Pim-1 associates with protein complexes necessary for mitosis

Chromosoma. 2002 Jul;111(2):80-95. doi: 10.1007/s00412-002-0192-6. Epub 2002 May 15.

Abstract

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
  • Cell Cycle Proteins
  • DNA Primers
  • Fluorescent Antibody Technique, Indirect
  • Humans
  • Mice
  • Microscopy, Confocal
  • Mitosis / drug effects
  • Mitosis / physiology*
  • Nocodazole / pharmacology
  • Nuclear Matrix-Associated Proteins
  • Nuclear Proteins / metabolism
  • Phosphorylation
  • Protein Binding
  • Protein-Serine-Threonine Kinases / metabolism*
  • Protein-Serine-Threonine Kinases / physiology
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins / physiology
  • Proto-Oncogene Proteins c-pim-1
  • Recombinant Fusion Proteins / metabolism
  • Spindle Apparatus
  • Tumor Cells, Cultured

Substances

  • Antigens, Nuclear
  • Cell Cycle Proteins
  • DNA Primers
  • NUMA1 protein, human
  • Nuclear Matrix-Associated Proteins
  • Nuclear Proteins
  • Numa1 protein, mouse
  • Proto-Oncogene Proteins
  • Recombinant Fusion Proteins
  • PIM1 protein, human
  • Pim1 protein, mouse
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-pim-1
  • Nocodazole