Ultanir Sila K, Yadav Smita, Hertz Nicholas T, Oses-Prieto Juan A, Claxton Suzanne, Burlingame Alma L, Shokat Kevan M, Jan Lily Y, Jan Yuh-Nung
Departments of Physiology, Biochemistry, and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA; Medical Research Council, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
Departments of Physiology, Biochemistry, and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA.
Neuron. 2014 Dec 3;84(5):968-82. doi: 10.1016/j.neuron.2014.10.025. Epub 2014 Nov 13.
Mammalian Sterile 20 (Ste20)-like kinase 3 (MST3) is a ubiquitously expressed kinase capable of enhancing axon outgrowth. Whether and how MST3 kinase signaling might regulate development of dendritic filopodia and spine synapses is unknown. Through shRNA-mediated depletion of MST3 and kinase-dead MST3 expression in developing hippocampal cultures, we found that MST3 is necessary for proper filopodia, dendritic spine, and excitatory synapse development. Knockdown of MST3 in layer 2/3 pyramidal neurons via in utero electroporation also reduced spine density in vivo. Using chemical genetics, we discovered thirteen candidate MST3 substrates and identified the phosphorylation sites. Among the identified MST3 substrates, TAO kinases regulate dendritic filopodia and spine development, similar to MST3. Furthermore, using stable isotope labeling by amino acids in culture (SILAC), we show that phosphorylated TAO1/2 associates with Myosin Va and is necessary for its dendritic localization, thus revealing a mechanism for excitatory synapse development in the mammalian CNS.
哺乳动物不育 20（Ste20）样激酶 3（MST3）是一种广泛表达的激酶，能够促进轴突生长。MST3 激酶信号是否以及如何调节树突丝状伪足和脊柱突触的发育尚不清楚。通过在发育中的海马培养物中利用 shRNA 介导的 MST3 缺失和激酶失活的 MST3 表达，我们发现 MST3 对于正常的丝状伪足、树突棘和兴奋性突触发育是必需的。通过子宫内电穿孔在 2/3 层锥体神经元中敲低 MST3 也会降低体内的棘密度。使用化学遗传学，我们发现了 13 种候选 MST3 底物并确定了磷酸化位点。在已鉴定的 MST3 底物中，TAO 激酶与 MST3 类似，调节树突丝状伪足和脊柱发育。此外，使用培养物中氨基酸稳定同位素标记（SILAC），我们表明磷酸化的 TAO1/2 与肌球蛋白 Va 相关联，并且是其树突定位所必需的，从而揭示了哺乳动物中枢神经系统中兴奋性突触发育的机制。
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