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. 2023 Jun 6;122(11):2301-2310. doi: 10.1016/j.bpj.2023.01.041. Epub 2023 Feb 2.
Previous studies have documented the formation of a heterodimer between the two protein kinases PDK1 and PKCα on a lipid bilayer containing their target lipids. This work investigates the association-dissociation kinetics of this PDK1:PKCα heterodimer. The approach monitors the two-dimensional diffusion of single, membrane-associated PDK1 molecules for diffusivity changes as PKCα molecules bind and unbind. In the absence of PKCα, a membrane-associated PDK1 molecule exhibits high diffusivity (or large diffusion constant, D) because its membrane-contacting PH domain binds the target PIP lipid headgroup with little bilayer penetration, yielding minimal frictional drag against the bilayer. In contrast, membrane-associated PKCα contacts the bilayer via its C1A, C1B, and C2 domains, which each bind at least one target lipid with significant bilayer insertion, yielding a large frictional drag and low diffusivity. The present findings reveal that individual fluor-PDK1 molecules freely diffusing on the membrane surface undergo reversible switching between distinct high and low diffusivity states, corresponding to the PDK1 monomer and the PDK1:PKCα heterodimer, respectively. The observed single-molecule diffusion trajectories are converted to step length time courses, then subjected to two-state, hidden Markov modeling and dwell time analysis. The findings reveal that both the PDK1 monomer state and the PDK1:PKCα heterodimer state decay via simple exponential kinetics, yielding estimates of rate constants for state switching in both directions. Notably, the PDK1:PKCα heterodimer has been shown to competitively inhibit PDK1 phosphoactivation of AKT1, and is believed to play a tumor suppressor role by limiting excess activation of the highly oncogenic PDK1/AKT1/mTOR pathway. Thus, the present elucidation of the PDK1:PKCα association-dissociation kinetics has important biological and medical implications. More broadly, the findings illustrate the power of single-molecule diffusion measurements to reveal the kinetics of association-dissociation events in membrane signaling reactions that yield a large change in diffusive mobility.
先前的研究已经记录了两种蛋白激酶 PDK1 和 PKCα 在含有其靶脂质的脂质双层上形成异二聚体。这项工作研究了这种 PDK1:PKCα 异二聚体的缔合-解离动力学。该方法监测单个膜相关 PDK1 分子的二维扩散，以观察 PKCα 分子结合和解离时扩散率的变化。在没有 PKCα 的情况下，膜相关 PDK1 分子表现出高扩散率（或大扩散常数，D），因为其膜接触 PH 结构域与靶 PIP 脂质头基结合，几乎没有双层穿透，对双层的摩擦阻力最小。相比之下，膜相关的 PKCα 通过其 C1A、C1B 和 C2 结构域与双层接触，每个结构域都与至少一个靶脂质结合，具有显著的双层插入，产生大的摩擦阻力和低扩散率。本研究发现，在膜表面自由扩散的单个荧光 PDK1 分子经历可逆切换，分别对应于 PDK1 单体和 PDK1:PKCα 异二聚体，处于不同的高和低扩散率状态之间。观察到的单个分子扩散轨迹被转换为步长时间历程，然后进行两态、隐马尔可夫建模和停留时间分析。研究结果表明，PDK1 单体状态和 PDK1:PKCα 异二聚体状态均通过简单指数动力学衰减，从而得出两个方向的状态转换速率常数的估计值。值得注意的是，PDK1:PKCα 异二聚体已被证明可以竞争性抑制 PDK1 对 AKT1 的磷酸化激活，并且通过限制高度致癌的 PDK1/AKT1/mTOR 途径的过度激活，被认为发挥肿瘤抑制作用。因此，目前对 PDK1:PKCα 缔合-解离动力学的阐明具有重要的生物学和医学意义。更广泛地说，这些发现说明了单分子扩散测量揭示膜信号反应中缔合-解离事件动力学的能力，这些反应导致扩散迁移率发生大的变化。
