| Protein Tyrosine Kinase (PTK) is a kind of kinase which exists in cellular membrane or cytoplasm. Having typical carboxyl-terminal, PTK can phosphorylate downstream molecules and also phosphorylate itself. The molecules Doks (downstream of tyrosine kinase) are substrates of tyrosine kinases and involved in the recruitment and assembly of specific signal transduction molecules.Glial cell line-derived neurotrophic factor (GDNF) was originally isolated based on its potent and specific ability to promote the survival and morphological differentiation of do-paminergic neurons and motoneurons in embryonic midbrain cultures. In addition, GDNF also support the survival and regulate the differentiation of many peripheral neurons, including sympathetic, parasympathetic, sensory and enteric neurons. GDNF also plays a crucial role outside the nervous system, as a morphogenetic factor in kidney development and as a regulator of spermatogonia differentiation. The neurotrophic and morphogenic activities of GDNF are mediated by its interaction with a multicomponent receptor complex formed by the RET receptor tyrosine kinase and a glycosylphosphatidylinositol (GPI)-an-chored "accessory" receptor, GDNF family receptor alpha-1 (GFRal). GFRal is an extracellular protein that is attached to the outer cell membrane by GPI-anchor and could not mediate signaling directly. According to the original model, a GDNF dimer first binds to either monomeric or dimeric GFRal. Then the GDNF-GFRal complexes interact with two RET molecules and induce their homodimerization and tyrosine autophosphorylation. In 2001 Grimms identified the new family member Dok-5, which is coexpressed with c-Ret in various neuronal tissues acting as substrates for the c-Ret receptor tyrosine kinase, inducing ligand-dependent axonal outgrowth of PC12 cells. However, in contrast to Dok1, Dok-5 does not associate with rasGAP or Nck but enhance c-Ret-dependent activation of mitogen-activated protein kinase. Currently the function of Dok5 is poorly understood. The knowledge of functional mechanism for the Dok5 in differentiation induced by GDNF will provide strong basis for further investigating biological function of GDNF, which may be useful for further study the mechanism of other neurotrophic factors and for treatment of neurodegenerative diseases.The PC12-cell line has been extensively used as a model for peripheral neuronal differentiation. In serum-containing medium, PC-12 cells undergo mitosis and display properties of adrenal chromaffin cells. Addition of NGF or FGF induces transformation of PC-12 cellsto non-dividing sympathetic neuron-like cells. Treatment with IGF- I or EGF instead stimulates proliferation of PC-12 cells but not differentiation. PC-12 cells does not express endogenous GFRal and only express low levels of RET.In the study, Dok5 was inserted into a pEYFP vector. The vector pEYFP-Nl-Dok5 were transfected into PC12-GFRal-RET cells to examine the cellular responses after GD-NF stimulation. And the percentage cells bearing neurites assays were performed to examine the function. In addition, its functional mechanism was successfully performed applying different inhibitor.The main results are as follows:1. Construction of pEYFP-Nl-Dok5 pcDNA3. 1 ( + )-Dok5 and generation of PC12-GFRal-RET-Dok5 transfectantsTo study its biological activity, the mammal expression plasmid pEYFP-Nl-Dok5 and pcDNA3. 1 ( + )-Dok5 were successfully constructed and transfected into PC12-GFRal-RET cells by lipofectamine. The pEYFP-Dok5 fusion protein could obviously locate in the cell membrane of PC12-GFRal-RET cells.2. Promotion function of Dok5 in PC12-GFRal-RET cells bearing neurites induced by GDNFPC12-GFRal-RET cells was transfected with pEYFP-Nl-Dokl or pEYFP-Nl-Dok5 bearing neurites induced by GDNF. PC12-GFRal-RET-Dok5 transfectants were cultured for 4-5days in low-serum-containing medium and neurite outgrowth of PC12-GFRal-RET cells was quantitated by scoring cells with neurites longer than the sizes of two cell bodies.GDNF protein could significantl...
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