Department of Biology, Stanford University, Stanford, CA 94305, USA.
Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, M5G 1X5, Canada.
J Cell Sci. 2023 Apr 15;136(8). doi: 10.1242/jcs.260353. Epub 2023 Apr 24.
Calcineurin, or protein phosphatase 2B (PP2B), the Ca2+ and calmodulin-activated phosphatase and target of immunosuppressants, has many substrates and functions that remain uncharacterized. By combining rapid proximity-dependent labeling with cell cycle synchronization, we mapped the spatial distribution of calcineurin in different cell cycle stages. While calcineurin-proximal proteins did not vary significantly between interphase and mitosis, calcineurin consistently associated with multiple centrosomal and/or ciliary proteins. These include POC5, which binds centrins in a Ca2+-dependent manner and is a component of the luminal scaffold that stabilizes centrioles. We show that POC5 contains a calcineurin substrate motif (PxIxIT type) that mediates calcineurin binding in vivo and in vitro. Using indirect immunofluorescence and ultrastructure expansion microscopy, we demonstrate that calcineurin colocalizes with POC5 at the centriole, and further show that calcineurin inhibitors alter POC5 distribution within the centriole lumen. Our discovery that calcineurin directly associates with centriolar proteins highlights a role for Ca2+ and calcineurin signaling at these organelles. Calcineurin inhibition promotes elongation of primary cilia without affecting ciliogenesis. Thus, Ca2+ signaling within cilia includes previously unknown functions for calcineurin in maintenance of cilia length, a process that is frequently disrupted in ciliopathies.
钙调神经磷酸酶，或蛋白磷酸酶 2B（PP2B），是钙离子和钙调蛋白激活的磷酸酶，也是免疫抑制剂的靶标，具有许多尚未确定的底物和功能。通过将快速邻近依赖性标记与细胞周期同步化相结合，我们绘制了钙调神经磷酸酶在不同细胞周期阶段的空间分布。虽然钙调神经磷酸酶邻近蛋白在间期中和有丝分裂中没有显著变化，但钙调神经磷酸酶始终与多个中心体和/或纤毛蛋白相关。其中包括 POC5，它以 Ca2+依赖性方式结合中心蛋白，是稳定中心体的腔支架的组成部分。我们表明，POC5 含有钙调神经磷酸酶结合基序（PxIxIT 型），介导体内和体外钙调神经磷酸酶结合。通过间接免疫荧光和超微结构扩展显微镜，我们证明钙调神经磷酸酶与 POC5 在中心体上共定位，并进一步表明钙调神经磷酸酶抑制剂改变 POC5 在中心体腔内的分布。我们发现钙调神经磷酸酶直接与中心体蛋白相关联，这凸显了 Ca2+和钙调神经磷酸酶信号在这些细胞器中的作用。钙调神经磷酸酶抑制剂促进初级纤毛的伸长，而不影响纤毛发生。因此，纤毛内的 Ca2+信号包括钙调神经磷酸酶在维持纤毛长度方面的以前未知的功能，在纤毛病中，该过程经常受到破坏。
