Is SUMO Bringing EphB4 into the Nucleus of Prostate Cancer Cells?

Overview

Topic status: We're looking for students to study this topic.

The receptor tyrosine kinase EphB4 is increased on the surface of cancer cells and has been shown to contribute to tumour development by promoting angiogenesis, increasing cancer cell survival, and facilitating invasion and migration. Whilst exploring cellular localisation of EphB4 in prostate cancer cells, we made the highly novel observation of immunoreactive EphB4 in the nucleus of LNCaP and DU145 PCa cells. We confirmed this result by preparing a nuclear protein extract from LNCaP cells and successfully immunoprecipitating full length EphB4 protein from this. More recently we have shown that the nuclear transportation protein SUMO-1 co-localises with EphB4 in PCa cell lines. Sumoylation (attachment of SUMO-1 to another protein) mediates translocation to the nucleus so we have now identified a potential mechanism for moving EphB4 into the nucleus. EphB4 contains a predicted sumoylation motif at K616, the second amino acid in the kinase domain of EphB4 and only two amino acids downstream of Y614, which is phosphorylated in kinase active EphB4.

Hypothesis: Nuclear localisation of EphB4 requires sumoylation at K616 and this is regulated by kinase activity and phosphorylation of Y614.

Aim 1: Use co-immunoprecipitation to determine whether EphB4 is sumoylated

Aim 2: Use site directed mutagenesis to produce constructs for expression of EphB4 with a mutated sumoylation site (K616R), EphB4 lacking the upstream tyrosine (Y614F) and kinase dead EphB4 K647R

Aim 3: Over-express the mutant and wild type EphB4 proteins in the breast cancer cell
line MCF10A and the prostate cancer cell lines 22Rv1 and LNCaP

Aim 4: Determine the cellular localisation of the mutant EphB4 proteins using
immunofluorescence and confocal microscopy

Methods and techniques that will be developed in the course of this project:

  • Site directed mutagenesis
  • Cell culture (cancer cells)
  • Transfection
  • Confocal microscopy
  • Western blotting and immunoprecipitation
Study level
Honours
Supervisors
QUT
Organisational unit

Science and Engineering Faculty

Research area

Cell and Molecular Biosciences

Contact

Please contact the supervisor.