CELL GROWTH REGULATION VIA THE MEKK/SAPK CASCADE

我们之前已经将MEKK1蛋白激酶描述为应激激活蛋白激酶级联的上游介质。由于应激激活蛋白激酶（SAPK，也称为JNK）级联参与了许多生理和病理刺激，特别是炎症细胞因子，因此最近被密切研究。同样重要的是，MEKK1在癌变中的作用尚未明确。应激激活级联是由DNA损伤剂刺激导致程序性细胞死亡（凋亡），通常用于化疗策略。人们普遍认为MEKK1和SAPK在细胞凋亡反应中发挥作用，尽管我们最近的数据表明，MEKK1对细胞凋亡的影响是复杂的，可能对细胞死亡起到积极或消极的作用。通过MEKK1信号传导的核心问题是：1)它的激活方式；2)它将“应激信号”传递给细胞生长机制的机制。MEKK1是一种200 kD的蛋白，具有一个大的氨基末端调节结构域，具有未表征的功能。虽然MEKK1在催化域中被磷酸化，但我们最近的研究结果表明，磷酸化并不是调节MEKK1功能的主要机制。相反，我们最近获得了两种极不寻常的机制调节MEKK1功能的证据。第一个是MEKK1的靶向蛋白水解。我们已经确定了MEKK1上三个不同的蛋白水解裂解位点，并假设不同的刺激事件以功能上可区分的方式激活MEKK1。在细胞凋亡过程中，被称为caspases的蛋白酶切割MEKK1，并将其活性从抗凋亡的NFkappaB途径转移，转而促进sapk介导的AP1转录因子途径的激活。其次，我们发现活性醌是SAPK通路的有效激活剂或抑制剂（取决于浓度），双电子醌还原酶NQO1对MEKK1上游应激信号通路的传递至关重要。我们提供的数据表明，巯基氧化还原状态的调节是MEKK1激活的主要手段，并假设MEKK1本身是通过其氨基端进行氧化还原调节的靶标。考虑到应激信号的下游功能，我们发现SAPK蛋白激酶在细胞周期转变期间（G2/M）被激活。此外，MEKK1的表达或用各种应激剂处理细胞会导致与细胞周期蛋白B相关的CDK激酶活性的抑制。我们提出表征应激信号与细胞周期蛋白B/cdc2激酶相互作用的机制。

We have previously characterized the MEKK1 protein kinase as an upstream mediator of the Stress Activated Protein Kinase cascade. Stress Activated Protein Kinase (SAPK, alternatively known as JNK) cascade has recently been closely investigated owing to its participation in many physiological and pathologic stimuli, particularly inflammatory cytokines. Also of central importance is the yet unclarified role of MEKK1 in carcinogenesis. The Stress-Activated cascade is stimulated by DNA damaging agents that lead to programmed cell death (apoptosis) and are commonly used in chemotherapeutic strategies. MEKK1 and SAPK are broadly assumed to play a role in the apoptotic response, though our recent data suggest that effects of MEKK1 on apoptosis are complex and can be either positive or negative towards cell death. Central questions in signaling through MEKK1 are 1) its means of activation, and 2) its mechanism of transmitting the "stress signal" through to cell growth machinery. MEKK1 is a 200 kD protein with a large amino terminal regulatory domain that has uncharacterized function. While MEKK1 is phosphorylated within the catalytic domain, our recent results indicate that phosphorylation is not the major mechanism of regulation of MEKK1 function. We have instead obtained recent evidence that two highly unusual mechanisms regulate MEKK1 functions. The first of these is targeted proteolysis of MEKK1. We have identified three distinct proteolytic cleavage sites on MEKK1, and hypothesize that different stimulatory events activate MEKK1 in functionally distinguishable ways. During apoptosis, proteases termed caspases cleave MEKK1 and redirect its activity away from the anti-apoptotic NFkappaB pathway, and instead promote activation of the SAPK-mediated AP1 transcription factor pathway. Secondly, we have identified reactive quinones as potent activators or inhibitors of the SAPK pathway (depending on concentration) and the two electron quinone reductase NQO1 as critical for transmission of stress signaling pathways upstream of MEKK1. We present data leading to the conclusion that regulation of sulfhydryl redox status is a prime means of MEKK1 activation, and hypothesize that MEKK1 itself is a target for redox regulation via its amino terminus. Considering the downstream function of stress signaling, we have found that the SAPK protein kinase becomes activated during cell cycle transition, during G2/M. Additionally, expression of MEKK1 or treatment of cells with various stress agents leads to inhibition of CDK kinase activity associated with cyclin B. We propose to characterize the mechanism by which stress signals interact with the cyclin B/cdc2 kinase.