Fig. 4: AURKA^T288V supports AURKA function in maintaining the mitotic state in CSF-arrested Xenopus oocytes.

a In the ovary, oocytes are arrested in prophase of meiosis I. At the time of ovulation and upon hormone stimulation (progesterone), oocytes re-enter into meiosis and reach the second meiotic division where they arrest in metaphase II, awaiting fertilization (CSF arrest). Upon Gwl immunodepletion (∆Gwl in red), the extracts exit the “mitotic state”. Complementation of the extract with recombinant hyperactive Gwl^K72M kinase (+Gwl^K72M in green) restores the mitotic state, if the extracts contain a functional Bora, AURKA, Plk1 pathway. GVBD: germinal vesicle breakdown. The green and red area represents the high and low CyclinB-Cdk1 (MPF) activity. b Network representing the pathways controlling Cyclin-Cdk activity and maintaining the “mitotic state” in CSF-arrested Xenopus egg extracts. c Flow chart of the experimental approach used to test the functionality of AURKA mutants in maintaining the “mitotic state” in CSF-arrested Xenopus oocytes extracts. Endogenous AURKA and Gwl were sequentially immunodepleted from CSF egg extracts and then complemented with recombinant human AURKA^WT, catalytically dead AURKA^D274N, or downregulated mimic AURKA^T288V and hyperactive Gwl^K72M kinase (K72M) (top panel). CSF extracts non-depleted or sequentially depleted of AURKA (∆AURKA) and then Greatwall (∆Gwl) were separated by SDS-PAGE and analyzed by Western blot using Xenopus Gwl and AURKA antibodies. Asterisks denote non-specific bands. d CSF egg extracts sequentially depleted of AURKA (∆AURKA) and then Gwl (∆Gwl) were supplemented at time 0 with recombinant AURKA^WT, or catalytically dead AURKA^D274N or AURKA^T288V in the presence of hyperactive Gwl^K72M. A fraction of the extracts was collected at the indicated time-points (0, 20, 40, 60, 90 min) and analyzed by Western blot using specific antibodies to monitor the levels of huGreatwall, Hu/Xe AURKA, Plk1, Cdc25, as well as the phosphorylation of Plx1 on T201 (pPlk1), PP1 phosphatase on T320 (pPP1), and Cdk on Y15 (pTyr). The green and red area represents the high and low CyclinB-Cdk1 (MPF) activity (“mitotic state”).