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

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 spectrometry, nuclear magnetic resonance spectroscopy and cell biology techniques to provide a comprehensive post-translational modification 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

Structural and Functional Investigation of SAM68 and T-STAR, with an Emphasis on the SAM68 and T-STAR Complex

The progression of some diseases can be linked to the dysregulation of protein activity. SAM68 and T-STAR are members of the STAR family of proteins which have been shown to link signal transduction with RNA metabolism: including RNA transport, translational regulation, and pre-mRNA alternative splicing. Overexpression of SAM68 and T-STAR has been linked to some cancers and, furthermore, the dysregulation of these proteins can alter pre-mRNA alternative splicing outcomes. SAM68 and T-STAR can form homodimers and they have been shown to interact with each other to form a SAM68/T-STAR complex. However, the structural basis and functional significance of the SAM68/T-STAR complex is unknown. The structure of the complex was studied by 2D NMR using a fusion protein of the T-STAR and SAM68 QUA1-KH domains. These results show that the SAM68/T-STAR complex can form via the QUA1-KH domains and that the complex contains a mixture of SAM68-SAM68, T-STAR-T-STAR, and SAM68-T-STAR interactions which is possible by the formation of higher-order multimers. Single-molecule studies also showed the presence of higher-order multimers for the full-length SAM68 and T-STAR proteins and multimers were further driven by the addition of Bcl-x and G8-5 RNA. These results contrast with previous studies showing that the free and RNA-bound SAM68 and T-STAR proteins form homodimers. Therefore, the results here illustrate a new mechanism for the formation of SAM68- and T-STAR-containing complexes and could provide a new basis for understanding the function of these proteins.</p