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Okadaic acid inhibits a protein phosphatase activity involved in formation of the mitotic spindle of GH 4 rat pituitary cells
Author(s) -
Van Dolah Frances M.,
Ramsdell John S.
Publication year - 1992
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1041520124
Subject(s) - okadaic acid , phosphatase , mitosis , biology , microbiology and biotechnology , biochemistry , chemistry , phosphorylation
Abstract Okadaic acid, a selective inhibitor of serine/threonine protein phosphatases, was utilized to investigate the requirement for phosphatases in cell cycle progression of GH 4 rat pituitary cells. Okadaic acid inhibited GH 4 cell proliferation in a concentration‐dependent manner with a half‐maximal inhibition (IC 50 ) of approximately 5 nM. Treatment of GH 4 cells with 10 nM okadaic acid resulted in a 40–60% decrease in phosphatase activity and an increase in the proportion of phosphorylated retinoblastoma (RB) protein. Cell cycle analysis indicated that okadaic acid increased the percentage of cells in G 2 ‐M, decreased proportionally the percentage of cells in G 1 phase, and had little effect on the percentage of cells in S‐phase. The absence of a change in the proportion of S‐phase cells indicates that G 1 ‐specific phosphatases responsible for dephosphorylation of RB protein were not inhibited by 10 mM okadaic acid. Mitotic index revealed that 10 nM okadaic acid decreased proliferation of GH 4 cells specifically by slowing the progression through mitosis. Immunostaining with anti‐tubulin demonstrated that 10 nM okadaic acid‐treated mitotic cells contained mitotic spindles; however, the spindle apparatus in these cells frequently contained multiple poles. These results suggest that the organization of spindle microtubules during prometaphase requires a protein phosphatase that is sensitive to nanomolar concentrations of okadaic acid. Chromosomes in 10 nM okadaic acid‐treated cells appear to be attached to spindle microtubules and the nuclear envelope is absent. The effects of okadaic acid on the spindle differ from those elicited by the calcium channel blocker, nimodipine, indicating that this okadaic acid sensitive phosphatase is not part of the calcium signalling events which participate in mitotic progression. © 1992 Wiley‐Liss, Inc.