Bird T A, Schule H D, Delaney P, de Roos P, Sleath P, Dower S K, Virca G D
Department of Biochemistry, Immunex Corporation, Seattle, WA 98101.
FEBS Lett. 1994 Jan 24;338(1):31-6. doi: 10.1016/0014-5793(94)80111-8.
In KB cells, interleukin-1 (IL-1), epidermal growth factor and phorbol ester transiently activated both MAP kinase and a serine kinase which phosphorylated the heat shock protein hsp27. Extracts made from IL-1-stimulated KB cells phosphorylated recombinant hsp27, in vitro, on serine residues 78 and 82 which are contained within Arg-X-X-Ser motifs similar to those phosphorylated by the ribosomal protein S6 kinases. Upon size exclusion chromatography, however, hsp27 kinase eluted as a single peak of activity at 50-60 kDa, clearly separated from ribosomal protein S6 kinases. Treatment of partially purified hsp27 kinase with protein phosphatase-2a reduced its activity by 80%. De-phosphorylated hsp27 kinase could be approximately 50% reactivated by a factor present in IL-1-treated cell extracts in the presence of ATP. This factor co-eluted with MAP kinase after partial purification by DEAE-cellulose, phenyl Sepharose, and size exclusion chromatography. Purified sea star p44mpk and recombinant ERK2 MAP kinases were also capable of re-activating hsp27 kinase to a similar extent. These data suggest that hsp27 kinase is downstream from, and probably a direct target of MAP kinase.
在KB细胞中，白细胞介素-1（IL-1）、表皮生长因子和佛波酯可瞬时激活丝裂原活化蛋白激酶（MAP激酶）和一种能使热休克蛋白hsp27磷酸化的丝氨酸激酶。从经IL-1刺激的KB细胞制备的提取物，在体外可使重组hsp27的丝氨酸残基78和82磷酸化，这些残基位于与核糖体蛋白S6激酶磷酸化的基序相似的精氨酸- X - X - 丝氨酸基序内。然而，经体积排阻色谱分析，hsp27激酶以一个50 - 60 kDa的单一活性峰洗脱，与核糖体蛋白S6激酶明显分离。用蛋白磷酸酶-2a处理部分纯化的hsp27激酶，其活性降低80%。在ATP存在的情况下，IL-1处理的细胞提取物中的一种因子可使去磷酸化的hsp27激酶重新激活约50%。经DEAE - 纤维素、苯基琼脂糖和体积排阻色谱部分纯化后，该因子与MAP激酶共洗脱。纯化的海星p44mpk和重组ERK2 MAP激酶也能在相似程度上重新激活hsp27激酶。这些数据表明，hsp27激酶位于MAP激酶的下游，可能是MAP激酶的直接作用靶点。
