Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, West Bengal, India.
Biochimie. 2012 Apr;94(4):991-1000. doi: 10.1016/j.biochi.2011.12.020. Epub 2012 Jan 2.
We have recently reported that α(2)β(1) and α(1)β(1) isozymes of Na(+)/K(+)-ATPase (NKA) are localized in the caveolae whereas only the α(1)β(1) isozyme of NKA is localized in the non-caveolae fraction of pulmonary artery smooth muscle cell membrane. It is well known that different isoforms of NKA are regulated differentially by PKA and PKC, but the mechanism is not known in the caveolae of pulmonary artery smooth muscle cells. Herein, we examined whether this regulation occurs through phospholemman (PLM) in the caveolae. Our results suggest that PKC mediated phosphorylation of PLM occurs only when it is associated with the α(2) isoform of NKA, whereas phosphorylation of PLM by PKA occurs when it is associated with the α(1) isoform of NKA. To investigate the mechanism of regulation of α(2) isoform of NKA by PKC-mediated phosphorylation of PLM, we have purified PLM from the caveolae and reconstituted into the liposomes. Our result revealed that (i) in the reconstituted liposomes phosphorylated PLM (PKC mediated) stimulate NKA activity, which appears to be due to an increase in the turnover number of the enzyme; (ii) phosphorylated PLM did not change the affinity of the pump for Na(+); and (iii) even after phosphorylation by PKC, PLM still remains associated with the α(2) isoform of NKA.
我们最近报道称，Na(+)/K(+)-ATPase（NKA）的α(2)β(1)和α(1)β(1)同工酶定位于质膜窖中，而 NKA 的α(1)β(1)同工酶仅定位于肺动脉平滑肌细胞膜的非质膜窖部分。众所周知，不同同工型的 NKA 被 PKA 和 PKC 差异调节，但在肺动脉平滑肌细胞的质膜窖中，其机制尚不清楚。在此，我们研究了这种调节是否通过质膜窖中的磷酸烯醇式丙酮酸羧激酶（PLM）发生。我们的结果表明，PKC 介导的 PLM 磷酸化仅发生在其与 NKA 的α(2)同工型相关联时，而 PKA 介导的 PLM 磷酸化发生在其与 NKA 的α(1)同工型相关联时。为了研究 PKC 介导的 PLM 磷酸化对 NKA 的α(2)同工型的调节机制，我们从质膜窖中纯化了 PLM 并将其重新构成到脂质体中。我们的结果表明：（i）在重新构成的脂质体中，PKC 介导的磷酸化 PLM 刺激 NKA 活性，这似乎是由于酶的周转率增加所致；（ii）磷酸化 PLM 未改变泵对 Na(+)的亲和力；（iii）即使在 PKC 磷酸化后，PLM 仍与 NKA 的α(2)同工型相关联。
