Davare M A, Horne M C, Hell J W
Department of Pharmacology, University of Wisconsin, Madison, Wisconsin 53706-1532, USA.
J Biol Chem. 2000 Dec 15;275(50):39710-7. doi: 10.1074/jbc.M005462200.
Phosphorylation by cAMP-dependent protein kinase (PKA) regulates a vast number of cellular functions. An important target for PKA in brain and heart is the class C L-type Ca(2+) channel (Ca(v)1.2). PKA phosphorylates serine 1928 in the central, pore-forming alpha(1C) subunit of this channel. Regulation of channel activity by PKA requires a proper balance between phosphorylation and dephosphorylation. For fast and specific signaling, PKA is recruited to this channel by an protein kinase A anchor protein (Davare, M. A., Dong, F., Rubin, C. S., and Hell, J. W. (1999) J. Biol. Chem. 274, 30280-30287). A phosphatase may be associated with the channel to effectively balance serine 1928 phosphorylation by channel-bound PKA. Dephosphorylation of this site is mediated by a serine/threonine phosphatase that is inhibited by okadaic acid and microcystin. We show that immunoprecipitation of the channel complex from rat brain results in coprecipitation of PP2A. Stoichiometric analysis indicates that about 80% of the channel complexes contain PP2A. PP2A directly and stably binds to the C-terminal 557 amino acids of alpha(1C). This interaction does not depend on serine 1928 phosphorylation and is not altered by PP2A catalytic site inhibitors. These results indicate that the PP2A-alpha(1C) interaction constitutively recruits PP2A to the channel complex rather than being a transient substrate-catalytic site interaction. Functional assays with the immunoisolated class C channel complex showed that channel-associated PP2A effectively reverses serine 1928 phosphorylation by endogenous PKA. Our findings demonstrate that both PKA and PP2A are integral components of the class C L-type Ca(2+) channel that determine the phosphorylation level of serine 1928 and thereby channel activity.
环磷酸腺苷依赖性蛋白激酶（PKA）介导的磷酸化作用调控着大量的细胞功能。PKA在脑和心脏中的一个重要作用靶点是C类L型钙通道（Ca(v)1.2）。PKA使该通道中心的成孔α(1C)亚基上的丝氨酸1928发生磷酸化。PKA对通道活性的调控需要磷酸化与去磷酸化之间保持适当平衡。为实现快速且特异的信号传导，一种蛋白激酶A锚定蛋白会将PKA招募至该通道（达瓦雷，M. A.，董，F.，鲁宾，C. S.，以及赫尔，J. W.（1999年）《生物化学杂志》274卷，30280 - 30287页）。一种磷酸酶可能与该通道相关联，以有效平衡通道结合的PKA对丝氨酸1928的磷酸化作用。该位点的去磷酸化由一种丝氨酸/苏氨酸磷酸酶介导，冈田酸和微囊藻毒素可抑制这种磷酸酶。我们发现，从大鼠脑内免疫沉淀通道复合物会导致PP2A共沉淀。化学计量分析表明，约80%的通道复合物含有PP2A。PP2A直接且稳定地结合至α(1C)的C末端557个氨基酸。这种相互作用不依赖于丝氨酸1928的磷酸化，且不受PP2A催化位点抑制剂的影响。这些结果表明，PP2A与α(1C)的相互作用持续地将PP2A招募至通道复合物，而非短暂的底物 - 催化位点相互作用。对免疫分离得到的C类通道复合物进行的功能分析表明，与通道相关的PP2A能有效逆转内源性PKA对丝氨酸1928的磷酸化作用。我们的研究结果表明，PKA和PP2A都是C类L型钙通道的组成成分，它们决定了丝氨酸1928的磷酸化水平，进而决定通道活性。
