RESUMO
Regions of hypoxia are a hallmark of solid tumors. Tumor cells modulate the regulation of specific genes allowing adaptation and survival in the harsh hypoxic environment. We have identified SKIP3, a novel human kinase-like gene, which is overexpressed in multiple human tumors and is regulated by hypoxia. SKIP3 is an ortholog of the Drosophila tribbles, rat NIPK, dog C5FW, and human C8FW genes. Drosophila tribbles is involved in slowing cell-cycle progression during Drosophila development, but little is known regarding the function or tissue distribution of the vertebrate orthologs. We show that the normal tissue expression of SKIP3 is confined to human liver, while multiple primary human lung, colon, and breast tumors express high levels of SKIP3 transcript. Endogenous SKIP3 protein accumulates within 48 h under hypoxic growth conditions in HT-29 and PC-3 cells, with upregulation of the SKIP3 mRNA transcript by 72 h. We identified activating transcription factor 4 (ATF4) as a SKIP3-binding partner using the yeast-two-hybrid assay. Coexpression of SKIP3 and ATF4 showed that SKIP3 is associated with the proteolysis of ATF4, which can be blocked using a proteosome inhibitor. These results indicate that SKIP3 may be an important participant in tumor cell growth.
Assuntos
Hipóxia Celular/fisiologia , Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Ciclo Celular/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Primers do DNA , Proteínas de Ligação a DNA , Drosophila/crescimento & desenvolvimento , Proteínas de Drosophila/genética , Humanos , Masculino , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Diester Fosfórico Hidrolases , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas Serina-Treonina Quinases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
The family of human Nek (NIMA Related Kinase) kinases currently contains 11 members. We have identified Nek8 as a new member of the Nek kinase family. For many of the Nek family members, primary tumor expression data and function have been limited. However, all of the Nek family proteins share considerable homology with the Never In Mitosis, gene A (NIMA) kinase from the filamentous fungus Aspergillus nidulans. NIMA, as well as its most closely related human ortholog, Nek2, are required for G(2)/M progression and promote centrosome maturation during mitosis. We isolated Nek8 from a primary human colon cDNA library, and found it to be highly homologous to murine Nek8. Recently, a previously named Nek8 sequence was renamed Nek9/Nercc1 in Genbank due to its lack of homology to murine Nek8 and its high homology to murine Nek9. Interestingly, in our study, phylogenetic analysis suggests that human Nek8 and Nek9 form a subfamily within the Nek family. Nek8 has high homology to the Nek family kinase domain as well as to a regulator of chromosome condensation domain (RCC1), which is also present in Nek9. The open reading frame of human Nek8 encodes a 692 amino-acid protein with a calculated molecular weight of 75 kDa. Nek8 is differently expressed between normal human breast tissue and breast tumors. Overexpression of a mutated kinase domain Nek8 in U2-0S cells led to a decrease in actin protein, and a small increase in the level of cdk1/cyclinB1. Our data demonstrate for the first time that Nek8 is a novel tumor associated gene, and shares considerable sequence homology with the Nek family of protein kinases and may be involved in G(2)/M progression.