Identification of the major phosphorylation sites of the Raf-1 kinase.
Identification of the major phosphorylation sites of the Raf-1 kinase.
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发表时间：
1993-08
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通讯作者：
D. Morrison;G. Heidecker;U. Rapp;T. Copeland
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作者：
D. Morrison;G. Heidecker;U. Rapp;T. Copeland
用各种生长因子和有丝分裂原处理细胞导致Raf-1激酶的快速过度磷酸化和活化。为了确定磷酸化事件是否影响Raf-1活性，我们已经开始实验以鉴定Raf-1的磷酸化位点。在这份报告中，我们发现，Ser 43，Ser 259和Ser 621是Raf-1的主要位点，在哺乳动物细胞和Sf 9昆虫细胞感染的重组杆状病毒编码的人Raf-1的磷酸化。缺乏激酶活性的突变体Raf-1蛋白在体内也在这些位点上磷酸化，表明这些磷酸化事件不是自磷酸化的结果。此外，我们发现，Thr 268是主要的Raf-1残基磷酸化在体外自激酶测定。此外，我们还研究了杆状病毒表达的Raf-1蛋白在这些磷酸化位点含有突变的生物化学活性。在体外蛋白激酶测定中，Ser 259突变蛋白的活性是野生型Raf-1的2倍，Ser 621突变蛋白作为激酶是无活性的。对Ser 259和Ser 621周围残基的分析表明，RSXSXP可能是负责Raf-1在这些位点磷酸化的激酶的共有序列。有趣的是，这些RSXSXP序列在所有Raf家族成员的进化过程中是完全保守的。
Treatment of cells with various growth factors and mitogens results in the rapid hyperphosphorylation and activation of the Raf-1 kinase. To determine if phosphorylation events affect Raf-1 activity, we have initiated experiments to identify the phosphorylation sites of Raf-1. In this report, we find that Ser43, Ser259, and Ser621 are the major sites of Raf-1 which are phosphorylated in mammalian cells and in Sf9 insect cells infected with a recombinant baculovirus encoding human Raf-1. Mutant Raf-1 proteins lacking kinase activity are also phosphorylated on these sites in vivo, indicating that these phosphorylation events are not a consequence of autophosphorylation. Furthermore, we find that Thr268 is the predominant Raf-1 residue phosphorylated in in vitro autokinase assays. In addition, we have examined the biochemical activity of baculovirus-expressed Raf-1 proteins containing mutations at these phosphorylation sites. In in vitro protein kinase assays Ser259 mutant proteins were 2-fold more active than wild-type Raf-1 and Ser621 mutant proteins were inactive as kinases. Analysis of the residues surrounding Ser259 and Ser621 indicates that RSXSXP may be a consensus sequence for the kinase responsible for phosphorylation of Raf-1 at these sites. Interestingly, these RSXSXP sequences are completely conserved throughout evolution in all Raf family members.