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Luke F. Peterson,1,
Youhong Zhang,2
Andrew S. Kraft,2 and
Dong-Er Zhang1*
Division of Oncovirology, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037,1 Hollings Cancer Center, Medical University of South Carolina, 86 Jonathan Lucas Street, Charleston, South Carolina 294252
Received 6 April 2006/ Returned for modification 10 May 2006/ Accepted 25 July 2006
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B (37). The AML1 protein is composed of a DNA binding runt homology domain located in the amino terminus followed by a transcriptional activation domain and a negative regulatory C-terminal domain (15, 20, 29). AML1 was initially identified during the study of breakpoint t(8;21), which is a common chromosomal translocation in acute myeloid leukemia (23). The association of AML1 with blood cell development is shown by the disruption of the AML1 gene through multiple chromosomal translocations, deletions, point mutations, or amplification in approximately 30% of human myeloid leukemias and myelodysplastic syndrome patients and a significant number of lymphoid leukemia patients (21, 24, 27, 32). Furthermore, no detectable definitive hematopoiesis is observed in Aml1 knockout mice (26, 40). The importance of AML1 in nonhematopoietic cells has also been recognized in angiogenesis, muscle function, and neurogenesis (6, 12, 14, 35, 41). AML1 is detected as a serine and threonine phosphorylated protein (9). Previous work has suggested that AML1 activity may be regulated by phosphorylation (34, 45). Phosphorylation at specific serine-proline or threonine-proline sites in AML1 appears to be necessary for normal activity (45). It has also been suggested that phosphorylation releases AML1 from an association with the nuclear matrix mediated by sin3A, in turn leading to both increased activity and an increased rate of degradation (11).
We have now shown that AML1 phosphorylation by cyclin-dependent kinases (CDKs) affects the overall stability of AML1 as well as the ability of certain ubiquitin ligase complexes, such as Cdc20-anaphase-promoting complex (APC), to target AML1 for degradation.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R1 endothelial lines and 293T lines expressing AML1 were established in the same manner. Nocodazole (Sigma-Aldrich, St. Louis, MO) was added to the 293T cells 8 to 16 h after transfection at a concentration of 1 µg/ml. Roscovitine, alsterpaullone, and Cdk2 inhibitor II (Calbiochem, San Diego, CA) were added to 293T cells at a final concentration of 50 µM 8 to 16 h after transfection. Cdk4 inhibitor (Calbiochem) was added to a final concentration of 5 µM. To label proteins with [32P]orthophosphate, 24 h after transfection cells were placed in phosphate-free Dulbecco's modified Eagle's medium (Cellgro, Herndon, VA) supplemented with 10% dialyzed fetal bovine serum and HEPES buffer, pH 7.5. A total of 100 µCi per ml of [32P]orthophosphate was added to the medium, and the cells were incubated for 4 to 6 h.
Plasmids.
Full-length AML1B was cloned into the HindIII site of pFLAG-CMV2 (Invitrogen, Carlsbad, CA) to generate FLAG-tagged AML1. To generate the glutathione S-transferase (GST)-AML1(267-315) mammalian expression construct, AML1B coding sequences from amino acids 267 to 315 were amplified by PCR and then cloned into the EcoRI and NotI sites of pGEX-4T-2 (Promega, Madison, WI). A BstBI-NotI fragment from this plasmid was then cloned into the pEBG vector, which contains the human elongation factor EF-1 promoter to drive expression (a gift from B. Mayer). BstBI cuts within the GST coding sequence. pcDNA3-AML1 (wild type and 4A) have been described previously (45). Similar expression vectors for AML1 containing two mutations (serines 276 and 303 or serine 293 and threonine 300) or single mutations were generated in the same manner as described previously (45). To construct GST-AML1(267-315) mammalian expression vectors with mutations, PCR was used to amplify amino acids 267 to 315 coding the sequence from full-length AML1 DNA carrying the appropriate mutations. These sequences were fused to GST and cloned into pEBG as described above. The pcDNA3-HA-Cdh1 (where HA is hemagglutinin) expression plasmid was received from Michele Pagano (New York University), the pCS2-HA-Cdc20 plasmid was received from Guowei Fang (Stanford University), and the pcDNAB-Myc-Skp2 plasmid was from Xiaohua Wu (The Scripps Research Institute).
Cell labeling, immunoprecipitation, Western blotting, and Northern blotting. Labeling of cells with [32P]orthophosphate and immunoprecipitation of FLAG-tagged AML1 were performed as described previously (45) and as above. GST-AML1(267-315) protein was purified from the lysate of 32P-labeled cells in a similar fashion, but glutathione-S agarose was used. Western blotting was performed as described previously (4). Phospho-AML1 antibodies were raised against AML1 peptides containing either phosphor-S276 or phosphor-S303 (Biosource) (39). Total RNA was prepared from cells using RNA-Bee (Tel-Test, Inc.) and the manufacturer's protocol. Northern blotting was performed as described previously (45).
In vitro kinase assays.
GST-AML1(267-315) constructs (wild type and mutants) were expressed in Escherichia coli from pGEX-4T-2 (Pfizer-Pharmacia, New York, NY) and bound to glutathione agarose. The GST-AML1 substrate was incubated in kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 2 mM dithiothreitol, 10 µM ATP) with [
-32P]ATP and purified active Cdk1/cyclin B (Calbiochem), Cdk2/cyclin A (Upstate Biotechnologies), Cdk6/cyclin D3 (Upstate Biotechnologies), or Cdk4/cyclin D1 (Biaffin GmbH and Co. KG, Kassel, Germany). The retinoblastoma fusion protein used as a control for Cdk4/cyclin D1 activity was purchased from Santa Cruz Biotechnology. After incubation for 10 min at 30°C, the in vitro kinase reaction mixtures were placed on ice and then washed three times with phosphate-buffered saline (PBS). The GST-AML1(267-315) on glutathione beads was then prepared for analysis on sodium dodecyl sulfate (SDS) gels by boiling in SDS sample buffer.
Coimmunoprecipitation. Lysates were prepared following transfection of 293T cells expressing either AML1, AML1-2xDBM (AML1 with a mutation in two D box sequences), AML1-4A, or AML1-4D with or without myc-Skp2, HA-Cdc20, or HA-Cdh1 in 400 µl of PBS-1 mM EDTA-0.5% Triton X-100; samples were sonicated twice for 6 s, and debris was removed by centrifugation at 17.000 x g for 30 min at 4°C. A total of 200 µg of lysate was immunoprecipitated overnight with either the anti-myc or anti-HA antibody with 15 µl of a 50% slurry of protein G-Sepharose in PBS. Following washes with the lysis buffer, the beads were boiled in 10 µl of 2x Laemmli buffer, resolved on an 8 to 10 to 12% gradient SDS-PAGE gel, electroblotted to nitrocellulose, and blocked in 4% milk-PBS-0.2% Tween 20. The blots were hybridized first with rabbit anti-AML1 and donkey anti-rabbit antibody-horseradish peroxidase, and detection was performed by chemiluminescence (NEN). Following stripping with 2% SDS-PBS-0.2% Tween 20 for 20 min at 50°C, the blots were washed for 30 min with various exchanges of wash buffer PBS-0.2% Tween 20 and blocked as above; the blots were then hybridized with either mouse anti-myc (clone 9E10) or anti-HA (Babco) and donkey anti-mouse antibody-horseradish peroxidase, and detection was performed as above. The input samples were 10 µg of total protein of each sample resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and manipulated as described above for the detection of AML1, myc-Skp2, HA-Cdc20, or HA-Cdh1.
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RESULTS |
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To determine whether CDKs phosphorylate AML1 in vivo, we first transfected 293T cells with AML1 and then treated the cells with the CDK inhibitor roscovitine. Roscovitine inhibits the activity of Cdk1, Cdk2, and Cdk5 but not ERK (1). As shown in Fig. 1A, roscovitine inhibits in vivo phosphorylation of full-length AML1.
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The data shown in Fig. 1 suggest that CDK inhibitors block phosphorylation of AML1 serines 276 and 303. To confirm this, Western blotting was performed using antibodies that specifically recognize AML1 phosphorylated on serine 276 or serine 303 (see Materials and Methods). The phosphospecific AML1 antibodies were used to examine phosphorylation of wild-type AML1 (FLAG-tagged). FLAG-AML1 was immunoprecipitated from transfected 293T cells; the immunoprecipitates were then used for Western blotting with anti-AML1 (P-303) or anti-AML1 (P-276). As shown in Fig. 2, treatment with CDK inhibitors blocks phosphorylation of both serine 279 and serine 303.
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In contrast to the first three CDK/cyclin complexes tested, Cdk4/cyclin D1 phosphorylation of wild-type GST-AML1(267-315) was not above the background phosphorylation observed for GST-AML1(267-315)-4A. This was not due to lack of Cdk4/cyclin D1 activity, because a control substrate (a fusion protein containing amino acids 769 to 921 of the retinoblastoma protein) was highly phosphorylated (Fig. 3B).
Although all four residues (serines 276, 293, and 303 and threonine 300) appear to be sites for phosphorylation by some CDK/cyclin complexes in vitro, different CDK/cyclin complexes exhibit subtle preferences for particular substrates. The presence of all four sites in the wild-type substrate often results in the highest level of phosphorylation, suggesting the possibility of cooperative interaction between sites. Our observations also suggest that serines 276 and 303 are much more highly phosphorylated in vivo. This may reflect the fact that in vitro phosphorylation conditions are more permissive than in vivo conditions. As discussed below, serine 293 and/or threonine 300 do appear to be weakly phosphorylated in vivo.
Phosphorylation of AML1 as cells progress through the cell cycle and cross talk between phosphorylation sites. As cells progress from the G1 phase of the cell cycle to S phase, Cdk2/cyclin E is replaced by Cdk2/cyclin A, while in G2 Cdk1/cyclin B and Cdk1/cyclin A are active (25). We examined phosphorylation of GST-AML1(267-315) as cells progressed through the cell cycle to determine whether specific CDK/cyclin complexes have greater AML1 phosphorylation activity.
To synchronize cells expressing GST-AML1(267-315), 293T cells were transfected with GST-AML1(267-315) and treated for 16 to 20 h with hydroxyurea (G1/S block) or with nocodazole (G2/M block). The cells were released from the block when the medium containing hydroxyurea or nocodazole was removed and replaced by fresh medium. Samples were collected 0, 4, and 8 h after removal of the blocks, and synchronization was confirmed by labeling with propidium iodide and by flow cytometry (Fig. 4A). To examine GST-AML1(267-315) phosphorylation, cells were labeled with [32P]orthophosphate for 4 h before the collection of time points. GST-AML1(267-315) was then isolated from the 32P-labeled cells and analyzed by Western blotting and autoradiography. As shown in Fig. 4B, there is a slight decrease in GST-AML1(267-315) phosphorylation in cells arrested with hydroxyurea and a slight increase when they reenter the cell cycle. This is consistent with the observation that hydroxyurea activates a cell cycle checkpoint which involves down-regulation of Cdk2 activity (7). The relatively small decrease in GST-AML1(267-315) phosphorylation in hydroxyurea-treated cells may be due to the fact that some Cdk2 activity remains (7) or to low levels of phosphatases which might dephosphorylate GST-AML1(267-315). Overall, the changes in GST-AML1(267-315) phosphorylation as cells progress from late G1 to G2/M are not drastic. This relatively steady level of phosphorylation over the cell cycle is similar to that observed for other transcription factors believed to be CDK/cyclin substrates (19, 31) and involved in the regulation of cell proliferation (19). It is thus possible that cells maintain a fairly even level of total CDK/cyclin activity during the cell cycle, even though individual CDK/cyclin complexes appear and disappear.
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Subsequent analysis (Fig. 4C) revealed that mutation of serine 293 and threonine 300 to alanine in GST-AML1(267-315) results in the loss of the nocodazole-specific phosphorylated species and results in increased phosphorylation at serines 276 and 303. Interestingly, when serines 276 and 303 are both mutated to alanine, this nocodazole-specific phosphorylation on serine 293/threonine 300 cannot occur (Fig. 4C, right). These results suggest a complex interaction between AML1 phosphorylation at serines 276, 293, and 303 and threonine 300, which is diagrammed in Fig. 4D.
The results described above indicate that mutation of serines 276 and 303 is equivalent to mutation of all four sites (serines 276, 293, and 303 and threonine 300) on AML1 phosphorylation. We have used AML1-4A, the full-length AML1 containing mutations in all four sites, for subsequent analysis.
Mutations in AML1 phosphorylation sites affect the stability of the protein and targeting by the APC.
To determine whether expression of AML1 with mutations in phosphorylation sites had different in vivo effects compared to wild-type AML1, AEL-R1 endothelial cells or NIH 3T3 cell lines stably expressing three types of AML1 were established. The AEL-R1 cell line was established by the immortalization of endothelial cells derived from the aorta-gonad-mesonephros region of a Runx1-null embryo (12). Wild-type AML1, AML1-4A, or AML1-4D were cloned into MSCV retroviral vectors, which were then used to produce virus and to infect cells. The AML1-4D protein is a phospho-mimic mutant, with serines 276, 293 and 303 and threonine 300 mutated to glutamic acid. Three pools of AEL-R1 and two to three pools of NIH 3T3 cells were infected with empty MSCV vector or vector expressing wild-type (WT) AML1, AML1-4A, or AML1-4D. After each infected pool was subjected to puromycin selection, the pools were tested for AML1 expression. Figure 5A and B show the expression of AML1 protein and mRNA in two pools each of empty vector AEL-R1 controls or cells expressing AML1-WT, AML1-4A, or AML1-4D. Figure 5A clearly shows that AML1-4A accumulates to a higher level than AML1-WT, while the AML1-4D protein does not. The Northern blot (Fig. 5B) shows that the level of AML1 mRNA expressed from the MSCV construct bears no relationship to the level of protein present in the various lines. Figure 5C and D show a similar pattern of expression in NIH 3T3 cell lines (AML1-4D pool not shown).
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To determine whether Chd1, Cdc20, or Skp2 could promote the degradation of AML1, we first constructed 293T cell lines stably expressing wild-type AML1, AML1-4A, or AML1-4D using the MSCV vectors described above. These 293T-AML1 cell lines were then transfected with either empty expression plasmid (control) or with plasmids expressing Cdh1, Cdc20, or Skp2. An example of such an experiment is shown in Fig. 7A, where the 293T-AML1 cell lines were transfected with empty vector, the Cdh1 expression vector, or the Cdc20 expression vector. The transfected cells were then used to prepare lysates for Western blot analysis with anti-AML1 antibodies to determine the level of AML1 present. Three independent transfection experiments were performed with Cdh1, Cdc20, and Skp2 expression vectors, and the level of AML1 present in each sample was measured by Western blotting followed by densitometry. The results of these experiments are summarized in the graphs shown in Fig. 7B. These experiments show that Cdh1 can promote the degradation of all three forms of AML1 to approximately the same extent. Cdc20, on the other hand, is unable to promote degradation of AML1-4A but is active against AML1-4D. This suggests that Cdc20 preferentially targets the phosphorylated form of AML1. The pattern displayed by Skp2 is similar to that seen with Cdc20, although Skp2 is less active in promoting the degradation of AML1-4D.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Not all ubiquitin ligase complexes that target AML1 recognize the phosphorylation state of AML1. The Cdh1-APC complex is able to promote the degradation of AML1-4A almost as efficiently as AML1-4D or wild-type AML1. This suggests that AML1 may contain more than one recognition element for ubiquitin ligase complexes and that AML1 phosphorylation could control access to some elements but not others. The Cdc20 and Cdh1 proteins both serve as substrate-targeting subunits for the APC (38, 42). Cdc20-APC mediates the degradation of proteins such as cyclin B1 and securin, allowing cells to exit mitosis. Cdh1-APC is then activated and is believed to maintain the G1 state by continuing the degradation of cyclin B1 and also proteins necessary for the initiation of S phase, such as Skp2. When S phase is initiated, Cdh1 is phosphorylated and inactivated, allowing the accumulation of Skp2 and the formation of the SCF-Skp2 complex (38). These facts are summarized in Fig. 10 and suggest that AML1 phosphorylation could be more important for the regulation of AML1 stability at specific points during the cell cycle, depending upon which ubiquitin ligase complexes are active. In addition to APC/C-Cdc20, APC/C-Cdh1, and SCF-Skp2, AML1 may be targeted for degradation by other as yet unidentified ubiquitin ligase complexes. If most ubiquitin ligase complexes which target AML1 require AML1 phosphorylation, this would explain why AML1-4A is more stable than wild-type AML1 and accumulates to higher levels. If APC/C-Cdh1 is exceptional in its ability to promote the degradation of both phosphorylated and nonphosphorylated forms of AML1, this fact might explain the observation that cellular AML1 levels are lowest during the G1 phase of the cell cycle, when APC/C-Cdh1 is active (3).
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Cdc20 recognizes a substrate amino acid motif known as the destruction box (reviewed in references 42 and 38), while Cdh1 can interact with a variety of motifs, including the destruction box (D box), the KEN box, the GEN box, and the A box (16, 38, 42). AML1 contains two potential D box motifs, beginning at amino acids 207 and 250 (AML1B/AML1c amino acid numbering). AML1 contains no matches for the other motifs known to assist in substrate recognition by Cdh1 and Cdc20. We have determined that mutation of both putative AML1 D box sequences does not greatly affect coprecipitation of Cdh1 with AML1. Mutation of the D boxes also fails to inhibit Cdh1-mediated degradation of AML1 (J. Biggs, unpublished data). In contrast, mutation of the D boxes greatly reduces the interaction between AML1 and Cdc20. This observation suggests that while Cdc20 recognizes AML1 through the D box motifs, Cdh1 utilizes some other motif to target AML1 for degradation.
The precise mechanism by which AML1 phosphorylation promotes degradation by APC/C-Cdc20 is not yet clear. Phosphorylation does not appear to be necessary for Cdc20 or Skp2 to coprecipitate with AML1, which may suggest that lack of phosphorylation does not necessarily prevent physical interaction between AML1 and Cdc20. Phosphorylation may promote proper association of the complete APC/C-Cdc20 complex with AML1 or some other step of the degradation process. Alternatively, previous studies have suggested that the nonphosphorylated form of AML1 interacts more strongly with the nuclear matrix (4, 11). It is possible that AML1 interaction with the matrix might block access of some (but not all) ubiquitin ligase complexes to AML1 or hinder placement of ubiquitin on AML1 lysine residues. This model might explain why all forms of AML1 (wild-type, 4A, and 4D) coimmunoprecipitate with Cdh1, Cdc20, and Skp2. Immunoprecipitation experiments must be performed with AML1 that has been detached from the nuclear matrix by the lysis process. Cdh1, Cdc20, and Skp2 may all bind equally to AML1 after detachment from the matrix but may not find matrix-associated AML1 equally accessible; this may especially be true for Cdc20 or Skp2. It is possible that matrix association hinders access by Cdc20 to the AML1 D box motif, but the unidentified motif recognized by Cdh1 is still accessible. Other proteins which bind to AML1 in this region, such as CBFß (10), sin3A (11), and cyclin D (28), might also regulate access to AML1 by Chd1 or Cdc20.
Expression of AML1 may either enhance cell growth (33) or suppress cell growth (44), depending on the cell type. AML1 suppresses mouse embryonic fibroblast proliferation in the presence of a functional p53-p19ARF pathway but promotes growth in the absence of p53 (44). AML1 also inhibits transcriptional elongation by binding to the elongation factor P-TEFb (13). Inhibition of elongation is usually associated with suppression of growth. Loss of AML1 function is associated with the development of acute myeloid leukemia, but amplification of AML1 is associated with B-cell acute lymphoblastic leukemia (reviewed in reference 21). Clearly, AML1 has both growth-enhancing and growth-inhibitory effects, and which effect is predominant depends on the cellular environment.
The level of AML1 in cells may also be critical for normal development. The Notch/AML1 pathway has been identified as a key component of the process of generating adult hematopoietic stem cells (5). These observations suggest that AML1 is required at a specific time during development for the appearance of adult hematopoietic stem cells. Recently, evidence has emerged that AML1 may also be required for proliferation of selected populations of neural progenitors (6, 14, 35), for the prevention of skeletal muscle atrophy (41), and for angiogenesis by epithelial cells from the aorta-gonad-mesonephros region (12).
As might be expected for a protein which can affect cell proliferation, AML1 expression can alter the way cells move through the cell cycle. AML1 has been shown to affect the transition through G1 (2, 33). It has also recently been shown that AML1 forms a complex with cyclin D (28). AML1 has been shown to activate the cyclin D promoter (17) and to repress the p21 (WAF1/Cip1) promoter (18, 35). It has been suggested that AML1 may shorten the G1 phase of the cell cycle by the up-regulation of proteins such as cyclin D (33). The fact that AML1 can alter cell cycle progression while the CDKs which are sequentially activated during the cell cycle phosphorylate AML1 and affect AML1 stability suggests that phosphorylation by CDKs may be a way to fine-tune AML1 activity over the course of the cell cycle, ensuring that a precise level of AML1 activity is present at the required time.
The RUNX2/AML3 protein has substantial amino acid sequence homology to RUNX1/AML1, but the reported phosphorylation patterns of the two proteins appears somewhat different (34, 43, 45). One RUNX2/AML3 phosphorylation site, serine 451, was recently shown to be a target of Cdk1/cyclin B (30). Mutation of this site resulted in loss of RUNX2/AML3 DNA binding and reduced stimulation of anchorage-independent growth in endothelial cells (30). Previous studies (4) have indicated that mutation of the RUNX1/AML1 phosphorylation sites in AML1-4A has little effect on DNA binding; this might suggest that CDK phosphorylation of different sites in the RUNX proteins can regulate different processes.
The overall effect of altering AML1 stability on AML1 target gene expression remains to be determined. AML1 may act as both a transcriptional activator and repressor (21) and may also inhibit transcriptional elongation by binding to cyclin T (13). Experiments using promoter-luciferase reporter constructs indicate that AML1-4A has lower transcriptional activation activity than wild-type AML1 on most, but not all, tested AML1 target promoters (45). The variable effect of AML1 phosphorylation on its transactivation may also dependent on its cooperation with other transcription factors, such as C/EBP, in different regulatory elements of gene expression. Further investigation is necessary to determine precisely which set of AML1 target genes is affected by mutations that alter AML1 stability and what effect this has on overall cell properties.
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ACKNOWLEDGMENTS |
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This work was supported by National Institutes of Health grants CA42533 (to A.S.K.) and CA72009 (to D.E.Z.). The Stein Endowment Fund has partially supported the MEM departmental molecular biology service laboratory for DNA sequencing and oligonucleotide synthesis.
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FOOTNOTES |
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This is paper number 17930-MEM from The Scripps Research Institute.
J.R.B. and L.F.P. contributed equally to this work.
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