Ma Kewei, Cheung Samuel M, Marshall Aaron J, Duronio Vincent
Deparment of Medicine, University of British Columbia and Vancouver Coastal Health Research Institute, Vancouver, BC, Canada.
Cell Signal. 2008 Apr;20(4):684-94. doi: 10.1016/j.cellsig.2007.12.004. Epub 2007 Dec 8.
The PI3K-PKB pathway is an important and widely studied pathway in cell signaling. The enzyme activity of PI3K produces D-3 phosphoinositides, including the lipid second messengers PI(3,4,5)P3 and PI(3,4)P2. PI(3,4,5)P3 has been deemed to be the most important second messenger for triggering PKB phosphorylation. PKB has two regulatory phosphorylation sites, Thr308 and Ser473, both of which contribute to its full activity. The direct relationship between PI3K lipid products and PKB phosphorylation is still not entirely clear. Our previous study showed that PI(3,4)P2 has a specific role in contributing to PKB phosphorylation on Ser473 sites in mast cells. In this study, we used two strategies to further elucidate this question in a well-established B cell system. First, by SHIP overexpression, we examined PKB activation under conditions where PI(3,4,5)P3 accumulation is largely suppressed. Second, we used dose response of different forms of B-cell receptor ligands to manipulate the relative levels of PI(3,4,5)P3 and PI(3,4)P2. Our results demonstrate a close relationship between PI(3,4,5)P3 levels and Thr308 phosphorylation levels, and PI(3,4)P2 levels and Ser473 phosphorylation levels, respectively. Furthermore, overall PKB activity, primarily consisting of cytosolic enzyme, was dependent upon levels of PI(3,4)P2, while only membrane-associated PKB activity was dependent upon PI(3,4,5)P3 levels. We conclude that PI(3,4,5)P3 and PI(3,4)P2 have distinct roles in determining PKB phosphorylation and activity. Thus, when investigating PI3K-PKB pathways, the importance of both lipids must be considered.
PI3K-PKB信号通路是细胞信号传导中一条重要且被广泛研究的信号通路。PI3K的酶活性产生D-3磷酸肌醇，包括脂质第二信使PI(3,4,5)P3和PI(3,4)P2。PI(3,4,5)P3被认为是触发PKB磷酸化的最重要的第二信使。PKB有两个调节性磷酸化位点，即苏氨酸308（Thr308）和丝氨酸473（Ser473），二者都对其充分活性有贡献。PI3K脂质产物与PKB磷酸化之间的直接关系仍不完全清楚。我们之前的研究表明，PI(3,4)P2在促进肥大细胞中Ser473位点的PKB磷酸化方面具有特定作用。在本研究中，我们采用两种策略在一个成熟的B细胞系统中进一步阐明这个问题。首先，通过SHIP过表达，我们在PI(3,4,5)P3积累被大幅抑制的条件下检测PKB的激活情况。其次，我们使用不同形式的B细胞受体配体的剂量反应来调控PI(3,4,5)P3和PI(3,4)P2的相对水平。我们的结果分别证明了PI(3,4,5)P3水平与Thr308磷酸化水平以及PI(3,4)P2水平与Ser473磷酸化水平之间存在密切关系。此外，主要由胞质酶组成的总体PKB活性依赖于PI(3,4)P2的水平，而只有膜相关的PKB活性依赖于PI(3,4,5)P3的水平。我们得出结论，PI(3,4,5)P3和PI(3,4)P2在决定PKB磷酸化和活性方面具有不同作用。因此，在研究PI3K-PKB信号通路时，必须考虑这两种脂质的重要性。
