SIGNAL TRANSDUCTION PATHWAYS IN NORMAL AND AGED CELLS

本项目的重点是信号转导途径介导细胞对细胞外刺激的反应在正常和老年细胞。过去一年的研究主要集中在两个主题上。(1) MAP激酶磷酸酶-1（MKP-1）结构/功能分析。MAP激酶磷酸酶是一组由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MAP激酶级联反应的反馈控制中起重要作用。已经证明，这类磷酸酶的一些成员可以对特定的MAP激酶亚家族表现出选择性。本研究的目的是了解MKP-1的底物特异性以及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JNK、ERK和p38，并分析了MKP- 1对它们的去磷酸化作用。我们发现MKP-1优先失活p38> JNK> 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，我们确定了MKP-1跨越磷酸酶结构域的177个氨基酸区域足以实现最佳的JNK结合和失活。这个区域对于p38的最佳结合和去磷酸化是必需的，但不是充分的。此外，我们在MKP-1的极端羧基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，也解释了为什么MKP-1在另一组的早期研究中对ERK表现出大量的磷酸酶活性。我们的研究有力地支持了MKP-1的主要功能是反馈控制JNK和p38通路的观点。(2)大鼠肝细胞信号通路的年龄相关改变。先前我们证明了衰老与生长因子处理后ERK MAP激酶和p70 S6激酶活性的降低相关。两种激酶活性的下降表明，衰老细胞可能在这些途径共同的早期上游事件中表现出改变。我们现在比较了年轻和老年肝细胞中EGF刺激反应中发生的最早信号事件。与年轻细胞相比，衰老细胞中MEK和Ras的活性也有所减弱，且可持续性较差。在年轻肝细胞中，EGF触发EGFR和Shc的快速酪氨酸磷酸化，并增强EGFR-Shc复合物的形成。尽管EGFR和Shc的酪氨酸磷酸化没有差异，但在衰老细胞中，EGF应答信号复合物的形成明显减少。生长因子受体复合物的改变可能导致衰老细胞增殖能力下降。-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