Montoya Valentina, Gutierrez Christina, Najera Omar, Leony Denisse, Varela-Ramirez Armando, Popova Juliana, Rasenick Mark M, Das Siddhartha, Roychowdhury Sukla
The Department of Biological Sciences, University of Texas El Paso, El Paso, Texas 79968, USA.
Cell Motil Cytoskeleton. 2007 Dec;64(12):936-50. doi: 10.1002/cm.20234.
The betagamma subunit of G proteins (Gbetagamma) is known to transfer signals from cell surface receptors to intracellular effector molecules. Recent results suggest that Gbetagamma also interacts with microtubules and is involved in the regulation of the mitotic spindle. In the current study, the anti-microtubular drug nocodazole was employed to investigate the mechanism by which Gbetagamma interacts with tubulin and its possible implications in microtubule assembly in cultured PC12 cells. Nocodazole-induced depolymerization of microtubules drastically inhibited the interaction between Gbetagamma and tubulin. Gbetagamma was preferentially bound to microtubules and treatment with nocodazole suggested that the dissociation of Gbetagamma from microtubules is an early step in the depolymerization process. When microtubules were allowed to recover after removal of nocodazole, the tubulin-Gbetagamma interaction was restored. Unlike Gbetagamma, however, the interaction between tubulin and the alpha subunit of the Gs protein (Gsalpha) was not inhibited by nocodazole, indicating that the inhibition of tubulin-Gbetagamma interactions during microtubule depolymerization is selective. We found that Gbetagamma also interacts with gamma-tubulin, colocalizes with gamma-tubulin in centrosomes, and co-sediments in centrosomal fractions. The interaction between Gbetagamma and gamma-tubulin was unaffected by nocodazole, suggesting that the Gbetagamma-gamma-tubulin interaction is not dependent on assembled microtubules. Taken together, our results suggest that Gbetagamma may play an important and definitive role in microtubule assembly and/or stability. We propose that betagamma-microtubule interaction is an important step for G protein-mediated cell activation. These results may also provide new insights into the mechanism of action of anti-microtubule drugs.
G蛋白的βγ亚基（Gβγ）已知可将信号从细胞表面受体传递至细胞内效应分子。最近的研究结果表明，Gβγ还与微管相互作用，并参与有丝分裂纺锤体的调节。在本研究中，使用抗微管药物诺考达唑来研究Gβγ与微管蛋白相互作用的机制及其在培养的PC12细胞微管组装中的可能影响。诺考达唑诱导的微管解聚极大地抑制了Gβγ与微管蛋白之间的相互作用。Gβγ优先与微管结合，诺考达唑处理表明Gβγ从微管上的解离是解聚过程中的早期步骤。去除诺考达唑后微管恢复时，微管蛋白 - Gβγ相互作用得以恢复。然而，与Gβγ不同，微管蛋白与Gs蛋白的α亚基（Gsα）之间的相互作用不受诺考达唑抑制，这表明微管解聚过程中微管蛋白 - Gβγ相互作用的抑制具有选择性。我们发现Gβγ还与γ-微管蛋白相互作用，在中心体中与γ-微管蛋白共定位，并在中心体组分中共沉降。Gβγ与γ-微管蛋白之间的相互作用不受诺考达唑影响，这表明Gβγ - γ-微管蛋白相互作用不依赖于组装好的微管。综上所述，我们的结果表明Gβγ可能在微管组装和/或稳定性中发挥重要且决定性的作用。我们提出βγ-微管相互作用是G蛋白介导的细胞激活的重要步骤。这些结果也可能为抗微管药物的作用机制提供新的见解。
