Programmed cell death 4 protein (Pdcd4) and homeodomain-interacting protein kinase 2 (Hipk2) antagonistically control translation of Hipk2 mRNA.

The tumor suppressor protein programmed cell death 4 (Pdcd4) is a highly conserved RNA-binding protein that inhibits the translation of specific mRNAs. Here, we have identified the homeobox-interacting protein kinase-2 (Hipk2) mRNA as a novel translational target of Pdcd4. Unlike most other protein kinases Hipk2 is constitutively active after being synthesized by the ribosome and its expression and activity are thought to be mainly controlled by modulation of the half-life of the kinase. Our work provides the first evidence that Hipk2 expression is also controlled on the level of translation. We show that Hipk2 stimulates the translation of its own mRNA and that Pdcd4 suppresses the translation of Hipk2 mRNA by interfering with this auto-regulatory feedback mechanism. We also show that the translation of the related kinase Hipk1 is controlled by a similar feedback loop and that Hipk2 also stimulates the translation of Hipk1 mRNA. Taken together, our work describes a novel mechanism of translational suppression by Pdcd4 and shows for the first time that Hipk2 controls its own synthesis by an auto-regulatory feedback mechanism. Furthermore, the effect of Hipk2 on the translation of Hipk1 RNA suggests that Hipk2 and Pdcd4 can act in similar manner to control the translation of other mRNAs.

An evolutionarily conserved interaction of tumor suppressor protein Pdcd4 with the poly(A)-binding protein contributes to translation suppression by Pdcd4.

The tumor suppressor protein programmed cell death 4 (Pdcd4) has been implicated in the translational regulation of specific mRNAs, however, the identities of the natural Pdcd4 target mRNAs and the mechanisms by which Pdcd4 affects their translation are not well understood. Pdcd4 binds to the eukaryotic translation initiation factor eIF4A and inhibits its helicase activity, which has suggested that Pdcd4 suppresses translation initiation of mRNAs containing structured 5'-untranslated regions. Recent work has revealed a second inhibitory mechanism, which is eIF4A-independent and involves direct RNA-binding of Pdcd4 to the target mRNAs. We have now identified the poly(A)-binding protein (PABP) as a novel direct interaction partner of Pdcd4. The ability to interact with PABP is shared between human and Drosophila Pdcd4, indicating that it has been highly conserved during evolution. Mutants of Pdcd4 that have lost the ability to interact with PABP fail to stably associate with ribosomal complexes in sucrose density gradients and to suppress translation, as exemplified by c-myb mRNA. Overall, our work identifies PABP as a novel functionally relevant Pdcd4 interaction partner that contributes to the regulation of translation by Pdcd4.

Interaction and cooperation of the CCAAT-box enhancer-binding protein ß (C/EBPß) with the homeodomain-interacting protein kinase 2 (Hipk2).

CCAAT box/enhancer-binding protein ß (C/EBPß) 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/EBPß cooperates with the coactivator p300 through a novel mechanism that involves the C/EBPß-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/EBPß, p300, and Hipk2 in more detail. We show that Hipk2 and C/EBPß 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/EBPß. In addition to phosphorylating p300 recruited to C/EBPß, Hipk2 also phosphorylates C/EBPß at sites that have previously been shown to plays key roles in the regulation of C/EBPß activity. Silencing of Hipk2 expression disrupts adipocyte differentiation of 3T3-L1 cells, a physiological C/EBPß-dependent differentiation process indicating that the cooperation of C/EBPß 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/EBPß, 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/EBPß and implicate different protein kinases in the cooperation of p300 with C/EBPß and C/EBPα.

Association of Tumor Suppressor Protein Pdcd4 With Ribosomes Is Mediated by Protein-Protein and Protein-RNA Interactions.

The Pdcd4 (programmed cell death gene 4) gene has been implicated as a novel tumor suppressor gene in the development of several types of human cancer. The Pdcd4 protein is believed to act as a translation suppressor of mRNAs containing structured 5' UTRs. Pdcd4 contains 2 copies of so-called MA3 domains that mediate tight interactions with the translation initiation factor eIF4A, resulting in the inhibition of the eIF4A helicase activity. The N-terminal part of Pdcd4, which has been less well characterized, binds RNA in vitro, but as yet, it has not been clear whether RNA binding by Pdcd4 plays a role in vivo. Here, the authors have identified 2 highly conserved clusters of basic amino acid residues that are essential for the RNA binding activity of Pdcd4. They also show that a substantial fraction of Pdcd4 is present, together with small ribosomal subunits, in translation preinitiation complexes. Using mutants that disrupt RNA binding or the Pdcd4-eIF4A interaction, they demonstrate that the ribosomal association of Pdcd4 is dependent on its RNA binding activity as well as on its ability to interact with eIF4A. Their work provides the first direct evidence for an essential role of the Pdcd4 RNA binding activity in vivo and suggests that RNA binding is required for recruiting Pdcd4 to the translation machinery.