在ㄧ些與細胞過度增生有關的疾病（例如：牛皮癬）當中，角質細胞中的角質素十六（keratin 16，K16）會有過度表現的情形。而由本實驗室先前對keratin 16的研究可知，在角質細胞（HaCaT cells）中，表皮生長因子（EGF）會活化Ras，造成c-Jun及c-Fos生合成增加，而增加的AP1核蛋白一方面會結合到keratin 16啟動區上的AP1區域，另ㄧ方面也會透過與Sp1的交互作用而結合到keratin 16啟動區上的Sp1區域，於是透過這兩個response elements來增加keratin 16基因表現；此外，EGF也會藉由MEK-ERK訊息傳遞路徑來增加p300聚集到keratin 16啟動區上，共同增加keratin 16基因的表現。而此調控機制，至少有一部分是藉由增加c-Jun以及組織蛋白H3之乙醯化（acetylation）所致。根據之前文獻的報告，在in vitro，p300純化蛋白的C端 (a.a. 1572-2370) 會受到活化態的ERK2所磷酸化，進而增加其轉錄活性。然而，在in vivo，p300會受到何種蛋白激酶磷酸化，且磷酸化的確切位置，及其如何影響p300的功能，還不是很清楚。於是，本研究主要是要探討p300磷酸化參與EGF誘導角質細胞中keratin 16基因表現之調控。由實驗結果可知，在 in vivo及in vitro狀態下，EGF會隨著劑量及時間增加而增加p300的磷酸化，此機制是藉由活化ERK訊息傳導路徑而造成的。進一步藉由in vitro kinase assay及單點突變實驗，則在p300上定出六個磷酸化位點，分別是三個蘇氨酸及三個絲氨酸。接下來在in vivo狀態下，進一步去分析這六個位點是否確實會被磷酸化。由實驗結果得知，在 in vivo狀態下，三個絲氨酸(Ser2279、2315、2366)為EGF主要的標的，這三個位置磷酸化後會增加p300聚集到角質素16啟動區上，增強其和Sp1的交互作用，另一方面也會增加p300本身的酵素活性進而增加角質素16基的表現。因此，如果將這三個絲氨酸(Ser2279、2315、2366)同時突變掉，也會造成EGF所誘導的角質素16基因表現被抑制。綜合以上結果可知，ERK2訊息傳導路徑所媒介的p300絲氨酸(Ser2279、2315、2366)磷酸化在EGF所誘導的角質素16基因表現中扮演一個非常關鍵的角色，而這也是第一篇研究指出在in vivo狀態下，p300在哪些確切位點被ERK2訊息傳導路徑磷酸化。

Overexpression of keratin 16 in keratinocytes has been found to be associated with hyperproliferative skin diseases such as psoriasis. In our previous studies on gene regulation of keratin 16, we have reported that transcriptional coactivator p300 interacts with Sp1 and AP1 proteins, and is involved in the regulation of epidermal growth factor (EGF)-induced keratin 16 gene expression in HaCaT cells at least in part through acetylation of c-Jun and histone H3. The recruitment of p300 to keratin 16 promoter by EGF is mediated by ERK signaling pathway. Although one study shows that activated ERK2 can phosphorylate p300 (a.a. 1572-2370) in vitro and increase its transcriptional activity, however, it remains to prove that ERK can phosphorylate p300 in vivo and to identify the exact phosphorylation sites. More importantly, the functional links between the specific phosphorylation events and the downstream gene regulation also remain to be explored. Therefore, the specific aim is to study the role of p300 phosphorylation in the regulation of EGF-induced keratin 16 gene expression in keratinocytes. Here, our data showed that EGF dramatically induced time- and dose-dependent phosphorylation of p300, both in vitro and in vivo, through the activation of ERK2. Six ERK2 phosphorylation sites on p300, including three threonine and three serine residues, were first identified in vitro. The data from in vivo phosphorylation assay further indicated that Ser2279, Ser2315, and Ser2366 on the C-terminus of p300 were the major phosphorylated targets in response to EGF. Furthermore, these serine phosphorylations increased p300 HAT activity and interaction with Sp1, thereby enhancing p300 recruitment to the keratin 16 promoter. Substitution of these three serine residues with alanine blocked EGF-induced keratin 16 gene expression. Taken together, our results not only first reported the specific p300 phosphorylation sites by ERK2 in vivo but also explored the critical roles of these phosphorylations in regulating keratin 16 expression in response to EGF.

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