Signaling Initiated by Overexpression of the Fibroblast Growth Factor Receptor-1 Investigated by Mass Spectrometry

Abstract

Overexpression of the fibroblast growth factor receptor-1 (FGFR-1), a prototypic receptor tyrosine kinase, is a feature of several human tumors. In human 293 cells overexpression of the FGFR-1 leads to constitutive activation of the receptor with concomitant sustained high increase in the cellular level of phosphotyrosine-containing proteins. Here we use mass spectrometry to study the tyrosinephosphorylated proteins induced by overexpression of the FGFR-1. Several well known components of FGFR-1 signaling were identified along with two novel candidates: NS-1-associated protein-1 and target of Myb 1-like protein. We subsequently applied mass spectrometry precursor ion scanning to identify 22 tyrosine phosphorylation sites distributed on six substrate proteins of the FGFR-1 or downstream tyrosine kinases. Novel in vivo tyrosine phosphorylation sites were found in the FGFR-1, phospholipase C␥, p90 ribosomal S6 kinase, cortactin, and NS-1-associated protein-1 as a result of sustained FGFR-1 signaling, and we propose these as functional links to downstream molecular and cellular processes. Receptor tyrosine kinases convey extracellular stimuli to intracellular signaling. When activated they induce tyrosine phosphorylation of specific cytosolic substrates and thereby create highly specific binding sites for the Src homology 2 (SH2) 1 or phosphotyrosine (Tyr(P)) binding domains in proteins that will further promote intracellular signaling cascades (1). The human genome encodes 58 different receptor tyrosine kinases (2), which are involved in events such as proliferation, differentiation, metabolic control, and migration.Overexpression of receptor tyrosine kinases can lead to constitutive activation as first described for the ErbB2 receptor (3) and to concomitant elevated cellular tyrosine phosphorylation status. Overexpression and amplification of the fibroblast growth factor receptor-1 (FGFR-1), a prototypic receptor tyrosine kinase, have been found in tumor samples from human pancreas (4, 5), breast (6, 7), brain (8, 9), prostate (10), thyroid gland (11), and salivary gland (12). To understand the role of FGFR-1 in cancer it is vital to define the signaling pathways that confer the oncogenic potential and how they differ from the "healthy" situation.In a recent study, immunoprecipitation with anti-Tyr(P) antibodies combined with SDS-PAGE and mass spectrometry was used to identify proteins that were tyrosine-phosphorylated as a result of epidermal growth factor stimulation of HeLa cells (13). Two novel candidates in the extensively studied epidermal growth factor receptor signaling pathway were isolated, and substrates present in femtomole amounts were identified. Techniques for tracing phosphopeptides in complex mixtures by MS have been developed and are now finding applications in phosphorylation studies of in vivo samples (14). These developments, however, have primarily been used in the characterization of serine and threonine phosphorylations due to (i) their relative abundance (Ͼ99.9%) compared with tyrosine pho...

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