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.proteomics ͉ syndecan ͉ mass spectrometry ͉ comodulation T yrosine (Y) phosphorylation is a key mechanism of cell regulation, and tyrosine kinases are frequently activated in cancers (1). FGF receptor-3 (FGFR3) is a receptor tyrosine kinase (RTK) and drug target in a subset of human multiple myelomas (MM) that contain the t(4;14) translocation, which is responsible for the aberrant expression of FGFR3 in these tumors (2). Mutations that activate FGFR3 are prevalent in bladder cancer, have been observed in t(4;14) MM, cervical and colon cancers, and are associated with skeletal dysplasias (3). A fundamental question in MM is the nature of phosphotyrosine (pY) signaling networks in tumors that express FGFR3.RTKs such as FGFR3 function to a large extent through ligandinduced autophosphorylation, which facilitates the activation of downstream effectors (4). Autophosphorylation of FGFR1 proceeds sequentially, involving one, and then both adjacent tyrosines in the kinase domain activation loop (AL), which causes an approximately 50-fold and 1,000-fold activation of kinase activity, respectively, followed by the modification of other receptor tyrosines (5). FGFR3 also contains tandem tyrosines in its AL (6), and has four additional Y sites including pY760 and pY724, which are linked to downstream signaling effector proteins (7,8). Systematic in vitro investigations of pY-dependent prote...

Quantitative Proteomic Analysis of Tumor Reversion in Multiple Myeloma Cells

“…[24][25][26][27][28] In recent years, different proteomic approaches have been taken to investigate various aspects of MM and have provided new insights into the pathogenesis of MM. [29][30][31] In this study, the SILAC method was employed to compare the protein profiles of parental MM cells and revertants. In total, 379 differentially expressed proteins (DEPs) were identified, most of which interact in some way with STAT3, suggesting that the STAT3 pathway may play a vital role in myeloma reversion.…”

Inhibitory effect of SPE‐39 due to tyrosine phosphorylation and ubiquitination on the function of Vps33B in the EGF‐stimulated cells

“…An anti-SPE-39 rabbit polyclonal antibody was raised against peptides having an additional cysteine residue at the N-terminal end of the SPE-39 amino acid sequence, KGDEEEYWNSSKF (amino acids [5][6][7][8][9][10][11][12][13][14][15][16][17]. The peptides were coupled to keyhole limpet hemocyanin and used to immunize rabbits.…”

“…Tyr-11 was identified as the site of phosphorylation in fibroblast growth factor-3 (FGF3)-stimulated cells, as well as in pervanadate-and calyculin-treated T-cell leukemia cells [9][10][11].…”

“…However, the precise mechanisms by which this occurs remain obscured. SPE-39 (also known as FLJ12707 or C14ORF133) was originally identified through tyrosine phosphoproteomics, and it has been reported that Tyr-11 is phosphorylated following certain stimuli in cultured cells [9][10][11]. However, further characteristics of its tyrosine phosphorylation have yet to be conducted.…”

Structure and function of the human parvulins Pin1 and Par14/17

“…Three different phosphate-proteomic screens found phosphorylation on Tyr 122 within Par14 upon the stimuli of the receptor tyrosine kinases FGFR1/3 and CSF-1R by their ligands (Kasyapa et al, 2009;St-Germain et al, 2009;Knowlton et al, 2010). The influence of this PTM on the structure and function of hPar14 remains elusive.…”