Department of Pharmacology and Experimental Therapy, Institute of Experimental and Clinical Pharmacology and Toxicology, Eberhard Karls University Hospitals and Clinics, and Interfaculty Center of Pharmacogenomics and Pharmaceutical Research, University of Tübingen, 72074 Tübingen, Germany.
Proc Natl Acad Sci U S A. 2009 Dec 1;106(48):20312-7. doi: 10.1073/pnas.0905506106. Epub 2009 Nov 11.
Class I(B) phosphoinositide 3-kinase gamma (PI3Kgamma) elicits various immunologic and cardiovascular responses; however, the molecular basis for this signal heterogeneity is unclear. PI3Kgamma consists of a catalytic p110gamma and a regulatory p87(PIKAP) (p87, also p84) or p101 subunit. Hitherto p87 and p101 are generally assumed to exhibit redundant functions in receptor-induced and G protein betagamma (Gbetagamma)-mediated PI3Kgamma regulation. Here we investigated the molecular mechanism for receptor-dependent p87/p110gamma activation. By analyzing GFP-tagged proteins expressed in HEK293 cells, PI3Kgamma-complemented bone marrow-derived mast cells (BMMCs) from p110gamma(-/-) mice, and purified recombinant proteins reconstituted to lipid vesicles, we elucidated a novel pathway of p87-dependent, G protein-coupled receptor (GPCR)-induced PI3Kgamma activation. Although p101 strongly interacted with Gbetagamma, thereby mediating PI3Kgamma membrane recruitment and stimulation, p87 exhibited only a weak interaction, resulting in modest kinase activation and lack of membrane recruitment. Surprisingly, Ras-GTP substituted the missing Gbetagamma-dependent membrane recruitment of p87/p110gamma by direct interaction with p110gamma, suggesting the indispensability of Ras for activation of p87/p110gamma. Consequently, interference with Ras signaling indeed selectively blocked p87/p110gamma, but not p101/p110gamma, kinase activity in HEK293 and BMMC cells, revealing an important crosstalk between monomeric and trimeric G proteins for p87/p110gamma activation. Our data display distinct signaling requirements of p87 and p101, conferring signaling specificity to PI3Kgamma that could open up new possibilities for therapeutic intervention.
I 类（B）磷酸肌醇 3-激酶γ（PI3Kγ）引发各种免疫和心血管反应；然而，这种信号异质性的分子基础尚不清楚。PI3Kγ 由催化 p110γ 和调节 p87（PIKAP）（p87，也称为 p84）或 p101 亚基组成。迄今为止，p87 和 p101 通常被认为在受体诱导和 G 蛋白βγ（Gβγ）介导的 PI3Kγ 调节中具有冗余功能。在这里，我们研究了受体依赖性 p87/p110γ 激活的分子机制。通过分析在 HEK293 细胞中表达的 GFP 标记蛋白、来自 p110γ（-/-）小鼠的补充 PI3Kγ 的骨髓来源肥大细胞（BMMC）以及重新组装到脂质体中的纯化重组蛋白，我们阐明了一种新的途径，即 p87 依赖性、G 蛋白偶联受体（GPCR）诱导的 PI3Kγ 激活。尽管 p101 与 Gβγ 强烈相互作用，从而介导 PI3Kγ 膜募集和刺激，但 p87 仅表现出较弱的相互作用，导致激酶激活程度较低且缺乏膜募集。令人惊讶的是，Ras-GTP 通过与 p110γ 的直接相互作用替代了 p87/p110γ 中缺失的 Gβγ 依赖性膜募集，这表明 Ras 对于 p87/p110γ 的激活是必不可少的。因此，干扰 Ras 信号确实选择性地阻断了 HEK293 和 BMMC 细胞中的 p87/p110γ，但不阻断 p101/p110γ 的激酶活性，揭示了单体和三聚体 G 蛋白之间对于 p87/p110γ 激活的重要串扰。我们的数据显示了 p87 和 p101 的不同信号要求，为 PI3Kγ 赋予了信号特异性，这为治疗干预开辟了新的可能性。
