The Assembly Of Microtubule Is Regulated By Non-receptor Tyrosine Kinase C-Abl

The non-receptor tyrosine kinase c-Abl in mammalian whose homeotic gene in murine leukemia virus is v-abl proto-oncogene, is widely expressed in mammalian cells. There are many functional domains in its structure, of those are Src Homology 3(SH3) and Src Homology 2(SH2). SH3 and SH2 domains can particularly recognize proline-rich protein regions and tyrosine serials which can be phosphorylated respectively. It can shuttle between nucleus and cytoplasm through its NLS and NES structure, and locate itself in different subcellular structures. Most cytoplasmic c-Abl is so jointed with G/F-actin by its C terminal that regulates cytoskeleton changing which is dependent to F-actin. C-Abl performs many biological functions on normal physiological and pathological conditions. It is involved in cell proliferation regulation, cell adhesion and migration, cell apoptosis, and plays important roles in oxidative stresss and DNA repair after damages.The microtubule, one of the cytoskeletons, consists ofα/β-tubulin dipolymers. It functions in sustaining cell structure and fixing the positions of the organelles, and is consisted in cilias and flagellas, and axon of nerve cells. Besides, microtubules are important parts of centrosomes and spindles, those play very important roles in mitosis. The spindles pull the chromosome and mediate the separation of sister chromosomes in mitosis and introduce the contractile ring formation to promote cytokinesis.PLK1 and FOXM1 are two proteins involved in cell cycle, and they express dependent on cell cycle. FOXM1, which belongs to FOX family, is a class of transcription factors with winged helix in its DNA binding domains. FOXM1 plays a role in regulation of cell cycle particular genes and promotion of cell proliferation, whose expression and activity are regulated by proliferation and anti-proliferation signals. PLK1 is a member of Polo-like kinases which is widely expressed in eukaryotic cells. As a serine/threonine kinase, PLK1 plays important roles in cell cycle, cell proliferation, tumorigenesis and other aspects. They are both involved in the formation of centrosomes and spindles. FOXM1 which is one of the substrates of PLK1 kinase, can regulate the express of PLK1. They regulate series of cell cycle proteins and factors by interactions to influence cell cycle.In this study, we demonstrated that c-Abl interacted with PLK1 and regulated its phosphorylation through immunobloting and co-immunoprecipiation, so PLK1 is a substrate of c-Abl. We synchronized cells and found that the interaction between c-Abl and PLK1 and the phosphorylation of PLK1 changed with the cell cycle. We demonstrated that c-Abl could promote the transcription of PLK1 through Fluorescence Quantitative PCR and Report Gene Detection technology; c-Abl could inhibit the degradation of FOXM1 through Pulse-chase; c-Abl could enhance the protein level of FOXM1 which was dependent on its activity through Westernblot. Thus c-Abl kinase influenced the expression and protein stability of FOXM1 indirectly by interacting with and phosphorylation of one of its substrate-PLK1.In this study, we separated the polymerized and soluble tubulins, and tested their protein levels by immunobloting. We found that the proportion of soluble ones increased and the polymerized ones decreased after c-Abl was knockdown. Then we found that the polymerization extent was decreased sharply when c-Abl was knockdown after separated the microtubules in physiological state, which showed that c-Abl could inhibit the depolymerization of microtubules. Through immuno-fluorescent assay, we found that the microtubule structure was dispersed when c-Abl was knockdown, which showed the assembly of microtubules was influenced by c-Abl. Besides, we compared the tubulin levels between wild type cell line and the c-Abl-knockdown one. We found that the tubulin level decreased sharply after c-Abl was knockdown, which showed that c-Abl could inhibit the degradation of tubulins.The influence of c-Abl on the polymerization and assembly of microtublues inevitably affected the assembly of spindles. We stained the spindles in mitosis by immunofluorescent technology and found that the assembly of spindles defect after c-Abl was knockdown. We also found that there were many polykaryocytes in c-Abl-knockdown cell lines, which showed that c-Abl could influence the cytokinesis.We found a new c-Abl substrate-PLK1, and demonstreted preliminarily that c-Abl regulated cell cycle through many ways by interacting with and phosphorylation of PLK1. C-Abl regulated the transcription of G2/M particular genes through regulating the expression and activity of transcription factor-FOXM1; c-Abl regulated mitosis through regulating the spindle assembly-associated substrate of PLK1; c-Abl regulated cytokinesis through regulating the cytokinesis-associated substrate of PLK1. This study provides a new basis for the function study of c-Abl family in cell cycle regulation, seeing the important roles in mitosis which the assembly of microtubules play.