for pharmacodynamic monitoring by label-free quantitative phospho-proteomics.