PTEN Regulates PC12 Cell Differentiation Via ERK5 Signaling Pathway

Introduction Neural cell aberrant regeneration, differentiation or degeneration causes diverse neural disorders such as dementia senilis, Werdnig-Hoffman spinal muscular atrophy, Huntington's chorea. PTEN and MAPKs signaling play positive and negative roles respectively in regulationg cell growth and differentiation, keeping a balance between cell survival and apoptosis, proliferation and differentiation. As the regulator of Akt signalling transduction pathway, PTEN is not only a phosphatase but also is an important tumor suppressor. PTEN had been widely researched since it had been found as a tumor suppressor gene in 1997. One of the most famous functions is that the PTEN can regulate the growth and proliferation of intracellular signals, especially inhibit excessive proliferation of the malignant cells. In other words, PTEN plays a negative regulatory role in cell growth and proliferation. PTEN not only has the phospholipid phosphorylation activity which dephosphorylate IP3 to PIP2, inhibiting the activity of PI3K/AKT signaling pathway but also has protein dephosphorylation activity. Studies have showed that PTEN dephosphorylated substrates at ser/thr and Tyr residues. FAK(focal adhesion kinase) was the first discovered protein substrate of PTEN, thus PTEN is also known as dual-phosphatase. Contrary to the negative regulation of PTEN, MAPK family belongs to positive regulation in promoting cell growth. In the classical MAPKs, ERK5 was also known as BMK1 and found later than the other MAPK family proteins. Its molecular weight is relatively larger, which is composed of 816 amino acids and the kinase domain consist of half of the amino acid residues near the amino terminus. ERK5 also includes two carboxy terminal proline-rich regions, a nuclear localization signal and an auto-phosphorylation region near the end carboxy terminal. ERK5 signaling pathways can be induced by stress signaling, growth factor and cytokine signaling, and involved in cell growth, differentiation and apoptosis. ERK5 is activated by MEKK2/3—MEK5 cascade, and then directly activates other transcription factors after translocating to the nucleus. Thus, PTEN may have a wide range of substrates in cells, and the activity of ERK5 may be directly or indirectly regulated by PTEN. The PC12 cells are rat adrenal pheochromocytoma tumor cell lines, there are NGF reptors on the membrane. The stop dividing, growprojections and differentiated to the cells which have the characteristics of sympathetic neurons after NGF induced. So the PC12 are commonly used in the nervous system in vitro studies. Therefore, we may discovery new therapeutic target sites in the cross-talk between ERK5 signaling pathways and PTEN and genes related to neuronal differentiation. This would be clinical applications with a very great significance.Objective Our research focuses on exploring the mechanism of ERK5 regulated by PTEN, identifying the interactions between PTEN and Cot/Tpl-MEKK2/3-MEK5-ERK5 cascade, and searching for the possible synergy molecules, transcription factors or molecules involved in cell differentiation. It is expected to clarify cross-talk between ERK5 signaling pathways and PTEN.Method Interactions between PTEN and Cot/Tpl-MEKK2/3-MEK5-ERK5 signaling pathway were analysed by Co-IP?GST-pulldown. After obtaining the PTEN over-expression and PTEN knocked-down HEK 293 T cell strains by lentiviral vector stable transfection. The phosphorylation levels of molecules mediating this signaling pathway had been detected to determine the target protein dephophorylated by PTEN. To identify the target protein interacting with PTEN, we expressed the GST-PTEN protein in Escherichia, then pull down the target protein in PC12 lysates. Moreover, we examined effects of PTEN regulating Cot/Tpl-MEKK2/3-MEK5-ERK5 signaling pathway on HEK 293 T cell proliferation. And monitoring the relative phosphorylation levels of receptor tyrosine kinase may be regulated by PTEN over-expression and knock-down via Ray Biotech Human RTKs Phosphorylation Antibody Array.Result After obtaining the stable transfection HEK 293 T cell lines with PTEN over-expression or PTEN silencing, we found MEK5 and ERK5 phosphorylation levels increased after PTEN silencing and PTEN could bind to MEK5 in experiments of western-blot, Co-IP and GST-pulldown. Cell growth curves showed that HEK 293 T cell lines with PTEN silencing grown faster compared to PTEN over-expression. Compared to the normal PC12 cells, the neurite out growth of the PTEN silenced PC12 was significantly by culture. We found the phosphorylation levels of receptor tyrosine kinase Eph B3, HGFR, MUSK and VEGFR2 were up-regulated in PTEN silencing cells compared to random RNA control cells. Furthermore,phosphorylation levels of receptor tyrosine kinase insulin receptor and VEGF receptor2 increased in the PTEN silencing cells compared to PTEN over-expression cells.Conclusion 1. PTEN down-regulates the activity of ERK5 signaling pathway by interating with MEK5 or targets ERK5 directly; 2. PTEN can promote the growth of the HEK 293 T cells after PTEN knock-down; 3. Compared to the normal PC12 cells, the neurite out growth of the PTEN silenced PC12 was significantly by culture. 4. Eph B3, HGFR, MUSK, VEGFR2, Insulin R and VEGFR2 are probably the target protein receptors of PTEN, but this required further research.