Yang Lin, Doshi Darshan, Morrow John, Katchman Alexander, Chen Xiang, Marx Steven O
Division of Cardiology, Department of Medicine, College of Physicians and Surgeons,Columbia University, New York, New York 10032, USA.
Biochemistry. 2009 Jul 21;48(28):6674-83. doi: 10.1021/bi900322a.
The regulation of Ca(2+) influx through the phosphorylation of the L-type Ca(2+) channel, Ca(v)1.2, is important for the modulation of excitation-contraction (E-C) coupling in the heart. Ca(v)1.2 is thought to be the target of multiple kinases that mediate the signals of both the renin-angiotensin and sympathetic nervous systems. Detailed biochemical information regarding the protein phosphorylation reactions involved in the regulation of Ca(v)1.2 is limited. The protein kinase C (PKC) family of kinases can modulate cardiac contractility in a complex manner, such that contractility is either enhanced or depressed and relaxation is either accelerated or slowed. We have previously reported that Ser(1928) in the C-terminus of alpha(1c) was a target for PKCalpha, -zeta, and -epsilon phosphorylation. Here, we report the identification of seven PKC phosphorylation sites within the alpha(1c) subunit. Using phospho-epitope specific antibodies to Ser(1674) and Ser(1928), we demonstrate that both sites within the C-terminus are phosphorylated in HEK cells in response to PMA. Phosphorylation was inhibited with a PKC inhibitor, bisindolylmaleimide. In Langendorff-perfused rat hearts, both Ser(1674) and Ser(1928) were phosphorylated in response to PMA. Phosphorylation of Ser(1674), but not Ser(1928), is PKC isoform specific, as only PKCalpha, -betaI, -betaII, -gamma, -delta, and -theta, but not PKCepsilon, -zeta, and -eta, were able to phosphorylate this site. Our results identify a molecular mechanism by which PKC isoforms can have different effects on channel activity by phosphorylating different residues.
通过对L型钙通道Ca(v)1.2进行磷酸化来调节Ca(2+)内流，对于调节心脏的兴奋-收缩（E-C）偶联至关重要。Ca(v)1.2被认为是多种激酶的作用靶点，这些激酶介导肾素-血管紧张素和交感神经系统的信号。关于参与Ca(v)1.2调节的蛋白质磷酸化反应的详细生化信息有限。蛋白激酶C（PKC）家族的激酶可以以复杂的方式调节心脏收缩力，使得收缩力增强或减弱，舒张加速或减慢。我们之前报道过，α(1c) C末端的Ser(1928)是PKCα、-ζ和-ε磷酸化的靶点。在此，我们报告在α(1c)亚基内鉴定出七个PKC磷酸化位点。使用针对Ser(1674)和Ser(1928)的磷酸表位特异性抗体，我们证明C末端的这两个位点在HEK细胞中对佛波酯（PMA）有磷酸化反应。磷酸化被PKC抑制剂双吲哚马来酰胺抑制。在Langendorff灌注的大鼠心脏中，Ser(1674)和Ser(1928)对PMA均有磷酸化反应。Ser(1674)的磷酸化具有PKC同工型特异性，而Ser(1928)没有，因为只有PKCα、-βI、-βII、-γ、-δ和-θ能够磷酸化该位点，而PKCε、-ζ和-η则不能。我们的结果确定了一种分子机制，通过该机制PKC同工型可以通过磷酸化不同残基对通道活性产生不同影响。
