Stemmer Christian, Leeming Diana J, Franssen Lars, Grimm Rudi, Grasser Klaus D
Institute of Life Sciences, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark.
Biochemistry. 2003 Apr 1;42(12):3503-8. doi: 10.1021/bi027350d.
In plants, a variety of chromatin-associated high mobility group (HMG) proteins belonging to the HMGB family have been identified. We have examined the phosphorylation of the HMGB proteins from the monocotyledonous plant maize and the dicotyledonous plant Arabidopsis by protein kinase CK2alpha. Maize CK2alpha phosphorylates the maize HMGB1 and HMGB2/3 proteins and the Arabidopsis HMGB1, HMGB2/3, and HMGB4 proteins. Maize HMGB4 and HMGB5 and Arabidopsis HMGB5 are not phosphorylated by CK2alpha. Depending on the HMGB protein up to five amino acid residues are phosphorylated in the course of the phosphorylation reaction. The HMGB1 proteins from both plants are markedly more slowly phosphorylated by CK2alpha than the other HMGB substrate proteins, indicating that certain HMGB proteins are clearly preferred substrates for CK2alpha. The rate of the phosphorylation reaction appears to be related to the ease of interaction between CK2alpha and the HMGB proteins, as indicated by chemical cross-linking experiments. MALDI/TOF mass spectrometry analyses demonstrate that the HMGB1 and HMGB2/3 proteins occur in various phosphorylation states in immature maize kernels. Thus, HMGB1 exists as monophosphorylated, double-phosphorylated, triple-phosphorylated, and tetraphosphorylated protein in kernel tissue, and the tetraphosphorylated form is the most abundant version. The observed in vivo phosphorylation states indicate that protein kinase(s) other than CK2alpha contribute(s) to the modification of the plant HMGB proteins. The fact that the HMGB proteins are phosphorylated to various extents reveals that the existence of differentially modified forms increases the number of distinct HMGB protein variants in plant chromatin that may be adapted to certain functions.
在植物中，已鉴定出多种属于HMGB家族的与染色质相关的高迁移率族（HMG）蛋白。我们研究了蛋白激酶CK2α对单子叶植物玉米和双子叶植物拟南芥中HMGB蛋白的磷酸化作用。玉米CK2α使玉米HMGB1和HMGB2/3蛋白以及拟南芥HMGB1、HMGB2/3和HMGB4蛋白发生磷酸化。玉米HMGB4和HMGB5以及拟南芥HMGB5不会被CK2α磷酸化。在磷酸化反应过程中，根据HMGB蛋白的不同，有多达五个氨基酸残基会被磷酸化。两种植物的HMGB1蛋白被CK2α磷酸化的速度明显比其他HMGB底物蛋白慢，这表明某些HMGB蛋白显然是CK2α更偏好的底物。如化学交联实验所示，磷酸化反应的速率似乎与CK2α和HMGB蛋白之间相互作用的难易程度有关。基质辅助激光解吸电离飞行时间（MALDI/TOF）质谱分析表明，HMGB1和HMGB2/3蛋白在未成熟玉米籽粒中以多种磷酸化状态存在。因此，HMGB1在籽粒组织中以单磷酸化、双磷酸化、三磷酸化和四磷酸化蛋白的形式存在，其中四磷酸化形式最为丰富。观察到的体内磷酸化状态表明，除CK2α外的其他蛋白激酶也参与了植物HMGB蛋白的修饰。HMGB蛋白在不同程度上被磷酸化这一事实表明，差异修饰形式的存在增加了植物染色质中可能适应某些功能的不同HMGB蛋白变体的数量。
