We found that both YAP and CREB gave strong signals and colocaliz

We found that both YAP and CREB gave strong signals and colocalized in both HCC cells (Fig. 4A) and HCC tissues (Fig. 4D) by IF assays. To confirm the interaction between YAP and CREB, we performed reciprocal coimmunoprecipitation (Co-IP) experiments and found that exogenous CREB-HA could be readily pulled down by YAP-Flag and vice versa (Fig. 4B). Co-IP for endogenous YAP and CREB proteins in Bel-7402 and HepG2 cells also demonstrated that these two proteins readily coimmunoprecipitated

(Fig. 4C). To further uncover the relationship between YAP and CREB, we performed IHC using TMA on greater than 400 human liver cancer samples. We MI-503 molecular weight found that both CREB and YAP proteins are highly expressed in a subset of human liver cancers and closely correlated with each other (Fig. 4E,F). Taken together, these experiments establish a close relationship between YAP and CREB in liver cancer. To reveal how YAP regulates CREB at the protein level, the mechanism underlying CREB degradation needed to be elucidated. We found that CREB protein could be down-regulated by both of the two phosphoinositide 3 kinase (PI3K) inhibitors, LY294002 and wortmannin. Surprisingly, the phosphorylated form of CREB STA-9090 research buy at Ser133 was up-regulated (Fig. 5A). Using PI3K activator hEGF, an opposite expression pattern of CREB and p-CREB was observed (Supporting Fig. 3A). pTEN is known as an endogenous PI3K inhibitor. In HepG2 cells

with pTEN overexpressed, we found that, inconsistent with the above-mentioned two chemical inhibitors, p-CREB was up-regulated, whereas total CREB was down-regulated (Supporting Fig. 3B). On the contrary, reduction of p-CREB with induction of total CREB was detected in Bel-7402 cells with pTEN knocked down (Supporting Fig. 3C),

suggesting that PI3K inhibits CREB phosphorylation, but protects CREB from degradation. As reported previously, CREB can be phosphorylated at ser133 by p38.[14] By Co-IP assays, we found 上海皓元医药股份有限公司 that p-p38 and CREB interacted with each other in HepG2 cells (Supporting Fig. 4). Such interaction was enhanced by both LY294002 and wortmannin, as detected by IF (Fig. 5B) and Co-IP assays (Fig. 5C), respectively. However, in HepG2 cells under treatment of either LY294002 or wortmannin, p-p38 was slightly up-regulated, whereas total p38 was slightly down-regulated (Fig. 5A). To investigate whether p38 contributes to the phosphorylation and degradation of CREB, two specific p38 inhibitors (SB203580 and SB202190) were used. We found that p-CREB was inhibited, whereas total CREB was induced (Fig. 5D). Consistently, reduced p-CREB with induced total CREB was detected in HepG2 cells with p38 knocked down (Fig. 5E). By contrast, opposite results were observed in HepG2 cells when p38 was overexpressed (Fig. 5F), suggesting that p38 phosphorylates CREB, which, ultimately, leads to CREB degradation.

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