Padilla-Benavides Teresita, Nasipak Brian T, Paskavitz Amanda L, Haokip Dominic T, Schnabl Jake M, Nickerson Jeffrey A, Imbalzano Anthony N
From the Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605 and.
the Department of Pediatrics, University of Massachusetts Medical School, Worcester, Massachusetts 01655.
J Biol Chem. 2017 Nov 10;292(45):18592-18607. doi: 10.1074/jbc.M117.799676. Epub 2017 Sep 22.
Transcriptional regulation is modulated in part by chromatin-remodeling enzymes that control gene accessibility by altering chromatin compaction or nucleosome positioning. Brahma-related gene 1 (Brg1), a catalytic subunit of the mammalian SWI/SNF chromatin-remodeling enzymes, is required for both myoblast proliferation and differentiation, and the control of Brg1 phosphorylation by calcineurin, PKCβ1, and p38 regulates the transition to differentiation. However, we hypothesized that Brg1 activity might be regulated by additional kinases. Here, we report that Brg1 is also a target of casein kinase 2 (CK2), a serine/threonine kinase, in proliferating myoblasts. We found that CK2 interacts with Brg1, and mutation of putative phosphorylation sites to non-phosphorylatable (Ser to Ala, SA) or phosphomimetic residues (Ser to Glu, SE) reduced Brg1 phosphorylation by CK2. Although BRG1-deleted myoblasts that ectopically express the SA-Brg1 mutant proliferated similarly to the parental cells or cells ectopically expressing wild-type (WT) Brg1, ectopic expression of the SE-Brg1 mutant reduced proliferation and increased cell death, similar to observations from cells lacking Brg1. Moreover, pharmacological inhibition of CK2 increased myoblast proliferation. Furthermore, the promoter, which controls expression of a key transcription factor required for myoblast proliferation, was in an inaccessible chromatin state in the SE-Brg1 mutant, suggesting that hyperphosphorylated Brg1 cannot remodel chromatin. WT-, SA-, and SE-Brg1 exhibited distinct differences in interacting with and affecting expression of the SWI/SNF subunits Baf155 and Baf170 and displayed differential sub-nuclear localization. Our results indicate that CK2-mediated phosphorylation of Brg1 regulates myoblast proliferation and provides insight into one mechanism by which composition of the mammalian SWI/SNF enzyme complex is regulated.
转录调控部分受染色质重塑酶的调节，这些酶通过改变染色质压缩或核小体定位来控制基因的可及性。哺乳动物SWI/SNF染色质重塑酶的催化亚基Brahma相关基因1（Brg1）对于成肌细胞的增殖和分化都是必需的，而钙调神经磷酸酶、蛋白激酶Cβ1（PKCβ1）和p38对Brg1磷酸化的控制调节着向分化的转变。然而，我们推测Brg1的活性可能受其他激酶的调节。在此，我们报告Brg1也是增殖性成肌细胞中丝氨酸/苏氨酸激酶酪蛋白激酶2（CK2）的作用靶点。我们发现CK2与Brg1相互作用，将假定的磷酸化位点突变为不可磷酸化（丝氨酸突变为丙氨酸，SA）或模拟磷酸化的残基（丝氨酸突变为谷氨酸，SE）会降低CK2对Brg1的磷酸化。虽然异位表达SA-Brg1突变体的Brg1缺失成肌细胞的增殖与亲代细胞或异位表达野生型（WT）Brg1的细胞相似，但SE-Brg1突变体的异位表达会降低增殖并增加细胞死亡，这与缺乏Brg1的细胞的观察结果相似。此外，CK2的药理学抑制增加了成肌细胞的增殖。此外，在SE-Brg1突变体中，控制成肌细胞增殖所需关键转录因子表达的启动子处于不可及的染色质状态，这表明过度磷酸化的Brg1无法重塑染色质。WT-Brg1、SA-Brg1和SE-Brg1在与SWI/SNF亚基Baf155和Baf170相互作用及影响其表达方面表现出明显差异，并显示出不同的亚核定位。我们的结果表明，CK2介导的Brg1磷酸化调节成肌细胞增殖，并为哺乳动物SWI/SNF酶复合物组成的调控机制提供了见解。
