Promyelocytic leukemia (PML) is a nuclear protein that functions as a regulator of transcription, cell proliferation, apoptosis and myeloid cell differentiation. PML is subjected to post-translational modifications such as sumoylation and phosphorylation. However, the physiological significance of these modifications, especially for myeloid cell differentiation, remains unclear. In this report, we found that four serine residues in the PML C-terminal region are highly phosphorylated in a myeloid cell line. Wild-type PML accelerated G-CSF-induced granulocytic differentiation, but a phosphorylation-deficient PML mutant failed. PML interacted with C/EBPɛ, a transcription factor essential for granulopoiesis, activated C/EBPɛ-mediated transcription in concert with p300 and accelerated C/EBPɛ-induced granulocytic differentiation. Phosphorylation of PML was required for stimulating C/EBPɛ-dependent transcription and accelerating C/EBPɛ-induced granulocytic differentiation. We also found that PML phosphorylation was required for stimulation of PU.1-dependent transcription and acceleration of PU.1-induced granulocytic differentiation. These results suggest that phosphorylation plays essential roles in the regulation of PML to accelerate granulocytic differentiation through multiple pathways.
Promyelocytic leukemia (PML) is a nuclear protein that plays a role in growth suppression, apoptosis, premature senescence and myeloid cell differentiation. PML concentrates in speckled subnuclear structures, termed PML nuclear bodies (NBs)/ND10/PODs, together with many other proteins, including Sp100, p53, pRb, Daxx and p300/CBP.1 These facts suggest that PML plays a role in transcriptional regulation. The PML gene is involved in the chromosomal translocation t(15;17) and fuses to the retinoic acid receptor α (RARα) gene in the majority of cases of acute promyelocytic leukemia (APL), which is characterized by disruption of NBs into abnormal microspeckle structures.2 In APL, the fusion gene product PML-RARα has been thought to block granulopoiesis by dominant-negative inhibition of both PML and RARα functions. PML is important for terminal differentiation of granulocytes, as shown by impaired granulopoiesis in PML-deficient mice.3 Although PML plays a role in granulopoiesis, at least in part, by its modulation of the retinoic acid pathway,3 it does not fully explain the role of PML in granulopoiesis, suggesting that other PML actions should be considered for myelopoiesis in the physiological condition.4
PML function is regulated by at least two distinct modifications, specifically, phosphorylation and sumoylation. Sumoylation is required for NB formation and enhancement of PML-dependent apoptosis.5 Phosphorylation of PML is induced by ATR or Chk1/2 after DNA damage and it regulates p53-dependent and -independent apoptosis.6, 7 extracellular signal-regulated kinases (ERK)-mediated phosphorylation of PML increases sumoylation and enhances apoptosis in response to arsenic trioxde.8 CK2-mediated phosphorylation leads to ubiquitin-dependent degradation of PML.9 Thus, these two modifications are important for regulating PML-dependent apoptosis and PML stability. We previously reported that PML sumoylation might have an impact on granulocytic differentiation,10 but the role of PML phosphorylation in regulating granulocytic differentiation has not yet been addressed.
Granulopoiesis is tightly controlled by lineage-specific transcription factors. CCAAT/enhancer-binding protein ɛ (C/EBPɛ) is expressed exclusively in granuloid cells and is essential for terminal differentiation of committed granulocyte progenitors.10 Although C/EBPɛ can activate or repress target genes depending on its associated protein,11 the essential partner in terminal granulocytic differentiation remains to be explored. PU.1 is also expressed exclusively in hematopoietic cells, and it is indispensable for the terminal differentiation of myeloid cells.12 Recently, we reported that PML promotes the association of PU.1 with p300 to form the active transcriptional complex,13 but the regulatory mechanism of their interaction remains to be elucidated.
L-G is an interleukin-3 (IL-3)-dependent myeloid cell line that can be differentiated into mature granulocytes in response to granulocyte-colony stimulating factor (G-CSF).14 We found that PML is highly phosphorylated in L-G cells and the phosphorylation of PML is essential for accelerating G-CSF-induced granulocytic differentiation. We also found that PML associates with C/EBPɛ. PML activated C/EBPɛ-mediated transcription in cooperation with p300 and accelerated C/EBPɛ-induced granulocytic differentiation in a phosphorylation-dependent manner. These effects of phosphorylation on the PML-dependent regulation of granulopoiesis and transcription were also observed in the case of PU.1 regulation. Taken together, these findings suggest an essential role of PML phosphorylation in transcriptional regulation during the terminal differentiation of granulocytes.
Materials and methods
The expression vectors for PML isoform IV, pLPCX-HA-PML and pLPCX-FLAG-PML, pMT-PU.1 and pLNCX–PU.1 were described previously.10, 13 C/EBPɛ cDNA encoding a 32-kDa protein was generated as described previously15 and subcloned into pHM6, pLNCX and pMT vectors. Phosphorylation-deficit PML-4A or phosphorylation-mimic PML-4D mutants were generated by site-specific mutagenesis with overlapping extension PCR. Four serine residues at codons 505, 518, 527 and 530 were substituted to alanines or aspartic acids (IndexTermTCC508SerGCC508Ala, -GAC505Asp; IndexTermTCA518Ser-GCA518Ala, -IndexTermGAC518Asp; IndexTermAGC527Ser-GCC527Ala, -IndexTermGAC527Asp; IndexTermAGC530Ser-GCC503Ala, -IndexTermGAC530Asp), respectively. The construction of sumoylation-deficient mutant PML-3R has been previously described.10 A PML-dSP mutant lacking the serine- and proline-rich (SP) region (aa 502–554) was generated by appropriate restriction enzymes and PCR. All constructs were verified by DNA sequencing.
Construction of stable clones and retrovirus
First, 1 × 107 L-G cells were electroporated with pMT-C/EBPɛ or pMT–PU.1 plasmid, and stable clones were selected with 1 μg/ml of G418. Expression of C/EBPɛ or PU.1 was induced by adding 100 μM ZnSO4 to the medium containing IL-3. Wild-type PML or its mutants were transduced by retrovirus infection as described previously,10 and stable infectants were selected by 1 μg/ml of puromycin.
Identification of phosphorylation sites in the PML protein
FLAG–PML proteins purified from L-G cells were subjected to liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) analysis as previously described.10 Phosphopeptides were identified using TurboSEQUEST software.
Immunoprecipitation and western blotting
Immunoprecipitation and western blotting analysis were performed as previously described.10
Primary antibodies used in this study were as follows: anti-FLAG (M2, Sigma, St Louis, MO, USA), anti-HA (3F10, Roche, Mannheim, Germany), anti-human C/EBPɛ (C-22, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-human PML (1B9, MBL, Nagoya, Japan; H238, Santa Cruz Biotechnology), anti-human p300 (NM11, BD Bioscience, San Jose, CA, USA), anti-human PU.1 (T-21, Santa Cruz Biotechnology) and anti-mouse TFIIB (C-18, Santa Cruz Biotechnology).
Cells, in vitro phosphatase treatment, immunofluorescence, luciferase reporter assay, quantitive reverse transcription PCR (qRT-PCR)
Technical details are available in Supplementary Information.
Identification of phosphorylation sites in PML protein
The primary structure of PML predicts putative phosphorylation sites within the N-terminal proline-rich (Pro) region and the C-terminal serine- and proline-rich (SP) region.16 We first investigated the post-translational modification of PML stably expressed in L-G cells (Figure 1a). Western blot analysis showed that PML migrates with variable electrophoretic mobility. Four distinct bands were observed after the treatment of PML proteins with alkaline phosphatase (CIAP), indicating that PML is modified by phosphorylation as well as sumoylation in L-G cells.
To determine phosphorylation sites, exogenously expressed PML was purified from L-G cells and analyzed by LC/MS/MS. Four serines at codons 505, 518, 527 and 530 in the SP region of PML were identified as phosphorylation sites (Figure 1b). A mutant in which these serines were substituted to alanines (PML-4A) migrated to a similar position to that of phosphatase-treated wild-type PML, indicating that the four serine residues were mainly phosphorylated in L-G cells (compare Figures 1a and c).
Phosphorylation and sumoylation of PML are essential for acceleration of G-CSF-induced granulocytic differentiation
To elucidate the significance of PML phosphorylation and sumoylation in granulocytic differentiation, we also constructed phosphorylation-mimic PML-4D mutant with substitutions of serines 505, 518, 527 and 530 by aspartic acids, sumoylation-deficient PML-3R mutant with substitutions of lysines 65, 160 and 490 by arginines, or PML-dSP mutant with a deletion of the SP region containing the phosphorylation sites (Figure 1b). Then, we introduced these mutants as well as wild-type and PML-4A into L-G cells by retrovirus infection and tested their effects on the differentiation of L-G cells. Equivalent levels of wild-type and mutant PML proteins were expressed in L-G cells (Figure 1c). In the presence of IL-3, all of these infectants remained in immature myeloblasts (Figure 2a). After treatment with G-CSF for 5 days, an increased population of mature granulocytes was observed in PML-WT and PML-4D infectants when compared with vector-transduced cells (Figures 2a and b). However, the majority of PML-4A, -dSP and -3R infectants still remained at the myelocyte or metamyelocyte stage and only a small population of mature granulocytes was observed. To objectively evaluate the effects of PML mutants on cell differentiation, we used qRT-PCR to quantify the expression of neutrophil gelatinase (NG), a gene encoding a secondary granule protein which is upregulated in mature granulocytes (Figure 2c). Compared to vector-transduced cells, PML-WT and -4D, but not PML-4A, -dSP and -3R, enhanced the increase in expression of NG after treatment with G-CSF. These results indicate that, in addition to sumoylation, phosphorylation in the SP region is essential for PML to accelerate G-CSF-induced granulocytic differentiation.
PML associates with C/EBPɛ
Since PML is a transcriptional coregulator, the above results suggest that phosphorylation and sumoylation may be crucial for its regulatory action on some transcription factors involved in granulocytic differentiation. It has been demonstrated that C/EBPɛ functions during the G-CSF-induced granulocytic differentiation.17 To examine the interaction between PML and C/EBPɛ, co-immunoprecipitation assays were performed. FLAG-PML and HA-C/EBPɛ were transiently coexpressed in Bosc23 cells, and immunoprecipitants with anti-FLAG antibody were analyzed by western blot with anti-HA antibody, showing co-precipitation of C/EBPɛ with PML (Figure 3a). Reciprocally, HA-PML was also co-precipitated with FLAG-C/EBPɛ. In HL60 cells, endogenous PML and p300 were co-precipitated with C/EBPɛ whose expression was immediately increased after differentiation induced by all-trans retinoic acid (ATRA) treatment (Figure 3b). Notably, the amount of p300 that co-precipitated with C/EBPɛ was significantly increased within 2 days, demonstrating an accumulation of p300 in the C/EBPɛ/PML complex. To further confirm the association of C/EBPɛ and PML, HA–C/EBPɛ and PML were coexpressed in NIH3T3 cells, and double immunofluorescent staining was performed using anti-HA or anti-PML antibodies (Figure 3c). Without co-transfection of PML, C/EBPɛ dispersed throughout nuclei. When PML was coexpressed, C/EBPɛ accumulated in small dot-like structures, which coincided with NBs. Taken together, these results indicate that PML interacts with C/EBPɛ.
Essential role of PML phosphorylation for regulating C/EBPɛ activity
We generated an L-G/pMT-C/EBPɛ cell line, in which C/EBPɛ expression could be induced by exposure to ZnSO4. The L-G/pMT-C/EBPɛ cells differentiated into mature granulocytes with segmented nuclei even in the presence of IL-3 within 6 days after exposure to ZnSO4 (data not shown). To examine the effects of PML and its modifications on the C/EBPɛ-induced granulocytic differentiation, the cells were further infected with retroviruses encoding PML constructs or control vector, and then C/EBPɛ expression was induced (Figure 4a). The induced C/EBPɛ expression suppressed cell proliferation, which was enhanced by coexpression of PML-WT (Figure 4b). Compared to vector-transduced cells, an increased population of mature granulocytes was observed 4 days after PML-WT infectants were treated with ZnSO4 (Figures 4c and d). Similarly, PML-4D inhibited cell proliferation and accelerated cell differentiation, but neither PML-4A nor -dSP did. Unexpectedly, PML-3R inhibited cell proliferation and accelerated cell differentiation as strongly as PML-WT. The increased expression of NG after ZnSO4 treatment was enhanced by PML-WT, -4D and -3R, but not by PML-4A and -dSP (Figure 4e). A similar result was observed for the expression of lactoferrin (LTF), a gene that encodes a protein that is present in the secondary granules and is directly activated by C/EBPɛ.18 These results indicate that PML accelerates C/EBPɛ-induced granulocytic differentiation and that phosphorylation, but not sumoylation, of PML is required for the effect.
We also examined whether the PML mutations affected the interaction and colocalization of PML with C/EBPɛ and p300 (supplementary figure). However, neither mutation affected these interactions and colocalizations. To test the effect of these modifications on C/EBPɛ-dependent transcription, we performed a luciferase reporter assay by co-transfecting plasmids for C/EBPɛ, p300 and wild-type or mutant PML together with a luciferase reporter containing the G-CSF receptor promoter (G-CSFR-luc), which contains a binding site for C/EBP family members (Figure 4f). While p300 alone modestly stimulated the transcriptional activity of C/EBPɛ, the coexpression of PML-WT further enhanced the C/EBPɛ-mediated transcription. PML-4D and -3R also stimulated transcription. However, PML-4A was less potent, and PML-dSP was completely silent on the C/EBPɛ/p300-mediated transcription. It is particularly noteworthy that these effects of PML-WT and PML mutants on the C/EBPɛ-mediated transcription were correlated with their abilities to accelerate C/EBPɛ-induced granulocytic differentiation, suggesting that the activation of C/EBPɛ transcription by the phosphorylated, but not the sumoylated, form of PML plays an important role in granulopoiesis.
Requirement of phosphorylation for PML-dependent regulation of PU.1
Recently, we demonstrated that the transcriptional activity of PU.1 is also positively regulated by interaction with PML.12 Therefore, we investigated the roles of PML modifications in PU.1-mediated transcription. A reporter assay showed that PML-WT, -4D and -3R activated PU.1-dependent transcription while PML-4A and -dSP did not (Figure 5a). To analyze the effects of PML modifications on PU.1-induced differentiation, we transduced PML constructs into L-G/pMT-PU.1 cells and then induced differentiation by ZnSO4 treatment to express PU.1 (Figure 5b). PML-WT, -4D and -3R suppressed proliferation and accelerated granulocytic differentiation, whereas PML-4A did not (Figures 5c–e). The expression of NG was further increased in PML-WT, -4D and -3R infectants, but not PML-4A infectants, after treatment with ZnSO4 (Figure 5f). These results indicate that PU.1-mediated transcription and granulocytic differentiation are also regulated by phosphorylated PML.
PML accelerates granulocytic differentiation
One role of PML in terminal myeloid differentiation has been demonstrated in PML-deficient mice, which experience impaired granulopoiesis.3, 13 In the present study, we found that PML accelerates G-CSF-induced granulocytic differentiation. A previous study17 and our results (data not shown) demonstrate that G-CSF stimulation induces the expression of C/EBPɛ followed by granulocytic differentiation. These findings prompted us to determine whether PML regulates C/EBPɛ transcriptional activity to accelerate granulocytic differentiation. The current data illustrate that PML interacts with C/EBPɛ to activate its transcriptional activity and accelerates the granulocytic differentiation induced by overexpression of C/EBPɛ. Previously, we found that PML also accelerates PU.1-induced granulocytic differentiation.13 Thus, PML appears to contribute to the regulation of granulopoiesis through interactions with C/EBPɛ and PU.1.
Phosphorylation of PML in myeloid cells
It has been suggested that the functions of PML are regulated at least in part by phosphorylation and sumoylation.5, 6, 7, 8, 9 However, the role of PML phosphorylation in myeloid cell differentiation has not previously been addressed. In the present study, we found that four serine residues within the SP region of PML are highly phosphorylated in L-G cells. PML also contains several other serine residues in the N- and C-terminal regions that have been reported to be phosphorylated by ERK or CK2.8, 9 However, we did not detect these modifications by LC/MS/MS. Furthermore, alanine mutations of the phosphorylation sites did not affect the electrophoretic mobility of PML in L-G cells (data not shown). Thus, the SP region of PML is the main target of phosphorylation in L-G myeloid cells. While the upstream kinase that phosphorylates PML during differentiation of L-G cells is unknown, kinases such as ERK and HIPK2, which phosphorylate serine residues within PxSP or SP sequences, interact with PML.8, 19, 20 Since the overexpression of these kinases increases the phosphorylation of PML,8, 19 it is possible that they are involved in the phosphorylation of PML during the differentiation of L-G cells.
Role of PML modifications in granulocytic differentiation
In C/EBPɛ-induced granulocytic differentiation, we showed that the phosphorylation of PML is required for the acceleration of cell differentiation and the further increase in the expression of secondary granule protein gene including LTF, the product of a C/EBPɛ target gene. Although the mechanism by which PML regulates transcription is not sufficiently understood, it has been shown that PML promotes the interaction between transcription factors and coregulators such as p300.10, 13 In the present study, we found that p300 accumulates in the C/EBPɛ/PML complex during granulocytic differentiation. Despite the phosphorylation-independent association and colocalization of PML with C/EBPɛ and p300, the phosphorylation of PML is required for the synergistic effect of PML and p300 on the activation of C/EBPɛ-dependent transcription. Therefore, the phosphorylation of PML contributes to the acceleration of granulocytic differentiation, at least in part, by enhancing the effect of p300 on C/EBPɛ-dependent transcription.
The role of PML sumoylation in granulopoiesis remains unclear. In the present study, sumoylation of PML was not required for the acceleration of C/EBPɛ- and PU.1-induced granulocytic differentiation; however, sumoylation was required for induction by G-CSF, which suggests that the sumoylation of PML may contribute to the regulation of factors other than C/EBPɛ and PU.1 to accelerate G-CSF-induced granulocytic differentiation. These results suggest that G-CSF signaling induces cell differentiation through multiple PML-regulated pathways.
We conclude that both phosphorylation and sumoylation are essential for the ability of PML to accelerate granulocytic differentiation. Elucidating the regulatory mechanism of these modifications may help the development of therapeutic agents that induce differentiation of leukemia cells.
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We thank Ms Yukiko Aikawa and Noriko Aikawa (National Cancer Center Research Institute) for technical assistance. This research was supported in part by a grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by a grant from the Leukemia Study Group of the Ministry of Health, Labour and Welfare of Japan.
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Tagata, Y., Yoshida, H., Nguyen, L. et al. Phosphorylation of PML is essential for activation of C/EBPɛ and PU.1 to accelerate granulocytic differentiation. Leukemia 22, 273–280 (2008). https://doi.org/10.1038/sj.leu.2405024
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