RESUMO
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.
Assuntos
Antígenos de Diferenciação/genética , Genes , Camundongos/genética , Sequência de Aminoácidos , Animais , Antígenos de Diferenciação/fisiologia , Sequência de Bases , Sítios de Ligação , Galinhas/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 19/genética , Cães , Evolução Molecular , Humanos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Polimorfismo Conformacional de Fita Simples , Coelhos , Alinhamento de Sequência , Homologia de Sequência , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Antígenos de Diferenciação/metabolismo , Divisão Celular , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas/metabolismo , Antígenos de Diferenciação/química , Apoptose , Sítios de Ligação , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Antígeno Nuclear de Célula em Proliferação/química , Proteínas/antagonistas & inibidores , Proteínas/química , Técnicas do Sistema de Duplo-Híbrido , Proteínas GADD45RESUMO
Towards dissecting the regulation of terminal differentiation, including growth arrest and apoptosis, myeloid differentiation primary response (MyD) genes, induced in the absence of de novo protein synthesis following induction of M1 myeloblastic leukemia cells for terminal differentiation have been isolated. MyD118 was one of the novel MyD genes cloned, subsequently observed also to be a primary response gene to TGF-beta, which induces M1 cells for growth arrest and apoptosis uncoupled from differentiation. The MyD118 encoded protein was observed to be remarkably similar to the protein encoded by Gadd45, a growth arrest and DNA damage induced gene, regulated in part by the tumor suppressor p53. Though evidence has accumulated that MyD118 functions as an important modulator of negative growth control both in hematopoietic and non-hematopoietic cells, its mechanism of action is unknown. To better understand the role(s) of MyD118 in negative growth control, we have analysed the expression and biological characteristics of the MyD118 protein, compared to the Gadd45 protein, in distinct pathways of growth arrest and apoptosis, including p53 dependent and independent pathways either coupled or uncoupled from differentiation. It is shown that MyD118 and Gadd45 differentially accumulated upon induction of distinct pathways of growth arrest and apoptosis; notably, MyD118, but not Gadd45, was induced by TGF-beta, whereas Gadd45, but not MyD118, was induced by activating wild type (wt) p53 function. It is also shown that MyD118 is a nuclear protein, which regardless of the pathway induced, predominantly localized within the cell nucleus, and interacted with the DNA replication and repair protein PCNA and the cyclin dependent kinase inhibitor P21WAF1/CIP1. MyD118 also modestly stimulated DNA repair in vitro. All of these characteristics were shared with Gadd45. Finally, it is demonstrated that MyD118, Gadd45 and p21 synergized in the suppression of colony formation by NIH3T3 cells. Taken together, these findings demonstrate that MyD118 and Gadd45 are representative of a new protein family that share remarkable functional similarities in the control of distinct pathways of negative growth, including the suppression of cellular growth and programmed cell death.
