PIPP在小鼠受精卵早期发育过程中对PKB/Akt和PKC的调控

体细胞中信号蛋白的PH结构域可以通过与信号分子如PtdIns(3,4,5)P3或PKC的结合参与细胞内的信号转导，而肌醇多磷酸5-磷酸酶（PIPP）却可以水解PtdIns(3,4,5)P3上5位磷酸根抑制这种结合。蛋白激酶B（PKB/Akt）即是具有PH结构域的信号蛋白之一，我们在对小鼠受精卵的研究中发现Akt1可以使有丝分裂促进因子（MPF）活化，但PIPP却可以通过水解PtdIns(3,4,5)P3降低Akt1-Ser473的磷酸化从而抑制卵裂。最近我们发现在小鼠受精卵的G2/M期PKC活性增高，而且PIPP也可以降低PKC的活性，本课题将构建失活型的PIPP-S691A并利用现有的HA-PIPP、PIPP-H557A以及PIPP siRNA深入研究其5-磷酸酶结构域通过干扰PH结构域降低Akt以及PKC活性的机制，同时使用Akt和PKC的促效剂和抑制剂观察PH结构域对它们表达与活性的影响。本研究将进一步揭示PH结构域在小鼠受精卵中作用的分子机制。

In somatic cells, PH domains in signaling proteins take part in cellular signal transduction via binding with PtdIns(3,4,5)P3 or PKC,but proline-rich inositol polyphosphate 5-phosphatase (PIPP), a novel regulator in the phosphoinositide 3-kinase(PI3K)/Akt pathway, may hydrolyze the 5-position phosphate from PtdIns(3,4,5)P3 to form PtdIns(3,4)P2 and inhibit the binding. Protein kinase B(PKB/Akt) is one of the signaling proteins containing a PH domain. Previously, we showed that in fertilized mouse eggs, Akt1 can promote the activity of Mitosis-phase Promoting Factor (MPF), but PIPP can decrease Ser473-Akt phosphorylation by hydrolyzing PtdIns(3,4,5)P3, resulting in the inhibition of cleavages. More recently, we observed that the activity of PKC was also increased in G2/M phase and its activity was decreased by PIPP. In this study, we will explore in depth the effect of PIPP on activity of Akt and PKC via the PH domain using a constructed inactive form of PIPP by siRNA and/or mutating residue serine 691 to alanine within PIPP, along with the agonist and/or inhibitor of Akt and PKC. We will determine the effect of PH domain on expression and activity of Akt and PKC in cellular signalling pathway. Outcomes of this study will uncover the function of PH domain during early development of fertilized mouse eggs.