Center for Translational Medicine, Thomas Jefferson University, Philadelphia, PA 19107, USA; Reproductive Medicine Center, The Affiliated Drum Tower Hospital of Nanjing University, Nanjing, China.
Int J Cardiol. 2013 Oct 9;168(4):3291-9. doi: 10.1016/j.ijcard.2013.04.045. Epub 2013 May 3.
Mammalian sterile 20-like kinase 1 (Mst1) is a mammalian homolog of Hippo kinase from Drosophila and it is a critical component of the Hippo signaling pathway, which regulates a variety of biological processes ranging from cell contact inhibition, organ size control, apoptosis and tumor suppression in mammals. Mst1 plays essential roles in heart disease since its activation causes cardiomyocyte apoptosis and dilated cardiomyopathy. However, the mechanism underlying Mst1 activation in the heart is not known.
To identify novel cardiac proteins that may regulate Mst1 activity in the heart under pathophysiological conditions, a yeast two-hybrid screening of a human heart cDNA library with a dominant-negative Mst1 (K59R) mutant used as bait was performed. As a result, protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) was identified as an Mst1-interacting protein. The interaction of PCMT1 with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK293 cells and native cardiomyocytes, in which PCMT1 interacted with the kinase domain of Mst1, but not with its C-terminal regulatory domain. Overexpression of PCMT1 did not affect the Mst1 expression, but significantly attenuated the Mst1 activation and its apoptotic effects in response to the hypoxia/reoxygenation induced injury in cardiomyocytes. Indeed, upregulation of PCMT1 by CGP3466B, a compound related to the anti-Parkinson's drug R-(-)-deprenyl with potent antiapoptotic effects, inhibited the hypoxia/reoxygenation induced Mst1 activation and cardiomyocyte apoptosis.
These findings implicate PCMT1 as a novel inhibitor of Mst1 activation in cardiomyocytes and suggest that targeting PCMT1 may prevent myocardial apoptosis through inhibition of Mst1.
哺乳动物无活性丝氨酸/苏氨酸激酶 1（Mst1）是果蝇 Hippo 激酶的哺乳动物同源物，是 Hippo 信号通路的关键组成部分，该通路调节从细胞接触抑制、器官大小控制、凋亡和肿瘤抑制等多种生物学过程。Mst1 在心脏病中发挥重要作用，因为其激活会导致心肌细胞凋亡和扩张型心肌病。然而，Mst1 在心脏中的激活机制尚不清楚。
为了鉴定在病理生理条件下可能调节心脏中 Mst1 活性的新的心脏蛋白，使用显性负性 Mst1（K59R）突变体作为诱饵，对人心脏 cDNA 文库进行酵母双杂交筛选。结果，蛋白-L-异天冬氨酸（D-天冬氨酸）O-甲基转移酶（PCMT1）被鉴定为与 Mst1 相互作用的蛋白。在共转染的 HEK293 细胞和天然心肌细胞中，通过共免疫沉淀证实了 PCMT1 与 Mst1 的相互作用，其中 PCMT1 与 Mst1 的激酶结构域相互作用，但不与 C 端调节结构域相互作用。PCMT1 的过表达不会影响 Mst1 的表达，但显著减弱了心肌细胞缺氧/复氧诱导损伤时 Mst1 的激活及其凋亡作用。事实上，CGP3466B（一种与抗帕金森病药物 R-(-)-deprenyl 相关的化合物，具有很强的抗凋亡作用）上调 PCMT1 可抑制缺氧/复氧诱导的 Mst1 激活和心肌细胞凋亡。
这些发现表明 PCMT1 是心肌细胞中 Mst1 激活的一种新型抑制剂，并表明靶向 PCMT1 可能通过抑制 Mst1 来防止心肌细胞凋亡。
