PhosY-secretome profiling combined with kinase-substrate interaction screening defines active c-Src-driven extracellular signaling
Sarah J. Backe,
SarahBeth D. Votra,
Matthew P. Stokes,
Endre Sebestyén,
Matteo Castelli,
Luca Torielli,
Giorgio Colombo,
Mark R. Woodford,
Mehdi Mollapour,
Dimitra Bourboulia
Affiliations
Sarah J. Backe
Department of Urology, SUNY Upstate Medical University, Syracuse, NY 13210, USA; Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, NY 13210, USA
SarahBeth D. Votra
Department of Urology, SUNY Upstate Medical University, Syracuse, NY 13210, USA; Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, NY 13210, USA
Matthew P. Stokes
Cell Signaling Technology, Inc., Danvers, MA 01923, USA
Endre Sebestyén
The Bioinformatics CRO, Inc., Orlando, FL, USA
Matteo Castelli
Dipartimento di Chimica, Università di Pavia, 27100 Pavia, Italy
Luca Torielli
Dipartimento di Chimica, Università di Pavia, 27100 Pavia, Italy
Giorgio Colombo
Dipartimento di Chimica, Università di Pavia, 27100 Pavia, Italy
Mark R. Woodford
Department of Urology, SUNY Upstate Medical University, Syracuse, NY 13210, USA; Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY 13210, USA; Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, NY 13210, USA
Mehdi Mollapour
Department of Urology, SUNY Upstate Medical University, Syracuse, NY 13210, USA; Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY 13210, USA; Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, NY 13210, USA
Dimitra Bourboulia
Department of Urology, SUNY Upstate Medical University, Syracuse, NY 13210, USA; Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY 13210, USA; Upstate Cancer Center, SUNY Upstate Medical University, Syracuse, NY 13210, USA; Corresponding author
Summary: c-Src tyrosine kinase is a renowned key intracellular signaling molecule and a potential target for cancer therapy. Secreted c-Src is a recent observation, but how it contributes to extracellular phosphorylation remains elusive. Using a series of domain deletion mutants, we show that the N-proximal region of c-Src is essential for its secretion. The tissue inhibitor of metalloproteinases 2 (TIMP2) is an extracellular substrate of c-Src. Limited proteolysis-coupled mass spectrometry and mutagenesis studies verify that the Src homology 3 (SH3) domain of c-Src and the P31VHP34 motif of TIMP2 are critical for their interaction. Comparative phosphoproteomic analyses identify an enrichment of PxxP motifs in phosY-containing secretomes from c-Src-expressing cells with cancer-promoting roles. Inhibition of extracellular c-Src using custom SH3-targeting antibodies disrupt kinase-substrate complexes and inhibit cancer cell proliferation. These findings point toward an intricate role for c-Src in generating phosphosecretomes, which will likely influence cell-cell communication, particularly in c-Src-overexpressing cancers.
