Regulation of the Nuclear MAP Kinase Phosphatases

该计划侧重于核MAP激酶磷酸酶的调控。有丝分裂原活化蛋白（MAP）激酶是信号转导途径的关键组成部分，介导细胞对各种细胞外刺激的反应，从生长因子到环境胁迫。到目前为止，至少有10个MAP激酶家族成员在哺乳动物细胞中被鉴定出来，包括细胞外信号调节激酶（ERK）、c-Jun n -末端激酶（JNK）和p38。ERK1和ERK2是ERK亚家族的原型，可被生长因子高度激活。相反，JNK和p38亚家族优先被包括紫外线、热休克和脂多糖在内的应激激活，因此也被称为应激激活蛋白激酶。一旦被激活，MAP激酶可以从细胞质转移到细胞核，导致大量转录因子磷酸化和基因表达改变。各种MAP激酶亚家族的生理功能在许多系统中得到了广泛的研究。一般来说，ERK的激活与细胞增殖、分化和细胞应激后细胞存活的增强密切相关，尽管在某些情况下，如最近描述的顺铂治疗，ERK的激活是执行细胞凋亡所必需的。另一方面，JNK和p38的激活通常与细胞凋亡的增强和炎症细胞因子的产生有关，尽管也有明显的例外，JNK/p38的激活是细胞增殖和分化所必需的。由于MAP激酶通路在调节许多关键的细胞过程中发挥重要作用，因此这些信号蛋白的精确调控对于维持细胞稳态至关重要。所有MAP激酶的活性都是通过特定MAP激酶和蛋白磷酸酶对其三肽特征基序中保守的苏氨酸和酪氨酸残基的可逆磷酸化来调节的。在哺乳动物细胞中，MAP激酶的失活主要是由一个双特异性MAP激酶磷酸酶（MKPs）家族完成的，它可以作用于磷苏氨酸和磷酪氨酸残基。到目前为止，已经确定了9种不同的哺乳动物MAP激酶磷酸酶家族成员。根据它们的亚细胞定位和转录调控模式，这些磷酸酶大致可以分为两类。第一类包括MKP-3/Pyst-1、Pyst-2、MKP-4、MKP-5和M3/6，它们主要定位于细胞质中，因此被认为主要控制细胞质中发生的MAP激酶调节事件。第二组酶包括MKP-1 （CL100/3CH134）、MKP-2、PAC-1和B23，它们主要定位于核室。由即时早期基因编码，这些核MAP激酶磷酸酶被许多同样激活MAP激酶的刺激迅速诱导。因此，有人认为这些MAP激酶磷酸酶在细胞核内MAP激酶信号的反馈控制中起重要作用。最近，有报道称，几种细胞质MAP激酶磷酸酶可以与其底物MAP激酶相互作用，这种相互作用大大增加了它们的催化活性。这些研究表明，通过底物结合介导的MAP激酶磷酸酶的催化活化可能在决定它们的底物偏好中起重要作用。然而，并非所有的细胞质MAP激酶磷酸酶在与其底物相互作用时都经历催化活化。例如，尽管MKP-5通过其n端结构域的一个基本基序与其底物MAP激酶相互作用，但这种相互作用对其催化活性几乎没有影响。尽管许多核MAP激酶磷酸酶的底物特异性已经被广泛研究，但它们与底物相互作用的能力却知之甚少。对于相互作用的机制，或者这些相互作用对其生化和生理功能的影响，人们所知的就更少了。尽管核MAP激酶磷酸酶的诱导已在多种生物系统中得到证实，但介导其转录调节的潜在机制尚不清楚。在过去的一年里，我们的研究集中在两个主题上。(1) MAP激酶对MKP-2的催化活化。MKP-2已被证明优先灭活ERK和JNK MAP激酶亚家族。为了了解这种底物选择性的分子基础，我们研究了MKP-2与不同MAP激酶亚家族的相互作用和催化激活。我们发现，ERK和JNK显著增强了MKP-2的催化活性，而p38对其影响很小。相比之下，p38和ERK结合MKP-2的亲和力比较强，而JNK和MKP-2的相互作用非常弱。通过定点诱变，我们将ERK/p38结合位点定义为MKP-2 n端结构域的一簇精氨酸残基。基本基序的突变取消了它与ERK和p38的相互作用，并严重损害了这些激酶对MKP-2的催化活性。出乎意料的是，这些突变对jnk触发的催化活化几乎没有影响。在体外和体内，野生型MKP-2都能有效地灭活ERK2，而不能与ERK/p38结合的MKP-2突变体则不能。最后，除了作为ERK和p38的对接位点外，MKP-2基本基序还在调节其核定位中发挥作用。我们的研究为MKP-2的底物偏好提供了机制解释，并表明MKP-2与底物结合时的催化活化对其功能至关重要。(2)组蛋白H3磷酸化/乙酰化在MKP-1诱导应激反应中的作用。我们已经证明MKP-1 mRNA被亚砷酸盐和紫外光强烈诱导，并被热休克和过氧化氢适度增加。有趣的是，亚砷酸盐还在诱导MKP-1 mRNA表达之前，在全局水平上显著诱导组蛋白H3的磷酸化/乙酰化。p38 MAP激酶抑制剂SB203580可部分阻止MKP-1的转录诱导、组蛋白H3修饰和MKP-1 mRNA的升高，提示p38途径参与了这些过程。最后，染色质免疫沉淀（ChIP）实验显示，亚砷酸盐诱导与MKP-1基因相关的组蛋白H3磷酸化/乙酰化，并增强RNA聚合酶II与MKP-1染色质的结合。暴露于其他应激剂后的ChIP分析显示MKP-1染色质上的组蛋白H3有不同程度的修饰。p38和ERK MAP激酶在不同应激因子介导MKP-1诱导中的差异作用进一步说明了应激诱导MKP-1表达的复杂性和多用途性。我们的研究结果强烈表明，应激后染色质重塑有助于MKP-1的转录诱导。

This program focuses on the regulation of nuclear MAP kinase phosphatases. The mitogen-activated protein (MAP) kinases are critical components of the signal transduction pathways that mediate the cellular response to a variety of extracellular stimuli, ranging from growth factors to environmental stresses. So far, at least ten MAP kinase family members have been identified in mammalian cells, including the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38. ERK1 and ERK2 are the archetypes of the ERK subfamily that are highly activated by growth factors. In contrast, the JNK and p38 subfamilies are preferentially activated by stress including ultraviolet light, heat shock, and lipopolysaccharide, and therefore, are also referred to as stress-activated protein kinases. Once activated, MAP kinases can translocate from the cytoplasm to the nucleus, leading to the phosphorylation of a multitude of transcription factors and altered gene expression. The physiological functions of various MAP kinase subfamilies have been extensively studied in a large number of systems. In general, ERK activation is closely associated with cell proliferation, differentiation and enhanced cell survival after cellular stress, although in certain situations, such as recently described for cisplatin treatment, ERK activation is required for the execution of apoptosis. On the other hand, activation of JNK and p38 is usually associated with enhanced apoptosis and production of inflammatory cytokines, although there are notable exceptions in which JNK/p38 activation is necessary for cell proliferation and differentiation. Since MAP kinase pathways play an important role in regulating many critical cellular processes, the precise regulation of these signaling proteins is crucial for the maintenance of cellular homeostasis. The activities of all MAP kinases are regulated via reversible phosphorylation of the conserved threonine and tyrosine residues in their tripeptide signature motifs by specific MAP kinase kinases and protein phosphatases. In mammalian cells, inactivation of MAP kinases is primarily accomplished by a family of dual-specificity MAP kinase phosphatases (MKPs) that can act on both the phosphothreonine and phosphotyrosine residues. So far, nine distinct mammalian MAP kinase phosphatase family members have been characterized. According to their subcellular localization and patterns of transcriptional regulation, these phosphatases can be roughly divided into two groups. The first group includes MKP-3/Pyst-1, Pyst-2, MKP-4, MKP-5, and M3/6, which are predominantly localized in the cytosol and are therefore thought to mainly control the MAP kinase-regulated events that occur in the cytosol. The second group of enzymes includes MKP-1 (CL100/3CH134), MKP-2, PAC-1, and B23, which are primarily localized in the nuclear compartment. Encoded by immediate early genes, these nuclear MAP kinase phosphatases are rapidly induced by many of the same stimuli that also activate MAP kinases. For this reason, it has been suggested that these MAP kinase phosphatases play an important role in the feedback control of MAP kinase signaling in the nucleus. Recently, it has been reported that several cytosolic MAP kinase phosphatases can interact with their substrate MAP kinases and such an interaction substantially increases their catalytic activities. These studies suggest that catalytic activation of MAP kinase phosphatases mediated through substrate-binding may play an important role in determining their substrate preferences. However, not all cytosolic MAP kinase phosphatases undergo catalytic activation upon interaction with their substrates. For example, although MKP-5 interacts with its substrate MAP kinases through a basic motif in its N-terminal domain, this interaction has little effect on its catalytic activity. Despite the fact that the substrate specificities for many nuclear MAP kinase phosphatases have been studied extensively, relatively little is known about their abilities to interact with their substrates. Even less is known about the mechanisms underlying the interactions, or the effects these interactions have on their biochemical and physiological functions. Although the induction of nuclear MAP kinase phosphatases has been documented in a variety of biological systems, the underlying mechanisms that mediate their transcriptional cregulation remain unclear. Our studies over the past year have concentrated on 2 topics. (1) the catalytic activation of MKP-2 by MAP kinases. MKP-2 has been shown to preferentially inactivate ERK and JNK MAP kinase subfamilies. In order to understand the molecular basis for this substrate selectivity, we have examined MKP-2's interaction with, and catalytic activation by, distinct MAP kinase subfamilies. We found that MKP-2's catalytic activity was dramatically enhanced by ERK and JNK but was only minimally affected by p38. By contrast, p38 and ERK bound MKP-2 with comparably strong affinities, while JNK and MKP-2 interacted very weakly. Through site-directed mutagenesis, we defined the ERK/p38-binding site as a cluster of arginine residues in the N-terminal domain of MKP-2. Mutation of the basic motif abrogated its interaction with both ERK and p38 and severely compromised MKP-2's catalytic activation by these kinases. Unexpectedly, such mutations had little effect on JNK-triggered catalytic activation. Both in vitro and in vivo, wild type MKP-2 effectively inactivated ERK2 while MKP-2 mutants incapable of binding to ERK/p38 did not. Finally, in addition to its role as a docking site for ERK and p38, the MKP-2 basic motif plays a role in regulating its nuclear localization. Our studies provided a mechanistic explanation for MKP-2's substrate preference and suggest that catalytic activation of MKP-2 upon binding to its substrates is crucial for its function. (2) The role of histone H3 phosphorylation/acetylation in mediating MKP-1 induction in response to stress. We have shown that MKP-1 mRNA was potently induced by arsenite and ultraviolet light, and modestly increased by heat shock and hydrogen peroxide. Interestingly, arsenite also dramatically induces phosphorylation/acetylation of histone H3 at a global level which precedes the induction of MKP-1 mRNA. The transcriptional induction of MKP-1, histone H3 modification, and elevation in MKP-1 mRNA in response to arsenite are all partially prevented by the p38 MAP kinase inhibitor SB203580, suggesting that the p38 pathway is involved in these processes. Finally, chromatin immunoprecipitation (ChIP) assays reveal that arsenite induces phosphorylation/acetylation of histone H3 associated with the MKP-1 gene and enhances binding of RNA polymerase II to MKP-1 chromatin. ChIP assays following exposure to other stress agents reveal varying degrees of histone H3 modification at the MKP-1 chromatin. The differential contribution of p38 and ERK MAP kinases in mediating MKP-1 induction by different stress agents further illustrates the complexity and versatility of stress-induced MKP-1 expression. Our results strongly suggest that chromatin remodeling after stress contributes to the transcriptional induction of MKP-1.