PYK2Is Invoved In The Spindle Assembly During The Maturation Of Mouse Oocytes

PYK2is a kind of nonreceptor tyrosine kinase, which belongs to focal adhesionkinase(FAK) family members. The tyrosine kinase has: an N-Terminal FERM domain,proline-rich domains, the C-terminal domain which acts as focal adhesion targeting (FATdomain) and FRNK domain that acts as a negative regulator. PYK2have three tyrosinephosphorylation sites; Tyr402,579,580. When cells are stimulated by surface factors,autophosphorylation of PYK2(Tyr402) in the catalytic domain will occur in the first. ThenTyr579and Tyr580are activated by dimerization and transphosphorylation. Tyr579and Tyr580get the kinase activity in a calcium-dependent way, which differ from Tyr402. Recent studiesfind that PYK2can participate in many cell signal ways and play important roles in cellproliferation, migration and apoptosis.In our research, we focus on activated PYK2’s function in spindle assembly during mouseoocyte maturation by using such as cell immunofluorescence, inhibitor treatment andWesternblot. The results showed that there were obvious autophoshorylated PYK2(Tyr402)clusters around the nucleus(also be called germinal vesicle) at the GV stage. The tyrosinekinase PYK2was distrubuted in cytoplasm when germinal vesicle breaking down. When theoocytes came to meiosisⅡ,the tyrosine kinase were detected in the cell poles. Then wedouble-stain PYK2(Tyr402) and α-Tubulin were double stained and the results showed thatmicrotubules gathered around chromosomes just like a microtubule ball while PYK2(Tyr402)co-located in the place where microtubules occurred. Chromosomes aliened to the metaphaseplate gradually and microtubules elongated to form a bipolar structure. At the same time,PYK2(Tyr402) left away and move towards the spindle ploes. PYK2(Tyr402) became two fociand located in the spindle poles colse to the cortex at MⅡ. It could be found that PYK2(Tyr402)colocated with γ-Tubulin during the process of oocyte maturation for double staining ofPYK2(Tyr402)and γ-Tubulin. γ-Tubulin was a MTOC protein which regulated microtubule nucleation and γ-Tubulin contained phosphorylation sites and could be activated by specificproteins.So, it was supposed that PYK2(Tyr402) might paticipate in spindle assemble as aMTOC related protein.When the PYK2activity was suppressed by the typical chemical inhibitor Tyrphostin A9,the spindle could not assemble in the right way. Contrary to the control group containing abipolar spindle, the spindle could not polymerize well. Some spindles were abnormal in lengthor area. The oocytes development was arrested before MⅡ stage. The microtubules expressionwas increased when tested in western blotting if the kinase was inhibited. The first pole bodycould not be extruded normally. The rate of the first pole body extrution decreased in a dosedependent manner. It come to a conclusion that when the PYK2activity was inhibited,α-Tubulin was affected and the structure and function of the spindle would become disordered.The results demonstrated PYK2influenced spindle assembly actually.In short, we propose that the activated PYK2play an important role in spindle assemblingduring mouse oocytes meiosis. It may function as a member of microtubule organizing centerthat can direct microtubules to assemble spindle. When the PYK2are inhibited, the progress ofmouse maturation will be affected and arrested.