Lees J A, Buchkovich K J, Marshak D R, Anderson C W, Harlow E
Cold Spring Harbor Laboratory, NY 11724.
EMBO J. 1991 Dec;10(13):4279-90. doi: 10.1002/j.1460-2075.1991.tb05006.x.
The retinoblastoma gene product (pRB) is a nuclear phosphoprotein that is thought to play a key role in the negative regulation of cellular proliferation. pRB is phosphorylated in a cell cycle dependent manner, and studies in both actively dividing and differentiated cells suggest that this modification may be essential for cells to progress through the cell cycle. Using tryptic phosphopeptide mapping we have shown that pRB is phosphorylated on multiple serine and threonine residues in vivo and that many of these phosphorylation events can be mimicked in vitro using purified p34cdc2. Using synthetic peptides corresponding to potential cdc2 phosphorylation sites, we have developed a strategy which has allowed the identification of five sites. S249, T252, T373, S807 and S811 are phosphorylated in vivo, and in each case these sites correspond closely to the consensus sequence for phosphorylation by p34cdc2. This and the observation that pRB forms a specific complex with p34cdc2 in vivo suggests that p34cdc2 or a p34cdc2-related protein is a major pRB kinase.
视网膜母细胞瘤基因产物（pRB）是一种核磷蛋白，被认为在细胞增殖的负调控中起关键作用。pRB以细胞周期依赖性方式被磷酸化，对活跃分裂细胞和分化细胞的研究表明，这种修饰对于细胞通过细胞周期可能至关重要。利用胰蛋白酶磷酸肽图谱分析，我们已证明pRB在体内多个丝氨酸和苏氨酸残基上被磷酸化，并且其中许多磷酸化事件在体外使用纯化的p34cdc2可以模拟。使用对应于潜在cdc2磷酸化位点的合成肽，我们开发了一种策略，该策略已能够鉴定出五个位点。S249、T252、T373、S807和S811在体内被磷酸化，并且在每种情况下这些位点都与p34cdc2磷酸化的共有序列紧密对应。这一点以及pRB在体内与p34cdc2形成特异性复合物的观察结果表明，p34cdc2或一种与p34cdc2相关的蛋白是主要的pRB激酶。
