HIPK2 modulates p53 activity towards pro-apoptotic transcription.
BACKGROUND
Activation of p53-mediated gene transcription is a critical cellular response to DNA damage and involves a phosphorylation-acetylation cascade of p53. The discovery of differences in the response to different agents raises the question whether some of the p53 oncosuppressor functions might be exerted by different posttranslational modifications. Stress-induced homeodomain-interacting protein kinase-2 (HIPK2) phosphorylates p53 at serine-46 (Ser46) for p53 apoptotic activity; p53 acetylation at different C-terminus lysines including p300-mediated lysine-382 (Lys382) is also required for full activation of p53 transcriptional activity. The purpose of the current study was to evaluate the interplay among HIPK2, p300, and p53 in p53 acetylation and apoptotic transcriptional activity in response to drug by using siRNA interference, p300 overexpression or deacetylase inhibitors, in cancer cells.


RESULTS
Knockdown of HIPK2 inhibited both adriamycin-induced Ser46 phosphorylation and Lys382 acetylation in p53 protein; however, while combination of ADR and zinc restored Ser46 phosphorylation it did not recover Lys382 acetylation. Chromatin immunoprecipitation studies showed that HIPK2 was required in vivo for efficient p300/p53 co-recruitment onto apoptotic promoters and that both p53 modifications at Ser46 and Lys382 were necessary for p53 apoptotic transcription. Thus, p53Lys382 acetylation in HIPK2 knockdown as well as p53 apoptotic activity in response to drug could be rescued by p300 overexpression. Similar effect was obtained with the Sirt1-inhibitor nicotinamide. Interestingly trichostatin A (TSA), the inhibitor of histone deacetylase complexes (HDAC) did not have effect, suggesting that Sirt1 was the deacetylase involved in p53 deacetylation in HIPK2 knockdown.


CONCLUSIONS
These results reveal a novel role for HIPK2 in activating p53 apoptotic transcription. Our results indicate that HIPK2 may regulate the balance between p53 acetylation and deacetylation, by stimulating on one hand co-recruitment of p300 and p53Lys382 on apoptotic promoters and on the other hand by inhibiting Sirt1 deacetylase activity. We attempted to reactivate p53 apoptotic transcriptional activity by rescuing both Ser46 and Lys382 modification in response to drug. Our data propose combination strategies for the treatment of tumors with dysfunctional p53 and/or HIPK2 that include classical chemotherapy with pharmacological or natural agents such as Sirt1-deacetylase inhibitors or zinc, respectively.
Puca
Rosa
R
Department of Experimental Oncology, Molecular Oncogenesis Laboratory, National Cancer Institute Regina Elena, Rome, Italy. puca@ifo.it
Nardinocchi
Lavinia
L
Sacchi
Ada
A
Rechavi
Gideon
G
Givol
David
D
D'Orazi
Gabriella
G
D000107:Acetylation / Q000187:drug effects; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D002352:Carrier Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000494:pharmacology; D050881:E1A-Associated p300 Protein / Q000378:metabolism; D017353:Gene Deletion; D055785:Gene Knockdown Techniques; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D008239:Lysine / Q000378:metabolism; D009536:Niacinamide / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D056564:Sirtuin 1 / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D015032:Zinc / Q000494:pharmacology; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D002352:Carrier Proteins; D006877:Hydroxamic Acids; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D009536:Niacinamide; C012589:trichostatin A; D004317:Doxorubicin; D050881:E1A-Associated p300 Protein; C494360:EP300 protein, human; C115114:HIPK2 protein, human; D017346:Protein Serine-Threonine Kinases; C447939:SIRT1 protein, human; D056564:Sirtuin 1; D015032:Zinc; D008239:Lysine