Gadd45a (Gadd45), Gadd45b (MyD118), and Gadd45g (CR6) constitute a family of evolutionarily conserved, small, acidic, nuclear proteins, which have been implicated in terminal differentiation, growth suppression, and apoptosis. How Gadd45 proteins function in negative growth control is not fully understood. Recent evidence has implicated Gadd45a in inhibition of cdc2/cyclinB1 kinase and in G2/M cell cycle arrest. Yet, whether Gadd45b and/or Gadd45g function as inhibitors of cdc2/cyclinB1 kinase and/or play a role in G2/M cell cycle arrest has not been fully established. In this work, we show that Gadd45b and Gadd45g specifically interact with the Cdk1/CyclinB1 complex, but not with other Cdk/Cyclin complexes, in vitro and in vivo. Data also has been obtained that Gadd45b and Gadd45g, as well as GADD45a, interact with both Cdk1 and cyclinB1, resulting in inhibition of the kinase activity of the Cdk1/cyclinB1 complex. Inhibition of Cdk1/cyclinB1 kinase activity by Gadd45b and Gadd45a was found to involve disruption of the complex, whereas Gadd45g did not disrupt the complex. Moreover, using RKO lung carcinoma cell lines, which express antisense Gadd45 RNA, data has been obtained, which indicates that all three Gadd45 proteins are likely to cooperate in activation of S and G2/M checkpoints following exposure of cells to UV irradiation.
Transforming growth factor- (TGF-)-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--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- and that the proximal Gadd45b promoter is activated by TGF- 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--dependent p38 activation and apoptosis. Furthermore, we also show that TGF- 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--induced apoptosis by acting upstream of p38 activation.
MyD118 and Gadd45 are related genes encoding for proteins that play important roles in negative growth control, including growth suppression and apoptosis. MyD118 and Gadd45 are related proteins that previously were shown to interact with proliferating cell nuclear antigen (PCNA), implicated in DNA replication, DNA repair, and cell cycle progression. To establish the role of MyD118 and Gadd45 interactions with PCNA, in this work we sought to identify the interacting domains and analyze the significance of this interaction in negative growth control. Using complementary in vivo and in vitro interaction assays the N-terminal (1-46) and middle (100 -127) regions of PCNA were identified as harboring MyD118-and Gadd45 interacting domains, whereas PCNA interacting domains within MyD118 and Gadd45 were localized to the C termini of these proteins (amino acids 114 -156 and 137-165, respectively). These findings provide first evidence that similar domains within MyD118 and Gadd45 mediate interactions with PCNA. Importantly, ectopic expression of MyD118 or Gadd45 N-terminal peptides, lacking the PCNA interacting domain, was found to suppress colony formation or induce apoptosis more efficiently than the full-length proteins. These findings suggest that interaction of MyD118 or Gadd45 with PCNA, in essence, serves to impede negative growth control.
The MyD118 (Gadd45beta) protein is a member of a family of structurally related proteins, including Gadd45 (Gadd45alpha) and CR6 (Gadd45gamma), that have critical roles in regulating growth arrest and apoptosis. The MyD118 and other members of its family display distinct patterns of expression in response to stimuli that induce differentiation, growth arrest, or apoptosis. Species-blot analysis showed that MyD118 is an evolutionarily conserved gene, and comparative sequence analysis showed that MyD118 has a gene structure similar to that of other members of its gene family. Comparison of putative transcription factor-binding sites found in sequences of this gene family provides evidence that p53 is involved in regulating the expression of MyD118 and that NF-kappaB may play a role in differential expression of MyD118 and Gadd45(Gadd45alpha). Fluorescence in situ hybridization localized the MyD118 gene to mouse chromosome band 10B5.3, correcting a previous assignment to mouse chromosome 9.
Gadd45g/CR6, Gadd45b/MyD118, and Gadd45a/Gadd45 are members of a gene family that displays distinct patterns of gene expression in response to stimuli that induce differentiation, growth arrest, and/or apoptosis. All three of these highly conserved proteins interact with a number of critical cell cycle and cell survival regulatory proteins such as PCNA, p21(WAF1/CIP1), CDK1 (cdc2-p34), and MTK1/MEKK4, and have been reported to influence the activity of the p38 and JNK kinases. Species-blot analysis showed that Gadd45g is an evolutionarily conserved gene and sequence analysis showed that Gadd45g has a gene structure conserved with that of other members of its gene family. A comparison of the putative transcription factor binding sites found in the sequences of the gene family members suggests, that like Gadd45b, NF-kappaB and STATs may be responsible for the differences in regulation of expression observed between Gadd45g and Gadd45a. Analysis of the Gadd45b/MyD118 promoter shows that there are three different enhanceosome-like regions that may allow cell-type specific responses to TGF-beta1 by the Gadd45b/MyD118 promoter. Fluorescent in situ hybridization (FISH) confirmed the localization of the Gadd45g gene to mouse chromosome band 13A5-B, which has been reported to contain a quantitative trait locus that regulates body weight in mice. This suggests that alleles of the Gadd45g gene may function in the regulation of body weight, in addition to its currently recognized roles in differentiation and stress responses.
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