Dataset Information

Cdk1-mediated threonine phosphorylation of Sam68 modulates its RNA binding, alternative splicing activity, and cellular functions

ABSTRACT: Sam68 is a member of the STAR family of proteins that directly link signal transduction with post-transcriptional gene regulation. Sam68 controls the alternative splicing of many oncogenic proteins and its role is modulated by post-translational modifications, including serine/threonine phosphorylation, that differ at various stages of the cell cycle. However, the molecular basis and mechanisms of these modulations remain largely unknown. Here, we combined mass spectrometry, NMR spectroscopy, and cell biology techniques to provide a comprehensive post-translational modification (PTM) mapping of Sam68 at different stages of the cell cycle in HEK293 and HCT116 cells. We established that Sam68 is specifically phosphorylated at T33 and T317 by Cdk1, and demonstrated that these phosphorylation events reduce the binding of Sam68 to RNA, control its cellular localization, and reduce its alternative splicing activity, leading to a reduction in the induction of apoptosis and an increase in the proliferation of HCT116 cells.

INSTRUMENT(S): LTQ Orbitrap Velos, Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Andrew Bottrill

LAB HEAD: Dr Cyril Dominguez

PROVIDER: PXD032716 | Pride | 2023-03-11

REPOSITORIES: Pride

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Publications

Cdk1-mediated threonine phosphorylation of Sam68 modulates its RNA binding, alternative splicing activity and cellular functions.

Malki Idir I Liepina Inara I Kogelnik Nora N Watmuff Hollie H Robinson Sue S Lightfoot Adam A Gonchar Oksana O Bottrill Andrew A Fry Andrew M AM Dominguez Cyril C

Nucleic acids research 20221201 22

Sam68, also known as KHDRBS1, is a member of the STAR family of proteins that directly link signal transduction with post-transcriptional gene regulation. Sam68 controls the alternative splicing of many oncogenic proteins and its role is modulated by post-translational modifications, including serine/threonine phosphorylation, that differ at various stages of the cell cycle. However, the molecular basis and mechanisms of these modulations remain largely unknown. Here, we combined mass spectromet ...[more]

PMID: 36537190

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Project description:SUMO modification of proteins (sumoylation) is essential for mitotic progression from yeast to humans, but only a limited number of sumoylated proteins with functions in mitosis have been discovered. Vertebrates express three SUMO paralogs, SUMO-1, SUMO-2 and SUMO-3, but only SUMO-2 and -3 are chromosome-associated in mitosis. In this study, we used chromosome spreads to more precisely define the localization of endogenous SUMO-2 and -3 to the centromere as well as the chromosome protein scaffold. Furthermore, we developed methodologies for the immunopurification of endogenous SUMO-2 and -3 modified proteins from cell extracts. Using LC-MS/MS, we analyzed proteins immunopurified from mitotic chromosome fractions and G0 nuclear fractions. We identified 296 putative sumoylated proteins, with 138 being modified specifically in mitosis. We identified proteins known to localize to the centromere and kinetochore and the chromosome protein scaffold, consistent with SUMO-2/3 localization. Furthermore, we demonstrate that utilizing cell synchronization and fractionation allowed for the discovery of low abundance sumoylated proteins that are not detected in large asynchronous analyses. Our results provide a foundation for further characterization of the roles of sumoylation in regulating diverse aspects of chromosome segregation in mitosis. Thermo .raw files were uploaded to the ProHits (Liu et al, 2010) analytical suite and converted to .mzXML format using ReAdW software. Data were searched using X!Tandem (Craig & Beavis, 2004) against human ORFs (RefSeq v45). Search parameters specified a parent MS tolerance of +/- 15ppm, and an MS/MS tolerance of 0.4 Da, with up to two missed cleavages for trypsin. Oxidation of methionine and tryptophan, ubiquitylation of lysine, and alkylation of cysteine (by NEM) were allowed as variable modifications. Statistical validation of the results was performed using Peptide Prophet and Protein Prophet (Keller et al, 2002; Nesvizhskii et al, 2003) as part of the trans-proteomic pipeline. For each search, the Protein Prophet probability at a 1% false discovery rate was used as a cutoff value to generate SAINT-compatible input files. SAINT parameters were as follows: 5000 iterations, low mode Off (0), minFold 1 and normalization (1) (Choi et al, 2011). SAINT cutoff values of 0.75 were used to generate a high-confidence list of protein identifications.

Dataset Information

Cdk1-mediated threonine phosphorylation of Sam68 modulates its RNA binding, alternative splicing activity, and cellular functions

Publications

Cdk1-mediated threonine phosphorylation of Sam68 modulates its RNA binding, alternative splicing activity and cellular functions.

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