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.
Cancer Prevention and Research Center, WSU, Pullman, WA 99164-4234, USA.
Nandini
N
Bhattacharya
Zeping
Z
Wang
Christine
C
Davitt
Ian F C
IF
McKenzie
Pei-Xiang
PX
Xing
Nancy S
NS
Magnuson
D016475:3T3 Cells; D000818:Animals; D034961:Antigens, Nuclear; D001483:Base Sequence; D018797:Cell Cycle Proteins; D000090266:Chromobox Protein Homolog 5; D017931:DNA Primers; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal; D008938:Mitosis / Q000187:drug effects / Q000502:physiology*; D015739:Nocodazole / Q000494:pharmacology; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism* / Q000502:physiology; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000378:metabolism* / Q000502:physiology; D051573:Proto-Oncogene Proteins c-pim-1; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D008941:Spindle Apparatus; D014407:Tumor Cells, Cultured
D034961:Antigens, Nuclear; C000715593:CBX1 protein, human; D018797:Cell Cycle Proteins; D017931:DNA Primers; C000715470:MAS1 protein, human; C050045:NUMA1 protein, human; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins; C489080:Numa1 protein, mouse; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D000090266:Chromobox Protein Homolog 5; C496634:PIM1 protein, human; C496636:Pim1 protein, mouse; D017346:Protein Serine-Threonine Kinases; D051573:Proto-Oncogene Proteins c-pim-1; D015739:Nocodazole