Thompson Jill L, Shuttleworth Trevor J
Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY, 14642, USA.
J Physiol. 2015 Feb 1;593(3):559-72. doi: 10.1113/jphysiol.2014.284182. Epub 2015 Jan 7.
Although both the calcium store-dependent CRAC channels and the store-independent ARC channels are regulated by the protein STIM1, CRAC channels are regulated by STIM1 in the endoplasmic reticulum, whilst ARC channels are regulated by the STIM1 constitutively resident in the plasma membrane. We now demonstrate that activation of the ARC channels, but not CRAC channels, is uniquely dependent on phosphorylation of a single residue (T389) in the extensive cytosolic domain of STIM1 by protein kinase A. We further demonstrate that the phosphorylation of the T389 residue by protein kinase A is mediated by the association of plasma membrane STIM1 with the scaffolding protein AKAP79. Together, these findings indicate that the phosphorylation status of this single residue in STIM1 represents a key molecular determinant of the relative activities of these two co-existing Ca(2+) entry channels that are known to play critical, but distinct, roles in modulating a variety of physiologically relevant activities.
The low-conductance, highly calcium-selective channels encoded by the Orai family of proteins represent a major pathway for the agonist-induced entry of calcium associated with the generation and modulation of the key intracellular calcium signals that initiate and control a wide variety of physiologically important processes in cells. There are two distinct members of this channel family that co-exist endogenously in many cell types: the store-operated Ca(2+) release-activated CRAC channels and the store-independent arachidonic acid-regulated ARC channels. Although the activities of both channels are regulated by the stromal-interacting molecule-1 (STIM1) protein, two distinct pools of this protein are responsible, with the major pool of STIM1 in the endoplasmic reticulum membrane regulating CRAC channel activity, whilst the minor pool of plasma membrane STIM1 regulates ARC channel activity. We now show that a critical feature in determining this selective activation of the two channels is the phosphorylation status of a single threonine residue (T389) within the extensive (∼450 residue) cytosolic domain of STIM1. Specifically, protein kinase A (PKA)-mediated phosphorylation of T389 of STIM1 is necessary for effective activation of the ARC channels, whilst phosphorylation of the same residue actually inhibits the ability of STIM1 to activate the CRAC channels. We further demonstrate that the PKA-mediated phosphorylation of T389 occurs at the plasma membrane via the involvement of the anchoring protein AKAP79, which is constitutively associated with the pool of STIM1 in the plasma membrane. The novel mechanism we have described provides a means for the cell to precisely regulate the relative activities of these two channels to independently modulate the resulting intracellular calcium signals in a physiologically relevant manner.
尽管钙库依赖性CRAC通道和钙库非依赖性ARC通道均受蛋白STIM1调控,但CRAC通道在内质网中受STIM1调控,而ARC通道则受组成性驻留在质膜中的STIM1调控。我们现在证明,ARC通道而非CRAC通道的激活唯一地依赖于蛋白激酶A对STIM1广泛胞质结构域中单个残基(T389)的磷酸化。我们进一步证明,蛋白激酶A对T389残基的磷酸化是由质膜STIM1与支架蛋白AKAP79的结合介导的。这些发现共同表明,STIM1中这个单个残基的磷酸化状态代表了这两种共存的Ca(2+)进入通道相对活性的关键分子决定因素,已知这两种通道在调节多种生理相关活动中发挥关键但不同的作用。
由Orai蛋白家族编码的低电导、高度钙选择性通道是激动剂诱导钙内流的主要途径,与关键细胞内钙信号的产生和调节相关,这些信号启动并控制细胞中多种生理重要过程。该通道家族有两个不同的成员,在许多细胞类型中内源性共存:钙库操纵的Ca(2+)释放激活的CRAC通道和钙库非依赖性花生四烯酸调节的ARC通道。尽管这两种通道的活性均受基质相互作用分子1(STIM1)蛋白调控,但该蛋白的两个不同池负责调控,内质网膜中STIM1的主要池调节CRAC通道活性,而质膜STIM1的次要池调节ARC通道活性。我们现在表明,决定这两种通道选择性激活的一个关键特征是STIM1广泛(约450个残基)胞质结构域内单个苏氨酸残基(T389)的磷酸化状态。具体而言,蛋白激酶A(PKA)介导的STIM1的T389磷酸化是ARC通道有效激活所必需的,而同一残基的磷酸化实际上抑制了STIM1激活CRAC通道的能力。我们进一步证明,PKA介导的T389磷酸化通过锚定蛋白AKAP79在质膜上发生,AKAP79与质膜中STIM1的池组成性相关。我们描述的新机制为细胞提供了一种手段,以精确调节这两种通道的相对活性,从而以生理相关的方式独立调节由此产生的细胞内钙信号。