Human SAD1 kinase is involved in UV-induced DNA damage checkpoint function
Human SAD1 kinase is involved in UV-induced DNA damage checkpoint function
复制标题
DOI：
10.1074/jbc.m404728200
复制
发表时间：
2004-07-23
影响因子：
4.8
通讯作者：
Nakanishi, M
中科院分区：
文献类型：
--
作者：
Lu, R;Niida, H;Nakanishi, M
G（2）期DNA损伤导致的检查点激活阻止了细胞周期蛋白B/Cdc 2复合物的激活，因此，有丝分裂进入受阻。尽管已知G2期阻滞的启动和维持受至少两种不同的信号通路调节，包括高等真核生物中的p38 MAPK和共济失调毛细血管扩张相关（ATM）-和Rad 3相关（ATR）-Chk 1，但参与这种调节的信号通路的实际数目仍然是难以捉摸的。在本研究中，我们确定了人类SAD 1（hsSAD 1）通过搜索序列数据库。预测的hsSAD 1蛋白由778个氨基酸组成，与分裂酵母Cdr 2（一种有丝分裂调节激酶）和秀丽隐杆线虫SAD 1（一种神经元细胞极性调节因子）具有显着的同源性。HsSAD 1转录本广泛表达，在脑和睾丸中表达水平最高。HsSAD 1在体外分别特异性磷酸化Ser-642、Ser-216和Ser-361上的Wee 1A、Cdc 25-C和- B。过表达hsSAD 1导致Cdc 25 C在体内Ser-216上的磷酸化增加。DNA损伤诱导的UV或甲磺酸甲酯，但不是由IR增强内源性hsSAD 1激酶活性的咖啡因敏感的方式，并导致其蛋白质从细胞质易位到细胞核。过表达野生型hsSAD 1可诱导HeLa S2细胞发生G（2）/M期阻滞。此外，紫外线诱导的G（2）/M停滞部分取消了减少表达的hsSAD 1使用小干扰RNA。这些结果表明，hsSAD 1作为检查点激酶在紫外线或甲基甲磺酸诱导的DNA损伤。这种新激酶的鉴定表明，除了ATR-Chk 1和p38 MAPK之外，还存在另一种检查点途径。
Checkpoint activation by DNA damage during G(2) prevents activation of cyclin B/Cdc2 complexes, and as a consequence, mitotic entry is blocked. Although initiation and maintenance of G2 arrest are known to be regulated by at least two distinct signaling pathways, including those of p38MAPK and ataxia-telangiectasiamutated (ATM)- and Rad3-related (ATR)-Chk1 in higher eukaryotes, the actual number of signaling pathways involved in this regulation is still elusive. In the present study, we identified human SAD1 (hsSAD1) by searching a sequence data base. The predicted hsSAD1 protein comprises 778 amino acids and shares significant homology with the fission yeast Cdr2, a mitosis-regulatory kinase, and Caenorhabditis elegans SAD1, a neuronal cell polarity regulator. HsSAD1 transcript was expressed ubiquitously with the highest levels of expression in brain and testis. HsSAD1 specifically phosphorylated Wee1A, Cdc25-C, and - B on Ser-642, Ser-216, and Ser-361 in vitro, respectively. Overexpression of hsSAD1 resulted in an increased phosphorylation of Cdc25C on Ser-216 in vivo. DNA damage induced by UV or methyl methane sulfonate but not by IR enhanced endogenous hsSAD1 kinase activity in a caffeine-sensitive manner and caused translocation of its protein from cytoplasm to nucleus. Overexpression of wild-type hsSAD1 induced G(2)/M arrest in HeLa S2 cells. Furthermore, UV-induced G(2)/M arrest was partially abrogated by the reduced expression of hsSAD1 using small interfering RNA. These results suggest that hsSAD1 acts as checkpoint kinase upon DNA damage induced by UV or methyl methane sulfonate. The identification of this new kinase suggests the existence of an alternative checkpoint pathway other than those of ATR-Chk1 and p38MAPK.