Phosphorylation of Runx1 by Cyclin-Dependent Kinases Regulates Its Interaction with HDAC1 and HDAC3.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1381-1381 ◽  
Author(s):  
Hong Guo ◽  
Alan D. Friedman
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Hematopoietic Cells ◽  
Cyclin D3 ◽  
Mrna Levels ◽  
Cycle Progression ◽  
Hematopoietic Stem ◽  
Protein Levels ◽  
Cdk Phosphorylation ◽  
Regulation Of Cell Cycle

Abstract Runx1/AML1 is a key regulator of hematopoiesis and leukemic transformation, as RUNX1(−/−) mice do not develop definitive hematopoietic stem cells, and sever alleukemic oncogenes, e.g. AML1-ETO, CBFβ-SMMHC, or TEL-AML1, inhibit Runx1activities. We have investigated regulation of cell cycle progression by Runx1. Runx1stimulates G1 to S cell cycle progression in hematopoietic cell lines and in transduced myeloid progenitors, and inhibition of Runx1 by CBFβ-SMMHC or AML1-ETO slows G1 progression. Runx1 induces cdk4 and cyclin D3 transcription, and exogenous cdk4, cyclin D2, or c-Myc overcomes inhibition of G1 progression by CBF oncoproteins. In addition to regulating cell cycle progression, Runx1 protein levels are themselves increased as hematopoietic cells progress from G1 to S to G2/M, though mRNA levels remain constant. Runx1 contains three consensus cdk sites, (S/T)PX(R/K), S48, S303, and S424, and using phospho-specific antisera we find that each of these is modified in hematopoietic cells. Mutation of these serines to aspartic acid, mimicking phosphorylation, increases trans-activation of a reporter containing four CBF sites or the TCRβ promoter, whereas mutation to alanine reduces trans-activation. p300 interacts similarly with Runx1(tripleA) and Runx1(tripleD). We have now evaluated interaction of HDACs1–8 with these variants and Runx1 and find that both HDAC1 and HDAC3 have reduced affinity for RUNX1(tripleD), as assessed by co-immunoprecipitation from transiently transfected 293T cells. Evaluation of single serine residue mutants (S48D, S303D, and S424D) demonstrates reduced affinity of HDAC1 or HDAC3 specifically for the Runx1(S424D) mutant, consistent with previous mapping of the Runx1:HDAC1 and Runx1:HDAC3 interactions to this region of Runx1. Thus, cdk phosphorylation of Runx1 S424 reduces affinity for HDAC1 and HDAC3, increasing Runx1 trans-activation potency. Regulation of Runx1 activity by cdks may control key developmental processes, including expansion of definitive HSC during development and regulation of the balance between adult HSC quiescence and proliferation.

RUNX1/AML1 Is Phosphorylated at Both Its N- and C-Terminus by cdk6/cyclin D3 or cdk1/cyclin B.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1360-1360
Author(s):  
Florence Bernardin Fried ◽  
Alan D. Friedman
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Cyclin B ◽  
Cyclin D3 ◽  
Phosphorylation Sites ◽  
Runt Domain ◽  
Cycle Progression ◽  
Hematopoietic Stem ◽  
Exogenous Dna ◽  
Cdk Phosphorylation

Abstract RUNX1/AML1 is a key transcriptional mediator of hematopoiesis and leukemogenesis. AML1 regulates myeloid and lymphoid differentiation via activation of lineage-specific genes such as those encoding myeloperoxidase or the T cell receptor δ and participates in apoptotic response pathways via its ability to tranactivate the p14/p19ARF gene. In addition, AML1 accelerates G1 to S cell cycle progression, via activation of the cyclin D3 and potentially the cdk4 genes. CBF oncoproteins such as AML1-ETO or CBFβ-SMMHC interfere with the activities of AML1 and block myeloid differentiation and slow cell cycle progression, and mutations such as loss of p16 which accelerate G1 prevent cell cycle inhibition and cooperate with CBF oncoproteins to induce acute leukemia in mice. In addition to regulation of the cell cycle by AML1, we have been interested in how AML1 activities vary and may be regulated during cell cycle progression. We recently reported that endogenous AML1 levels increase in hematopoietic cell lines as they progress from G1 to S and then diminish again at the end of mitosis (Bernardin-Fried et al J. Biol. Chem.279:15678, 2004). RNA levels did not vary, but exogenous AML1 mimicked the behaviour of the endogenous protein, suggesting regulation at the level of protein stability. Mutation of two Ras-dependent phosphorylation sites, S276 and S293, to alanine did not prevent cell cycle variation. We have therefore set out to evaluate whether AML1 stability might be regulated by cyclin-dependent kinase (cdk) phosphorylation. AML1 contains 480 amino acids and binds DNA via its N-terminal Runt domain. Both the cdk6/cyclin D3 and the cdk1/cyclin B complex, expressed from baculovirus vectors, phosphorylated GST-AML1(1-290) and GST-AML1(290–480). The Runt domain alone, in GST-AML1(86–217), was not phosphorylated. Interestingly, exogenous DNA-binding domain alone did not vary during the cell cycle. This is the first demonstration that a specific kinase phosphorylates AML1 in vitro. There are three (S/T)PX(K/R) cdk consensus sites in AML1, with serines at residues 48, 303, and 424. Mutation of S424 to alanine did not prevent phosphorylation of GST-AML1(290–480). Additional mutations of these and other serines or threonines adjacent to proline are being generated to further map the cdk phosphorylation sites and to enable in vivo experiments designed to evaluate the effects of these mutations on cell cycle-specific AML1 expression. We propose a model in which accumulated phosphorylation of AML1 during the S and G2/M cell cycle phases leads to ubiquitin-mediated AML1 destabilization at the end of mitosis. The increased stability of AML1 in the presence of proteosome inhibitors supports this model. Phosphorylation-mediated destabilization of AML1 may complement the recent finding that direct interaction of cyclin D3 with AML1 inhibits its activity as a transcriptional activator. Each of these mechanisms may help regulate the proliferation of hematopoietic stem/progenitor cells. Finally, perhaps loss of destabilizing C-terminal phosphorylation sites in the AML1-ETO oncoprotein increases its ability to dominantly repress AML1-target genes during myeloid leukemogenesis.

Homing efficiency, cell cycle kinetics, and survival of quiescent and cycling human CD34+ cells transplanted into conditioned NOD/SCID recipients

Blood ◽  
2002 ◽  
Vol 99 (5) ◽  
pp. 1585-1593 ◽  
Author(s):  
Anna Jetmore ◽  
P. Artur Plett ◽  
Xia Tong ◽  
Frances M. Wolber ◽  
Robert Breese ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Hematopoietic Cells ◽  
Cycle Progression ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Nonobese Diabetic ◽  
Cd34 Cells ◽  
G1 Cells ◽  
Active Proliferation

Differences in engraftment potential of hematopoietic stem cells (HSCs) in distinct phases of cell cycle may result from the inability of cycling cells to home to the bone marrow (BM) and may be influenced by the rate of entry of BM-homed HSCs into cell cycle. Alternatively, preferential apoptosis of cycling cells may contribute to their low engraftment potential. This study examined homing, cell cycle progression, and survival of human hematopoietic cells transplanted into nonobese diabetic severe combined immunodeficient (NOD/SCID) recipients. At 40 hours after transplantation (AT), only 1% of CD34+ cells, or their G0(G0CD34+) or G1(G1CD34+) subfractions, was detected in the BM of recipient mice, suggesting that homing of engrafting cells to the BM was not specific. BM of NOD/SCID mice receiving grafts containing approximately 50% CD34+ cells harbored similar numbers of CD34+ and CD34− cells, indicating that CD34+ cells did not preferentially traffic to the BM. Although more than 64% of human hematopoietic cells cycled in culture at 40 hours, more than 92% of cells recovered from NOD/SCID marrow were quiescent. Interestingly, more apoptotic human cells were detected at 40 hours AT in the BM of mice that received xenografts of expanded cells in S/G2+M than in recipients of G0/G1 cells (34.6% ± 5.9% and 17.1% ± 6.3%, respectively; P < .01). These results suggest that active proliferation inhibition in the BM of irradiated recipients maintains mitotic quiescence of transplanted HSCs early AT and may trigger apoptosis of cycling cells. These data also illustrate that trafficking of transplanted cells to the BM is not selective, but lodgment of BM-homed cells may be specific.

Cdk1-Dependent Phosphorylation ofNPM Overrides G2/M Checkpoint and Increases Leukemic Blasts in Mice

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1322-1322
Author(s):  
Wei Du ◽  
Yun Zhou ◽  
Suzette Pike ◽  
Qishen Pang
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Cell Cycle Control ◽  
Xenograft Model ◽  
Phosphorylation Sites ◽  
M Cell ◽  
Cycle Progression ◽  
Hematopoietic Stem ◽  
Cycle Arrest ◽  
Regulation Of Cell Cycle

Abstract An elevated level of nucleophosmin (NPM) is often found in actively proliferative cells including human tumors. To identify the regulatory role for NPM phosphorylation in proliferation and cell cycle control, a series of mutants targeting the consensus cyclin-dependent kinase (CKD) phosphorylation sites was created to mimic or abrogate either single-site or multi-site phosphorylation. Cells expressing the phosphomimetic NPM mutants showed enhanced proliferation and G2/M cell-cycle transition; whereas nonphosphorylatable mutants induced G2/M cell-cycle arrest. Simultaneous inactivation of two CKD phosphorylation sites at Ser10 and Ser70 (S10A/S70A, NPM-AA) induced phosphorylation of Cdk1 at Tyr15 (Cdc2Tyr15) and increased cytoplasmic accumulation of Cdc25C. Strikingly, stress-induced Cdk1Tyr15 and Cdc25C sequestration were completely suppressed by expression of a double phosphomimetic NPM mutant (S10E/S70E, NPM-EE). Further analysis revealed that phosphorylation of NPM at both Ser10 and Ser70 sites were required for proper interaction between Cdk1 and Cdc25C in mitotic cells. Moreover, the NPM-EE mutant directly bound to Cdc25C and prevented phosphorylation of Cdc25C at Ser216 during mitosis. Finally, NPM-EE overrided stress-induced G2/M arrest, increased peripheral-blood blasts and splenomegaly in a NOD/SCID xenograft model, and promoted leukemia development in Fanconi mouse hematopoietic stem/progenitor cells. Thus, these findings reveal a novel function of NPM on regulation of cell-cycle progression, in which Cdk1-dependent phosphorylation of NPM controls cell-cycle progression at G2/M transition through modulation of Cdc25C activity.

scholarly journals Regulation of Skp2 Expression and Activity and Its Role in Cancer Progression

2010 ◽  
Vol 10 ◽  
pp. 1001-1015 ◽  
Author(s):  
Chia-Hsin Chan ◽  
Szu-Wei Lee ◽  
Jing Wang ◽  
Hui-Kuan Lin
Keyword(s):  
Cell Cycle ◽  
Cancer Progression ◽  
Cell Cycle Progression ◽  
Cycle Progression ◽  
Protein Levels ◽  
Human Cancers ◽  
Attractive Therapeutic Target ◽  
Skp2 Protein ◽  
Regulation Of Cell Cycle ◽  
F Box Protein

The regulation of cell cycle entry is critical for cell proliferation and tumorigenesis. One of the key players regulating cell cycle progression is the F-box protein Skp2. Skp2 forms a SCF complex with Skp1, Cul-1, and Rbx1 to constitute E3 ligase through its F-box domain. Skp2 protein levels are regulated during the cell cycle, and recent studies reveal that Skp2 stability, subcellular localization, and activity are regulated by its phosphorylation. Overexpression of Skp2 is associated with a variety of human cancers, indicating that Skp2 may contribute to the development of human cancers. The notion is supported by various genetic mouse models that demonstrate an oncogenic activity of Skp2 and its requirement in cancer progression, suggesting that Skp2 may be a novel and attractive therapeutic target for cancers.

Cyclin-Dependent Kinase Phosphorylation of RUNX1/AML1 on Three Sites Increases Trans-Activation Potency and Stimulates Cell Proliferation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3351-3351
Author(s):  
Alan D. Friedman ◽  
Linsheng Zhang ◽  
Florence Bernardin Fried
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Aspartic Acid ◽  
Cell Cycle Progression ◽  
Hematopoietic Cell ◽  
Cyclin D3 ◽  
Cycle Progression ◽  
Inhibition Of Proliferation ◽  
Cdk Phosphorylation ◽  
Stimulation Of

RUNX1/AML1 regulates lineage-specific genes during hematopoiesis and also stimulates G1 cell cycle progression. CBFβ-SMMHC or AML1-ETO dominantly inhibit RUNX1 and slow G1 progression in hematopoietic cell lines or in murine or human marrow progenitors, cdk4, cyclin D2, or c-Myc overcome inhibition of proliferation by these CBF oncoproteins, exogenous RUNX1 stimulates G1 progression, and stimulation of G1 via deletion of p16INK4a or expression of E7 cooperates with CBFβ-SMMHC or TEL-AML1 to induce acute leukemia in mice. Induction of cdk4 or cyclin D3 transcription may underlie stimulation of G1 progression by RUNX1. Remarkably, the C. elegans ortholog of RUNX1, RNT-1, also stimulates G1 progression and couples stem cell proliferation with differentiation. Not only does RUNX1 regulate cell cycle progression, but in addition RUNX1 levels increase as hematopoietic cells progress from G1 to S and from S to G2/M. Within RUNX1, S48, S303, and S424 fit the cdk phosphorylation consensus, (S/T)PX(R/K). Phosphorylation of RUNX1 by cyclin dependent kinases on serine 303 was shown to mediate destabilization of RUNX1 in G2/M. We now find that S48 and S424 are also phosphorylated by cdk1 or cdk6. S48, S303, or S424 phosphopeptide antiserum that we developed specifically recognized kinased GST-RUNX1 and interacted with RUNX1 expressed in 293T cells or in the Ba/F3 hematopoietic cell line. S48 phosphorylation of RUNX1 paralleled total RUNX1 levels during cell cycle progression, S303 was more effectively phosphorylated in G2/M, and S424 in G1. Single, double, and triple mutation to alanine or to the partially phosphomimetic aspartic acid progressively diminished or increased trans-activation, such that the tripleA mutant activated a RUNX1 reporter 5-fold less potently than the tripleD mutant. Aspartic acid does not perfectly mimic serine phosphorylation, as illustrated by the much greater affinity of our antisera for wild-type RUNX1 versus RUNX1(tripleD), suggesting that the biologic effect of RUNX1 cdk phosphorylation is even more significant. The p300 co-activator retained interaction with the tripleA variant. The tripleD RUNX1 mutant rescued Ba/F3 cells from inhibition of proliferation by CBFβ-SMMHC more effectively than the tripleA mutant. Cdk phosphorylation of RUNX1 on three sites increases its ability to active transcription and to stimulate proliferation, potentially coupling entry of stem/progenitors into cycle with induction of genes required for hematopoietic lineage progression, such as those encoding myeloperoxidase, neutrophil elastase, the M-CSF receptor, and PU.1.

scholarly journals POD-1/TCF21Reduces SHP Expression, AffectingLRH-1Regulation and Cell Cycle Balance in Adrenocortical and Hepatocarcinoma Tumor Cells

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Monica Malheiros França ◽  
Bruno Ferraz-de-Souza ◽  
Antonio Marcondes Lerario ◽  
Maria Candida Barisson Villares Fragoso ◽  
Claudimara Ferini Pacicco Lotfi
Keyword(s):  
Cell Cycle ◽  
Tumor Cells ◽  
Cell Cycle Progression ◽  
Mrna Levels ◽  
Adrenocortical Tumor ◽  
Cycle Progression ◽  
Cyclin E1 ◽  
Protein Levels ◽  
E1 Protein ◽  
E Box

POD-1/TCF21may play a crucial role in adrenal and gonadal homeostasis and repressesSf-1/SF-1 expression in adrenocortical tumor cells. SF-1 and LRH-1 are members of the Fzt-F1 subfamily of nuclear receptors. LRH-1 is involved in several biological processes, and both LRH-1 and its repressor SHP are involved in many types of cancer. In order to assess whether POD-1 can regulate LRH-1 via the same mechanism that regulates SF-1, we analyzed the endogenous mRNA levels ofPOD-1,SHP, andLRH-1in hepatocarcinoma and adrenocortical tumor cells using qRT-PCR. Hereafter, these tumor cells were transiently transfected with pCMVMycPod-1, and the effect of POD-1 overexpression on E-box elements in theLRH-1andSHPpromoter region were analyzed by ChIP assay. Also, Cyclin E1 protein expression was analyzed to detect cell cycle progression. We found that POD-1 overexpression significantly decreasedSHP/SHP mRNA and protein levels through POD-1 binding to the E-box sequence in theSHPpromoter. DecreasedSHPexpression affectedLRH-1regulation and increased Cyclin E1. These findings show that POD-1/TCF21regulates SF-1 and LRH-1 by distinct mechanisms, contributing to the understanding of POD-1 involvement and its mechanisms of action in adrenal and liver tumorigenesis, which could lead to the discovery of relevant biomarkers.

PTTG has a Dual Role of Promotion-Inhibition in the Development of Pituitary Adenomas

2019 ◽  
Vol 26 (11) ◽  
pp. 800-818
Author(s):  
Zujian Xiong ◽  
Xuejun Li ◽  
Qi Yang
Keyword(s):  
Cell Cycle ◽  
Pituitary Adenoma ◽  
Pituitary Adenomas ◽  
Cell Cycle Progression ◽  
Key Factors ◽  
Cycle Progression ◽  
Sister Chromatids ◽  
Regulation Of Cell Cycle ◽  
Late Stages

Pituitary Tumor Transforming Gene (PTTG) of human is known as a checkpoint gene in the middle and late stages of mitosis, and is also a proto-oncogene that promotes cell cycle progression. In the nucleus, PTTG works as securin in controlling the mid-term segregation of sister chromatids. Overexpression of PTTG, entering the nucleus with the help of PBF in pituitary adenomas, participates in the regulation of cell cycle, interferes with DNA repair, induces genetic instability, transactivates FGF-2 and VEGF and promotes angiogenesis and tumor invasion. Simultaneously, overexpression of PTTG induces tumor cell senescence through the DNA damage pathway, making pituitary adenoma possessing the potential self-limiting ability. To elucidate the mechanism of PTTG in the regulation of pituitary adenomas, we focus on both the positive and negative function of PTTG and find out key factors interacted with PTTG in pituitary adenomas. Furthermore, we discuss other possible mechanisms correlate with PTTG in pituitary adenoma initiation and development and the potential value of PTTG in clinical treatment.

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