Protein phosphatase inhibitors induce the selective breakdown of stable microtubules in fibroblasts and epithelial cells.
In many cell types, a small subset of microtubules (MTs) are unusually long-lived compared with the majority of the MTs. These "stable" MTs may be important mediators of differentiative events since they are usually found aligned with developing asymmetries of cells undergoing morphogenesis. In addition to their longevity, the stable MTs are more resistant to drug depolymerization and are enriched in post-translationally detyrosinated tubulin (Glu-tubulin). To determine the role of protein phosphorylation in the regulation of these stable MTs, we treated NIH 3T3 fibroblasts and TC-7 monkey kidney epithelial cells with okadaic acid (OA) and calyculin A, potent inhibitors of protein phosphatases 1 and 2A (PP1 and PP2A), and then localized dynamic MTs and stable MTs with antibodies specific for tyrosinated tubulin (Tyrtubulin) and Glu-tubulin, respectively. OA at 0.1-10 microM caused a rapid and complete breakdown of Glu-MTs (MTs enriched in Glu-tubulin) in both cell types without substantially affecting the number of Tyr-MTs. While all concentrations of OA over this range resulted in a complete loss of Glu-MTs, the onset of Glu-MT breakdown was proportional to the logarithm of the OA concentration. The inactive analog of OA, 1-norokadaone, had no effect at any concentration. Calyculin A also caused a selective loss of Glu-MTs but was effective at 10 nM, consistent with its more potent inhibition of PP1. That the loss of Glu-MTs reflected the loss of stable MTs from the cells was shown by the absence of nocodazole-resistant MTs in OA-treated cells. OA did not appear to activate a MT-severing activity, since no MT fragments were observed after OA treatment of cells pretreated with taxol. These results suggest that PP1 and perhaps PP2A are involved in the regulation of MT stability in cells and show that the dynamic and stable subsets of MTs are regulated differentially by protein phosphorylation.
Gurland
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Department of Pathology, Columbia University, College of Physicians and Surgeons, New York, NY 10032.
Gundersen
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GG
D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D004848:Epithelium / Q000187:drug effects / Q000648:ultrastructure; D004988:Ethers, Cyclic / Q000633:toxicity*; D005347:Fibroblasts / Q000187:drug effects / Q000648:ultrastructure; D007668:Kidney; D007700:Kinetics; D008387:Marine Toxins; D051379:Mice; D008870:Microtubules / Q000187:drug effects* / Q000648:ultrastructure; D015739:Nocodazole / Q000633:toxicity*; D019319:Okadaic Acid; D010080:Oxazoles / Q000633:toxicity*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D013997:Time Factors; D014404:Tubulin / Q000187:drug effects / Q000378:metabolism
D004988:Ethers, Cyclic; D008387:Marine Toxins; D010080:Oxazoles; D014404:Tubulin; D019319:Okadaic Acid; C059041:calyculin A; D010749:Phosphoprotein Phosphatases; D015739:Nocodazole