p53 is phosphorylated by CDK7-cyclin H in a p36MAT1-dependent manner.

The tumor suppressor protein p53 acts as a transcriptional activator that can mediate cellular responses to DNA damage by inducing apoptosis and cell cycle arrest. p53 is a nuclear phosphoprotein, and phosphorylation has been proposed to be a means by which the activity of p53 is regulated. The cyclin-dependent kinase (CDK)-activating kinase (CAK) was originally identified as a cellular kinase required for the activation of a CDK-cyclin complex, and CAK is comprised of three subunits: CDK7, cyclin H, and p36MAT1. CAK is part of the transcription factor IIH multiprotein complex, which is required for RNA polymerase II transcription and nucleotide excision repair. Because of the similarities between p53 and CAK in their involvement in the cell cycle, transcription, and repair, we investigated whether p53 could act as a substrate for phosphorylation by CAK. While CDK7-cyclin H is sufficient for phosphorylation of CDK2, we show that p36MAT1 is required for efficient phosphorylation of p53 by CDK7-cyclin H, suggesting that p36MAT1 can act as a substrate specificity-determining factor for CDK7-cyclin H. We have mapped a major site of phosphorylation by CAK to Ser-33 of p53 and have demonstrated as well that p53 is phosphorylated at this site in vivo. Both wild-type and tumor-derived mutant p53 proteins are efficiently phosphorylated by CAK. Furthermore, we show that p36 and p53 can interact both in vitro and in vivo. These studies reveal a potential mechanism for coupling the regulation of p53 with DNA repair and the basal transcriptional machinery.

Candidate tumor suppressor B-cell translocation gene 3 impedes neoplastic progression by suppression of AKT.

BTG3 (B-cell translocation gene 3) is a p53 target that also binds and inhibits E2F1. Although it connects two major growth-regulatory pathways functionally and is downregulated in human cancers, whether and how BTG3 acts as a tumor suppressor remain largely uncharacterized. Here we present evidence that BTG3 binds and suppresses AKT, a kinase frequently deregulated in cancers. BTG3 ablation results in increased AKT activity that phosphorylates and inhibits glycogen synthase kinase 3ß. Consequently, we also observed elevated ß-catenin/T-cell factor activity, upregulation of mesenchymal markers, and enhanced cell migration. Consistent with these findings, BTG3 overexpression suppressed tumor growth in mouse xenografts, and was associated with diminished AKT phosphorylation and reduced ß-catenin in tissue specimens. Significantly, a short BTG3-derived peptide was identified, which recapitulates these effects in vitro and in cells. Thus, our study provides mechanistic insights into a previously unreported AKT inhibitory pathway downstream of p53. The identification of an AKT inhibitory peptide also unveils a new avenue for cancer therapeutics development.

Loss of the candidate tumor suppressor BTG3 triggers acute cellular senescence via the ERK-JMJD3-p16(INK4a) signaling axis.

The B-cell translocation gene 3 (BTG3) is a member of the antiproliferative BTG gene family and a downstream target of p53. BTG3 also binds and inhibits E2F1. Although it connects functionally two major growth-regulatory pathways, the physiological role of BTG3 remains largely uncharacterized. Here, we present evidence that loss of BTG3 in normal cells induced cellular senescence, which was correlated with enhanced ERK-AP1 signaling and elevated expression of the histone H3K27me3 demethylase JMJD3/KDM6B, leading to acute induction of p16(INK4a). Importantly, we also found that BTG3 expression is specifically downregulated in prostate cancer, thus providing a physiological link with human cancers. Our data suggest that BTG3 may have a fail-safe role against tumorigenic progression.

p53-Mediated transactivation of LIMK2b links actin dynamics to cell cycle checkpoint control.

The p53 tumor suppressor protein is widely known for its role as a sequence-specific transcription factor that regulates the expression of stress response genes. Here, we report the identification of LIMK2, which encodes a kinase that regulates actin dynamics through phosphorylation of cofilin, as a p53 target upregulated by DNA damage. Interestingly, the splice variant LIMK2b, but not LIMK2a, was induced in a p53-dependent manner through an intronic consensus p53-binding site. Depletion of LIMK2b leads to early exit of G2/M arrest after DNA damage, whereas its overexpression prolongs the arrest. These responses are recapitulated by ectopic expression of the active cofilin S3A mutant and the inactive cofilin S3D mutant, respectively, suggesting that LIMK2b may modulate G2/M arrest through cofilin phosphorylation. Furthermore, in support of its potential role as a tumor suppressor, LIMK2b was downregulated in esophageal and thyroid cancers, as well as in a number of established cancer cell lines, and its expression suppresses cancer cell migration. Taken together, our results unveil a novel pathway whereby LIMK2b, acting downstream of p53, ensures proper execution of checkpoint arrest by modulating the dynamics of actin polymerization.

Human FEM1B is required for Rad9 recruitment and CHK1 activation in response to replication stress.

Human checkpoint kinase 1 (CHK1) is an essential kinase required to preserve genome stability, and is activated by DNA replication blockage through the ataxia-telangiectasia-mutated-and-Rad3-related (ATR)/ATRIP-signaling pathway. In this report, we show that a novel CHK1-interacting protein, FEM1B (human homologue of the Caenorhabditis elegans sex determination fem1 protein), identified by a yeast two-hybrid screen, is involved in the activation of CHK1 by replication stress. Depletion of FEM1B by small interfering RNA in cancer cells impairs the activation of CHK1 kinase activity and attenuates the induction of CHK1 Ser345 phosphorylation upon replication interference. It is to be noted that, CHK2 Thr68 phosphorylation is not altered by FEM1B downregulation. By fractionation, we further demonstrated that FEM1B is able to associate with chromatin, and such association facilitates chromatin loading of the Rad9 protein. Consistently, ATR activity is poorly maintained in FEM1B knockdown cells; and FEM1B-ablated cells are as sensitive to replication block as CHK1-depleted cells. Our study has uncovered an adaptor protein FEM1B, which acts as a bridge linking CHK1 and Rad9, thus facilitating checkpoint signaling induced by replication stress.

The cell cycle checkpoint kinase CHK2 mediates DNA damage-induced stabilization of TTK/hMps1.

Cell cycle progression is monitored constantly to ensure faithful passage of genetic codes and genome stability. We have demonstrated previously that, upon DNA damage, TTK/hMps1 activates the checkpoint kinase CHK2 by phosphorylating CHK2 at Thr68. However, it remains to be determined whether and how TTK/hMps1 responds to DNA damage. In this report, we present evidence that TTK/hMps1 can be induced by DNA damage in normal human fibroblasts. Interestingly, the induction depends on CHK2 because CHK2-targeting small interfering RNA or a CHK2 inhibitor abolishes the increase. Such induction is mediated through phosphorylation of TTK/hMps1 at Thr288 by CHK2 and requires the CHK2 SQ/TQ cluster domain/forkhead-associated domain. In cells, TTK/hMps1 phosphorylation at Thr288 is induced by DNA damage and forms nuclear foci, which colocalize partially with gamma-H2AX. Reexpression of TTK/hMps1 T288A mutant in TTK/hMps1-knockdown cells causes a defect in G(2)/M arrest, suggesting that phosphorylation at this site participates in the proper checkpoint execution. Our study uncovered a regulatory loop between TTK/hMps1 and CHK2 whereby DNA damage-activated CHK2 may facilitate the stabilization of TTK/hMps1, therefore maintaining the checkpoint control.

Cell-specific and ubiquitous factors are responsible for the enhancer activity of the rat insulin II gene.

Pancreatic beta-cell-specific expression of the insulin gene is mediated, at least in part, by an enhancer element termed the rat insulin promoter element 3 (RIPES) found within the rat insulin II gene between positions -126 and -86. Here we identify three distinct factors interacting with RIPE3, namely 3a1, 3a2, and 3b1, which bind to the sequences between -100 to -90, -108 to -99, and -115 to -107, respectively. Factors 3a1 and 3b1 are beta-cell specific whereas 3a2 is ubiquitously distributed. The 3a1 site contains the consensus binding sequence (CANNTG) for a group of DNA-binding proteins called basic-helix-loop-helix proteins. We showed in this study that the 3a1 binding activity contains E12/E47, a member of the basic-helix-loop-helix protein family, or an E12/E47-like protein. Sequence comparison of the 3a2 and 3b1 binding sites suggest that they are unique and may bind to novel transcription factors. Mutation analysis of each individual binding site in transient expression experiments indicates that all of the three binding sites contribute to the enhancer activity of the RIPE3 in beta-cells. Mutation in any one of the three binding sites not only disrupts binding of the corresponding factor but decreases RIPE3 enhancer activity by 4-7-fold. The results suggest that interactions among the 3a1, 3a2, and 3b1 factors are required for maximum enhancer activity of the RIPE3 in insulin-producing cells.

DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2.

DNA-damaging agents signal to p53 through as yet unidentified posttranscriptional mechanisms. Here we show that phosphorylation of human p53 at serine 15 occurs after DNA damage and that this leads to reduced interaction of p53 with its negative regulator, the oncoprotein MDM2, in vivo and in vitro. Furthermore, using purified DNA-dependent protein kinase (DNA-PK), we demonstrate that phosphorylation of p53 at serines 15 and 37 impairs the ability of MDM2 to inhibit p53-dependent transactivation. We present evidence that these effects are most likely due to a conformational change induced upon phosphorylation of p53. Our studies provide a plausible mechanism by which the induction of p53 can be modulated by DNA-PK (or other protein kinases with similar specificity) in response to DNA damage.

Molecular characterization of the rat insulin enhancer-binding complex 3b2. Cloning of a binding factor with putative helicase motifs.

Cell-specific expression of the rat insulin II gene is in part mediated through an element located in the 5'-flanking region. The element, termed RIPE3b (-126 to -101), confers beta-cell-specific expression in conjunction with an adjacent element RIPE3a (-110 to -86). Here we report the characterization of one of the RIPE3b-binding complexes, 3b2. UV cross-linking analysis demonstrated that it is composed of at least three polypeptides: p58, p62, and p110. Furthermore, a cDNA was isolated via expression screening for binding to RIPE3b. Sequence analysis reveals that the encoded protein, designated Rip-1, possessed putative helicase motifs and a potential transcription activation domain. Overexpression of Rip-1 in cells greatly enhances the 3b2 binding complex, suggesting that Rip-1 is involved in the binding of 3b2.

DNA damage-inducible phosphorylation of p53 at N-terminal sites including a novel site, Ser20, requires tetramerization.

Upon DNA damage, p53 has been shown to be modified at a number of N-terminal phosphorylation sites including Ser15 and -33. Here we show that phosphorylation is induced as well at a novel site, Ser20. Phosphorylation at Ser15, -20 and -33 can occur within minutes of DNA damage. Interestingly, while the DNA-binding activities of p53 appear to be dispensable, efficient phosphorylation at these three sites requires the tetramerization domain of p53. Substitution of an artificial tetramerization domain for this region also permits phosphorylation at the N-terminus, suggesting that oligomerization is important for DNA damage-induced signalling to p53.

The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites.

Upon DNA damage, the amino terminus of p53 is phosphorylated at a number of serine residues including S20, a site that is particularly important in regulating stability and function of the protein. Because no known kinase has been identified that can modify this site, HeLa nuclear extracts were fractionated and S20 phosphorylation was followed. We discovered that a S20 kinase activity copurifies with the human homolog of the Schizosaccharomyces pombe checkpoint kinase, Chk1 (hCHK1). We confirmed that recombinant hCHK1, but not a kinase-defective version of hCHK1, can phosphorylate p53 in vitro at S20. Additional inducible amino- and carboxy-terminal sites in p53 are also phosphorylated by hCHK1, indicating that this is an unusually versatile protein kinase. It is interesting that hCHK1 strongly prefers tetrameric to monomeric p53 in vitro, consistent with our observation that phosphorylation of amino-terminal sites in vivo requires that p53 be oligomeric. Regulation of the levels and activity of hCHK1 in transfected cells is directly correlated with the levels of p53; expression of either a kinase-defective hCHK1 or antisense hCHK1 leads to reduced levels of cotransfected p53, whereas overexpression of wild-type hCHK1 or the kinase domain of hCHK1 results in increased levels of expressed p53 protein. The human homolog of the second S. pombe checkpoint kinase, Cds1 (CHK2/hCds1), phosphorylates tetrameric p53 but not monomeric p53 in vitro at sites similar to those phosphorylated by hCHK1 kinase, suggesting that both checkpoint kinases can play roles in regulating p53 after DNA damage.

Expression of the trans-active factors that stimulate insulin control element-mediated activity appear to precede insulin gene transcription.

Cell type-specific expression of the major differentiated products of alpha (glucagon) and beta (insulin) cells are regulated by sequences found within their 5'-flanking region. Specific transcription of the insulin gene appears to be principally controlled by a single cis-acting DNA element, termed the insulin control element (ICE). The ICE activator acts in combination with other positive regulatory factors that interact within this region to generate the correct, cell type-specific expression. In the present study, we show that the ICE activator is not only present but is functionally active in the islet glucagon-producing alpha cell line, alpha TC6. Analysis of the expression of various transfected insulin enhancer expression plasmids demonstrated that the insulin enhancer is active in alpha TC6 cells, although at a lower level than in beta cells. The reduced transcription from these constructs appears to be a consequence of the lack of other essential positive regulator(s). The alpha TC6 cells were also shown to display neuronal-like properties. Since islet cells appear to evolve from an alpha-like precursor cell that transiently expresses neuronal cell markers, these results would indicate that the ICE activator factor is induced before transcription of the insulin gene in the developing islet.

A role for ATR in the DNA damage-induced phosphorylation of p53.

Phosphorylation at Ser-15 may be a critical event in the up-regulation and functional activation of p53 during cellular stress. In this report we provide evidence that the ATM-Rad3-related protein ATR regulates phosphorylation of Ser-15 in DNA-damaged cells. Overexpression of catalytically inactive ATR (ATRki) in human fibroblasts inhibited Ser-15 phosphorylation in response to gamma-irradiation and UV light. In gamma-irradiated cells, ATRki expression selectively interfered with late-phase Ser-15 phosphorylation, whereas ATRki blocked UV-induced Ser-15 phosphorylation in a time-independent manner. ATR phosphorylated p53 at Ser-15 and Ser-37 in vitro, suggesting that p53 is a target for phosphorylation by ATR in DNA-damaged cells.

E1A signaling to p53 involves the p19(ARF) tumor suppressor.

The adenovirus E1A oncogene activates p53 through a signaling pathway involving the retinoblastoma protein and the tumor suppressor p19(ARF). The ability of E1A to induce p53 and its transcriptional targets is severely compromised in ARF-null cells, which remain resistant to apoptosis following serum depletion or adriamycin treatment. Reintroduction of p19(ARF) restores p53 accumulation and resensitizes ARF-null cells to apoptotic signals. Therefore, p19(ARF) functions as part of a p53-dependent failsafe mechanism to counter uncontrolled proliferation. Synergistic effects between the p19(ARF) and DNA damage pathways in inducing p53 may contribute to E1A's ability to enhance radio- and chemosensitivity.