Dataset Information

MudPIT analysis of STRN4 interacting proteins from HEK TER cells expressing either SV40 ST or GFP

ABSTRACT: HEK TER cells expressing either SV40 ST or GFP (30 x 15-cm diameter plates) were harvested with lysis buffer (20 mM imidazole HCl, 2 mM EDTA, 2 mM EGTA, pH 7.0 with 10 ug/mL each of aprotinin, leupeptin, pepstatin, 1 mM benzamidine, and 1 mM PMSF). The clarified cell extract was incubated overnight at 4C with 20-100 ug of STRN4 antibodies (Abcam, ab177155) crosslinked to 30 mg protein A agarose beads (Thermo Scientific) by dimethyl pimelimidate (DMP). Beads were washed 5 times with high salt lysis buffer (containing 300 mM NaCl), washed with TBS 2 times, and then eluted with 0.2 M glycine pH 3 and neutralized with 1 M Tris-HCl pH 8.0. Proteins were precipitated with trichloroacetic acid (20% final concentration) overnight at 4C, washed with cold acetone and processed for subsequent MudPIT analysis. In brief, TCA-precipitated protein eluates were urea-denatured, reduced, alkylated, and digested with endoproteinase LysC followed by trypsin. The peptide mixtures were loaded onto microcapillary fused silica columns (100um i.d.), packed with C18 reverse phase (Aqua; Phenomenex), SCX (Luna; Phenomenex) and C18-RP, placed in-line with an Agilent 11000 quaternary pump, and analyzed by a 10-step MudPIT on linear ion traps. MS/MS datasets were searched using ProLuCID against a non-redundant human protein database (NCBI, 2019-12-03) containing 44,080 non-redundant human proteins, 426 usual contaminants, as well as the sequences for small and large T antigens from SV40 Macaca mulatta polyomavirus 1. To estimate false discover rates (FDRs), the amino acid sequence of each non-redundant protein was randomized (44,521 shuffled proteins) and added to the search space. Cysteine carboxylation was searched as a static modification while methionine oxidation was searched dynamically. Peptide/spectrum matches were sorted and selected using DTASelect in combination with an in-house software, swallow, to FDRs at the peptide and protein levels of less than 1%.

INSTRUMENT(S): LTQ

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Laurence Florens

PROVIDER: MSV000084662 | MassIVE | Thu Dec 05 10:28:00 GMT 2019

SECONDARY ACCESSION(S): PXD016628

REPOSITORIES: MassIVE

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Publications

STRIPAK directs PP2A activity toward MAP4K4 to promote oncogenic transformation of human cells.

Kim Jong Wook JW Berrios Christian C Kim Miju M Schade Amy E AE Adelmant Guillaume G Yeerna Huwate H Damato Emily E Iniguez Amanda Balboni AB Florens Laurence L Washburn Michael P MP Stegmaier Kim K Gray Nathanael S NS Tamayo Pablo P Gjoerup Ole O Marto Jarrod A JA DeCaprio James J Hahn William C WC

eLife 20200108

Alterations involving serine-threonine phosphatase PP2A subunits occur in a range of human cancers, and partial loss of PP2A function contributes to cell transformation. Displacement of regulatory B subunits by the SV40 Small T antigen (ST) or mutation/deletion of PP2A subunits alters the abundance and types of PP2A complexes in cells, leading to transformation. Here, we show that ST not only displaces common PP2A B subunits but also promotes A-C subunit interactions with alternative B subunits ...[more]

PMID: 31913126

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