MAP4K4 expression in cardiomyocytes: multiple isoforms, multiple phosphorylations and interactions with striatins.
MAP4K4 expression in cardiomyocytes: multiple isoforms, multiple phosphorylations and interactions with striatins.
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DOI：
10.1042/bcj20210003
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发表时间：
2021-06-11
期刊：
影响因子：
--
通讯作者：
Clerk A
中科院分区：
文献类型：
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作者：
Fuller SJ;Edmunds NS;McGuffin LJ;Hardyman MA;Cull JJ;Alharbi HO;Meijles DN;Sugden PH;Clerk A
与其他GCKIV激酶一样，Ser/Thr激酶MAP 4K 4具有N-末端激酶和C-末端柠檬酸同源（CNH）结构域。MAP 4K 4可以激活c-Jun N-末端激酶（JNKs），心脏研究表明它将氧化应激与JNKs和心力衰竭联系起来。在其他系统中，MAP 4K 4在纹状体蛋白相互作用磷酸酶和激酶（STRIPAK）复合物中受到调节，其中三种纹状体蛋白之一将PP 2A与激酶相邻，以保持其去磷酸化和失活。我们的目的是了解心肌细胞中MAP 4K 4是如何调节的。大鼠MAP 4K 4基因没有被正确定义。我们利用5′-RACE技术克隆了大鼠MAP 4K 4基因的第一个编码外显子，并克隆了全长序列，证实了MAP 4K 4在大鼠心肌细胞中存在选择性剪接。我们确定了一个额外的α-螺旋C-末端的激酶结构域重要的激酶活性。在进一步的研究中，FLAG-MAP 4K 4在HEK 293细胞或心肌细胞中表达。Ser/Thr蛋白磷酸酶抑制剂calyculin A（CalA）诱导MAP 4K 4过度磷酸化，激活环磷酸化，激酶和CNH结构域之间的接头广泛磷酸化。这需要激酶活性。在未处理的心肌细胞中，MAP 4K 4与肌球蛋白相关，并且这在CalA处理中丢失。FLAG-MAP 4K 4与心肌细胞中的所有三种纹状体蛋白相关，表明STRIPAK复合物内的调节，并与CalA的激活一致。计算分析表明，这种相互作用是直接的，并通过卷曲螺旋结构域介导。令人惊讶的是，FLAG-MAP 4K 4抑制了心肌细胞中H2 O2对JNK的激活，并增加了肌原纤维组织。我们的数据确定MAP 4K 4作为心肌细胞中的STRIPAK调节激酶，并表明它调节细胞骨架而不是激活JNK。
The Ser/Thr kinase MAP4K4, like other GCKIV kinases, has N-terminal kinase and C-terminal citron homology (CNH) domains. MAP4K4 can activate c-Jun N-terminal kinases (JNKs), and studies in the heart suggest it links oxidative stress to JNKs and heart failure. In other systems, MAP4K4 is regulated in striatin-interacting phosphatase and kinase (STRIPAK) complexes, in which one of three striatins tethers PP2A adjacent to a kinase to keep it dephosphorylated and inactive. Our aim was to understand how MAP4K4 is regulated in cardiomyocytes. The rat MAP4K4 gene was not properly defined. We identified the first coding exon of the rat gene using 5′-RACE, we cloned the full-length sequence and confirmed alternative-splicing of MAP4K4 in rat cardiomyocytes. We identified an additional α-helix C-terminal to the kinase domain important for kinase activity. In further studies, FLAG-MAP4K4 was expressed in HEK293 cells or cardiomyocytes. The Ser/Thr protein phosphatase inhibitor calyculin A (CalA) induced MAP4K4 hyperphosphorylation, with phosphorylation of the activation loop and extensive phosphorylation of the linker between the kinase and CNH domains. This required kinase activity. MAP4K4 associated with myosin in untreated cardiomyocytes, and this was lost with CalA-treatment. FLAG-MAP4K4 associated with all three striatins in cardiomyocytes, indicative of regulation within STRIPAK complexes and consistent with activation by CalA. Computational analysis suggested the interaction was direct and mediated via coiled-coil domains. Surprisingly, FLAG-MAP4K4 inhibited JNK activation by H2O2 in cardiomyocytes and increased myofibrillar organisation. Our data identify MAP4K4 as a STRIPAK-regulated kinase in cardiomyocytes, and suggest it regulates the cytoskeleton rather than activates JNKs.