Expression and activity of protein kinase D/protein kinase Cμ in myocardium:: Evidence for α1-adrenergic receptor- and protein kinase C-mediated regulation
Expression and activity of protein kinase D/protein kinase Cμ in myocardium:: Evidence for α1-adrenergic receptor- and protein kinase C-mediated regulation
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DOI：
10.1006/jmcc.2000.1143
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发表时间：
2000-06-01
影响因子：
5
通讯作者：
Avkiran, M
中科院分区：
文献类型：
--
作者：
Haworth, RS;Goss, MW;Avkiran, M
蛋白激酶D（PKD），也称为蛋白激酶C（PKC）μ，是一种新型的丝氨酸/苏氨酸激酶，其可以在各种细胞类型中与PKC平行或下游被激活。但其在心肌中的表达和调节尚未被表征。在本研究中，110和115 kDa的两种蛋白在大鼠心室肌中检测到使用针对极端的N-或C-末端的PKD的抗体。这两种蛋白质在胎儿心脏中高度表达，但在发育过程中丰度下降。分级研究表明，PKD在新生儿心脏中分布在心肌细胞和非心肌细胞组分之间，但在成人心脏中主要存在于非心肌细胞组分中。在培养的新生大鼠心室肌细胞，在体外激酶测定显示增加PKD的自磷酸化（EC 50 2.8 nM）响应佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）。暴露于去甲肾上腺素也诱导PKD自磷酸化的剂量依赖性增加（EC 50 0.6 μ M）。用α 1肾上腺素能受体（AR）拮抗剂哌唑嗪预处理可阻断去甲肾上腺素诱导的PKD自磷酸化，而β 1 AR拮抗剂阿替洛尔无作用，表明去甲肾上腺素通过α 1 AR激活PKD。通过将肌细胞暴露于α（1）-AR激动剂苯肾上腺素，证实了α（1）-AR的参与，苯肾上腺素诱导了与去甲肾上腺素相似的PKD自磷酸化特征（EC 50 0.6 μ M）。α（1）-AR刺激和PMA对PKD自磷酸化的作用均由PKC介导，因为这些作用可通过用PKC抑制剂双吲哚马来酰亚胺预处理心肌细胞而减弱。这些数据表明PKD在大鼠心室肌中表达，其表达受发育控制，并且心室肌细胞中PKD活性通过α（1）-AR和PKC介导的途径调节。(C)北京大学出版社.
Protein kinase D (PKD), which is also known as protein kinase C (PKC) mu is a novel serine/threonine kinase that can be activated in parallel with or downstream of PKC in various cell types. but its expression and regulation in myocardium have not been characterized. In the present study, two proteins of 110 and 115 kDa were detected in rat ventricular myocardium using antibodies directed at the extreme N- or C-terminus of PKD. Both proteins were highly expressed in the fetal heart but showed a developmental decline in abundance. Fractionation studies showed that PKD was distributed between myocyte and non-myocyte fractions in the neonatal heart, but was found predominantly in the non-myocyte fraction in the adult heart. In cultured neonatal rat ventricular myocytes, an in vitro kinase assay revealed increased autophosphorylation of PKD (EC50 2.8 nM) in response to phorbol-12-myristate-13-acetate (PMA). Exposure to norepinephrine also induced a dose-dependent increase in PKD autophosphorylation (EC50 0.6 mu M). Pretreatment with the alpha(1)-adrenergic receptor (AR) antagonist prazosin blocked norepinephrine-induced PKD autophosphorylation, while the beta(1)-AR antagonist atenolol had no effect, indicating that activation of PKD by norepinephrine occurred via the alpha(1)-AR. Involvement of the alpha(1)-AR was confirmed by exposure of myocytes to the alpha(1)-AR agonist phenylephrine, which induced a similar profile of PKD autophosphorylation to norepinephrine (EC50 0.6 mu M). The effects of both alpha(1)-AR stimulation and PMA on PKD autophosphorylation were mediated by PKC, since these effects could be attenuated by pretreatment of myocytes with the PKC inhibitor bisindolylmaleimide. These data show that PKD is expressed in rat ventricular myocardium, where its expression is subject to developmental control, and that PKD activity in ventricular myocytes is regulated through alpha(1)-AR-and PKC-mediated pathways. (C) 2000 Academic Press.