Role of HSP90 Family Chaperone Proteins in Cellular Sign

HSP 90和GRP 94是分别在胞质溶胶和内质网中发现的同源细胞伴侣。几年前，我们发现苯醌和安莎霉素类抗生素的成员，包括除莠霉素A和格尔德霉素（GA）与HSP 90和GRP 94结合，并破坏了这些蛋白质是其中一部分的某些多分子复合物。我们利用药物破坏HSP 90和GRP 94活性来研究这些分子伴侣在细胞信号转导中的功能。多种信号转导蛋白与这些charperones相互作用，包括kinasesz src，erbB 2和c-raf-1，以及突变的（但不是野生型）p53。分子伴侣/信号蛋白复合物的药理学破坏的一般后果是信号蛋白的所得显著不稳定性和不正确的亚细胞定位。不稳定性是由于分子伴侣解离后26 S蛋白酶体蛋白水解复合物刺激信号蛋白的靶向降解。在过去的一年中，我们已经取得了新的观察，热休克蛋白90与细胞溶质激酶RIP，导致NF κ B激活的TNF信号通路的关键组成部分。我们已经确定，格尔德霉素对RIP稳定性的破坏阻止了TNF对NF κ B的诱导，但不能阻止TNF向JNK的信号传导，从而使细胞对TNF的凋亡特性敏感。我们还观察到另一种与细胞存活相关的激酶Akt对格尔德霉素敏感。格尔德霉素阻断除TNF以外的多种刺激物（包括化疗药物和IL-1）对NF κ B的诱导。它这样做的能力可能与它对Akt的不稳定有关。目前正在进行实验来验证这一假设。苯醌安莎霉素（格尔德霉素）是唯一能够特异性干扰HSP 90/GRP 94功能的药物。最近，我们确定了根赤霉素代表一类新的天然产物能够结合到HSP 90。根赤霉素和安莎霉素都在氨基末端核苷酸口袋处与HSP 90结合。最近，我们已经确定了第三类天然产物新生霉素，它也结合到HSP 90，虽然在一个较低的亲和力比苯醌安莎霉素或根赤霉素。尽管如此，新生霉素似乎对“客户蛋白”产生与安莎霉素和根赤霉素相同的生物学效应。令人惊讶的是，新生霉素似乎与HSP 90上的羧基末端区域相互作用，这可能是以前未被识别的第二个核苷酸结合位点。初步的动物试验表明，新生霉素每天两次给药超过一个月后没有毒性。该方案在erbB 2驱动的乳腺癌转基因小鼠模型中显示出显著的抗肿瘤活性。最后，我们观察到格尔德霉素逆转黑色素瘤细胞中β-连环蛋白酪氨酸磷酸化，可能是由于这些细胞中erbB 2的快速丢失。在未处理的细胞中，erbB 2和β-连环蛋白可以容易地共沉淀。β-连环蛋白酪氨酸磷酸化的丧失导致与E-钙粘蛋白的关联增加和体外细胞运动性降低。这是黑色素瘤细胞中β连环蛋白酪氨酸磷酸化状态的调节与运动性降低相关的第一个迹象。β-连环蛋白酪氨酸磷酸化的事实似乎是介导的，在3/3的黑色素瘤细胞系检查，erbB 2-格尔德霉素敏感的酪氨酸激酶-表明格尔德霉素治疗可能是抗转移。该假设目前正在体内转移模型中进行测试。 受体酪氨酸激酶的ErbB家族包含四个成员。我们发现ErbB 2是该家族中唯一的无配体成员，是最敏感的格尔德霉素底物之一。由于ErbB是跨膜蛋白，它们可能与Hsp 90和Grp 94两者接触，并且一个或两个伴侣可能负责ErbB 2的格尔德霉素敏感性。我们目前的数据表明，ErbB 2的激酶结构域介导其格尔德霉素的反应性和热休克蛋白90结合到成熟蛋白的这个结构域。与此相反，成熟的ErbB 1，格尔德霉素比ErbB 2敏感得多，不与热休克蛋白90。格尔德霉素诱导的新生ErbB 2的不稳定性也是由其激酶结构域介导的，我们几乎没有发现证据支持Grp 94在ErbB 2成熟中的作用。

HSP90 and GRP94 are homologous cellular chaperones found in cytosol and endoplasmic reticulum, respectively. Several years ago, we discovered that members of the benzoquinone and ansamycin class of antibiotic, including herbimycin A and geldanamycin (GA) bound to HSP90 and GRP94 and disrupted certain multi-molecular complexes of which these proteins were a part. We have utilized pharmacologic disruption of HSP90 and GRP94 activity to study the function of these chaperones in cellular signal transduction. Multiple signal transduction proteins interact with these charperones, including the kinasesz src, erbB2 and c-raf-1, and mutated (but not wild type) p53. A general consequence of pharmacologic disruption of the chaperone/signal protein complex is the resultant marked instability and incorrect subcellular localization of the signalling protein. The instability is due to stimulation of targeted degradation of the signalling protein by the 26S proteasome proteolytic complex following chaperone dissociation. Within the last year, we have made the novel observation that HSP90 associates with the cytosolic kinase RIP, a key component of the TNF signalling pathway which leads to NFkB activation. We have determined that disruption of RIP stability by geldanamycin prevents NFkB induction by TNF, but not TNF signalling to Jnk, thus sensitizing cells to the apoptotic properties of TNF. We have additionally observed that another kinase associated with cell survival, Akt, is sensitive to geldanamycin. Geldanamycin blocks NFkB induction by a wide variety of stimuli other than TNF, including chemotherapeutic drugs and IL-1. Its ability to do this may relate to its destabilization of Akt. Experiments to test this hypothesis are currently underway. Benzoquinone ansamycins (geldanamycin) had been the only agents capable of specifically interfering in HSP90/GRP94 function. Recently, we identified radicicol as representing a novel class of natural product capable of binding to HSP90. Both radicicol and the ansamycins bind to HSP90 at an amino terminal nucleotide pocket. Most recently, we have identified a third class of natural product, novobiocin, which also binds to HSP90, although at a lower affinity than either benzoquinone ansamycins or radicicol. Nonetheless, novobiocin appears to cause the same biologic effects on "client proteins" as ansamycins and radicicol. Surprisingly, novobiocin appears to interact with a carboxyl terminal region on HSP90, which may be a previously unrecognized second nucleotide binding site. Preliminary animal testing has revealed no toxicity after twice daily administration of novobiocin for more than one month. This regimen demonstrates significant anti-tumor activity in a transgenic murine model of erbB2-driven breast cancer. Finally, we have observed that geldanamycin reverses beta-catenin tyrosine phosphorylation in melanoma cells, probably due to the rapid loss of erbB2 from these cells. In untreated cells, erbB2 and beta-catenin can be readily co-precipitated. Loss of beta-catenin tyrosine phosphorylation leads to an increased association with E-cadherin and decreased cell motility in vitro. This is the first indication that modulation of the tyrosine phosphorylation status of beta catenin in melanoma cells is associated with decreased motility. The fact that beta-catenin tyrosine phosphorylation seems to be mediated, in 3/3 melanoma cell lines examined, by erbB2 - a geldanamycin-sensitive tyrosine kinase - suggests that geldanamycin treatment may be anti-metastatic. This hypothesis is currently being tested in an in vivo metastasis model. The ErbB family of receptor tyrosine kinases contains four members. We have found that ErbB2, the only ligandless member of the family, is one of the most sensitive geldanamycin substrates. Since the ErbBs are transmembrane proteins, they are likely to come in contact with both Hsp90 and Grp94, and one or both chaperones may be responsible for ErbB2's geldanamycin sensitivity. Our current data demonstrate that the kinase domain of ErbB2 mediates its geldanamycin responsiveness and that Hsp90 binds to this domain in the mature protein. In contrast, mature ErbB1, much less sensitive to geldanamycin than ErbB2, does not associate with Hsp90. Geldanamycin-induced instability of nascent ErbB2 is also mediated by its kinase domain, and we can find little evidence to support a role for Grp94 in ErbB2 maturation.