The identification of phosphorylation sites of pp32 and biochemical purification of a cellular pp32-kinase
The identification of phosphorylation sites of pp32 and biochemical purification of a cellular pp32-kinase
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DOI：
10.1021/bi0493968
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发表时间：
2004-08-10
期刊：
影响因子：
2.9
通讯作者：
Chakravarti, D
中科院分区：
文献类型：
--
作者：
Hong, R;Macfarlan, T;Chakravarti, D
多功能磷蛋白pp 32参与重要的生理过程，包括细胞增殖、凋亡、mRNA转运和转录。我们以前曾报道，pp 32，通过组蛋白掩蔽，抑制组蛋白乙酰化和转录激活组蛋白乙酰转移酶。然而，pp 32本身是如何调控的，在很大程度上仍然未知。虽然pp 32是一种磷蛋白，但其磷酸化位点和细胞激酶均未被确定。在这份报告中，利用体外激酶测定和生化纯化方案，我们确定酪蛋白激酶II作为细胞pp 32-激酶。我们的缺失和位点特异性诱变研究确定丝氨酸158和204作为磷酸化位点。对磷酸化p32具有更高亲和力的抗体的产生和利用证明pp 32在这两个位点确实在体内被磷酸化。对pp 32的诱变研究表明丝氨酸158和204在其功能中的作用。pp 32激酶和pp 32磷酸化位点的鉴定以及对磷酸化p32具有更高亲和力的抗体的产生现在应该为今后pp 32调控的研究提供关键信息和工具。
The versatile phosphoprotein pp32 is involved in important physiological processes, including cell proliferation, apoptosis, mRNA transport, and transcription. We have previously reported that pp32, through histone masking, inhibits histone acetylation and transcriptional activation by histone acetyltransferases. However, how pp32 itself is regulated remained largely unknown. Although pp32 is a phosphoprotein, neither the phosphorylation sites nor the cellular kinase has been identified. In this report, utilizing an in vitro kinase assay and a biochemical purification scheme, we identify casein kinase II as a cellular pp32-kinase. Our deletion and site-specific mutagenesis studies identify serines 158 and 204 as the sites of phosphorylation. Generation and utilization of antibodies with higher affinity for phosphopp32 demonstrate that pp32 is indeed phosphorylated in vivo at these two sites. Mutagenesis studies on pp32 suggest a role for serines 158 and 204 in its function. The identification of the pp32 kinase and the sites of pp32 phosphorylation as well as the generation of antibodies with higher affinity for phosphopp32 should now provide key information and tools for future studies on pp32 regulation.