Sp1是一個廣泛存在於哺乳類動物細胞內的一個轉錄調控因子，它可以調控細胞內許多生理功能。過去對於Sp1的研究發現Sp1的後轉譯修飾可以調控Sp1的功能，而其中一個重要的後轉譯修飾為磷酸化，Sp1可以在絲氨酸或蘇氨酸上被磷酸化。在實驗室之前的研究發現Sp1在Thr739的位置會被磷酸化，我們發現在此時其DNA結合親和力會下降。此外我們也發現一個獨特的異構酶Pin1在有絲分裂期會與Sp1有交互作用，因此我們假設Sp1或許會受到Pin1的作用使構形改變，進而調控Sp1之DNA結合親和力。在DAPA分析中，可以看到WT和T739D突變的Sp1在有無Pin1的情況下，其DNA結合親和力沒有差異。接著在細胞外激酶磷酸化實驗中，發現Sp1在Pin1存在時其磷酸化程度會增加。因此Pin1可能可以調控Sp1的構形，使得CDK1可以用auto-priming的方式對Sp1進行過度磷酸化，此過度磷酸化可能是使Sp1之DNA結合親和力下降的原因。接下來我們利用定點突變將可能是過度磷酸化位置的絲氨酸或蘇氨酸突變為丙氨酸。在細胞外激酶磷酸化實驗中發現Sp1 S720A-6A的磷酸化會降低，因此可能的過度磷酸化位置就包含在其中。這個研究可以讓我們更加了解Sp1之DNA結合親和力以及轉錄調控因子在細胞週期進行中離開染色質之調控。

The transcription factor Sp1 is ubiquitously expressed in mammalian cells and is important in a variety of physiological processes. Previous studies have revealed that the posttranslational modifications of Sp1 could alter its transcriptional activity, DNA-binding affinity, or protein stability. Sp1 can be phosphorylated on serine or threonine residues. Phosphorylation of Sp1 can regulate functions of Sp1 in different physiological condition. Our previous studies indicated that Sp1 can be phosphorylated at Thr739. Sp1 might loss DNA bind affinity after being phosphorylated at Thr739 during mitosis. In addition, our preliminary data indicated the Pin1, a unique peptidyl-prolyl cis/trans isomerase, could interact with phosphor-Sp1 in mitotic stage. Therefore, we suppose that DNA binding activity of Sp1 might be regulated through pin1-dependent manner. In the DAPA assay, results indicated that DNA binding affinity is no different between Sp1 and Sp1 T739D mutant in the presence or absence Pin1. These results demonstrated that T739 phosphorylation or the Pin1 recruitment does not decrease the DNA binding activity of Sp1 significantly. In the in vitro kinase assay, we found an increased signal caused by phosphorylation in the presence of Pin1. Therefore Pin1 might modulate Sp1 conformation that result in CDK1 “auto-priming”, which leads further phosphorylation of Sp1. This hyperphosphorylation of Sp1 might decrease the DNA binding activity of Sp1. Next we focused on the hyperphosphorylation site(s) of Sp1 by CDK1 in the presence of Pin1. By using site-directed mutagenesis, we constructed the serine/threonine to alanine mutant on possible hyperphosphorylation site(s) of Sp1. In the in vitro kinase assay, we found that the phosphorylation of Sp1 was decreased in S720A-6A mutant. This data suggests that the key hyperphosphorylation site of Sp1 might locate between S720 and T739. This study will let us understand the regulation of DNA binding activity of Sp1 much clearly, and more transcription factors that might bind to and release from chromosome via this pathway during cell cycle progression.

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