Identification and characterization of zipper-interacting protein kinase as the unique vascular smooth muscle myosin phosphatase-associated kinase
Identification and characterization of zipper-interacting protein kinase as the unique vascular smooth muscle myosin phosphatase-associated kinase
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DOI：
10.1074/jbc.m403676200
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发表时间：
2004-10-01
影响因子：
4.8
通讯作者：
Mendelsohn, ME
中科院分区：
文献类型：
--
作者：
Endo, A;Surks, HK;Mendelsohn, ME
肌球蛋白轻链(MLC)磷酸化激活肌球蛋白轻链(MLC)，增加肌球蛋白肌动蛋白激活的ATPase活性，导致收缩。Rho相关蛋白或内源性SMPP-1M相关蛋白使MLC磷酸酶(SMPP-1M)磷酸化，抑制SMPP-1M，增强MLC的磷酸化和收缩。然而，SMPP-1M相关激酶的确切身份仍不清楚。生化证据有力地支持了这一观点，即SMPP-1M相关蛋白与人类丝氨酸/苏氨酸亮氨酸拉链相互作用蛋白激酶(HZIPK)有关，hZIPK在细胞凋亡中起重要作用，因此SMPP-1M相关蛋白激酶被称为ZIP样激酶(MacDonald，J.A.，Borman，M.A.，Murani，A.，Somlyo，A.V.，HartShorne，D.J.和Haystead，T.A.J.(2001)Proc.娜塔莉。阿卡德。SCI。美国A.98,2419-2424)。目前尚不清楚血管平滑肌SMPP-1M相关激酶是hZIPK的截短型、天然的hZIPK，还是hZIPK的独特同源物。在这里，我们发现只有天然的hZIPK mRNA和蛋白在人血管平滑肌细胞(VSMCs)中被检测到。对人主动脉SMPP-1M相关蛋白的cDNA文库进行高精度筛选，鉴定出18个阳性克隆，均为hZIPK的克隆。基于PCR的VSMC RNA研究发现了天然的hZIPK转录本，但没有证据表明hZIPK或ZIP样激酶的剪接变体。对多种血管和非血管组织RNA的Northern印迹研究表明，包括人膀胱RNA在内的多个血管和非血管组织RNA只预测了2.3kb的全长hZIPK。免疫印迹显示VSMC中有天然的52 kDa hZIPK全长表达。全长N-末端hZIPK与SMPP-1M肌球蛋白结合亚单位(MBS)的C-末端结构域(氨基酸681847)结合。HZIPK与SMPP-1M的MBS共沉淀，显性负性RhoA抑制hZIPK-MBS的相互作用。这些数据证实hZIPK是在人的囊泡平滑肌中表达的唯一的SMPP-1相关的激酶，并支持Rho在促进hZIPK-MBS相互作用中的作用。
Excitation-contraction coupling in smooth muscle involves activation of myosin light chain (MLC) phosphorylation, which increases activity of the myosin actin-activated ATPase, resulting in contraction. Phosphorylation of MLC phosphatase (SMPP-1M) by Rho-associated kinase or endogenous SMPP-1M-associated kinase inhibits SMPP-1M, enhancing MLC phosphorylation and contraction. However, the precise identity of SMPP-1M-associated kinase remains unclear. Biochemical evidence strongly supports the idea that SMPP-1M-associated kinase is related to the human serine/ threonine leucine zipper-interacting protein kinase (hZIPK), which is important in cell apoptosis, and the SMPP-1M-associated kinase has therefore been called ZIP-like kinase (MacDonald, J. A., Borman, M. A., Murani, A., Somlyo, A. V., Hartshorne, D. J., and Haystead, T. A. J. ( 2001) Proc. Natl. Acad. Sci. U. S. A. 98, 2419 - 2424). Whether the vascular smooth muscle SMPP-1M-associated kinase is a truncated version of hZIPK, native hZIPK, or a unique homologue of hZIPK is unclear. Here we show that only native hZIPK mRNA and protein are detectable in human vascular smooth muscle cells (VSMCs). High stringency screening of a human aortic cDNA library for the SMPP-1M-associated kinase identified 18 positive clones, all of which proved to be clones of hZIPK. PCR-based studies of VSMC RNA revealed native hZIPK transcripts but no evidence for splice variants of hZIPK or a ZIP-like kinase. Northern blotting studies of multiple vascular and non-vascular tissue RNAs, including human bladder RNA, showed only 2.3 kb of mRNA predicted for full-length hZIPK. Immunoblotting showed native full-length 52-kDa hZIPK expression in VSMCs. Full-length and N-terminal hZIPK bound the C-terminal domain ( amino acids 681 847) of the myosin binding subunit (MBS) of SMPP-1M. hZIPK immunoprecipitated with the MBS of SMPP-1M and dominant negative RhoA inhibited the hZIPK-MBS interaction. These data identify hZIPK as the unique SMPP-1-associated kinase expressed in human vesicular smooth muscle and support a role for Rho in promoting the hZIPK-MBS interaction.