Recruitment of p300 by C/EBPbeta triggers phosphorylation of p300 and modulates coactivator activity

Abstract

Transcriptional coactivators such as p300 act as crucial elements in the eukaryotic gene regulation network. These proteins bind to various transcription factors which recruit them to specific gene regions whose chromatin structure subsequently is remodeled. Previously, we have shown that C/EBPβ recruits p300 by interacting with the E1A‐binding site of the coactivator. We now show that C/EBPβ not only binds to p300 but also triggers massive phosphorylation of p300. This novel activity of C/EBPβ is dependent on the E1A‐binding region of p300 as well as on several subdomains of C/EBPβ, all of which are involved in the p300–C/EBPβ interaction. We have identified several sites of C/EBPβ‐inducible phosphorylation within the C‐terminal domain of p300. Mutation of these sites substantially impairs the activity of p300 as a coactivator of C/EBPβ. Interestingly, phosphorylation of p300 is also triggered by other C/EBP family members as well as by various other transcription factors that interact with the E1A‐binding domain of p300, suggesting that this novel phosphorylation mechanism may be of general relevance.

Transformation suppressor protein Pdcd4 interferes with JNK-mediated phosphorylation of c-Jun and recruitment of the coactivator p300 by c-Jun

“…To investigate whether Pdcd4 affects the Jun-induced phosphorylation of p300, we cotransfected expression vectors for c-Jun, the carboxyterminal domain of p300 and increasing amounts of Pdcd4 into a fibroblast cell line followed by SDS-PAGE and Western blotting to determine the mobility of p300. As expected from our previous work (Schwartz et al, 2003), in the presence of c-Jun, multiple slowermigrating forms of the p300 carboxyterminal domain accumulated, giving rise to a rather broad smear of bands ( Figure 7a). Adding increasing amounts of Pdcd4 resulted in a partial suppression of the appearance of these slower-migrating forms of p300, suggesting that Pdcd4 interferes with the interaction of p300 and c-Jun or with the subsequent phosphorylation of p300.…”

“…We have recently shown that recruitment of p300 by c-Jun and several other transcription factors triggers the phosphorylation of the carboxyterminal region of p300 (Schwartz et al, 2003). This phosphorylation can be easily monitored by analysing the electrophoretic mobility of the carboxyterminal domain of p300 in an SDS-polyacrylamide gel.…”

“…An expression vector for a Gal4/c-Jun(1-166) fusion protein was obtained from P Angel. The expression vectors for the C-terminal domain of p300 (pCDNA3-p300/1751-2370) and the p300/VP16 protein (containing amino acids 1752-1998 of human p300 fused to the VP16 transactivation domain) have been described (Mink et al, 1997;Schwartz et al, 2003). The expression vector for full-length p300 (pCMV-p300CHA) was obtained from R Eckner (Eckner et al, 1994).…”

The cooperation of B-Myb with the coactivator p300 is orchestrated by cyclins A and D1

“…GST fusion proteins and in vitro binding assay pGST/B-Myb-D3, pGST/B-Myb-DFsp, GST/p300(1-672), GST/p300(672-1193), GST/p300(1710-1891) and GST/ p300(1883-1927) have been described elsewhere (Horstmann et al, 2000a;Hasan et al, 2001;Schwartz et al, 2003). GST fusion protein expression was induced in logarithmically growing cultures of transformed bacteria (E. coli BL21-lys) by adding isopropyl-b-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM.…”

Elevated c-Jun expression in acute myeloid leukemias inhibits C/EBPα DNA binding via leucine zipper domain interaction

“…Most interestingly, protein-protein interactions are not only responsible for inactivation of C/EBPa in AML, but also C/EBPa itself seems to function via protein-protein interactions (McKnight, 2001). Thus, C/EBPa arrests cell proliferation through direct inhibition of Cdk2 and Cdk4 (Wang et al, 2001(Wang et al, , 2002, E2F repression by C/EBPa is required for adipogenesis and granulopoiesis (Porse et al, 2001), and C/EBPa binds to and leads to phosphorylation of p300 (Schwartz et al, 2003).…”