Phosphorylation of Mixed Lineage Leukemia 5 by Cdc2 Affects Its Cellular Distribution and Is Required for Mitotic Entry

Abstract

The human mixed lineage leukemia-5 (MLL5) gene is frequently deleted in myeloid malignancies. Emerging evidence suggests that MLL5 has important functions in adult hematopoiesis and the chromatin regulatory network, and it participates in regulating the cell cycle machinery. Here, we demonstrate that MLL5 is tightly regulated through phosphorylation on its central domain at the G 2 /M phase of the cell cycle. Upon entry into mitosis, the phosphorylated MLL5 delocalizes from condensed chromosomes, whereas after mitotic exit, MLL5 becomes dephosphorylated and re-associates with the relaxed chromatin. We further identify that the mitotic phosphorylation and subcellular localization of MLL5 are dependent on Cdc2 kinase activity, and Thr-912 is the Cdc2-targeting site. Overexpression of the Cdc2-targeting MLL5 fragment obstructs mitotic entry by competitive inhibition of the phosphorylation of endogenous MLL5. In addition, G 2 phase arrest caused by depletion of endogenous MLL5 can be compensated by exogenously overexpressed full-length MLL5 but not the phosphodomain deletion or MLL5-T912A mutant. Our data provide evidence that MLL5 is a novel cellular target of Cdc2, and the phosphorylation of MLL5 may have an indispensable role in the mitotic progression.Aberrations in chromosome 7, either due to monosomy or partial deletion of 7q, are commonly associated with myeloid malignancies, including myelodyplastic syndrome, acute myeloid leukemia, and therapy-induced myeloid neoplasms (1, 2). Cytogenetic studies have delineated 7q22 as a critical region in myeloid malignancies (3-5). A search for a candidate tumor suppressor gene within this region led to the identification of a novel gene, which was later categorized as the fifth MLL/trithorax member and designated MLL5 (6). MLL1 (also known as ALL-1, HRX, and Htrx), a frequent target of chromosomal translocation in myeloid and lymphoid leukemia, is the best characterized member of the MLL protein family (7-9). Mll1 and Mll2 knock-out mice are embryonic lethal (10, 11). Mice carrying mutant Mll3 genes are partially embryonic lethal, and surviving mice are stunted in overall growth (12). Recent studies on Mll5 knock-out mice have revealed that MLL5 plays a critical role in adult hematopoiesis and appears to be dispensable for embryonic development (13-16). Although Mll5 knockout mice do not have an increased incidence of tumors, they suffer from mild growth retardation, male infertility, and compromised immunity. Nonetheless, the physiological function of MLL5 and its cellular targets remain elusive.A common feature of the MLL protein family is the concomitant presence of a variable number of PHD 3 (plant homeodomain) zinc fingers and a single SET (Su (var) 3-9, enhancer-ofzeste and trithorax) domain. Reports have shown that PHD fingers are binding or recognition modules for histone modification (17), whereas the SET domain possesses methyltransferase activity (18,19). MLL1 and MLL2 form a protein complex containing menin, WDR5, and chromatin-remodeling components and are ...

Camptothecin-induced downregulation of MLL5 contributes to the activation of tumor suppressor p53

“…To study if the affinity of p53 to DNA was affected by downregulation of MLL5, cellular fractionation was performed in scrambled or MLL5-siRNAtransfected HCT116 cells. Consistent with the previous reports (Deng et al, 2004;Liu et al, 2010b), MLL5 was primarily detected in the chromatin-associated fraction (Figure 4e). Interestingly, a detectable translocation of p53 from the nucleoplasmic to the chromatin-associated fraction was observed upon MLL5 knockdown, an indication of an enhanced recruitment of p53 to its target site (Figure 4e).…”

“…Upon MLL5 knockdown, the entry of quiescent myoblasts into S-phase was delayed, but the completion of S-phase progression was hastened (Sebastian et al, 2009). Recently, we showed that phosphorylation of MLL5 by mitotic kinase Cdc2 is required for mitotic entry (Liu et al, 2010b). Taken together, these data imply that MLL5 have different regulatory roles throughout cell cycle.…”

“…Cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, L-glutamine (2 mM), penicillin (100 units/ml) and streptomycin (100 mg/ml). Generation of FLAG-tagged MLL5 deletion mutants and GFP-tagged MLL5 were described before (Deng et al, 2004;Liu et al, 2010b). The HA-tagged p53 deletion mutants were cloned into pXJ40 in frame with BamHI and NotI sites.…”

“…Phosphorylation of MLL5 by Cdc2 is required for mitotic entry, at least partly explained the G 2 /M phase arrest caused by knockdown of MLL5 (Liu et al, 2010b); however, the mechanism underlying the G 1 /S arrest remains obscure. The G 1 /S arrest is a typical response to the activation of G 1 /S checkpoint, which serves as an important defense mechanism against genomic instability, before the full commitment of cell division (Elledge, 1996;Bartek and Lukas, 2001).…”

Effect of Prostaglandin I2 Analogs on Cytokine Expression in Human Myeloid Dendritic Cells via Epigenetic Regulation

“…Interestingly, phosphorylation signaling has been shown to play an essential role in regulating the function and interaction between proteins, maintaining the stability and participating in the localization of the protein. Recently, it was suggested that the phosphorylation modification on Thr-912 residue of MLL protein controls its subcellular localization and is required for mitotic entry (41). In the present study, we showed that iloprost suppressed poly I:C-induced phosphorylation of MAPK-p38, and by using the MAPK-p38 inhibitor SB203580, we also showed that the poly I:C-induced translocation of MLL and WDR5 proteins from cytoplasm to nucleus was MAPKp38 dependent.…”