The GADD45 inhibition of Cdc2 kinase correlates with GADD45-mediated growth suppression

Cell cycle growth arrest is an important cellular response to genotoxic stress. Gadd45, a p53-regulated stress protein, plays an important role in the cell cycle G(2)-M checkpoint following exposure to certain types of DNA-damaging agents such as UV radiation and methylmethane sulfonate. Recent findings indicate that Gadd45 interacts with Cdc2 protein and inhibits Cdc2 kinase activity. In the present study, a series of Myc-tagged Gadd45 deletion mutants and a Gadd45 overlapping peptide library were used to define the Gadd45 domains that are involved in the interaction of Gadd45 with Cdc2. Both in vitro and in vivo studies indicate that the interaction of Gadd45 with Cdc2 involves a central region of the Gadd45 protein (amino acids 65-84). The Cdc2-binding domain of Gadd45 is also required for Gadd45 inhibition of Cdc2 kinase activity. Sequence analysis of the central Gadd45 region reveals no homology to inhibitory motifs of known cyclin-dependent kinase inhibitors, indicating that the Cdc2-binding and -inhibitory domains on Gadd45 are a novel motif. The peptide containing the Cdc2-binding domain (amino acids 65-84) disrupted the Cdc2-cyclin B1 protein complex, suggesting that dissociation of this complex results from a direct interaction between the Gadd45 and Cdc2 proteins. GADD45-induced cell cycle G(2)-M arrest was abolished when its Cdc2 binding motif was disrupted. Importantly, a short term survival assay demonstrated that GADD45-induced cell cycle G(2)-M arrest correlates with GADD45-mediated growth suppression. These findings indicate that the cell cycle G(2)-M growth arrest mediated by GADD45 is one of the major mechanisms by which GADD45 suppresses cell growth.     

UVB-Induced G2 Arrest of Human Melanocytes Involves Cdc2 Sequestration by Gadd45a in Nuclear Speckles

Exposure to solar UVB radiation is involved in the development of cutaneous melanoma. We previously showed that human melanocytes and melanoma cells respond to UVB radiation via a p53-independent pathway involving GADD45A activation. Here, we determined that UVB-induction of Gadd45a is necessary for G2 arrest and that Gadd45a and its partner p21Waf1 co-localize in nuclear bodies called Nuclear Speckles. We further observed that UVB-induced G2 arrest is associated with Cdc2 accumulation in these Nuclear Speckles. Knock-down of Gadd45a expression by RNA interference prevents both UVB-induced Cdc2 accumulation in Nuclear Speckles and G2 arrest. Our results demonstrate that UVB-induced G2 arrest of melanoma cells is Gadd45a-dependent. Furthermore, we show that Cdc2 sequestration by Gadd45a occurs in Nuclear Speckles, suggesting a new role for these nuclear bodies, so far only linked to RNA maturation.     

Gadd45 mutations are uncommon in human tumour cell lines

GADD45 is an evolutionarily conserved gene that encodes a small acidic, nuclear protein and is an example of a p53 responsive gene. Gadd45 protein has been shown to interact with PCNA and also p21^waf1. It has been implicated in growth arrest, DNA repair, chromatin structure and signal transduction. The confusing biochemical data has been clarified by the demonstration that Gadd45 null mice have a phenotype strikingly similar to that of p53 null mice, being tumour prone and showing marked genomic instability. We have tested the hypothesis that mutations in the GADD45 coding region might substitute for p53 abnormalities in tumour cell lines where p53 is wild type. After generating cDNA from mRNA in a panel of 24 cell lines we sequenced the GADD45 cDNA and have demonstrated that no mutations can be observed, even in the p53 wild type cell lines. Such data suggest that Gadd45 mutations are uncommon in human cancer. From this we postulate that, despite the phenotype of the GADD45 null mouse, GADD45 is unlikely to be the key mechanistic determinant of the tumour suppressor activity of the p53 pathway.
Note on nomenclature: We have employed GADD45 to designate the gene and Gadd45 to designate the encoded protein. This gene has also be denoted GADD45 α elsewhere in the literature.     

Identification of a functional domain in a GADD45-mediated G2/M checkpoint

Cell cycle checkpoints are essential for the maintenance of genomic stability in response to DNA damage. We demonstrated recently that GADD45, a DNA damage-inducible protein, activates a G(2)/M checkpoint induced by either UV radiation or alkylating agents. GADD45 can interact in vivo with the G(2) cell cycle-specific kinase, Cdc2, proliferating cell nuclear antigen (PCNA), and the cell cycle kinase inhibitor p21(waf1). The ability of GADD45 to induce a G(2)/M arrest may be caused in part by the inhibition of Cdc2 kinase activity. Here, we report the identification of a region of GADD45 that is involved in this G(2)/M checkpoint. Mutants of GADD45 that lacked either the first 35 or the last 80 residues still retained an ability to induce G(2)/M arrest. A mutant with a deletion of the central region (residues 50-76), which is conserved in the family members GADD45beta and GADD45gamma, lacked such activity. This mutant also lacked an ability to bind to Cdc2, PCNA, and p21(waf1) in vivo. Consistently, either GADD45beta or GADD45gamma bind to Cdc2 in vivo. However, unlike GADD45, neither GADD45beta nor GADD45gamma inhibited the Cdc2 kinase or induced G(2)/M arrest. The unique effect of GADD45 may be caused by the presence of a region containing DEDDDR residues. Alanine substitutions in the region abolished GADD45 induction of a G(2)/M arrest and its inactivation of the Cdc2 kinase but not its binding to Cdc2, PCNA, or p21(waf1). Therefore, the binding of GADD45 to Cdc2 was insufficient to induce a G(2)/M arrest, and additional activity contributed by the DEDDDR residues may be necessary to regulate the G(2)/M checkpoint.     

B23 regulates GADD45a nuclear translocation and contributes to GADD45a-induced cell cycle G2-M arrest

Gadd45a is an important player in cell cycle G2-M arrest in response to genotoxic stress. However, the underlying mechanism(s) by which Gadd45a exerts its role in the control of cell cycle progression remains to be further defined. Gadd45a interacts with Cdc2, dissociates the Cdc2-cyclin B1 complex, alters cyclin B1 nuclear localization, and thus inhibits the activity of Cdc2/cyclin B1 kinase. These observations indicate that Gadd45a nuclear translocation is closely associated with its role in cell cycle G2-M arrest. Gadd45a has been characterized as a nuclear protein, but it does not contain a classical nuclear localization signal, suggesting that Gadd45a nuclear translocation might be mediated through different nuclear import machinery. Here we show that Gadd45a associates directly with B23 (nucleophosmin), and the B23-interacting domain is mapped at the central region (61-100 amino acids) of the Gadd45a protein using a series of Myc tag-Gadd45a deletion mutants. Deletion of this central region disrupts Gadd45a association with B23 and abolishes Gadd45a nuclear translocation. Suppression of endogenous B23 through a short interfering RNA approach disrupts Gadd45a nuclear translocation and results in impaired Gadd45a-induced cell cycle G2-M arrest. These findings demonstrate a novel association of B23 and Gadd45a and implicate B23 as an important regulator in Gadd45a nuclear import.     

Gadd45a, a p53- and BRCA1-regulated stress protein, in cellular response to DNA damage

Mammalian cells exhibit complex, but intricate cellular responses to genotoxic stress, including cell cycle checkpoints, DNA repair and apoptosis. Inactivation of these important biological events may result in genomic instability and cell transformation, as well as alterations of therapeutic sensitivity. Gadd45a, a p53- and BRCA1-regulated stress-inducible gene, has been characterized as one of the important players that participate in cellular response to a variety of DNA damage agents. Interestingly, the signaling machinery that regulates Gadd45a induction by genotoxic stress involves both p53-dependent and -independent pathways; the later may employ BRCA1-related or MAP kinase-mediated signals. Gadd45a protein has been reported to interact with multiple important cellular proteins, including Cdc2 protein kinase, proliferating cell nuclear antigen (PCNA), p21Waf1/Cip1 protein, core histone protein and MTK/MEKK4, an up-stream activator of the JNK/SAPK pathway, indicating that Gadd45a may play important roles in the control of cell cycle checkpoint, DNA repair process, and signaling transduction. The importance of Gadd45a in maintaining genomic integrity is well manifested by the demonstration that disruption of endogenous Gadd45a in mice results in genomic instability and increased carcinogenesis. Therefore, Gadd45a appears to be an important component in the cellular defense network that is required for maintenance of genomic stability.     

Transforming growth factor-beta-induced apoptosis is mediated by Smad-dependent expression of GADD45b through p38 activation

Transforming growth factor-beta (TGF-beta)-dependent apoptosis is important in the elimination of damaged or abnormal cells from normal tissues in vivo. In this report, we identify GADD45b as an effector of TGF-beta-induced apoptosis. GADD45b has been shown to be a positive mediator of apoptosis induced by certain cytokines and oncogenes. We show that Gadd45b is an immediateearly response gene for TGF-beta and that the proximal Gadd45b promoter is activated by TGF-beta through the action of Smad2, Smad3, and Smad4. We show that ectopic expression of GADD45b in AML12 murine hepatocytes is sufficient to activate p38 and to trigger apoptotic cell death, whereas antisense inhibition of Gadd45b expression blocks TGF-beta-dependent p38 activation and apoptosis. Furthermore, we also show that TGF-beta can activate p38 and induce apoptosis in mouse primary hepatocytes from wild-type mice, but not from Gadd45b-/- mice. All of these findings suggest that GADD45b participates in TGF-beta-induced apoptosis by acting upstream of p38 activation.     

A polymorphism but no mutations in the GADD45 gene in breast cancers

The p53 gene product is part of a pathway regulating growth arrest at the G₁ checkpoint of the cell cycle. Mutation of other components of this pathway, including the products of the ataxia telangiectasia (AT), GADD45, mdm2, and p21^WAF1/CIP1 genes may have effects comparable to mutations in the p53 gene. The GADD45 gene is induced by ionizing radiation and several DNA-damaging xenobiotics. Induction requires the binding of wild-type p53 to an evoulutionarily highly conserved putative intronic p53 binding site in intron 3 of GADD45. We recently analyzed the entire coding region of the p53 gene in primary breast cancers of Midwestern white women and found 21 mutations among 53 tumors (39,6%). We now have shown by direct sequencing that there are no mutations in the intronic p53 binding site of the GADD45 gene in any of the 53 primary breast cancers and no mutations in the entire coding region of the GADD45 gene in a subset of 26 consecutive tumors (12 with p53 mutation and 14 without p53 mutation). The only sequence variation detected was a common polymorphism in intron 3. The absence of mutations in the GADD45 gene, including the putative p53-binding intronic site, suggests that this gene is not a frequent target of mutations in breast cancer. Although mutations of the p53 gene have been studied in a wide spectrum of human cancers, GADD45 has not been examined in any tumor or cell line to the best of our knowledge. Our results raise the possibility that mutation of the GADD45 gene alone is not functionally equivalent to loss of wild-type p53 activity. Received: 14 September 1995     

p21CIP1 and Cdc25A: competition between an inhibitor and an activator of cyclin-dependent kinases.

Cdc25A, a phosphatase essential for G1-S transition, associates with, dephosphorylates, and activates the cell cycle kinase cyclin E-cdk2. p21CIP1 and p27 are cyclin-dependent kinase (cdk) inhibitors induced by growth-suppressive signals such as p53 and transforming growth factor beta (TGF-beta). We have identified a cyclin binding motif near the N terminus of Cdc25A that is similar to the cyclin binding Cy (or RR LFG) motif of the p21CIP1 family of cdk inhibitors and separate from the catalytic domain. Mutations in this motif disrupt the association of Cdc25A with cyclin E- or cyclin A-cdk2 in vitro and in vivo and selectively interfere with the dephosphorylation of cyclin E-cdk2. A peptide based on the Cy motif of p21 competitively disrupts the association of Cdc25A with cyclin-cdks and inhibits the dephosphorylation of the kinase. p21 inhibits Cdc25A-cyclin-cdk2 association and the dephosphorylation of cdk2. Conversely, Cdc25A, which is itself an oncogene up-regulated by the Myc oncogene, associates with cyclin-cdk and protects it from inhibition by p21. Cdc25A also protects DNA replication in Xenopus egg extracts from inhibition by p21. These results describe a mechanism by which the Myc- or Cdc25A-induced oncogenic and p53- or TGF-beta-induced growth-suppressive pathways counterbalance each other by competing for cyclin-cdks.     

Human melanoma cell line UV responses show independency of p53 function.

UV radiation-induced mutation of the p53 gene is suggested as a causative event in skin cancer, including melanoma. We have analyzed here p53 mutations in melanoma cell lines and studied its stabilization, DNA-binding activity, and target gene activation by UVC. p53 was mutated in three of seven melanoma cell lines. However, high levels of p53 were detected in all cell lines, including melanoma cells with wild-type p53, with the exception of one line with a truncated form. Upon UV induction, p53 accumulated in lines with wild-type p53, and p53 target genes p21Cip1/Waf1, GADD45, and mdm2 were induced, but the induction of p21Cip1/Waf1 was significantly delayed as compared with the increase in p53 DNA-binding activity. However, despite p53 target gene induction, p53 DNA-binding activity was absent in one melanoma line with wild-type p53, and p53 target genes were induced also in cells with mutant p53. In response to UV, DNA replication ceased in all cell lines, and apoptosis ensued in four lines independently of p53 but correlated with high induction of GADD45. The results suggest that in melanoma, several p53 regulatory steps are dislodged; its basal expression is high, its activation in response to UV damage is diminished, and the regulation of its target genes p21Cip1/Waf1 and GADD45 are dissociated from p53 regulation.     

Interaction of CR6 (GADD45gamma ) with proliferating cell nuclear antigen impedes negative growth control

GADD45, MyD118, and CR6 (also termed GADD45alpha, beta, and gamma) comprise a family of genes that encode for related proteins playing important roles in negative growth control, including growth suppression. Data accumulated suggest that MyD118/GADD45/CR6 serve similar but not identical functions along different apoptotic and growth suppressive pathways. It is also apparent that individual members of the MyD118/GADD45/CR6 family are differentially induced by a variety of genetic and environmental stress agents. The MyD118, CR6, and GADD45 proteins were shown to predominantly localize within the cell nucleus. Recently, we have shown that both MyD118 and GADD45 interact with proliferating cell nuclear antigen (PCNA), a protein that plays a central role in DNA replication, DNA repair, and cell cycle progression, as well as with the universal cyclin-dependent kinase inhibitor p21. In this work we show that also CR6 interacts with PCNA and p21. Moreover, it is shown that CR6 interacts with PCNA via a domain that also mediates interaction of both GADD45 and MyD118 with PCNA. Importantly, evidence has been obtained that interaction of CR6 with PCNA impedes the function of this protein in negative growth control, similar to observations reported for MyD118 and GADD45.     

Loss of oncogenic H-ras-induced cell cycle arrest and p38 mitogen-activated protein kinase activation by disruption of Gadd45a

The activation of p53 is a guardian mechanism to protect primary cells from malignant transformation; however, the details of the activation of p53 by oncogenic stress are still incomplete. In this report we show that in Gadd45a(-/-) mouse embryo fibroblasts (MEF), overexpression of H-ras activates extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) but not p38 kinase, and this correlates with the loss of H-ras-induced cell cycle arrest (premature senescence). Inhibition of p38 mitogen-activated protein kinase (MAPK) activation correlated with the deregulation of p53 activation, and both a p38 MAPK chemical inhibitor and the expression of a dominant-negative p38alpha inhibited p53 activation in the presence of H-ras in wild-type MEF. p38, but not ERK or JNK, was found in a complex with Gadd45 proteins. The region of interaction was mapped to amino acids 71 to 96, and the central portion (amino acids 71 to 124) of Gadd45a was required for p38 MAPK activation in the presence of H-ras. Our results indicate that this Gadd45/p38 pathway plays an important role in preventing oncogene-induced growth at least in part by regulating the p53 tumor suppressor.     

Crystal structure of human Gadd45 reveals an active dimer

The human Gadd45 protein family plays critical roles in DNA repair, negative growth control, genomic stability, cell cycle checkpoints and apoptosis. Here we report the crystal structure of human Gadd45, revealing a unique dimer formed via a bundle of four parallel helices, involving the most conserved residues among the Gadd45 isoforms. Mutational analysis of human Gadd45 identified a conserved, highly acidic patch in the central region of the dimer for interaction with the proliferating cell nuclear antigen (PCNA), p21 and cdc2, suggesting that the parallel dimer is the active form for the interaction. Cellular assays indicate that: (1) dimerization of Gadd45 is necessary for apoptosis as well as growth inhibition, and that cell growth inhibition is caused by both cell cycle arrest and apoptosis; (2) a conserved and highly acidic patch on the dimer surface, including the important residues Glu87 and Asp89, is a putative interface for binding proteins related to the cell cycle, DNA repair and apoptosis. These results reveal the mechanism of self-association by Gadd45 proteins and the importance of this self-association for their biological function.     

Growth arrest and DNA damage-45 alpha (GADD45alpha)

Regulation of cell cycle and growth is integral for cell survival. The intricate mechanisms that control proliferation and cell cycle are numerous. The growth arrest and DNA damage (GADD)-inducible gene family is often up-regulated in response to various environmental stresses and drug therapies. GADD45alpha was the first stress-inducible gene determined to be up-regulated by p53 and is also a target for the p53 homologues, p63 and p73. When GADD45alpha is deleted or repressed, cells show uncontrolled proliferation. Furthermore, decreased GADD45alpha expression is also considered a survival mechanism, as cancer cells without this control can evade the apoptotic pathway leading to increased tumourigenesis. Drug therapies can act to directly or indirectly up-regulate GADD45alpha and promote apoptosis. As GADD45alpha is an essential component of many metabolic pathways that control proliferating cancer cells, it presents itself as an emerging drug target worthy of further investigation.