Phosphorylation of Nuclear MyoD Is Required for Its Rapid Degradation

ABSTRACT MyoD is a basic helix-loop-helix transcription factor involved in the activation of genes encoding skeletal muscle-specific proteins. Independent of its ability to transactivate muscle-specific genes, MyoD can also act as a cell cycle inhibitor. MyoD activity is regulated by transcriptional and posttranscriptional mechanisms. While MyoD can be found phosphorylated, the functional significance of this posttranslation modification has not been established. MyoD contains several consensus cyclin-dependent kinase (CDK) phosphorylation sites. In these studies, we examined whether a link could be established between MyoD activity and phosphorylation at putative CDK sites. Site-directed mutagenesis of potential CDK phosphorylation sites in MyoD revealed that S200 is required for MyoD hyperphosphorylation as well as the normally short half-life of the MyoD protein. Additionally, we determined that turnover of the MyoD protein requires the proteasome and Cdc34 ubiquitin-conjugating enzyme activity. Results of these studies demonstrate that hyperphosphorylated MyoD is targeted for rapid degradation by the ubiquitin pathway. The targeted degradation of MyoD following CDK phosphorylation identifies a mechanism through which MyoD activity can be regulated coordinately with the cell cycle machinery (CDK2 and CDK4) and/or coordinately with the cellular transcriptional machinery (CDK7, CDK8, and CDK9).

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p34Cdc28-mediated control of Cln3 cyclin degradation.

Molecular and Cellular Biology ◽

10.1128/mcb.15.2.731 ◽

1995 ◽

Vol 15 (2) ◽

pp. 731-741 ◽

Cited By ~ 193

Author(s):

J Yaglom ◽

M H Linskens ◽

S Sadis ◽

D M Rubin ◽

B Futcher ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

G1 Phase ◽

Phosphorylation Sites ◽

Nonpermissive Temperature ◽

Yeast Saccharomyces Cerevisiae ◽

Rapid Degradation ◽

Ubiquitin Conjugating Enzyme ◽

A Cell ◽

Cdc28 Kinase ◽

Beta Galactosidase

Cln3 cyclin of the budding yeast Saccharomyces cerevisiae is a key regulator of Start, a cell cycle event in G1 phase at which cells become committed to division. The time of Start is sensitive to Cln3 levels, which in turn depend on the balance between synthesis and rapid degradation. Here we report that the breakdown of Cln3 is ubiquitin dependent and involves the ubiquitin-conjugating enzyme Cdc34 (Ubc3). The C-terminal tail of Cln3 functions as a transferable signal for degradation. Sequences important for Cln3 degradation are spread throughout the tail and consist largely of PEST elements, which have been previously suggested to target certain proteins for rapid turnover. The Cln3 tail also appears to contain multiple phosphorylation sites, and both phosphorylation and degradation of Cln3 are deficient in a cdc28ts mutant at the nonpermissive temperature. A point mutation at Ser-468, which lies within a Cdc28 kinase consensus site, causes approximately fivefold stabilization of a Cln3-beta-galactosidase fusion protein that contains a portion of the Cln3 tail and strongly reduces the phosphorylation of this protein. These data indicate that the degradation of Cln3 involves CDC28-dependent phosphorylation events.

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Whi5 and Stb1 define redundant pathways through which the G1 cyclin Cln3 promotes cell division

10.1101/2021.11.04.467365 ◽

2021 ◽

Author(s):

Sangeet Honey ◽

Bruce Futcher

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Transcription Factors ◽

Cell Division ◽

Transcriptional Repression ◽

Phosphorylation Site ◽

Phosphorylation Sites ◽

G1 Cyclin ◽

Cdk Phosphorylation ◽

Commitment To Cell Division

In the budding yeast S. cerevisiae, commitment to cell division, Start, is promoted by a trio of G1 cyclins, Cln1, Cln2, and Cln3, that activate the CDK kinase Cdc28. The active kinases somehow activate two transcription factors, SBF and MBF, leading to induction of about 100 genes for budding, DNA synthesis, and other early cell cycle processes. Activation of the transcription factors is opposed by a repressive protein called Whi5, and also by a second repressive protein called Stb1. Both Whi5 and Stb1 contain many potential sites for phosphorylation by CDK kinase, and is thought that relief of transcriptional repression involves the phosphorylation of Whi5 and Stb1 by CDK. Phosphorylation site mutants have been studied for Whi5, but not for Stb1. Here, we create phosphorylation site mutants of Stb1, and combine them with site mutants of Whi5. We find that the G1 cyclin Cln3 activates cell cycle transcription effectively when at least one of these proteins has its phosphorylation sites. However, when both Whi5 and Stb1 simultaneously lack all consensus phosphorylation sites, Cln3 is unable, or almost unable, to induce any gene expression, or any advancement of Start. Thus the G1 cyclin signaling pathway to Start has a requirement for CDK phosphorylation sites on either Whi5 or Stb1.

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Differential induction of ‘metabolic genes’ after mitogen stimulation and during normal cell cycle progression

Journal of Cell Science ◽

10.1242/jcs.107.1.241 ◽

1994 ◽

Vol 107 (1) ◽

pp. 241-252 ◽

Cited By ~ 1

Author(s):

C. Burger ◽

M. Wick ◽

S. Brusselbach ◽

R. Muller

Keyword(s):

Cell Cycle ◽

Normal Cell ◽

Cell Cycle Progression ◽

S Phase ◽

Cycle Progression ◽

Genes Encoding ◽

A Cell ◽

Induced Genes ◽

Cycle Dependent ◽

Normal Cell Cycle

Mitogenic stimulation of quiescent cells not only triggers the cell division cycle but also induces an increase in cell volume, associated with an activation of cellular metabolism. It is therefore likely that genes encoding enzymes and other proteins involved in energy metabolism and biosynthetic pathways represent a major class of mitogen-induced genes. In the present study, we investigated in the non-established human fibroblast line WI-38 the induction by mitogens of 17 genes whose products play a role in different metabolic processes. We show that these genes fall into 4 different categories, i.e. non-induced genes, immediate early (IE) primary genes, delayed early (DE) secondary genes and late genes reaching peak levels in S-phase. In addition, we have analysed the regulation of these genes during normal cell cycle progression, using HL-60 cells separated by counterflow elutriation. A clear cell cycle regulation was seen with those genes that are induced in S-phase, i.e. thymidine kinase, thymidylate synthase and dihydrofolate reductase. In addition, two DE genes showed a cell cycle dependent expression. Ornithine decarboxylase mRNA increased around mid-G1, reaching maximum levels in S/G2, while hexokinase mRNA expression was highest in early G1. In contrast, the expression of other DE and IE genes did not fluctuate during the cell cycle, a result that was confirmed with elutriated WI-38 and serum-stimulated HL-60 cells. These observations suggest that G0-->S and G1-->S transition are distinct processes, exhibiting characteristic programmes of gene regulation, and merging around S-phase entry.

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

Blood ◽

10.1182/blood.v106.11.1360.1360 ◽

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.

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Differential Regulation of Basal and Cyclic Adenosine 3′,5′-Monophosphate-Induced Somatostatin Gene Transcription in Neural Cells by DNA Control Elements That Bind Homeodomain Proteins

Molecular Endocrinology ◽

10.1210/mend.12.9.0171 ◽

1998 ◽

Vol 12 (9) ◽

pp. 1280-1293

Author(s):

Petra T. Schwartz ◽

Mario Vallejo

Keyword(s):

Protein Complexes ◽

Regulatory Elements ◽

Site Directed Mutagenesis ◽

Neural Cells ◽

Homeodomain Protein ◽

Pancreatic Cells ◽

Endocrine Organs ◽

Genes Encoding ◽

A Cell ◽

Rat Forebrain

Abstract A number of genes encoding neuropeptides are expressed in the peripheral and central nervous systems, in different endocrine organs, and in specialized cells distributed along the gastrointestinal tract. Whether expression of the same neuropeptide gene in different tissues is regulated by similar transcriptional mechanisms or by mechanisms that differ in a cell-specific manner remains unclear. We report on promoter studies on the regulation of the somatostatin gene in immortalized neural precursor cells derived from developing rat forebrain. Expression of the somatostatin gene in these cells was determined by RT-PCR/Southern blot analysis, by immunocytochemistry, and by RIA. We show that in cerebrocortical and hippocampal cells, expression of the somatostatin gene is regulated by several negative and positive DNA cis-regulatory elements located throughout the promoter region. The somatostatin cAMP-response element appears to play a prominent role in neural somatostatin gene expression by acting as a strong enhancer even in the absence of cAMP stimulation. Site-directed mutagenesis followed by transient transfection assays indicated that SMS-TAAT1, SMS-TAAT2, and SMS-UE, three previously identified homeodomain protein-binding regulatory elements that enhance transcription in pancreatic cells, act as repressors of transcription in neural cells. Electrophoretic mobility shifts assays indicate that those elements bind protein complexes that differ between neural and pancreatic cells. Our results support the notion that expression of the somatostatin gene in neural cells occurs via transcriptional mechanisms that are different from those regulating expression of the same gene in pancreatic cells.

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Conserved Promoter Motif Is Required for Cell Cycle Timing of dnaX Transcription inCaulobacter

Journal of Bacteriology ◽

10.1128/jb.183.16.4860-4865.2001 ◽

2001 ◽

Vol 183 (16) ◽

pp. 4860-4865 ◽

Cited By ~ 10

Author(s):

Kenneth C. Keiler ◽

Lucy Shapiro

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Caulobacter Crescentus ◽

Transcriptional Networks ◽

Cellular Processes ◽

Genes Encoding ◽

Constitutive Gene Expression ◽

A Cell ◽

Replication Factors

ABSTRACT Cells use highly regulated transcriptional networks to control temporally regulated events. In the bacterium Caulobacter crescentus, many cellular processes are temporally regulated with respect to the cell cycle, and the genes required for these processes are expressed immediately before the products are needed. Genes encoding factors required for DNA replication, includingdnaX, dnaA, dnaN,gyrB, and dnaK, are induced at the G1/S-phase transition. By analyzing mutations in thednaX promoter, we identified a motif between the −10 and −35 regions that is required for proper timing of gene expression. This motif, named RRF (for repression of replication factors), is conserved in the promoters of other coordinately induced replication factors. Because mutations in the RRF motif result in constitutive gene expression throughout the cell cycle, this sequence is likely to be the binding site for a cell cycle-regulated transcriptional repressor. Consistent with this hypothesis, Caulobacter extracts contain an activity that binds specifically to the RRF in vitro.

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Acetylation of Mammalian ADA3 Is Required for Its Functional Roles in Histone Acetylation and Cell Proliferation

Molecular and Cellular Biology ◽

10.1128/mcb.00342-16 ◽

2016 ◽

Vol 36 (19) ◽

pp. 2487-2502 ◽

Cited By ~ 4

Author(s):

Shakur Mohibi ◽

Shashank Srivastava ◽

Aditya Bele ◽

Sameer Mirza ◽

Hamid Band ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Histone Acetylation ◽

Cell Cycle Progression ◽

Site Directed Mutagenesis ◽

Dependent Manner ◽

Cycle Progression ◽

Functional Roles ◽

A Cell ◽

Cycle Dependent

Alteration/deficiency in activation 3 (ADA3) is an essential component of specific histone acetyltransferase (HAT) complexes. We have previously shown that ADA3 is required for establishing global histone acetylation patterns and for normal cell cycle progression (S. Mohibi et al., J Biol Chem 287:29442–29456, 2012,http://dx.doi.org/10.1074/jbc.M112.378901). Here, we report that these functional roles of ADA3 require its acetylation. We show that ADA3 acetylation, which is dynamically regulated in a cell cycle-dependent manner, reflects a balance of coordinated actions of its associated HATs, GCN5, PCAF, and p300, and a new partner that we define, the deacetylase SIRT1. We use mass spectrometry and site-directed mutagenesis to identify major sites of ADA3 acetylated by GCN5 and p300. Acetylation-defective mutants are capable of interacting with HATs and other components of HAT complexes but are deficient in their ability to restore ADA3-dependent global or locus-specific histone acetylation marks and cell proliferation inAda3-deleted murine embryonic fibroblasts (MEFs). Given the key importance of ADA3-containing HAT complexes in the regulation of various biological processes, including the cell cycle, our study presents a novel mechanism to regulate the function of these complexes through dynamic ADA3 acetylation.

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Acm1 Is a Negative Regulator of the Cdh1-Dependent Anaphase-Promoting Complex/Cyclosome in Budding Yeast

Molecular and Cellular Biology ◽

10.1128/mcb.00603-06 ◽

2006 ◽

Vol 26 (24) ◽

pp. 9162-9176 ◽

Cited By ~ 46

Author(s):

Juan S. Martinez ◽

Dah-Eun Jeong ◽

Eunyoung Choi ◽

Brian M. Billings ◽

Mark C. Hall

Keyword(s):

Cell Cycle ◽

Budding Yeast ◽

Mitotic Exit ◽

Negative Regulator ◽

Phosphorylation Sites ◽

Anaphase Promoting Complex ◽

Uncharacterized Protein ◽

Delay Effects ◽

A Cell ◽

Lines Of Evidence

ABSTRACT Cdh1 is a coactivator of the anaphase-promoting complex/cyclosome (APC/C) and contributes to mitotic exit and G1 maintenance by facilitating the polyubiquitination and subsequent proteolysis of specific substrates. Here, we report that budding yeast Cdh1 is a component of a cell cycle-regulated complex that includes the 14-3-3 homologs Bmh1 and Bmh2 and a previously uncharacterized protein, which we name Acm1 (APC/C Cdh1 modulator 1). Association of Cdh1 with Bmh1 and Bmh2 requires Acm1, and the Acm1 protein is cell cycle regulated, appearing late in G1 and disappearing in late M. In acm1Δ strains, Cdh1 localization to the bud neck and association with two substrates, Clb2 and Hsl1, were strongly enhanced. Several lines of evidence suggest that Acm1 can suppress APC/CCdh1-mediated proteolysis of mitotic cyclins. First, overexpression of Acm1 fully restored viability to cells expressing toxic levels of Cdh1 or a constitutively active Cdh1 mutant lacking inhibitory phosphorylation sites. Second, overexpression of Acm1 was toxic in sic1Δ cells. Third, ACM1 deletion exacerbated a low-penetrance elongated-bud phenotype caused by modest overexpression of Cdh1. This bud elongation was independent of the morphogenesis checkpoint, and the combination of acm1Δ and hsl1Δ resulted in a dramatic enhancement of bud elongation and G2/M delay. Effects on bud elongation were attenuated when Cdh1 was replaced with a mutant lacking the C-terminal IR dipeptide, suggesting that APC/C-dependent proteolysis is required for this phenotype. We propose that Acm1 and Bmh1/Bmh2 constitute a specialized inhibitor of APC/CCdh1.

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Dual control by Cdk1 phosphorylation of the budding yeast APC/C ubiquitin ligase activator Cdh1

Molecular Biology of the Cell ◽

10.1091/mbc.e15-11-0787 ◽

2016 ◽

Vol 27 (14) ◽

pp. 2198-2212 ◽

Cited By ~ 15

Author(s):

Sebastian Höckner ◽

Lea Neumann-Arnold ◽

Wolfgang Seufert

Keyword(s):

Cell Cycle ◽

Ubiquitin Ligase ◽

Budding Yeast ◽

Cell Cycle Control ◽

Eukaryotic Cell ◽

Negative Control ◽

Nuclear Localization Sequence ◽

Phosphorylation Sites ◽

Localization Sequence ◽

Cdk Phosphorylation

The antagonism between cyclin-dependent kinases (Cdks) and the ubiquitin ligase APC/C-Cdh1 is central to eukaryotic cell cycle control. APC/C-Cdh1 targets cyclin B and other regulatory proteins for degradation, whereas Cdks disable APC/C-Cdh1 through phosphorylation of the Cdh1 activator protein at multiple sites. Budding yeast Cdh1 carries nine Cdk phosphorylation sites in its N-terminal regulatory domain, most or all of which contribute to inhibition. However, the precise role of individual sites has remained unclear. Here, we report that the Cdk phosphorylation sites of yeast Cdh1 are organized into autonomous subgroups and act through separate mechanisms. Cdk sites 1–3 had no direct effect on the APC/C binding of Cdh1 but inactivated a bipartite nuclear localization sequence (NLS) and thereby controlled the partitioning of Cdh1 between cytoplasm and nucleus. In contrast, Cdk sites 4–9 did not influence the cell cycle–regulated localization of Cdh1 but prevented its binding to the APC/C. Cdk sites 4–9 reside near two recently identified APC/C interaction motifs in a pattern conserved with the human Cdh1 orthologue. Thus a Cdk-inhibited NLS goes along with Cdk-inhibited APC/C binding sites in yeast Cdh1 to relay the negative control by Cdk1 phosphorylation of the ubiquitin ligase APC/C-Cdh1.

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Cumulative Effect of Phosphorylation of pRB on Regulation of E2F Activity

Molecular and Cellular Biology ◽

10.1128/mcb.19.5.3246 ◽

1999 ◽

Vol 19 (5) ◽

pp. 3246-3256 ◽

Cited By ~ 62

Author(s):

Vivette D. Brown ◽

Robert A. Phillips ◽

Brenda L. Gallie

Keyword(s):

Transcriptional Repression ◽

Susceptibility Gene ◽

Cumulative Effect ◽

Site Directed Mutagenesis ◽

Phosphorylation Sites ◽

Cyclin Dependent Kinases ◽

Transactivation Activity ◽

Nuclear Phosphoprotein ◽

Cdk Phosphorylation ◽

Phosphate Groups

ABSTRACT The product of the retinoblastoma susceptibility gene, pRB, is a nuclear phosphoprotein that controls cell growth by binding to and suppressing the activities of transcription factors such as the E2F family. Transactivation activity is inhibited when E2F is bound to hypophosphorylated pRB and released when pRB is phosphorylated by cyclin-dependent kinases (CDKs). To determine which of 16 potential CDK phosphorylation sites regulated the pRB-E2F interaction, mutant pRB proteins produced by site-directed mutagenesis were tested for the ability to suppress E2F-mediated transcription in a reporter chloramphenicol acetyltransferase assay. Surprisingly, no one CDK site regulated the interaction of pRB with E2F when E2F was bound to DNA. Instead, disruption of transcriptional repression resulted from accumulation of phosphate groups on the RB molecule.

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