Department of Biochemistry and Molecular Biology, Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
J Biol Chem. 2010 Dec 31;285(53):41686-700. doi: 10.1074/jbc.M110.179648. Epub 2010 Nov 1.
Although the function of protein kinase D1 (PKD) in cardiac cells has remained enigmatic, recent work has shown that PKD phosphorylates the nuclear regulators HDAC5/7 (histone deacetylase 5/7) and CREB, implicating this kinase in the development of dysfunction seen in heart failure. Additional studies have shown that PKD also phosphorylates multiple sarcomeric substrates to regulate myofilament function. Initial studies examined PKD through adenoviral vector expression of wild type PKD, constitutively active PKD (caPKD), or dominant negative PKD in cultured adult rat ventricular myocytes. Confocal immunofluorescent images of these cells reveal a predominant distribution of all PKD forms in a non-nuclear, Z-line localized, striated reticular pattern, suggesting the importance of PKD in Ca(2+) signaling in heart. Consistent with an established role of PKD in targeting cardiac troponin I (cTnI), caPKD expression led to a marked decrease in contractile myofilament Ca(2+) sensitivity with an unexpected electrical stimulus dependence to this response. This desensitization was accompanied by stimulus-dependent increases in cTnI phosphorylation in control and caPKD cells with a more pronounced effect in the latter. Electrical stimulation also provoked phosphorylation of regulatory site Ser(916) on PKD. The functional importance of this phospho-Ser(916) event is demonstrated in experiments with a phosphorylation-defective mutant, caPKD-S916A, which is functionally inactive and blocks stimulus-dependent increases in cTnI phosphorylation. Dominant negative PKD expression resulted in sensitization of the myofilaments to Ca(2+) and blocked stimulus-dependent increases in cTnI phosphorylation. Taken together, these data reveal that localized PKD may play a role as a dynamic regulator of Ca(2+) sensitivity of contraction in cardiac myocytes.
虽然蛋白激酶 D1（PKD）在心肌细胞中的功能仍然是一个谜，但最近的研究表明，PKD 可使核调节因子 HDAC5/7（组蛋白去乙酰化酶 5/7）和 CREB 磷酸化，表明这种激酶参与心力衰竭时出现的功能障碍的发生。此外的研究表明，PKD 还可使多个肌节底物磷酸化，以调节肌丝功能。最初的研究通过在培养的成年大鼠心室肌细胞中用腺病毒载体表达野生型 PKD、组成型激活的 PKD（caPKD）或显性负性 PKD 来检查 PKD。这些细胞的共聚焦免疫荧光图像显示所有 PKD 形式都主要分布在非核、Z 线定位的条纹状网状结构中，提示 PKD 在心脏 Ca（2+）信号转导中的重要性。与 PKD 靶向心肌肌钙蛋白 I（cTnI）的既定作用一致，caPKD 的表达导致收缩肌丝 Ca（2+）敏感性明显降低，并且对这种反应具有出乎意料的电刺激依赖性。这种脱敏伴随着控制和 caPKD 细胞中 cTnI 磷酸化的刺激依赖性增加，后者的效果更为明显。电刺激还引发了 PKD 调节位点 Ser（916）的磷酸化。在具有磷酸化缺陷突变体 caPKD-S916A 的实验中，证实了该磷酸化 Ser（916）事件的功能重要性，该突变体无功能并阻止了 cTnI 磷酸化的刺激依赖性增加。显性负性 PKD 的表达导致肌丝对 Ca（2+）的敏感性增加，并阻止了 cTnI 磷酸化的刺激依赖性增加。总之，这些数据表明局部 PKD 可能作为心肌细胞收缩 Ca（2+）敏感性的动态调节剂发挥作用。
