SIGNAL TRANSDUCTION PATHWAYS IN MAMMALIAN CELLS

本项目的重点是在正常细胞和衰老细胞中调节细胞对细胞外刺激反应的信号转导通路。过去一年的研究集中在两个主题上。(1)MKP-1的结构/功能分析。MAP激酶磷酸酶是一组受细胞外刺激诱导的双特异性磷酸酶，包括生长因子和应激。它们在MAPK级联的反馈控制中起着重要的作用。已经证明，这类磷酸酶中的一些成员可以对特定的MAP激酶亚家族表现出选择性(S)。本研究的目的是了解MKP-1的底物特异性以及导致这种特异性的结构基础。我们在同一细胞中同时表达和激活了JNK、ERK和p38，并用MKP-1分析了它们的去磷酸化。我们发现MKP-1优先失活p38&gt；JNK&gt；ERK。通过创建大量的MKP-1截断突变体并在MKP-1和MKP-3之间交换结构域，我们已经确定MKP-1的177个氨基酸组成的区域横跨磷酸酶结构域，足以与JNK最佳结合和失活。这个区域是p38最佳结合和去磷酸化所必需的，但不是充分的。此外，我们还在MKP-1的极端羧基末端发现了一个新的自抑制结构域。该结构域的缺失显著增强了其对JNK和ERK MAP激酶的磷酸酶活性。这些结果不仅确立了p38和JNK是MKP-1的生理靶点，而且也解释了为什么MKP-1对ERK有很强的磷酸酶活性。我们的研究有力地支持了MKP-1的主要功能是对JNK和p38通路的反馈控制的观点。(2)大鼠肝细胞内信号通路的增龄性改变。在此之前，我们证明了衰老与生长因子治疗后ERK蛋白激酶和p70S6激酶活性的降低有关。这两种激酶活性的下降表明，衰老的细胞可能在这些通路共同的早期上游事件中表现出变化。我们现在已经比较了EGF刺激对年轻和老年肝细胞最早发生的信号事件。与年轻细胞相比，衰老细胞中MEK和RAS的活性也在数量上减弱，且更难持续。在年轻的肝细胞中，EGF触发EGFR和Shc的快速酪氨酸磷酸化，并促进EGFR-Shc复合体的形成。在衰老的细胞中，这种响应EGF的信号复合体的形成显著减少，尽管在EGFR和Shc的酪氨酸磷酸化方面没有观察到差异。生长因子受体复合体的改变可能是衰老细胞增殖能力下降的原因之一。-信号转导、MAP激酶、EGF受体、Shc、MKP-1、衰老、应激

This project focuses on signal transduction pathways mediating the cellular response to extracellular stimuli in normal and aged cells. Studies over the past year have concentrated on two topics. (1) Structure/function analysis of MAP kinase phosphatase-1(MKP-1). MAP kinase phosphatases are a group of dual specificity phosphatases induced by extracellular stimuli including growth factors and stress. They play an important role in the feedback control of the MAP kinase cascades. It has been demonstrated that some members of this class of phosphatases can exhibit selectivity toward a specific MAP kinase subfamily(s). The goal of this study is to understand the substrate specificity of MKP-1 and the structural basis responsible for the specificity. We have simultaneously expressed and activated JNK, ERK, and p38 in the same cells, and analyzed their dephosphorylation by MKP- 1. We have found that MKP-1 preferentially inactivates p38> JNK> ERK. By creating numerous MKP-1 truncation mutants and swapping domains between MKP-1 and MKP-3, we have determined that a region of 177 amino acids of MKP-1 spanning the phosphatase domain is sufficient for optimal binding and inactivation of JNK. This region is required but not sufficient for optimal binding and dephosphorylation of p38. In addition, we have identified a novel auto-inhibitory domain in the extreme carboxyl-terminus of MKP-1. Deletion of this domain dramatically augments its phosphatase activity toward both JNK and ERK MAP kinases. These results not only establish p38 and JNK as the physiological targets of MKP-1 but also provide an explanation for why MKP-1 exhibited substantial phosphatase activity towards ERK in the earlier studies by another group. Our study strongly supports that idea that the main function of MKP-1 is feedback control of the JNK and p38 pathways. (2) Age-associated alteration in signaling pathways in rat hepatocytes. Previously we demonstrated that aging is correlated with decreases in both ERK MAP kinase and p70 S6 kinase activities following growth factor treatment. A decline in the activities of both kinases suggests that aged cells may display an alteration in an early upstream event common to these pathways. We have now compared the earliest signaling events that occur in response to EGF stimulation in young and aged hepatocytes. The activities of both MEK and Ras are also attenuated in magnitude and less sustainable in aged cells in comparison to those in the young cells. In young hepatocytes, EGF triggers rapid tyrosine phosphorylation of EGFR and Shc, and enhances EGFR-Shc complex formation. Formation of this signaling complex in response to EGF is significantly reduced in aged cells, although no difference in tyrosine-phosphorylation of either EGFR or Shc is observed. The alteration in the growth factor receptor complexes may contribute to the decline in proliferation capacity in aged cells. - Signal transduction, MAP kinase, EGF receptor, Shc, MKP-1, aging, stress