Abstract
CCAAT box/enhancer-binding protein β (C/EBPβ) is a bZip transcription factor that plays crucial roles in important cellular processes such as differentiation and proliferation of specific cell types. Previously, we showed that C/EBPβ cooperates with the coactivator p300 through a novel mechanism that involves the C/EBPβ-induced phosphorylation of multiple sites in the carboxyl-terminal domain of p300 by protein kinase Hipk2. We have now examined the interaction and cooperation of C/EBPβ, p300, and Hipk2 in more detail. We show that Hipk2 and C/EBPβ are direct physical binding partners whose interaction is mediated by sequences located in the amino-terminal and central domains of Hipk2 and the amino-terminal part of C/EBPβ. In addition to phosphorylating p300 recruited to C/EBPβ, Hipk2 also phosphorylates C/EBPβ at sites that have previously been shown to plays key roles in the regulation of C/EBPβ activity. Silencing of Hipk2 expression disrupts adipocyte differentiation of 3T3-L1 cells, a physiological C/EBPβ-dependent differentiation process indicating that the cooperation of C/EBPβ and Hipk2 is functionally relevant. Finally, we demonstrate that C/EBPα, a related C/EBP family member whose amino-terminal sequences differ significantly from that of C/EBPβ, is unable to interact and cooperate with Hipk2. Instead, our data suggest that C/EBPα cooperates with the protein kinase Jnk to induce phosphorylation of p300. Overall, our data identify Hipk2 as a novel regulator of C/EBPβ and implicate different protein kinases in the cooperation of p300 with C/EBPβ and C/EBPα.
Highlights
CCAAT box/enhancer-binding protein (C/EBP) is a bZip transcription factor that triggers phosphorylation of p300
Interaction of C/EBP with the Protein Kinase Hipk2—Our previous work has shown that the recruitment of the co-activator p300 by the transcription factor C/EBP is coupled to the phosphorylation of p300 at multiple sites in its carboxyl-terminal domain
Hipk2, we were interested to know whether C/EBP serves mainly as a scaffold that allows Hipk2 and p300 to come into close proximity or whether C/EBP is itself phosphorylated by Hipk2
Summary
C/EBP is a bZip transcription factor that triggers phosphorylation of p300. Results: Protein kinase Hipk interacts with and phosphorylates the longest isoform of C/EBP, thereby facilitating recruitment and subsequent phosphorylation of p300. CCAAT box/enhancer-binding protein  (C/EBP) is a bZip transcription factor that plays crucial roles in important cellular processes such as differentiation and proliferation of specific cell types. We demonstrate that C/EBP␣, a related C/EBP family member whose amino-terminal sequences differ significantly from that of C/EBP, is unable to interact and cooperate with Hipk. C/EBP␣ and C/EBP are members of the CCAAT box/enhancer-binding protein (C/EBP) family of basic-region-leucine-zipper transcription factors, which play central roles in fundamental cellular processes including differentiation, pro-. In addition to differential isoform expression, C/EBP, is regulated by posttranslational modifications, such as phosphorylation (16 –18), acetylation (19 –21), and sumoylation [22] as well as by the binding of specific cofactors via protein-protein interactions [23,24,25,26,27]. We show that two related C/EBP family members, C/EBP and C/EBP␣, differ in their ability to cooperate with Hipk
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