Jaiswal A S, Narayan S
UF Shands Cancer Center and Department of Anatomy and Cell Biology, College of Medicine, University of Florida, Gainesville, FL 32610, USA.
Int J Oncol. 2001 Sep;19(3):613-6. doi: 10.3892/ijo.19.3.613.
In response to DNA damage, p53 protein transiently stabilizes and accumulates in the nucleus, where it performs its role as a transcription factor. Phosphorylation of p53 increases its sequence-specific DNA-binding activity. In the present study, we have examined the effect of methylmethane sulfonate (MMS) to HCT-116 human colon cancer cells on the phosphorylation of p53. Results show that p53 protein becomes phosphorylated at serine 15 (Ser15) and Ser392 residues after treatment with MMS in a time-dependent manner. Increased levels of phospho-p53(Ser15) and phospho-p53(Ser392) were maintained up to 50 h of the MMS treatment. We also examined the involvement of probable kinase(s), which could be responsible for MMS-induced phosphorylation of p53 at Ser15 and Ser392. In vitro phosphorylation assay, carried out with the immunoprecipates of MMS-treated cells, showed an increased phosphorylation of p53 by c-Jun kinase 1 (JNK1) at early time points (2.5 h). However, with cyclin-dependent kinase (Cdk2) and TFIIH complex associated kinase CAK, the phosphorylation of p53 was increased at later time points (25 h). The phosphorylation of p53 by Cdc2 and MAPK (p38) kinases remained unaffected in the MMS-treated versus untreated cells. The MMS-induced phosphorylation of p53 correlates with our previous findings of p53's ability for increased sequence-specific DNA-binding and transcriptional activity in the cells treated with DNA alkylating agents.
响应DNA损伤时，p53蛋白会在细胞核中短暂稳定并积累，在细胞核中它发挥转录因子的作用。p53的磷酸化增加其序列特异性DNA结合活性。在本研究中，我们检测了甲磺酸甲酯（MMS）对HCT-116人结肠癌细胞中p53磷酸化的影响。结果显示，用MMS处理后，p53蛋白在丝氨酸15（Ser15）和丝氨酸392残基处发生磷酸化，且呈时间依赖性。在MMS处理长达50小时的过程中，磷酸化p53（Ser15）和磷酸化p53（Ser392）的水平持续升高。我们还检测了可能负责MMS诱导p53在Ser15和Ser392处磷酸化的激酶。用MMS处理细胞的免疫沉淀产物进行的体外磷酸化试验表明，在早期时间点（2.5小时），c-Jun激酶1（JNK1）使p53的磷酸化增加。然而，对于细胞周期蛋白依赖性激酶（Cdk2）和TFIIH复合物相关激酶CAK，在后期时间点（25小时）p53的磷酸化增加。在MMS处理的细胞与未处理的细胞中，Cdc2和丝裂原活化蛋白激酶（p38）激酶对p53的磷酸化没有影响。MMS诱导的p53磷酸化与我们之前关于在DNA烷化剂处理的细胞中p53增强序列特异性DNA结合和转录活性能力的研究结果相关。
