Volume 6 Issue 1 - October 10, 2008
Phosphorylation by c-Jun NH2-terminal Kinase 1 Regulates the Stability of Transcription Factor Sp1 during Mitosis
Jan-Jong Hung

Institute of Biosignal transduction, College of Bioscience and Biotechnology, National Cheng Kung University
petehung@mail.ncku.edu.tw

Molecular Biology of the Cell, Vol. 19, p. 1139-1151, 2008.

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Research data of the past few decades indicate that transcription factors play a critical role in many biological events by virtue of their regulation of gene expression. It is well established that specific combinations of transcription factors exert unique effects on individual gene promoters, allowing spatio-temporal specificity of gene expression using only a small number of transcription factors under widely varying physiological and pathological conditions. One transcription factor worthy of our attention is Sp1, because it is ubiquitously expressed in mammalian cells, and it is important in a variety of physiological processes, including cell cycle regulation, apoptosis, and differentiation.

The transcriptional activity of a transcription factor is determined at least by three factors: transactivational activity, DNA binding affinity, and protein level. Previous studies on the regulation of Sp1 activities focused mostly on transactivational activity, thereby allowing study of its interaction with other proteins and its DNA binding affinity. However, one of the apparent key elements regulating the activity of Sp1 is via its stability, which certainly needs to be explored and established. Recent studies revealed that the DNA binding ability, transactivational activity, and protein stability of Sp1 might be influenced by its posttranslational modifications such as sumoylation, glycosylation, ubiquitination, acetylation, and phosphorylation. Recent studies also indicate that the cell cycle might regulate the phosphorylation states of Sp1. When cells enter the S phase of the cell cycle, Sp1 is phosphorylated by cyclin-dependent kinase 2 (CDK2), thus increasing the expression of dihydrofolate reductase, a key enzyme in the production of thymidine. Whether Sp1 is also phosphorylated by other kinases during different phases of the cell cycle and why Sp1 must be modified as such are still not clear.

Although a previous study characterized the distribution of Sp1 during the cell cycle, we were interested in studying how Sp1 is able to occupy the same nuclear space as DNA in interphase cells but is devoid of DNA in metaphase cells. Therefore, we examined the relation between nuclear membrane disturbance and Sp1 distribution at different stages of the cell cycle (Figure 1). Chromatin and Sp1 were evenly distributed within the nuclear, and they were colocalized in interphase cells. Lamin, a marker of the nuclear membrane, was also observed at different stages. At the metaphase, the chromatin was even more condensed, but the Sp1 distributed throughout the cell because of the disappearance of the nuclear membrane. During telophase, the colocalization of chromatin, Sp1, and lamin A/C became evident. As such, Sp1 was present in every different phase with its level well maintained at different stages, and its distribution correlated with the integrity of the nuclear membrane. Together, these findings suggest that Sp1 was stably reserved during mitosis, and there was different Sp1 localization between interphase and mitotic periods.
Figure 1. Distribution of Sp1 during the cell cycle. HeLa cells were double-labeled with anti-Sp1 antibodies (Green, G–L) and anti-lamin A/C antibodies (Red, M–R). DNA was stained with DAPI (Blue, A–F).

Figure 2. (A)Sp1 was highly phosphorylated during the mitotic stage in HeLa, A549 and MDA-MB-231 cells. Cyclin B1 was used as M phase marker, and actin was used as an equal loading control. (B) Mitotic cell extracts were treated with alkaline phosphatase (CIP) to dephosphorylate Sp1, and incubated at 37°C.

To study how the level of Sp1 is shielded from the ubiquitin-dependent degradation pathway, HeLa cells, A549 cells, and MDA-MB-231 cells were divided into interphase and mitosis groups after nocodazole treatment. In the interphase, cyclin B, considered as a marker of mitosis, was expressed less. In Western blottings examining Sp1 at interphase, two bands were found, and the bottom band was the major band. When the cells entered the mitotic stage, however, the major signal shifted to the top band (Figure 2A). A CIP assay was done next to determine whether phosphorylation had caused this band-shift. The result revealed that the major band recognized by anti-Sp1 antibodies at the interphase had now shifted to the top band at the mitotic stage. When cell lysate from mitotic cells was incubated with alkaline phosphatase at 37°C, the major signal shifted to the bottom band (Figure 2B).

The c-Jun NH2-terminal kinase (JNK) is a major member of the mitogen-activated protein kinase family, and it is known to respond to stress stimuli such as UV irradiation, heat shock, and reactive oxygen species. In the present study, we found that JNK1 was activated during mitosis and then caused most of the Sp1 to be the phosphorylated form. However, it is interesting that, with the JNK inhibitor, SP600125, treatment, not only the phosphorylated level of Sp1 was reduced but also the Sp1 protein level was significantly reduced (Figure 3A). These results indicated that Sp1 might be shielded from ubiquitination-dependent degradation through a high phosphorylation by JNK1 in the mitotic stage. In addition, Sp1 is known to be increased in many tumor cells. To study whether the Sp1 accumulation is related to JNK activation during mitosis in tumorigenesis, the rat glioma C6 cells and rat primary glial cells were divided into mitosis and interphase groups by nocodazole treatment (Figure 3B). In glioma C6 cells, Sp1 was consistent with the previous result that had most of Sp1 to be phosphorylated form during mitosis and the level of Sp1 could be maintained. Here, we also found that JNK was activated in mitotic stage. However, in primary glial cells, the Sp1 protein level was significantly reduced, and JNK was inactivated during mitosis. In addition, we also found that the protein level of Sp1 was significantly increased human cervical tumor compared with normal cervical tissue (Figure 3C).
Figure 3. (A)JNK1 phosphorylated Sp1 and increaseedSp1 stabilization during mitosis. (B and C) JNK1 was activated during mitosis and Sp1 was accumulated in glioma C6 cells and cervical cancer tissue was compared with primary glial cells and normal cervical tissue

Figure 4. JNK activation correlates Sp1 accumulation on MNU-induced mammary tumorigenesis in female rats.

In an animal study, N-methyl-N-nitrosourea (MNU) injection is associated with the formation of mammary tumors, at an incidence of >90%, which are predominantly adenocarcinomas of a ductal origin with the presence of only very few benign tumors. In our study, tumors were raised in 12 of 17 rats within 8 wk after the MNU injection (Figure 4A). Furthermore, we found that amount of Sp1 was accumulated inside the MNU-induced tumors compared with that observed in mammary normal cells (Figure 4, B and C). In addition, the phosphorylation level of JNK1 was increased in the MNU-induced tumor (Figure 4C). Thus, a correlation may exist between the Sp1 accumulation and the JNK1 activity in mammary tumors.

Other studies have shown that abnormal Sp1 activation might augment the growth and metastatic potential of tumor cells through the overexpression of many downstream genes of Sp1, including VEGF. The role of Sp1 as an essential transcription factor for many genes that regulate cell growth, angiogenesis, and survival has been proved in pancreatic, gastric, and colorectal cancers. In this study, we found Sp1 accumulation and JNK1 phosphorylation, which might result from the increase in the total JNK1 level, indicating that the level of Sp1 may thus be important for tumor formation. In addition, JNK1 activation is necessary to phosphorylate Sp1 and to shield Sp1 from the ubiquitin-dependent degradation pathway during mitosis in tumor cell lines. However, in normal cells, the Sp1 protein level was significantly reduced, and JNK was inactivated during mitosis (Figure 5).
Figure 5. Our finding indicates that JNK1 activation is necessary to phosphorylate Sp1 and to shield Sp1 from the ubiquitin-dependent degradation pathway during mitosis in tumor cell lines, but, in normal cells, the JNK and Sp1 protein level were significantly reduced.
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