Photoaffinity-engineered protein scaffold for systematically exploring native phosphotyrosine signaling complexes in tumor samples

Abstract

Phosphotyrosine (pTyr)-regulated protein complexes play critical roles in cancer signaling. The systematic characterization of these protein complexes in tumor samples remains a challenge due to their limited access and the transient nature of pTyr-mediated interactions. We developed a hybrid chemical proteomics approach, termed Photo-pTyr-scaffold, by engineering Src homology 2 (SH2) domains, which specifically bind pTyr proteins, with both trifunctional chemical probes and genetic mutations to overcome these challenges. Dynamic SH2 domain-scaffolding protein complexes were efficiently cross-linked under mild UV light, captured by biotin tag, and identified by mass spectrometry. This approach was successfully used to profile native pTyr protein complexes from breast cancer tissue samples on a proteome scale with high selectivity, achieving about 100 times higher sensitivity for detecting pTyr signaling proteins than that afforded by traditional immunohistochemical methods. Among more than 1,000 identified pTyr proteins, receptor tyrosine kinase PDGFRB expressed on cancer-associated fibroblasts was validated as an important intercellular signaling regulator with poor expression correlation to ERBB2, and blockade of PDGFRB signaling could efficiently suppress tumor growth. The Photo-pTyr-scaffold approach may become a generic tool for readily profiling dynamic pTyr signaling complexes in clinically relevant samples.

Kindlin-2 promotes Src-mediated tyrosine phosphorylation of androgen receptor and contributes to breast cancer progression

“… A Volcano plots of the enriched native tyrosine phosphorylation proteins upon EGF stimulation in BT549 cells transfected with control siRNA (Ctrl siRNA) or Kindlin-2 siRNA (K2 siRNA) using Src superbinder Photo-pTyr-scaffold approach [ 86 ]. Red dots indicated the significantly enriched AR and Kindlin-2 (FERMT2).…”

“…Control or Kindlin-2 knockdown BT549 cells were cultured in serum-free DMEM media containing 100 ng/ml EGF or vehicle for 24 h. Cells were lysed by ice-cold NP-40 buffer (50 mM Tris-HCl, 150 mMNaCl, and 1% (v/v) NP-40, pH 7.4). The Photo-pTyr-scaffold operation was performed as described before [ 86 ]. In brief, the obtained cell lysates were sonicated for 5 s at 200 W 4 times with 10 s rest for each time.…”

High-Throughput Phosphotyrosine Protein Complexes Screening by Photoaffinity-Engineered Protein Scaffold-Based Forward-Phase Protein Array

“…The Photo-pTyr-scaffold was prepared by following our previous report . Briefly, the Src superbinder SH2 domain was expressed in E. coli which was inducted by isopropyl β-D-1-thiogalactopyranoside (IPTG) and purified using Glutathione Sepharose 4B beads (GE Healthcare).…”

“…However, the PLA assay requires high-quality primary monoclonal antibodies and is limited to only one pair of interacting protein per assay . Recently, we developed a hybrid chemical biology probe (termed Photo-pTyr-scaffold) to overcome the above challenges . Engineered Src superbinder SH2 domain is functionalized by a trifunctional probe through N -hydroxysuccinimide (NHS) ester group that reacts with the free primary amines of lysine residues without affecting the pTyr-binding activity and introduced with photoreactive groups and enrichment groups for covalently capturing pTyr proteins and pTyr-mediated protein complexes.…”

Chondroitin sulfate synthase 1 enhances proliferation of glioblastoma by modulating PDGFRA stability

“…For survival analysis, mice were intraperitoneally injected with 15 mg/kg of Cre or equivalent solvent and 5% of Gly (Sigma-Aldrich), on the day after GL261 transplantation for 7 serial days. The usage of Cre in our animal model was by referring to previous reports 47,48 . Mice were observed daily and were killed when they reached endpoints, including loss exceeding 20% of body weight or hunched posture.…”