Polizotto R S, Cyert M S
Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA.
J Cell Biol. 2001 Sep 3;154(5):951-60. doi: 10.1083/jcb.200104078.
Calcineurin is a conserved Ca2+/calmodulin-specific serine-threonine protein phosphatase that mediates many Ca2+-dependent signaling events. In yeast, calcineurin dephosphorylates Crz1p, a transcription factor that binds to the calcineurin-dependent response element, a 24-bp promoter element. Calcineurin-dependent dephosphorylation of Crz1p alters Crz1p nuclear localization. This study examines the mechanism by which calcineurin regulates the nuclear localization of Crz1p in more detail. We describe the identification and characterization of a novel nuclear localization sequence (NLS) in Crz1p, which requires both basic and hydrophobic residues for activity, and show that the karyopherin Nmd5p is required for Crz1p nuclear import. We also demonstrate that the binding of Crz1p to Nmd5p is dependent upon its phosphorylation state, indicating that nuclear import of Crz1p is regulated by calcineurin. Finally, we demonstrate that residues in both the NH2- and COOH-terminal portions of Crz1p are required for regulated Crz1p binding to Nmd5p, supporting a model of NLS masking for regulating Crz1p nuclear import.
钙调神经磷酸酶是一种保守的Ca2+/钙调蛋白特异性丝氨酸-苏氨酸蛋白磷酸酶，介导许多Ca2+依赖性信号事件。在酵母中，钙调神经磷酸酶使Crz1p去磷酸化，Crz1p是一种转录因子，可与钙调神经磷酸酶依赖性反应元件（一个24bp的启动子元件）结合。钙调神经磷酸酶依赖性的Crz1p去磷酸化改变了Crz1p的核定位。本研究更详细地研究了钙调神经磷酸酶调节Crz1p核定位的机制。我们描述了Crz1p中一个新的核定位序列（NLS）的鉴定和特征，该序列的活性需要碱性和疏水残基，并表明核转运蛋白Nmd5p是Crz1p核输入所必需的。我们还证明Crz1p与Nmd5p的结合取决于其磷酸化状态，表明Crz1p的核输入受钙调神经磷酸酶调节。最后，我们证明Crz1p的NH2端和COOH端部分的残基对于Crz1p与Nmd5p的调节结合都是必需的，支持了一种通过NLS掩盖来调节Crz1p核输入的模型。
