MacDonald Justin A, Sutherland Cindy, Carlson David A, Bhaidani Sabreena, Al-Ghabkari Abdulhameed, Swärd Karl, Haystead Timothy A J, Walsh Michael P
Department of Biochemistry and Molecular Biology, University of Calgary, Cumming School of Medicine, Calgary, Alberta, Canada (J.A.M., C.S., S.B., A.A.-G., M.P.W.); Department of Experimental Medical Science, Lund University, Lund, Sweden (K.S.); and Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina (D.A.C., T.A.J.H.)
Department of Biochemistry and Molecular Biology, University of Calgary, Cumming School of Medicine, Calgary, Alberta, Canada (J.A.M., C.S., S.B., A.A.-G., M.P.W.); Department of Experimental Medical Science, Lund University, Lund, Sweden (K.S.); and Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina (D.A.C., T.A.J.H.).
Mol Pharmacol. 2016 Jan;89(1):105-17. doi: 10.1124/mol.115.100529. Epub 2015 Oct 13.
A novel inhibitor of zipper-interacting protein kinase (ZIPK) was used to examine the involvement of ZIPK in the regulation of smooth muscle contraction. Pretreatment of de-endothelialized rat caudal arterial smooth muscle strips with the pyrazolo[3,4-d]pyrimidinone inhibitor 2-((1-(3-chlorophenyl)-4-oxo-4,5-dihydro-1H-pyrazolo [3,4-d]-pyrimidin-6-yl)thio)propanamide (HS38) decreased the velocity of contraction (time to reach half-maximal force) induced by the phosphatase inhibitor calyculin A in the presence of Ca(2+) without affecting maximal force development. This effect was reversed following washout of HS38 and correlated with a reduction in the rate of phosphorylation of myosin 20-kDa regulatory light chains (LC20) but not of protein kinase C-potentiated inhibitory protein for myosin phosphatase of 17 kDa (CPI-17), prostate apoptosis response-4, or myosin phosphatase-targeting subunit 1 (MYPT1), all of which have been implicated in the regulation of vascular contractility. A structural analog of HS38, with inhibitory activity toward proviral integrations of Moloney (PIM) virus 3 kinase but not ZIPK, had no effect on calyculin A-induced contraction or protein phosphorylations. We conclude that a pool of constitutively active ZIPK is involved in regulation of vascular smooth muscle contraction through direct phosphorylation of LC20 upon inhibition of myosin light chain phosphatase activity. HS38 also significantly attenuated both phasic and tonic contractile responses elicited by phenylephrine, angiotensin II, endothelin-1, U46619, and K(+)-induced membrane depolarization in the presence of Ca(2+), which correlated with inhibition of phosphorylation of LC20, MYPT1, and CPI-17. These effects of HS38 suggest that ZIPK also lies downstream from G protein-coupled receptors that signal through both Gα12/13 and Gαq/11.
一种新型的拉链相互作用蛋白激酶（ZIPK）抑制剂被用于研究ZIPK在平滑肌收缩调节中的作用。用吡唑并[3,4-d]嘧啶酮抑制剂2-((1-(3-氯苯基)-4-氧代-4,5-二氢-1H-吡唑并[3,4-d]嘧啶-6-基)硫代)丙酰胺（HS38）对去内皮的大鼠尾动脉平滑肌条进行预处理，在存在Ca(2+)的情况下，可降低磷酸酶抑制剂花萼海绵诱癌素A诱导的收缩速度（达到最大力一半所需的时间），而不影响最大力的产生。在洗脱HS38后，这种效应被逆转，并且与肌球蛋白20 kDa调节轻链（LC20）的磷酸化速率降低相关，但与17 kDa的蛋白激酶C增强的肌球蛋白磷酸酶抑制蛋白（CPI-17）、前列腺凋亡反应-4或肌球蛋白磷酸酶靶向亚基1（MYPT1）的磷酸化速率降低无关，所有这些都与血管收缩性的调节有关。HS38的一种结构类似物，对莫洛尼（PIM）病毒3激酶的前病毒整合具有抑制活性，但对ZIPK无抑制活性，对花萼海绵诱癌素A诱导的收缩或蛋白磷酸化没有影响。我们得出结论，一组组成型活性ZIPK通过在肌球蛋白轻链磷酸酶活性受到抑制时直接磷酸化LC20参与血管平滑肌收缩的调节。HS38还显著减弱了去甲肾上腺素、血管紧张素II、内皮素-1、U46619和K(+)诱导的膜去极化在存在Ca(2+)时引发的相位和张力收缩反应，这与抑制LC20、MYPT1和CPI-17的磷酸化相关。HS38的这些效应表明，ZIPK也位于通过Gα12/13和Gαq/11信号传导的G蛋白偶联受体的下游。
