ABSTRACT
The human TPIP (TPTE and PTEN homologous Inositol lipid Phosphatase) belongs to the PTEN (Phosphatase and TENsin homologue deleted on chromosome 10) family of dual-specific phosphatases and is expressed from the human chromosome 13 as multiple splice-variants, e.g., TPIPα, ß, γ mRNAs. PTEN is a well characterized tumor suppressor, which controls survival, adhesion, motility and migration of mammalian cells, its C2-domain plays crucial role in controlling these functions. However, role of isolated C2-domain protein in regulation of cell proliferation and apoptosis is not reported. We report sequence analysis and function of a novel human TPIP (TPIP-C2) cDNA encoding a 193 amino acid C2-domain in cell proliferation and apoptosis regulation. In silico analysis and homology modelling revealed that the C2-domain of TPIP-C2 is similar to that of PTEN but with short disorder sequences overlapping or adjacent to the post-translational modification sites. Overexpression of TPIP-C2 cDNA in human embryonic kidney (HEK-293) cells caused cell cycle arrest, inhibition of cell proliferation and induced apoptosis in an activated caspase 3 and PARP-dependent manner in comparison to overexpression of the full length human PTEN cDNA. TPIP-C2 overexpressed cells also showed S-phase cell cycle arrest. We suggest that C2-domain of TPIP-C2 may act as a dominant negative effector, which may bind to and arrest the cell proliferation signalling complex and isolated TPIP-C2-domain-like proteins expressed in mammalian cells/tissues may play important role in regulation of cell proliferation and apoptosis. The TPIP-C2 cDNA may be exploited for inducing cell cycle-inhibition and apoptosis in human cancer cells and tissues.
Subject(s)
Apoptosis/genetics , Cell Cycle Checkpoints/genetics , Dual-Specificity Phosphatases/genetics , Dual-Specificity Phosphatases/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolism , Amino Acid Sequence , Base Sequence , Cell Line , Cell Proliferation , Cell Survival , Dual-Specificity Phosphatases/chemistry , HEK293 Cells , Humans , Protein Isoforms/genetics , Protein Processing, Post-Translational , Protein Structure, Secondary , Sequence Alignment , Sequence Analysis, DNAABSTRACT
Alternative splicing of mRNAs is known to involve a major regulation of gene expression at RNA level in mammalian cells. The PTEN (Phosphatase and TENsin homologue deleted from the human chromosome 10), TPTE (Transmembrane Phosphatase with TEnsin homology) and TPIP (TPTE and PTEN homologous Inositol lipid Phosphatase) belong to a family of dual-specific lipid and protein phosphatases. PTEN is a well characterized tumor suppressor, which plays crucial role in cell survival, cell cycle regulation, cell proliferation as well as adhesion, motility and migration of cells. The C2-domain of PTEN is essential for PTEN-functions. We have isolated a novel 1019 bp human TPIP cDNA (TPIP-C2) from a human testis cDNA library. In silico analysis of the cDNA revealed that it is produced from the TPIP-locus on the human chromosome 13 by alternative RNA-splicing. It has a unique 5'-Alu sequence, a LINE sequence followed by a 582 bp Open Reading Frame (ORF) encoding a 193 aa polypeptide with a partial phosphatase domain and a C2-domain. TPIP-C2 mRNA is expressed in human testis and in mouse tissues. Mouse testis and brain showed higher levels of TPIP-C2 mRNA in comparison to the heart, liver and kidney under normal physiological conditions. TPIP-C2 mRNAs from human and mouse testes show extensive sequence identity. Over-expression of TPIP-C2 cDNA in HeLa cells strongly (up to 85%) inhibited cell growth/proliferation and caused apoptosis in a caspase 3-dependent manner. These findings suggest for the first time that a TPIP splice-variant mRNA with a partial phosphatase domain and a C2-domain is expressed in cells and tissues of human and murine origins under normal physiological conditions. Inhibition of cell growth/proliferation and induction of apoptosis by overexpression of TPIP-C2 mRNA in HeLa cells suggest that it may be involved in negative regulation of cell growth/proliferation.