Molecular Biophysics Program and Department of Biochemistry, University of Colorado, Boulder, Colorado.
Molecular Biophysics Program and Department of Biochemistry, University of Colorado, Boulder, Colorado.
Biophys J. 2021 Dec 21;120(24):5657-5673. doi: 10.1016/j.bpj.2021.10.015. Epub 2021 Oct 19.
Leukocyte migration is controlled by a leading-edge chemosensory pathway that generates the regulatory lipid phosphatidylinositol-3,4,5-trisphosphate (PIP), a growth signal, thereby driving leading-edge expansion up attractant gradients toward sites of infection, inflammation, or tissue damage. PIP also serves as an important growth signal in growing cells and oncogenesis. The kinases PDK1, AKT1 or PKB, and PKCα are key components of a plasma-membrane-based PIP and Ca signaling circuit that regulates these processes. PDK1 and AKT1 are recruited to the membrane by PIP, whereas PKCα is recruited to the membrane by Ca. All three of these master kinases phosphoregulate an array of protein targets. For example, PDK1 activates AKT1, PKCα, and other AGC kinases by phosphorylation at key sites. PDK1 is believed to form PDK1-AKT1 and PDK1-PKCα heterodimers stabilized by a PDK1-interacting fragment (PIF) interaction between the PDK1 PIF pocket and the PIF motif of the AGC binding partner. Here, we present the first, to our knowledge, single-molecule studies of full-length PDK1 and AKT1 on target membrane surfaces, as well as their interaction with full-length PKCα. These studies directly detect membrane-bound PDK1-AKT1 and PDK1-PKCα heterodimers stabilized by PIF interactions formed at physiological ligand concentrations. PKCα exhibits eightfold higher PDK1 affinity than AKT1 and can competitively displace AKT1 from PDK1-AKT1 heterodimers. Ensemble activity measurements under matched conditions reveal that PDK1 activates AKT1 via a cis mechanism by phosphorylating an AKT1 molecule in the same PDK1-AKT1 heterodimer, whereas PKCα acts as a competitive inhibitor of this phosphoactivation reaction by displacing AKT1 from PDK1. Overall, the findings provide insights into the binding and regulatory interactions of the three master kinases on their target membrane and suggest that a recently described tumor suppressor activity of PKC isoforms may arise from its ability to downregulate PDK1-AKT1 phosphoactivation in the PIP-PDK1-AKT1-mTOR pathway linked to cell growth and oncogenesis.
白细胞迁移受前沿化学感觉通路控制，该通路生成调节脂质磷脂酰肌醇-3,4,5-三磷酸（PIP），作为生长信号，从而驱动前沿在吸引物梯度上扩张，向感染、炎症或组织损伤部位移动。PIP 也是生长细胞和肿瘤发生中的重要生长信号。PDK1、AKT1 或 PKB 和 PKCα 激酶是基于质膜的 PIP 和 Ca 信号通路的关键组成部分，该通路调节这些过程。PDK1 和 AKT1 通过 PIP 募集到膜上，而 PKCα 通过 Ca 募集到膜上。这三种主要激酶通过磷酸化调节一系列蛋白靶标。例如，PDK1 通过磷酸化关键位点激活 AKT1、PKCα 和其他 AGC 激酶。PDK1 被认为形成 PDK1-AKT1 和 PDK1-PKCα 异二聚体，由 PDK1 相互作用片段（PIF）与 AGC 结合伴侣的 PIF 模体之间的相互作用稳定。在这里，我们首次在目标膜表面上进行全长 PDK1 和 AKT1 的单分子研究，以及它们与全长 PKCα 的相互作用。这些研究直接检测到在生理配体浓度下形成的 PIF 相互作用稳定的膜结合 PDK1-AKT1 和 PDK1-PKCα 异二聚体。PKCα 对 PDK1 的亲和力比 AKT1 高 8 倍，并且可以竞争性地将 AKT1 从 PDK1-AKT1 异二聚体中置换出来。在匹配条件下的整体活性测量表明，PDK1 通过磷酸化同一 PDK1-AKT1 异二聚体中的 AKT1 分子，以顺式机制激活 AKT1，而 PKCα 通过从 PDK1 中置换 AKT1 来充当该磷酸化反应的竞争性抑制剂。总的来说，这些发现提供了关于三种主要激酶在其靶膜上的结合和调节相互作用的见解，并表明最近描述的 PKC 同工型的肿瘤抑制活性可能源于其降低与细胞生长和肿瘤发生相关的 PIP-PDK1-AKT1-mTOR 途径中 PDK1-AKT1 磷酸化激活的能力。
