Evidence has indicated that loss of PTEN expression inside the absence of biallelic mutation occurs a lot more regularly. While possible mechanisms causing the lack of expression of PTEN in tumours retaining at the very least one particular wild sort Aurora B activation PTEN copy have been identified, just like promoter methylation, it appears that other, unknown, mechanisms might be acting in quite a few tumours. Understanding the mechanisms regulating PTEN expression seems to become especially critical, as, unlike a lot of tumour suppressors, sturdy evidence indicates that partial loss of PTEN expression can boost tumour development. It is clear that PTEN stability is usually regulated via the C terminal tail, that’s phosphorylated upon a cluster of serine and threonine residues, Ser380, Thr382, Thr383 and Ser385. This phosphorylation appears to stabilize the PTEN protein as well as to inhibit its biological activity. Also, a protein named PICT1/ GLTSCR2 has been described that binds to the C terminal tail of PTEN, knockdown of which by RNAi also leads to decreased PTEN protein stability. While PTEN ubiquitination and proteasomal degradation have been implicated previously, it has recently been shown that PTEN stability may be regulated by means of ubiquitination mediated from the NEDD4 1 ubiquitin ligase.
Although LDE225 it seems probably that C terminal cluster phosphorylation regulates PTEN stability through regulating a conformational alter within the protein, and therefore ubiquitination, additional mechanistic details are not yet clear. Two other phosphorylation sites inside the PTEN C terminal tail have already been identified, Ser370 and Thr366.
Ser370 was initially identified as a phosphorylation web site by metabolic labelling and mutational evaluation as well as by MS. It could possibly be phosphorylated efficiently in vitro by CK2. Thr366 was identified as a phosphorylation site based upon the combined utilization of MS, mutational analysis along with the utilization of phosphothreonine/ proline particular antibodies. It seems to become phosphorylated efficiently in vitro and likely in cells by GSK3 . In the present study, we have raised phospho particular antibodies to phospho Ser370 and phospho Thr366, and made use of these to analyse the phosphorylation of these internet sites by CK2 and GSK3 respectively. We show that, despite the fact that the phosphorylation of these websites doesn’t appear to alter PTEN activity in vitro or in cells, phosphorylation of Thr366 particularly can lead to destabilization on the PTEN protein. EXPERIMENTAL Cell culture U87MG glioblastoma cells and NIH 3T3 fibroblasts had been obtained from the ECACC and maintained inside the recommended media. Typical cell culture media, additives and sera were from Invitrogen/Gibco. Other chemicals were from Sigma. PTEN was expressed in U87MG cells using an adapted baculoviral delivery method. Adapted baculoviruses containing the PTEN cDNA downstream of a CMV promoter were prepared in SF9 cells, employing typical protocols developed for recombinant protein expression in insect cells, and added to low confluence U87MG cell cultures for 24 h at 5% culture volume.