Phosphorylation regulates the assembly of NuMA in a mammalian mitotic extract.
NuMA is a 236 kDa nuclear protein that is required for the organization of the mitotic spindle. To determine how NuMA redistributes in the cell during mitosis, we have examined the behavior of NuMA in a mammalian mitotic extract under conditions conducive to the reassembly of interphase nuclei. NuMA is a soluble protein in mitotic extracts prepared from synchronized cultured cells, but forms insoluble structures when the extract becomes non-mitotic (as judged by the inactivation of cdc2/cyclin B kinase and the disappearance of mpm-2-reactive antigens). These NuMA-containing structures are irregularly shaped particles of 1-2 microm in diameter and their assembly is specific because other nuclear components such as the lamins remain soluble in the extract under these conditions. NuMA is dephosphorylated during this assembly process, and the assembly of these NuMA-containing structures is catalyzed by protein dephosphorylation because protein kinase inhibitors enhance their formation and protein phosphatase inhibitors block their formation. Finally, immunodepletion demonstrates that NuMA is an essential structural component of these insoluble particles, and electron microscopy shows that the particles are composed of a complex interconnected network of foci. These results demonstrate that phosphorylation regulates the solubility of NuMA in a mammalian mitotic extract, and the spontaneous assembly of NuMA into extensive structures upon dephosphorylation supports the conclusion that NuMA serves a structural function.
Saredi
A
A
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.
Howard
L
L
Compton
D A
DA
D000818:Animals; D034961:Antigens, Nuclear; D016466:CHO Cells; D018797:Cell Cycle Proteins; D006224:Cricetinae; D019084:Fluorescent Antibody Technique, Indirect; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D012995:Solubility; D008941:Spindle Apparatus / Q000378:metabolism* / Q000648:ultrastructure
D034961:Antigens, Nuclear; D018797:Cell Cycle Proteins; C050045:NUMA1 protein, human; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins