Csermely P, Kahn C R
Research Division, Joslin Diabetes Center, Boston, Massachusetts.
J Biol Chem. 1991 Mar 15;266(8):4943-50.
The 90-kDa heat shock protein (hsp-90) is an abundant cytosolic protein believed to play a role in maintenance of protein trafficking and closely associated with several steroid hormone receptors. Incubation of highly purified hsp-90 with [gamma-32P]ATP results in its autophosphorylation on serine residues. There are several lines of evidence which suggest that this activity is due to a kinase intrinsic to hsp-90 rather than some closely associated protein kinases: 1) the phosphorylation persists after the removal of casein kinase II by heparin chromatography and after immunoprecipitation of hsp-90 with anti-hsp-90 antibodies. 2) The approximate kM for ATP of the reaction is 0.16 mM, higher than that of many other protein kinases. 3) Phosphorylation is not affected by a number of activators and inhibitors of other known kinases which might associate with hsp-90. 4) The phosphorylation displays a unique cation dependence being most active in the presence of Ca2+ and practically inactive with Mg2+, although the autophosphorylation in the presence of Mg2+ is activated by histones and polyamines. 5) The activity is remarkably heat-stable; incubation of hsp-90 for 20 min at 95 degrees C results in only a 60% decrease in autophosphorylation. 6) Finally, and most importantly, purified hsp-90 can be labeled with azido-ATP and it is able to bind to ATP-agarose. The binding shows similar cation dependence to the autophosphorylation. These data are in agreement with the presence of a consensus sequence for ATP binding sites in the primary structure of the protein similar to that observed in the 70-kDa heat-shock proteins. Our data suggest the 90-kDa heat shock protein possesses an enzymatic activity analogous in many respects to the similar activity of the 70-kDa heat shock proteins. This may represent an important, previously unrecognized function of hsp-90.
90千道尔顿热休克蛋白（hsp - 90）是一种丰富的胞质蛋白，被认为在维持蛋白质运输中发挥作用，并与几种类固醇激素受体密切相关。将高度纯化的hsp - 90与[γ-32P]ATP一起温育会导致其丝氨酸残基发生自磷酸化。有几条证据表明这种活性是由于hsp - 90自身固有的激酶，而不是某些紧密相关的蛋白激酶：1）通过肝素柱层析去除酪蛋白激酶II后以及用抗hsp - 90抗体免疫沉淀hsp - 90后，磷酸化仍持续存在。2）该反应中ATP的近似米氏常数（kM）为0.16 mM，高于许多其他蛋白激酶。3）磷酸化不受可能与hsp - 90相关的许多其他已知激酶的激活剂和抑制剂的影响。4）磷酸化表现出独特的阳离子依赖性，在Ca2+存在下最活跃，而在Mg2+存在下几乎无活性，尽管在Mg2+存在下的自磷酸化可被组蛋白和多胺激活。5）该活性具有显著热稳定性；hsp - 90在95℃温育20分钟只会导致自磷酸化降低60%。6）最后，也是最重要的，纯化的hsp - 90可用叠氮基ATP标记，并且它能够与ATP - 琼脂糖结合。这种结合表现出与自磷酸化相似的阳离子依赖性。这些数据与该蛋白一级结构中存在类似于在70千道尔顿热休克蛋白中观察到的ATP结合位点的共有序列一致。我们的数据表明90千道尔顿热休克蛋白具有一种在许多方面类似于70千道尔顿热休克蛋白的类似活性的酶活性。这可能代表了hsp - 90一种重要的、以前未被认识到的功能。
