Role of Rho GTPases and PTEN in the migration of human glioma cells
Rho GTPases play a key role in regulating the migration of many cell types including astrocytes. Astrocytcs are relatively poor migrating cells, whereas, gliomas, can be highly invasive, infiltrating the surrounding tissue and spreading difftisely in the brain due, in part, to their highly motile behaviour. This thesis investigates the aberrant migration of three human glioma cell lines (U373, U138, U87) and, using microinjection techniques, it is shown that the small GTPase Rac is essential for the migration of astrocytes as well as for the three glioma cell lines. In agreement with a higher rate of migration, it was demonstrated that the level of active Rac (Rac-GTP) is higher in U373 than in astrocytes. Surprisingly, however, the levels of Rac-GTP in the more motile cell lines, U138 and U87, are much lower than U373 cells. Rac activity in cells can be stimulated through increases in the levels of PI(3,4,5)P3 generated by the enzymeP I 3- kinase. PI(3,4,5)P3le vels are negatively controlled by the dual specificity phosphatase PTEN, which dephosphorylates PI(3,4,5)P3- In in vitro assays,P TEN has also been shown to have protein phosphatase activity. All three glioma cell lines lack PTEN. This work demonstrates that re-expression of wtPTEN inhibits the migration of all three glioma cell lines, without affecting the migration of astrocytes. Moreover, data is presented showing that the protein, not the lipid, phosphatase activity of PTEN, is essential for inhibiting migration. Most unexpectedly, however, additional mutagenesis studies reveal that it is the C2 domain of PTEN that mediatest he inhibitory effect on the migration of these tumour cell lines, and that in the full-length protein, the phosphorylation of residue Tbr383 controls the anti-migratory activity of the C2 domain. Finally, using the carboxy terminus of PTEN as bait to screen a brain yeast two-hybrid library, new potential binding partners of PTEN are identified.