Pim-1 associates with protein complexes necessary for mitosis.
The proto-oncogene pim-1 is a serine/threonine kinase the over-expression of which promotes lymphoma formation. Neither the normal function of Pim-1 nor the biochemical mechanism for cancer development mediated by the gene has been delineated, although recent studies have provided compelling evidence that Pim-1 is involved in differentiation and cell survival. ... We now provide the first evidence that Pim-1 may be involved in the proliferative process. By confocal microscopy, we observed a dynamic redistribution of Pim-1 during the cell cycle, the protein moving from the nucleus and cytoplasm in interphase to the spindle poles during mitosis. From a computer search for putative substrates of Pim-1 that are located in the spindle poles, we discovered that the nuclear mitotic apparatus (NuMA) protein has two peptide sequences that contain preferred phosphorylation sites for Pim-1 kinase. Recombinant glutathione-S-transferase-Pim-1 also readily phosphorylates immunoprecipitated NuMA. By confocal microscopy and co-immunoprecipitation we showed the interaction of the Pim-1 and NuMA proteins in HeLa cells that had been arrested during mitosis with nocodazole. Pim-1 also appeared to interact with heterochromatin-associated protein 1beta (HP1beta) and the cytoplasmic proteins dynein and dynactin via complex formation with NuMA. In our studies, overexpressed wild-type-Pim-1-GFP (green fluorescent protein) fusion protein was found to co-localize in the spindle pole with NuMA during mitosis. In contrast, the 'kinase-dead' mut-Pim-1-GFP fusion protein did not co-localize with NuMA, and appeared to promote apoptosis. Further evidence for apoptotic cell death was the observed blebbing and fragmentation of the chromosomes and a decrease in the level of NuMA protein detected by confocal microscopy. These results strongly suggest that Pim-1 kinase plays a role, most likely by phosphorylation, in promoting complex formation between NuMA, HP1beta, dynein and dynactin, a complex that is necessary for mitosis.
Mesh Terms:
3T3 Cells, Animals, Antigens, Nuclear, Base Sequence, DNA Primers, Fluorescent Antibody Technique, Indirect, Humans, Mice, Microscopy, Confocal, Mitosis, Mitotic Spindle Apparatus, Nocodazole, Nuclear Matrix-Associated Proteins, Nuclear Proteins, Phosphorylation, Protein Binding, Protein-Serine-Threonine Kinases, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-pim-1, Recombinant Fusion Proteins, Tumor Cells, Cultured
3T3 Cells, Animals, Antigens, Nuclear, Base Sequence, DNA Primers, Fluorescent Antibody Technique, Indirect, Humans, Mice, Microscopy, Confocal, Mitosis, Mitotic Spindle Apparatus, Nocodazole, Nuclear Matrix-Associated Proteins, Nuclear Proteins, Phosphorylation, Protein Binding, Protein-Serine-Threonine Kinases, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-pim-1, Recombinant Fusion Proteins, Tumor Cells, Cultured
Chromosoma
Date: Jul. 01, 2002
PubMed ID: 12111331
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