Department of Molecular Medicine, Cornell University, Ithaca, NY 14853, USA.
Stowers Institute for Medical Research, Kansas City, MO 64110, USA.
Genetics. 2022 Aug 30;222(1). doi: 10.1093/genetics/iyac102.
How serine/threonine phosphatases are spatially and temporally tuned by regulatory subunits is a fundamental question in cell biology. Ankyrin repeat, SH3 domain, proline-rich-region-containing proteins are protein phosphatase 1 catalytic subunit binding partners associated with cardiocutaneous diseases. Ankyrin repeat, SH3 domain, proline-rich-region-containing proteins localize protein phosphatase 1 catalytic subunit to cell-cell junctions, but how ankyrin repeat, SH3 domain, proline-rich-region-containing proteins localize and whether they regulate protein phosphatase 1 catalytic subunit activity in vivo is unclear. Through a Caenorhabditis elegans genetic screen, we find that loss of the ankyrin repeat, SH3 domain, proline-rich-region-containing protein homolog, APE-1, suppresses a pathology called "jowls," providing us with an in vivo assay for APE-1 activity. Using immunoprecipitations and mass spectrometry, we find that APE-1 binds the protein phosphatase 1 catalytic subunit called GSP-2. Through structure-function analysis, we discover that APE-1's N-terminal half directs the APE-1-GSP-2 complex to intercellular junctions. Additionally, we isolated mutations in highly conserved residues of APE-1's ankyrin repeats that suppress jowls yet do not preclude GSP-2 binding, implying APE-1 does more than simply localize GSP-2. Indeed, in vivo reconstitution of APE-1 suggests the ankyrin repeats modulate phosphatase output, a function we find to be conserved among vertebrate homologs.
丝氨酸/苏氨酸磷酸酶如何通过调节亚基进行时空调节是细胞生物学中的一个基本问题。锚蛋白重复序列、SH3 结构域、富含脯氨酸区的蛋白质是与心皮疾病相关的蛋白磷酸酶 1 催化亚基结合伴侣。锚蛋白重复序列、SH3 结构域、富含脯氨酸区的蛋白质将蛋白磷酸酶 1 催化亚基定位到细胞-细胞连接处，但锚蛋白重复序列、SH3 结构域、富含脯氨酸区的蛋白质如何定位以及它们是否在体内调节蛋白磷酸酶 1 催化亚基活性尚不清楚。通过对秀丽隐杆线虫的遗传筛选，我们发现锚蛋白重复序列、SH3 结构域、富含脯氨酸区的蛋白质同源物 APE-1 的缺失可抑制一种称为“下颌”的病理，为我们提供了一种体内 APE-1 活性的测定方法。通过免疫沉淀和质谱分析，我们发现 APE-1 与称为 GSP-2 的蛋白磷酸酶 1 催化亚基结合。通过结构功能分析，我们发现 APE-1 的 N 端半部分将 APE-1-GSP-2 复合物引导至细胞间连接处。此外，我们分离出 APE-1 的锚蛋白重复序列中高度保守残基的突变，这些突变可抑制下颌，但不排除 GSP-2 的结合，这表明 APE-1 的作用不仅仅是简单地定位 GSP-2。事实上，体内的 APE-1 重建表明锚蛋白重复序列调节磷酸酶的输出，我们发现这一功能在脊椎动物同源物中是保守的。
