CELL-CYCLE-REGULATED LOCALIZATION OF TYROSINE AND THREONINE PHOSPHOEPITOPES AT THE KINETOCHORES OF MITOTIC CHROMOSOMES

We have detected novel phosphotyrosine epitopes at the kinetochores of mitotic chromosomes in rat kangaroo PtK1 and mouse P388D1 tissue cult ure cells. Immunofluorescence labeling of detergent-resistant cytoskel etons reveals that these phosphotyrosine epitopes are tightly bound at the centrosomes and kinetochores of mitotic cells. These phosphoepito pes are found at the kinetochores during only prophase and prometaphas e. Inclusion of a mixture of phosphatase inhibitors in the cell extrac tion procedure was necessary to preserve these previously undetected p hosphotyrosine epitopes. The use of the phosphatase inhibitor mixture also improved the detection of the centrosome and kinetochore antigens recognized by the monoclonal antibody MPM-2. The MPM-2 antibody label s a subset of phosphothreonine-containing antigens found primarily dur ing M phase. Ultrastructural immunolabeling studies indicated that bot h the phosphotyrosine and the MPM-2 phosphoepitopes were contained in both the outer and the inner dense plaques of the kinetochore, We deve loped large-scale chromosome isolation procedures designed to maintain chromosome protein phosphorylation. Immunoblot analysis revealed that the phosphotyrosine and MPM-2 antibodies recognized a number of chrom osomal proteins, some of which were concentrated in the chromosome sca ffold fraction prepared by nuclease digestion and salt extraction of w hole chromosomes. The strictly regulated appearance of the phosphotyro sine and MPM-2 epitopes at the kinetochores of chromosomes during vari ous stages of mitosis suggests that these phosphoepitopes may be invol ved in signal transduction pathways controlling kinetochore assembly a nd function during mitosis. (C) 1995 Academic Press, Inc.