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SP600125 Selectively Inhibits Histone H3-Ser10 Phosphorylation Kamal D. Mehta*, Wei Huang, Sanjay Batra, and Sasi Korrapati Department of Molecular and Cellular Biochemistry, The Ohio State University College of Medicine and Public Health, 1645 Neil Avenue, Columbus, OH *[email protected] INTRODUCTION. The role played by histone modifications in transcriptional regulation is one recent area of interest in the study of gene expression. These modifications collectively influence a web of regulatory events, and their interconnectedness has led to the hypothesis that there is a “histone code” controlling chromatin dynamics (1). Many types of cancer are associated with translocations or mutations in chromatin modifying enzymes and regulatory proteins. The phosphorylation of histone H3 at serine 10 (histone H3-Ser10) is conserved through eukaryotes, and an increase in phosphorylation has been shown to correlate with gene activation and cell growth (2). In contrast to many studies on the structural and functional consequences of histone H3 acetylation, the impact of the phosphorylation is relatively unexplored due to the lack of pharmacological inhibitor. We report here for the first time that putative c-jun N-terminal kinase (JNK) inhibitor SP600125 inhibits histone H3-Ser10 phosphorylation, and the inhibitory effect is not a direct consequence of this kinase.. METHODS. JNK was assayed by a glutathione bead pull-down method and equal amounts of protein were analyzed by immunoblotting (3). RESULTS. We show that SP600125 dramatically reduced global histone H3Ser10 phosphorylation in a dose- and time-dependent manner, without affecting global acetylation of lysine 14 or its expression in human hepatoma cells (Fig. 1A). Similar reduction in histone H3-Ser10 phosphorylation by SP600125 was also observed in other cancer cells. A comparison of the kinetics of a reduction in histone H3-Ser10 phosphorylation and p46/54JNK inhibition by SP600125 (Fig. 2B) suggests that SP600125dependent loss of histone H3-Ser10 phosphorylation is possibly independent of the p46/54JNK pathway. A close match would be expected if SP600125 mediated its effect by inhibiting this kinase. SP600125 (µM), 1 h SP600125 (10 µM), h 0 .5 1 2 5 10 20 30.15 .33 .5 1 3 6 100 Phosphorylation 80 P-Ser10-H3 % Control JNK 60 40 Ac-Lys14-H3 20 Histone H3 0 0 5 10 15 SP600125 (µM) 20 (A) (B) Fig 1. (A) SP600125 inhibits histone H3-Ser10 in a dose- and time-dependent manner in human hepatoma cells. (B) A comparison of the kinetics of reduction in histone H3-Ser10 phosphorylation and JNK inhibition by SP600125. DISCUSSION. We report here for the first time, in any cell type, that SP600125 inhibits histone H3-Ser10 phosphorylation, and the inhibitory effect appears to be not a direct consequence of JNK inhibition. A perusal of the existing literature using SP600125 for the inhibition of p46/54JNKs indicates that this compound is typically used at concentrations ranging from 1 to 50 µM. Based on the studies reported herein, we anticipate that concentrations of SP600125 sufficient to inhibit p46/54JNK activities in cultured cells and tissues would also suppress histone H3-Ser10 phosphorylation. The extent to which histone H3-Ser10 phosphorylation suppression by SP600125 compromises its usefulness as a JNK inhibitor will be dependent on the nature of the study. In view of our results, data obtained with SP600125 must be interpreted with great caution. Furthermore, the ability to easily manipulate this modification in vivo by SP600125 provides an excellent strategy for elucidating the histone code and to assess functional role of this modification in various cellular events. A better understanding of the underlying mechanisms will help guide the development of more effective agents to treat cancer. ACKNOWLEDGEMENT. This work was supported by the National Institutes of Health Research Grants HL-742202 and HL-60000355 (to K.D.M). REFERENCES. 1. Jenuwein, T., and Allis, C. D. (2001) Translating the histone code. Science 293:1074-1080. 2. Cheung, P., Allis, P., and Sassone-Corci, P. (2000) Signaling to chromatin through histone modifications. Cell 103:263-271. 3. Huang, W., Batra, S., Atkins, B. A., Mishra, V., and Mehta, K. D. (2005) Increases in intracellular calcium dephosphorylate histone H3 at Serine 10 in human hepatoma cells: Potential role of protein phosphatase 2A0protein kinase CbII complex. J. Cellular Physiol. 205:37-46. 2