Yu J, Zhang Y, McIlroy J, Rordorf-Nikolic T, Orr G A, Backer J M
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Mol Cell Biol. 1998 Mar;18(3):1379-87. doi: 10.1128/MCB.18.3.1379.
We propose a novel model for the regulation of the p85/pl10alpha phosphatidylinositol 3'-kinase. In insect cells, the p110alpha catalytic subunit is active as a monomer but its activity is decreased by coexpression with the p85 regulatory subunit. Similarly, the lipid kinase activity of recombinant glutathione S-transferase (GST)-p110alpha is reduced by 65 to 85% upon in vitro reconstitution with p85. Incubation of p110alpha/p85 dimers with phosphotyrosyl peptides restored activity, but only to the level of monomeric p110alpha. These data show that the binding of phosphoproteins to the SH2 domains of p85 activates the p85/p110alpha dimers by inducing a transition from an inhibited to a disinhibited state. In contrast, monomeric p110 had little activity in HEK 293T cells, and its activity was increased 15- to 20-fold by coexpression with p85. However, this apparent requirement for p85 was eliminated by the addition of a bulky tag to the N terminus of p110alpha or by the growth of the HEK 293T cells at 30 degrees C. These nonspecific interventions mimicked the effects of p85 on p110alpha, suggesting that the regulatory subunit acts by stabilizing the overall conformation of the catalytic subunit rather than by inducing a specific activated conformation. This stabilization was directly demonstrated in metabolically labeled HEK 293T cells, in which p85 increased the half-life of p110. Furthermore, p85 protected p110 from thermal inactivation in vitro. Importantly, when we examined the effect of p85 on GST-p110alpha in mammalian cells at 30 degrees C, culture conditions that stabilize the catalytic subunit and that are similar to the conditions used for insect cells, we found that p85 inhibited p110alpha. Thus, we have experimentally distinguished two effects of p85 on p110alpha: conformational stabilization of the catalytic subunit and inhibition of its lipid kinase activity. Our data reconcile the apparent conflict between previous studies of insect versus mammalian cells and show that p110alpha is both stabilized and inhibited by dimerization with p85.
我们提出了一种调控p85/p110α磷脂酰肌醇3'-激酶的新模型。在昆虫细胞中，p110α催化亚基作为单体时具有活性，但其活性会因与p85调节亚基共表达而降低。同样，重组谷胱甘肽S-转移酶（GST）-p110α的脂质激酶活性在与p85进行体外重组后降低了65%至85%。将p110α/p85二聚体与磷酸酪氨酸肽一起孵育可恢复活性，但仅恢复到单体p110α的水平。这些数据表明，磷蛋白与p85的SH2结构域结合，通过诱导从抑制状态到去抑制状态的转变，激活了p85/p110α二聚体。相比之下，单体p110在HEK 293T细胞中活性很低，与p85共表达可使其活性提高15至20倍。然而，通过在p110α的N末端添加一个大标签或在30℃培养HEK 293T细胞，消除了对p85的这种明显需求。这些非特异性干预模拟了p85对p110α的影响，表明调节亚基通过稳定催化亚基的整体构象起作用，而不是诱导特定的活化构象。这种稳定性在代谢标记的HEK 293T细胞中得到直接证明，其中p85延长了p110的半衰期。此外，p85在体外保护p110免受热失活。重要的是，当我们在30℃的哺乳动物细胞中研究p85对GST-p110α的影响时，这种培养条件稳定了催化亚基且类似于用于昆虫细胞的条件，我们发现p85抑制了p110α。因此，我们通过实验区分了p85对p110α的两种作用：催化亚基的构象稳定和其脂质激酶活性的抑制。我们的数据调和了先前昆虫细胞与哺乳动物细胞研究之间明显的冲突，并表明p110α与p85二聚化时既被稳定又被抑制。
