Multiple myeloma phosphotyrosine proteomic profile associated with FGFR3 expression, ligand activation, and drug inhibition

Abstract

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.

Fig. 1.

Phosphorylation in MM cells, and modulation by pervanadate, FGF, and PD173074 (PD). (A) Anti-pY Western blot (WB) of lysates from MM-derived cells (as indicated) that differ in their expression of FGFR3: KMS12PE no expression; LP1, wild-type; OPM2, K650E; KMS11, Y373C. Lane 5 is the adenocarcinoma A431 that expresses approximately 1 M EGFR/cell. Lanes 6–8 are serum-starved KMS11 cells either untreated (lane 5), FGF-stimulated (lane 6), or treated with pervanadate (lane 7). (B) FGFR3 was isolated by immunoprecipitation from KMS11 cells that had been treated with PD173074, FGF, and pervanadate as indicated, and then Western blotted for pY (upper panel) or FGFR3 (lower panel), and then imaged for chemiluminescence. Shown are representative results of repeated experiments. (C) Venn diagram showing nonredundant pY containing peptides (and proteins in parentheses) from KMS11 cells treated without or with pervanadate (VO₄) to potentiate cellular protein tyrosine phosphorylation.

Fig. 2.

Label-free MS-based quantification of FGFR3 activation loop phosphorylation in KMS11 cells. Extracted MS ion currents for singly (left) and doubly (right) phosphorylated isoforms of the indicated FGFR3 activation loop tryptic peptide are shown (arrows). KMS11 cells were grown in medium with serum as a control (CON), treated with pervanadate (VO₄), or pervanadate (VO₄) plus PD173074 as indicated.