PEBP2-β/CBF-β–dependent phosphorylation of RUNX1 and p300 by HIPK2: implications for leukemogenesis

Abstract

The heterodimeric transcription factor RUNX1/PEBP2-␤ (also known as AML1/ CBF-␤) is essential for definitive hematopoiesis. Here, we show that interaction with PEBP2-␤ leads to the phosphorylation of RUNX1, which in turn induces p300 phosphorylation. This is mediated by homeodomain interacting kinase 2 (HIPK2), targeting Ser 249 , Ser 273 , and Thr 276 in RUNX1, in a manner that is also dependent on the RUNX1 PY motif. Importantly, we observed the in vitro disruption of this phosphorylation cascade by multiple leukemogenic genetic defects targeting RUNX1/CBFB. In particular, the oncogenic protein PEBP2-␤-SMMHC prevents RUNX1/p300 phosphorylation by sequestering HIPK2 to mislocalized RUNX1/ ␤-SMMHC complexes. Therefore, phosphorylation of RUNX1 appears a critical step in its association with and phosphorylation of p300, and its disruption may be a common theme in RUNX1-associated leukemogenesis. (Blood. 2008;112:3777-3787) IntroductionThe runt-related transcription factor RUNX1 (AML1; PEBP2-␣; CBF-␣) and its non-DNA binding partner PEBP2-␤/CBF-␤ are frequent targets of leukemogenic genetic changes. [1][2][3] The heterodimer plays a critical role in definitive hematopoiesis, and disruption of its normal function by chromosomal abnormalities or mutations is collectively the most common cause of acute myeloid leukemia (AML). Their central role in orchestrating proper differentiation of hematopoietic stem cells is underscored by the ablation of definitive hematopoiesis in Runx1 or Pebp2b Ϫ/Ϫ knockout mice [4][5][6] and an expanded HSC compartment in conditional Runx1-deficient mice. [7][8][9][10] However, although the overall biologic functions of RUNX1 are becoming clear, how these functions are controlled at the molecular level and the contribution of PEBP2-␤ remain to be fully determined.The evolutionarily conserved partner protein PEBP2-␤ is known to enhance DNA binding ability of all 3 mammalian RUNX proteins (RUNX1-3) by inducing allosteric change of DNA binding Runt domain without direct DNA contact. In addition, we have previously reported that PEBP2-␤ regulates RUNX1 metabolic stability by preventing its ubiquitin-mediated degradation. 11 It has also been speculated that PEBP2-␤ may also function to recruit other proteins to the target gene promoter, although only 2 cytoplasmic proteins, Crl-1 and filamin A, have ever been reported to interact with PEBP2-␤. 12-15 Notwithstanding our current incomplete understanding of molecular interaction between PEBP2-␤ and RUNX1, it is clear that PEBP2-␤ is essential for RUNX1 function in vivo, as Pebp2␤-deficient mice exhibit a very similar phenotype to Runx1 Ϫ/Ϫ mice. [4][5][6] Whereas early studies have implicated RUNX1 to be a transcriptional activator, subsequent studies revealed more diverse and intricate functions. It is now clear that RUNX1 can also act as a transcriptional repressor and functions as a molecular scaffold for the organization of partner transcription factors and cofactors at the regulatory regions of target genes. RUNX1 interacts with a growing lis...

Regulation of p53 activity by HIPK2: molecular mechanisms and therapeutical implications in human cancer cells

“…However, in that study, HIPK2 was found mutated in a very small percentage (2 out of 130 cases) so that it is difficult to justify a connection with p53 inactivation. HIPK2 cytoplasmic localization, similarly to what seen in the leukemogenesis model (Britos-Bray et al, 1998;Wee et al, 2008) (see above), was found related to high-mobility group A1 (HMGA1) overexpression leading to inhibition of p53-mediated apoptosis (Pierantoni et al, 2007). HMGA1 protein is frequently overexpressed in tumors and correlates strongly with cytoplasmic HIPK2 localization and low apoptotic index in wt p53-positive human breast cancer tissues (Pierantoni et al, 2007).…”

“…One important finding that supports the relationship between HIPK2, p53 and p300 for p53 apoptotic function comes from a leukemogenesis model in which the oncogene CBFb-SMMHC attenuates the p53 apoptotic function in response to DNA damage (that is, g-irradiation and VP-16) (Britos-Bray et al, 1998). CBFb-SMMHC prevents p300 phosphorylation by sequestering HIPK2 in the cytoplasm (Wee et al, 2008), which becomes the mechanistic explanation of the attenuation of the HIPK2/p53 apoptotic pathway in response to DNA damage.…”

Counteracting MDM2‐induced HIPK2 downregulation restores HIPK2/p53 apoptotic signaling in cancer cells

“…Indeed we previously showed a correlation between low HIPK2 expression and bad outcome in wild-type p53 expressing colon cancer tumors [14]. HIPK2 cytoplasmic localization may also affect p53 apoptotic function, following overexpression of oncogenes such as CBFb-SMMHC in a leukemogenesis model [22,23], high-mobility group A1 (HMGA1) [24] or b4 integrin in breast cancers [25]. HIPK2 can be downregulated by several E3 ubiquitin ligases such as WD40-repeat/SOCS box protein WSB-1, the RING family ligases Siah-1 and Siah-2 and, MDM2 oncogene [21].…”

Aurora kinase-induced phosphorylation excludes transcription factor RUNX from the chromatin to facilitate proper mitotic progression

“…1F). Both were previously shown to be phosphorylated (18)(19)(20). T173 phosphorylation has not been reported.…”