Site-specific phosphorylation of MCM4 during the cell cycle in mammalian cells

MCM4, a subunit of a putative replicative helicase, is phosphorylated during the cell cycle, at least in part by cyclin-dependent kinases (CDK), which play a central role in the regulation of DNA replication. However, detailed characterization of the phosphorylation of MCM4 remains to be performed. We examined the phosphorylation of human MCM4 at Ser3, Thr7, Thr19, Ser32, Ser54, Ser88 and Thr110 using anti-phosphoMCM4 sera. Western blot analysis of HeLa cells indicated that phosphorylation of MCM4 at these seven sites can be classified into two groups: (a) phosphorylation that is greatly enhanced in the G2 and M phases (Thr7, Thr19, Ser32, Ser54, Ser88 and Thr110), and (b) phosphorylation that is firmly detected during interphase (Ser3). We present data indicating that phosphorylation at Thr7, Thr19, Ser32, Ser88 and Thr110 in the M phase requires CDK1, using a temperature-sensitive mutant of mouse CDK1, and phosphorylation at sites 3 and 32 during interphase requires CDK2, using a dominant-negative mutant of human CDK2. Based on these results and those from in vitro phosphorylation of MCM4 with CDK2/cyclin A, we discuss the kinases responsible for MCM4 phosphorylation. Phosphorylated MCM4 detected using anti-phospho sera exhibited different affinities for chromatin. Studies on the nuclear localization of chromatin-bound MCM4 phosphorylated at sites 3 and 32 suggested that they are not generally colocalized with replicating DNA. Unexpectedly, MCM4 phosphorylated at site 32 was enriched in the nucleolus through the cell cycle. These results suggest that phosphorylation of MCM4 has several distinct and site-specific roles in the function of MCM during the mammalian cell cycle.     

糖原合成酶激酶-3对τ蛋白磷酸化作用的研究

糖原合成酶激酶 ３（ Ｇ Ｓ Ｋ ３）在 ３０℃与 τ蛋白保温 ４ ｈ 可催化 １７±０４ ｍ ｏｌ磷酸参入 １ ｍ ｏｌτ蛋白 将磷酸化的 τ蛋白经胰蛋白酶消化, Ｆｅ Ｃｌ３ 亲和柱分离及 Ｃ１８反相高压液相层析纯化后,再用高压电泳,手工 Ｅｄｍ ａｎ 降解及自动氨基酸序列分析等检测技术,对其磷酸化位点进行鉴定 结果发现： Ｇ Ｓ Ｋ ３ 可使 τ蛋白 Ｔｈｒ １８１, Ｓｅｒ １８４, Ｓｅｒ ２６２, Ｓｅｒ ３５６ 和 Ｓｅｒ ４００ 发生磷酸化 其中 Ｓｅｒ ２６２ 和 Ｓｅｒ ４００ 为 Ａｌｚｈｅｉｍ ｅｒ 病（ Ａ Ｄ）τ蛋白的异常磷酸化位点根据上述磷酸化作用仅轻度抑制τ蛋白生物学活性,推测： Ａ Ｄ τ蛋白 Ｓｅｒ ２６２ 和 Ｓｅｒ ４００ 的磷酸化可能不是决定其生物功能的关键性位点,单纯 Ｇ Ｓ Ｋ ３ 不能复制 Ａ Ｄ 样 τ蛋白的病理改变      

Thr2446 is a novel mammalian target of rapamycin (mTOR) phosphorylation site regulated by nutrient status

The mammalian target of rapamycin (mTOR) is a key regulator of protein translation. Signaling via mTOR is increased by growth factors but decreased during nutrient deprivation. Previous studies have identified Ser2448 as a nutrient-regulated phosphorylation site located in the mTOR catalytic domain, insulin stimulates Ser2448 phosphorylation via protein kinase B (PKB), while Ser2448 phosphorylation is attenuated with amino acid starvation. Here we have identified Thr2446 as a novel nutrient-regulated phosphorylation site on mTOR. Thr2446 becomes phosphorylated when CHO-IR cells are nutrient-deprived, but phosphorylation is reduced by insulin stimulation. Nutrient deprivation activates AMP-activated protein kinase (AMPK). To test whether this could be involved in regulating phoshorylation of mTOR, we treated cultured murine myotubes with 5'-aminoimidazole-4-carboxamide ribonucleoside (AICAR) or dinitrophenol (DNP). Both treatments activated AMPK and also caused a concomitant increase in phosphorylation of Thr2446 and a parallel decrease in insulin's ability to phosphorylate p70 S6 kinase. In vitro kinase assays using peptides based on the sequence in amino acids 2440-2551 of mTOR found that PKB and AMPK are capable of phosphorylating sites in this region. However, phosphorylation by PKB is restricted when Thr2446 is mutated to an acidic residue mimicking phosphorylation. Conversely, AMP-kinase-induced phosphorylation is reduced when Ser2448 is phosphorylated. These data suggest differential phosphorylation Thr2446 and Ser2448 could act as a switch mechanism to integrate signals from nutrient status and growth factors to control the regulation of protein translation.     

Ser787 in the proline-rich region of human MAP4 is a critical phosphorylation site that reduces its activity to promote tubulin polymerization

p34cdc2 kinase-phosphorylation sites in the microtubule (MT)-binding region of MAP4 were determined by peptide sequence of phosphorylated MTB3, a fragment containing the carboxy-terminal half of human MAP4. In addition to two phosphopeptides containing Ser696 and Ser787 which were previously indicated to be in vivo phosphorylation sites, two novel phosphopeptides, containing Thr892 or Thr901 and Thr917 as possible phosphorylation sites, were isolated, though only in in vitro phosphorylation. The role of phosphorylation at Ser696 and Ser787, which were differently phosphorylated during the cell cycle (Ookata et al., (1997). Biochemistry, 36: 15873-15883), was investigated in MT-polymerization, using MAP4 Ser to Glu mutants, which mimic phosphorylation at each site. Mutation of Ser787 to Glu strikingly reduced the MAP4's MT-polymerization activity, while Glu-mutation at Ser696 did not. These results suggest that Ser787 could be the critical phosphorylation site causing MTs to be dynamic at mitosis.