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MPM-2 antibody-reactive phosphorylations can be created in detergent-extracted cells by kinetochore-bound and soluble
L Renzi1, M S Gersch, M S Campbell
1Department of Cell Biology, University of Virginia, Charlottesville 22908, USA.
Abstract:
The MPM-2 antibody labels mitosis-specific and cell cycle-regulated phosphoproteins. The major phosphoproteins of mitotic chromosomes recognized by the MPM-2 antibody are DNA topoisomerase II (topoII) alpha and beta. In immunofluorescence studies of PtK1 cytoskeletons, prepared by detergent lysis in the presence of potent phosphatase inhibitors, the MPM-2 antibody labels phosphoproteins found at kinetochores, chromosome arms, midbody and spindle poles of mitotic cells. In cells extracted without phosphatase inhibitors, labeling of the MPM-2 antibodies at kinetochores is greatly diminished. However, in cytoskeletons this epitope can be regenerated through the action of kinases stably bound at the kinetochore. Various kinase inhibitors were tested in order to characterize the endogenous kinase responsible for these phosphorylations. We found that the MPM-2 epitope will not rephosphorylate in the presence of the broad specificity kinase inhibitors K-252a, staurosporine and 2-aminopurine. Several other inhibitors had no effect on the rephosphorylation indicating that the endogenous MPM-2 kinase at kinetochores is not p34cdc2, casein kinase II, MAP kinase, protein kinase A or protein kinase C. The addition of N-ethylmaleimide inactivated the endogenous kinetochore kinase; this allowed testing of several purified kinases in the kinetochore rephosphorylation assay. Active p34cdc2-cyclin B, casein kinase II and MAP kinase could not generate the MPM-2 phosphoepitope. However, bacterially expressed NIMA from Aspergillus and ultracentrifuged mitotic HeLa cell extract were able to catalyze the rephosphorylation of the MPM-2 epitope at kinetochores. Furthermore, fractionation of mitotic HeLa cell extract showed that kinases that create the MPM-2 epitope at kinetochores and chromosome arms are distinct. Our results suggest that multiple kinases (either soluble or kinetochore-bound), including a homolog of mammalian NIMA, can create the MPM-2 phosphoepitope. The kinetochore-bound kinase that catalyzes the formation of the MPM-2 phosphoepitope may play an important role in key events such as mitotic kinetochore assembly and sister chromatid separation at anaphase.
Insights
The MPM-2 antibody identifies cell cycle-specific phosphoproteins, primarily DNA topoisomerase II. Kinetochore-bound kinases, potentially including a NIMA homolog, regenerate the MPM-2 epitope, crucial for mitosis.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The MPM-2 antibody recognizes mitosis-specific and cell cycle-regulated phosphoproteins.
- Key phosphoproteins targeted by MPM-2 include DNA topoisomerase II alpha and beta in mitotic chromosomes.
Purpose of the Study:
- To characterize the endogenous kinase responsible for phosphorylating MPM-2 epitopes at kinetochores.
- To identify kinases capable of regenerating the MPM-2 epitope in vitro.
Main Methods:
- Immunofluorescence studies using MPM-2 antibody on PtK1 cytoskeletons.
- Inhibition assays with various kinase inhibitors and N-ethylmaleimide.
- Rephosphorylation assays using purified kinases and cell extracts.
Main Results:
- MPM-2 epitope labeling at kinetochores diminishes without phosphatase inhibitors but can be regenerated by endogenous kinases.
- Broad specificity kinase inhibitors and specific kinases like p34cdc2, CKII, and MAPK could not regenerate the epitope.
- Bacterially expressed NIMA and mitotic HeLa cell extracts successfully regenerated the MPM-2 epitope, indicating distinct kinases for kinetochores and chromosome arms.
Conclusions:
- Multiple kinases, including a mammalian NIMA homolog, can generate the MPM-2 phosphoepitope.
- A kinetochore-bound kinase is likely involved in mitotic kinetochore assembly and sister chromatid separation.