Identification of novel M phase phosphoproteins by expression cloning.
Using an expression cloning technique, we isolated cDNAs for eight M phase phosphoproteins (MPPs 4-11). We then used affinity-purified antibodies to fusion proteins to characterize the intracellular localization and some biochemical properties of these proteins and two others that we identified previously (MPPs 1-2). Each antibody immunoprecipitated one or two protein species of a characteristic size ranging from 17,000 to 220,000 Da. Each MPP, when immunoprecipitated from lysates of M phase cells, was reactive with MPM2, a monoclonal antibody that recognizes a group of related M phase phosphorylation sites, including F-phosphoT-P-L-Q. This reactivity indicated that all the MPPS encoded genuine M phase phosphoproteins. When antibodies to the MPPS were used for immunofluorescence microscopy, each anti-MPP gave a characteristic pattern of localization. In interphase, several of the MPPs were nuclear proteins, whereas others were cytoplasmic or distributed throughout the cell. Three MPPS were strikingly localized to interphase structures: MPP7 to centers of DNA replication, MPP9 to the Golgi complex, and MPP10 to nucleoli. In mitosis, most of the MPPs were distributed throughout the cells. Further studies of the 10 MPPs, most of which are previously undescribed, are expected to provide new understandings of the process of cell division.
Matsumoto-Taniura
N
N
Department of Cell Biology, Scripps Research Institute, La Jolla, California 92037, USA.
Pirollet
F
F
Monroe
R
R
Gerace
L
L
Westendorf
J M
JM
D000818:Animals; D000941:Antigens / Q000235:genetics; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D003001:Cloning, Molecular; D004261:DNA Replication; D018076:DNA, Complementary / Q000235:genetics / Q000276:immunology; D006056:Golgi Apparatus / Q000276:immunology / Q000378:metabolism; D006168:Guinea Pigs; D000882:Haplorhini; D006367:HeLa Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007106:Immune Sera; D007399:Interphase; D008938:Mitosis / Q000235:genetics* / Q000276:immunology*; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D013347:Subcellular Fractions
D000941:Antigens; D018076:DNA, Complementary; D007106:Immune Sera; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins