ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Endocrine Abstracts (2011) 25 P335

The use of tandem mass spectrometry to identify binding partners of PBF in thyroid cells

Neil Sharma, Robert Seed, Martin Read, Vicki Smith, Gregory Lewy, Gavin Ryan, Jim Fong, Perkin Kwan, Kristien Boelaert, Jayne Franklyn, Ashley Martin & Christopher McCabe

University of Birmingham, Birmingham, UK.

PBF is a proto-oncogene implicated in the aetiology of thyroid cancer. PBF binds to several proteins, including PTTG and NIS, but its full function is unknown. Understanding the range of interactions PBF has within thyroid cells is therefore vital; these were explored by tandem mass spectrometry (MS/MS), which relies on the predictable fragmentation pattern of an amino acid chain to identify the exact proteins present in a sample.

K1 and TPC1 papillary thyroid cancer (PTC) cells transfected with HA-tagged PBF were lysed and immunoprecipitated with anti-HA and passed through MS/MS (n=4). 2587 proteins were identified. Those appearing in vector-only controls were eliminated, as were those identified by only 1 peptide in a single run. Ten proteins were classed as being most likely to interact with PBF. Lyn, a Src tyrosine kinase, was identified by 2 peptides in the TPC1 immunoprecipitant but not the K1. This was supported by western blotting, where K1 cells were shown to have a low expression of Lyn compared to TPC1 cells. Cortactin, a substrate of Src, was identified by 8 peptides, as were FAK2 (2 peptides, 2 runs) and the serine/threonine kinases cMos (1 peptide, 3 runs) and SMG (9 peptides). Given that we have recently identified PBF as a phosphorylated protein, interaction with Lyn, cMos and SMG may thus be critical to PBF phosphorylation. Binding to Familial adenomatous polyposis (FAP) protein (9 peptides) and ERC1 (2 peptides) was also apparent. There is a well documented link between FAP and thyroid cancer and an ERC1/ret mutation is able to induce PTC. Additionally identified proteins were CIT (3 peptides, 2 runs), ARAP (1 peptide, 2 runs), UACA (2 peptides) and ADAMTS18 (2 peptides). Future work will seek to validate these interactions using pull-down assays and co-immunoprecipitation, and to evaluate their function alongside PBF in thyroid cancer.