Myosin VI (MVI) is an unconventional myosin that acts as a cargo transporter and plays a role in a variety of cellular processes such as mitosis. It is also overexpressed in ovarian, breast and prostate cancer. Due to its wide range of functions, MVI must have tight spatial and temporal regulation. The most common regulatory mechanisms are alternative isoform splicing, interaction with binding partners, and phosphorylation. In the literature, the least is known about phosphorylation, especially within the cargo binding domain (CBD), as most studies focus on the motor. There are several potential regulatory phosphorylation sites within the CBD, including Y1114. This is located between 2 motifs where binding partners interact, RRL and WWY, and so may play a role in regulating this interaction. This study aims to generate tools to investigate how phosphorylation within the CBD of MVI may regulate its interaction with binding partners, and therefore its function. A Y1114F phospho-deletion mutation was induced in a pET28-RFP-CBD plasmid via site-directed mutagenesis PCR. This mutated plasmid was recombinantly expressed and the mRFP-MVICBD mutant protein was analysed using SDS-PAGE. The pET28-RFP-CBD plasmid was successfully mutated to contain the Y1114F mutation, as shown by an amino acid change (Y à F) following analysis of sequencing results. The recombinant protein was expressed effectively, with clear bands present on the SDS-PAGE gel at ~55Ka. There was no difference in staining intensity between the mutant and wild-type bands, demonstrating that the Y1114F mutation does not hinder the expression of the mRFPMVI-CBD protein. This recombinant protein is a tool that can be used in future experiments to identify whether phosphorylation at Y1114 impacts the interaction of MVI with binding partners, such as nickel affinity chromatography. Further experiments utilising this tool could investigate the potential role of phosphorylation regulation in various pathological states, like cancer.
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