Princess Margaret Cancer Center, University Health Network, Toronto, Ontario M5G 1L7, Canada.
Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L7, Canada.
Sci Signal. 2020 Mar 31;13(625):eaaz0344. doi: 10.1126/scisignal.aaz0344.
KRAS4b is a small guanosine triphosphatase (GTPase) protein that regulates several signal transduction pathways that underlie cell proliferation, differentiation, and survival. KRAS4b function requires prenylation of its C terminus and recruitment to the plasma membrane, where KRAS4b activates effector proteins including the RAF family of kinases. The Ca-sensing protein calmodulin (CaM) has been suggested to regulate the localization of KRAS4b through direct, Ca-dependent interaction, but how CaM and KRAS4b functionally interact is controversial. Here, we determined a crystal structure, which was supported by solution nuclear magnetic resonance (NMR), that revealed the sequestration of the prenyl moiety of KRAS4b in the hydrophobic pocket of the C-terminal lobe of Ca-bound CaM. Our engineered fluorescence resonance energy transfer (FRET)-based biosensor probes (CaMeRAS) showed that, upon stimulation of Ca influx by extracellular ligands, KRAS4b reversibly translocated in a Ca-CaM-dependent manner from the plasma membrane to the cytoplasm in live HeLa and HEK293 cells. These results reveal a mechanism underlying the inhibition of KRAS4b activity by Ca signaling pathways.
KRAS4b 是一种小的鸟嘌呤三磷酸酶（GTPase）蛋白，可调节细胞增殖、分化和存活所必需的几种信号转导途径。KRAS4b 的功能需要其 C 末端的异戊烯化和募集到质膜，在质膜上 KRAS4b 激活包括 RAF 激酶家族在内的效应蛋白。钙感应蛋白钙调蛋白（CaM）被认为通过直接的 Ca 依赖性相互作用来调节 KRAS4b 的定位，但 CaM 和 KRAS4b 如何发挥功能相互作用仍存在争议。在这里，我们确定了一个晶体结构，该结构得到了溶液核磁共振（NMR）的支持，揭示了 KRAS4b 的异戊烯基部分被 Ca 结合的 CaM 的 C 端结构域的疏水性口袋隔离。我们设计的荧光共振能量转移（FRET）基于生物传感器探针（CaMeRAS）显示，在细胞外配体刺激 Ca 内流时，KRAS4b 在 Ca-CaM 依赖性方式下从质膜可逆地易位到活的 HeLa 和 HEK293 细胞的细胞质中。这些结果揭示了 Ca 信号通路抑制 KRAS4b 活性的机制。
