Functional characterisation of Sam68 post-translational modifications

Lead Research Organisation: University of Leicester
Department Name: Molecular and Cell Biology

Abstract

The STAR (signal transduction and activation of RNA) family of proteins are RNA binding proteins of which the best characterised is Sam68, which is predominantly a nuclear protein. Sam68 has previously been shown to be involved in alternative splicing of specific transcripts and is a target for a number of post-translational modifications. Examples of these modifications include serine/threonine phosphorylation, arginine methylation, lysine acetylation and sumoylation and can have an effect on both structure and function. Within this project we aim to characterise specific modified residues with a focus on serine/threonine phosphorylation. This will be achieved by utilising NMR spectroscopy. Mutational studies will then allow us to study the effects of phosphorylation of target residues in a structural and functional way.
Following on from the mapping of the modifications, we aim to perform NMR on cytoplasmic and nuclear extracts to examine similarities and differences in modifications in each subcellular location. We will use two types of nuclear extract; nuclear HeLa extract and nuclear extracts depleted of specific kinases. Additionally we aim to examine post-translational modifications in vivo on HeLa cells using NMR comparing between normal and cancerous cells, as overexpression of Sam68 is a characteristic of some types of colorectal and prostate cancers. From a functional perspective, we will aim to look at the effects of post-translational modifications on the splicing patterns of known Sam68 target genes by performing splicing assays. Finally we aim to address the effect of inhibiting enzymes that are responsible for the post-translational modifications of Sam68 within cytosolic and nuclear extracts. These inhibitors will be used in the splicing assays to examine changes in splicing patterns when specific modifications are inhibited.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M01116X/1 01/10/2015 30/09/2023
1645572 Studentship BB/M01116X/1 05/10/2015 30/09/2019 Adam Lightfoot
 
Description The brief conclusions of this project can be summarised as followed:
The STAR protein Sam68 is a target for the post-translational modification of serine and threonine phosphorylation. Previous work has identified that numerous enzymes known as kinases target Sam68 for serine and threonine phosphorylation but the precise amino acid residue of the protein, and the consequential effects of the phosphorylation are unknown. Using a combination of nuclear magnetic resonance and mass spectrometry, we have identified some specific residues of the N-terminal domain of Sam68 phosphorylation from the kinases Cdk1 and Erk1. These residues include S20 and T33 (Cdk1) and T84 (Erk1).
After identification of these residues, we went onto looking at how these modifications could affect the functionally of the protein. Using fluorescence polarisation, we examined if RNA binding by Sam68 was altered by phosphorylation by Erk1 and Cdk1. The results from these experiments suggests that this is not the case, as both specificity and affinity were not significantly altered following phosphorylation of the protein constructs examined. We do however provide evidence of RNA binding by the N-terminal domain however we were unable to characterise this binding in terms of if it assists with the main mediator of RNA binding of Sam68, the KH domain. Localisation of serine and threonine phosphorylation of this protein did not appear to alter localisation. In collaboration with Professor Yutaka Ito of Tokyo Metropolitan University, we were also able to visualise the STAR domain of Sam68 inside living cells using in-cell NMR.
Exploitation Route Identifying the specific residues of phosphorylation for Sam68 could provide useful information in explaining how the protein functions and how these functions can be altered in diseases such as cancer. Sam68 is a known splicing regulator of numerous genes that can have a big influence of the fate of the cell. It may be possible to try and influence splicing activities of Sam68 by manipulating its splicing patterns through alterations of phosphorylation or any other modification that can affect the function of this protein. Future work looking at how splicing patterns could be altered through phosphorylation of specific residues could utilise the point mutants that were designed during this project, to determine specific important targets in the cell signal transduction process. Further work will also look at the rest of the protein in a similar manner. Similar methodologies could also be used to look at other proteins in terms of post-translational modifications and they can affect the function of the protein.
Sectors Pharmaceuticals and Medical Biotechnology