Rodríguez-Asiain Arantza, Ruiz-Babot Gerard, Romero Walter, Cubí Roger, Erazo Tatiana, Biondi Ricardo M, Bayascas Jose R, Aguilera Jose, Gómez Nestor, Gil Carles, Claro Enrique, Lizcano Jose M
Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Barcelona, Spain.
Biochim Biophys Acta. 2011 Dec;1811(12):1124-35. doi: 10.1016/j.bbalip.2011.10.004. Epub 2011 Oct 12.
Brain specific kinases 1 and 2 (BRSK1/2, also named SAD kinases) are serine-threonine kinases specifically expressed in the brain, and activated by LKB1-mediated phosphorylation of a threonine residue at their T-loop (Thr189/174 in human BRSK1/2). BRSKs are crucial for establishing neuronal polarity, and BRSK1 has also been shown to regulate neurotransmitter release presynaptically. How BRSK1 exerts this latter function is unknown, since its substrates at the synaptic terminal and the mechanisms modulating its activity remain to be described. Key regulators of neurotransmitter release, such as SNARE complex proteins, are located at membrane rafts. Therefore we initially undertook this work to check whether BRSK1 also locates at these membrane microdomains. Here we show that brain BRSK1, but not BRSK2, is palmitoylated, and provide biochemical and pharmacological evidences demonstrating that a pool of BRSK1, but not BRSK2 or LKB1, localizes at membrane lipid rafts. We also show that raft-associated BRSK1 has higher activity than BRSK1 from non-raft environment, based on a higher T-loop phosphorylation at Thr-189. Further, recombinant BRSK1 activity increased 3-fold when assayed with small multilamellar vesicles (SMV) generated with lipids extracted from synaptosomal raft fractions. A similar BRSK1-activating effect was obtained with synthetic SMV made with phosphatidylcholine, cholesterol and sphingomyelin, mixed in the same molar ratio at which these three major lipids are present in rafts. Importantly, SMV also enhanced the activity of a constitutively active BRSK1 (T189E), underpinning that interaction with lipid rafts represents a new mechanism of BRSK1 activity modulation, additional to T-loop phosphorylation.
脑特异性激酶1和2（BRSK1/2，也称为SAD激酶）是在脑中特异性表达的丝氨酸 - 苏氨酸激酶，通过LKB1介导其T环中苏氨酸残基（人BRSK1/2中的Thr189/174）的磷酸化而被激活。BRSKs对于建立神经元极性至关重要，并且BRSK1还被证明在突触前调节神经递质释放。BRSK1如何发挥后一种功能尚不清楚，因为其在突触末端的底物以及调节其活性的机制仍有待描述。神经递质释放的关键调节因子，如SNARE复合蛋白，位于膜筏中。因此，我们最初开展这项工作以检查BRSK1是否也位于这些膜微结构域中。在这里，我们表明脑BRSK1而不是BRSK2被棕榈酰化，并提供生化和药理学证据证明一部分BRSK1而不是BRSK2或LKB1定位于膜脂筏。我们还表明，基于Thr-189处更高的T环磷酸化，与脂筏相关的BRSK1比来自非脂筏环境的BRSK1具有更高的活性。此外，当用从突触体脂筏级分中提取的脂质产生的小多层囊泡（SMV）进行测定时，重组BRSK1活性增加了3倍。用磷脂酰胆碱、胆固醇和鞘磷脂以与脂筏中这三种主要脂质相同的摩尔比混合制成的合成SMV也获得了类似的BRSK1激活效果。重要的是，SMV还增强了组成型活性BRSK1（T189E）的活性，这表明与脂筏的相互作用代表了BRSK1活性调节的一种新机制，这是除T环磷酸化之外的。
