Quantitation of class IA PI3Ks in mice reveals p110-free-p85s and isoform-selective subunit associations and recruitment to receptors.
Quantitation of class IA PI3Ks in mice reveals p110-free-p85s and isoform-selective subunit associations and recruitment to receptors.
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DOI：
10.1073/pnas.1803446115
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发表时间：
2018-11-27
影响因子：
11.1
通讯作者：
Hawkins PT
中科院分区：
文献类型：
--
作者：
Tsolakos N;Durrant TN;Chessa T;Suire SM;Oxley D;Kulkarni S;Downward J;Perisic O;Williams RL;Stephens L;Hawkins PT
IA类PI 3 Ks由调节性（p85α/p85β/p55γ）和催化性（p110α/β/δ）亚基组成，其构成信号传导脂质PIP 3，构成信号传导中的关键节点。许多因素构成了它们在PI 3 K抑制剂产生中的众多细胞作用和大量投资的基础。异源二聚体异构体（至少9个）的存在与不同的分布和性质和经常争论的存在“p110-游离调节亚基”作为调制器提供了系统的灵活性，冗余和异构体选择性功能。尽管这一努力的规模很大，但该系统的许多“交战规则”仍不为人知。在这里，我们证明了优先亚基协会，澄清了“p110-free-regulatory-subunit”的存在，表明它们具有可以允许它们调节通路活性的特性，并揭示了允许受体选择性激活PI 3 K α和β的机制。IA类PI 3 K在健康和疾病中具有许多作用。然而，支配亚基间和受体关联的规则仍不清楚。我们改造了小鼠品系，其中单个内源性IA类PI 3 K亚基的C末端标记有17个氨基酸，其可以在体内生物素化。使用这些工具，我们定量了链霉亲和素或PDGFR下拉和细胞裂解物中的PI 3 K亚基。这表明p85α和β与p110α或p110β的结合力相当，但p85α优先与p110δ结合。在许多情况下，包括MEFs（p85β，20%）和肝脏（p85α，30%）中，p85的摩尔含量超过p110。在血清饥饿的MEFs中，与异源二聚体p85相比，p110-free-p85优先与PDGFR结合，这与体外试验一致，该试验证明它们以更高的亲和力和协同性结合基于PDGFR的酪氨酸磷酸化肽;这表明它们可能起调节PI 3 K激活阈值的作用。p110α-异源二聚体被MEF中活化的PDGFR或MEF裂解物中基于PDGFR的酪氨酸磷酸化肽的募集效率比p110β-异源二聚体高5-6倍。这表明PI 3 K α对相关酪氨酸磷酸化基序的亲和力高于PI 3 K β。尽管如此，PI 3 K β在MEFs中对PIP 3和PKB的急性PDGF刺激有很大贡献，因为它通过其RBD被受体募集和小GTP酶（Rac/CDC 42）协同激活，并且可能是顺序激活的，而PI 3 K α的平行激活不依赖于其RBD。这些结果开始为IA类PI 3 K亚基在体内和过去描述“过量p85”、p85α作为肿瘤抑制因子以及PI 3 K α和PI 3 K β的不同受体激活的工作之间的接合规则提供分子清晰度。
The class IA PI3Ks, comprised of regulatory (p85α/p85β/p55γ) and catalytic (p110α/β/δ) subunits, which make the signaling lipid PIP3, constitute a key node in signaling. Many factors underlie their numerous cellular roles and large investments in the creation of PI3K-inhibitors. The existence of heterodimeric-isoforms (at least nine) with distinct distributions and properties and the often-debated existence of “p110-free-regulatory-subunits” as modulators provide the system with flexibility, redundancy and isoform-selective functions. Despite the scale of this endeavour, many of the system’s “rules of engagement” are unknown. Here we demonstrate preferential subunit associations, clarify the existence of “p110-free-regulatory-subunits”, show that they have properties that could allow them to modulate pathway activity, and reveal mechanisms that allow selective activation of PI3Kα and β by receptors. Class IA PI3Ks have many roles in health and disease. The rules that govern intersubunit and receptor associations, however, remain unclear. We engineered mouse lines in which individual endogenous class IA PI3K subunits were C-terminally tagged with 17aa that could be biotinylated in vivo. Using these tools we quantified PI3K subunits in streptavidin or PDGFR pull-downs and cell lysates. This revealed that p85α and β bound equivalently to p110α or p110β but p85α bound preferentially to p110δ. p85s were found in molar-excess over p110s in a number of contexts including MEFs (p85β, 20%) and liver (p85α, 30%). In serum-starved MEFs, p110-free-p85s were preferentially, compared with heterodimeric p85s, bound to PDGFRs, consistent with in vitro assays that demonstrated they bound PDGFR-based tyrosine-phosphorylated peptides with higher affinity and co-operativity; suggesting they may act to tune a PI3K activation threshold. p110α-heterodimers were recruited 5–6× more efficiently than p110β-heterodimers to activated PDGFRs in MEFs or to PDGFR-based tyrosine-phosphorylated peptides in MEF-lysates. This suggests that PI3Kα has a higher affinity for relevant tyrosine-phosphorylated motifs than PI3Kβ. Nevertheless, PI3Kβ contributes substantially to acute PDGF-stimulation of PIP3 and PKB in MEFs because it is synergistically, and possibly sequentially, activated by receptor-recruitment and small GTPases (Rac/CDC42) via its RBD, whereas parallel activation of PI3Kα is independent of its RBD. These results begin to provide molecular clarity to the rules of engagement between class IA PI3K subunits in vivo and past work describing “excess p85,” p85α as a tumor suppressor, and differential receptor activation of PI3Kα and PI3Kβ.
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影响因子：
64.5
作者：
Fritsch R;de Krijger I;Fritsch K;George R;Reason B;Kumar MS;Diefenbacher M;Stamp G;Downward J
通讯作者：
Downward J
通过p85α单体二聚体平衡对PI3K途径进行调节。
影响因子：
7.7
作者：
Cheung LW;Walkiewicz KW;Besong TM;Guo H;Hawke DH;Arold ST;Mills GB
通讯作者：
Mills GB
影响因子：
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作者：
Burke, John E;Williams, Roger L
通讯作者：
Williams, Roger L
DOI：
10.1073/pnas.0908899107
发表时间：
2010-03-23
影响因子：
11.1
作者：
Chagpar, Ryaz B.;Links, Philip H.;Anderson, Deborah H.
通讯作者：
Anderson, Deborah H.
影响因子：
56.9
作者：
Miled, Nabil;Yan, Ying;Williams, Roger L.
通讯作者：
Williams, Roger L.