To understand how protein phosphorylation modulates cytoskeletal organization, we used immunofluorescence microscopy to examine the effects of okadaic acid, a serine/threonine protein phosphatase inhibitor, and taxol, a microtubule-stabilizing agent, on stable (acetylated and detyrosinated) microtubules, vimentin intermediate filaments and other cytoskeletal elements in CV-1 cells. Okadaic acid caused major changes in both stable microtubules and vimentin intermediate filaments, but through independent mechanisms. At 300 nM, okadaic acid caused apparent fragmentation and loss of stable microtubules which was not prevented by prior exposure to K252a. In contrast, major reorganization of vimentin intermediate filaments elicited at 750 nM okadaic acid was prevented by prior exposure to K252a. Taxol pretreatment blocked the effects of okadaic acid on stable microtubules and vimentin intermediate filaments. Recent reports have revealed that taxol can activate cellular signal transduction pathways in addition to its known ability to promote microtubule stabilization, so the possibility that taxol-induced resistance of vimentin intermediate filaments to okadaic acid was through a microtubule-independent mechanism involving direct phosphorylation of intermediate filament proteins was explored. Vimentin immunoprecipitation from cytoskeletal extracts from 32P-labeled cells revealed that taxol (4 microM, 1 or 2 hours) caused about a 2-fold increase in vimentin phosphorylation. This phosphorylation was recovered exclusively in cytoskeletal vimentin, in contrast to the increased phosphorylation of soluble and cytoskeletal vimentin caused by exposure to 750 nM okadaic acid. Phosphorylation of soluble and cytoskeletal vimentin from cells exposed to taxol (4 microM, 1 hour) then okadaic acid (750 nM, 1 hour) was comparable to taxol-treatment alone. These findings demonstrate a novel new activity of taxol, induction of vimentin phosphorylation, that may impact on vimentin organization and stability.

REFERENCES

Blagosklonny
M. V.
,
Schulte
T.
,
Nguyen
P.
,
Trepel
J.
,
Neckers
L. M.
(
1996
).
Taxol-induced apoptosis and phosphorylation of Bcl-2 protein involves c-Raf-1 and represents a novel c-Raf-1 signal transduction pathway.
Cancer Res
56
,
1851
1854
Blankson
H.
,
Holen
I.
,
Seglen
P. O.
(
1995
).
Disruption of the cytokeratin cytoskeleton and inhibition of hepatocytic autophagy by okadaic acid.
Exp. Cell Res
218
,
522
530
Bulinski
J. C.
,
Gundersen
G. G.
(
1991
).
Stabilization and post-translational modification of microtubules during cellular morphogenesis.
BioEssays
13
,
285
293
Chou
Y.-H.
,
Bischoff
J. R.
,
Beach
D.
,
Goldman
R. D.
(
1990
).
Intermediate filament reorganization during mitosis is mediated by p34cdc2 phosphorylation of vimentin.
Cell
62
,
1063
1071
Chou
Y.-H.
,
Ngai
K. L.
,
Goldman
R.
(
1991
).
The regulation of intermediate filament reorganization in mitosis, p34cdc2 phosphorylates vimentin at a unique N-terminal site.
J. Biol. Chem
266
,
7325
7328
Chou
Y.-H.
,
Opal
P.
,
Quinlan
R. A.
,
Goldman
R. D.
(
1996
).
The relative roles of specific N-and C-terminal phosphorylation sites in the disassembly of intermediate filament in mitotic BHK-21 cells.
J. Cell Sci
109
,
817
826
Draberova
E.
,
Draber
P.
(
1993
).
A microtubule-interacting protein involved in coalignment of vimentin intermediate filaments with microtubules.
J. Cell Sci
106
,
1263
1273
Eriksson
J. E.
,
Brautigan
D. L.
,
Vallee
R.
,
Olmsted
J.
,
Fujiki
H.
,
Goldman
R. D.
(
1992
).
Cytoskeletal integrity in interphase cells requires protein phosphatase activity.
Proc. Nat. Acad. Sci. USA
89
,
11093
11097
Fiorentini
C.
,
Matarrese
P.
,
Battorossi
A.
,
Donelli
F.
(
1995
).
Okadaic acid induces changes in the organization of F-actin in intestinal cells.
Toxicon
24
,
937
945
Gliksman
N. R.
,
Parsons
S. F.
,
Salmon
E. D.
(
1992
).
Okadaic acid induces interphase to mitotic-like microtubule dynamic instability by inactivating rescue.
J. Cell Biol
119
,
1271
1276
Goldman
R. D.
,
Khuon
S.
,
Chou
Y. H.
,
Opal
P.
,
Steinert
P. M.
(
1996
).
The function of intermediate filaments in cell shape and cytoskeletal integrity.
J. Cell Biol
134
,
971
983
Gurland
G.
,
Gundersen
G. G.
(
1993
).
Protein phosphatase inhibitors induce the selective breakdown of stable microtubules in fibroblasts and epithelial cells.
Proc. Nat. Acad. Sci. USA
90
,
8827
8831
Gurland
G.
,
Gundersen
G. G.
(
1995
).
Stable, detyrosinated microtubules function to localize vimentin intermediate filaments in fibroblasts.
J. Cell Biol
131
,
1275
1290
Haldar
S.
,
Jena
N.
,
Croce
C. M.
(
1995
).
Inactivation of Bcl-2 by phosphorylation.
Proc. Nat. Acad. Sci. USA
92
,
4507
4511
Haldar
S.
,
Chintapalli
J.
,
Croce
C. M.
(
1996
).
Taxol induces bcl-2 phosphorylation and death of prostrate cancer cells.
Cancer Res
56
,
1253
1255
Hamm-Alvarez
S. F.
,
Kim
P. Y.
,
Sheetz
M. P.
(
1993
).
Regulation of vesicle transport in CV-1 cells and extracts.
J. Cell Sci
106
,
955
966
Hamm-Alvarez
S. F.
,
Wei
X.
,
Berndt
N.
,
Runnegar
M.
(
1996
).
Protein phosphatases independently regulate vesicle movement and microtubule subpopulations in hepatocytes.
Am. J. Physiol
271
,
929
–.
Haystead
T. A.
,
Sim
A. T.
,
Carling
D.
,
Honnor
R. C.
,
Tsukitani
Y.
,
Cohen
P.
,
Hardie
D. G.
(
1989
).
Effects of the tumour promoter okadaic acid on intracellular protein phosphorylation and metabolism.
Nature
337
,
78
81
Hedberg
K. K.
,
Birrell
G. B.
,
Habliston
D. L.
,
Griffith
O. H.
(
1990
).
Staurosporine induces dissolution of microfilament bundles by a protein kinase C-independent pathway.
Exp. Cell Res
188
,
199
208
Heins
S.
,
Aebi
U.
(
1994
).
Making heads and tails of intermediate filament assembly, dynamics and networks.
Curr. Opin. Cell Biol
6
,
25
33
Hirano
K.
,
Hartshorne
D. J.
(
1993
).
Phosphorylation of vimentin in the C-terminal domain after exposure to calyculin-A.
Eur. J. Cell Biol
62
,
59
65
Ku
N.-O.
,
Liao
J.
,
Omary
M. B.
(
1997
).
Apoptosis generates stable fragments of human type I keratins.
J. Biol. Chem
272
,
33197
33203
Lee
W. C.
,
Yu
J. S.
,
Yang
S. D.
,
Lai
Y. K.
(
1992
).
Reversible hyperphosphorylation and reorganization of vimentin intermediate filaments by okadaic acid in 9L rat brain tumor cells.
J. Cell Biochem
49
,
378
393
MacRae
T. H.
(
1997
).
Tubulin post-translational modifications- enzymes and their mechanisms of action.
Eur. J. Biochem
244
,
265
278
Maier
G. D.
,
Wright
M. A.
,
Lozano
Y.
,
Djordjevic
A.
,
Matthews
J. P.
,
Young
M. R. I.
(
1995
).
Regulation of cytoskeletal organization in tumor cells by protein phosphatases 1 and 2A.
Int. J. Cancer
61
,
54
61
Mandelkow
E.
,
Mandelkow
E. M.
(
1995
).
Microtubules and microtubule-associated proteins.
Curr. Opin. Cell Biol
7
,
72
81
Pletjushkina
O. J.
,
Ivanova
O. J.
,
Kaverina
I. N.
,
Vasiliev
J. M.
(
1994
).
Taxol-treated fibroblasts acquire an epithelioid shape and a circular pattern of actin bundles.
Exp. Cell Res
212
,
201
208
Sontag
E.
,
Nunbhakdi-Craig
V.
,
Bloom
G. S.
,
Mumby
M. C.
(
1995
).
A novel pool of protein phosphatase 2A is associated with microtubules and is regulated during the cell cycle.
J. Cell Biol
128
,
1131
1144
Svitkina
T. M.
,
Verkhovsky
A. B.
,
Borisy
G. G.
(
1996
).
Plectin sidearms mediate interaction of intermediate filaments with microtubules and other components of the cytoskeleton.
J. Cell Biol
135
,
991
1007
Takai
T.
,
Ogawara
M.
,
Tomono
Y.
,
Moritoh
C.
,
Imajoh-Ohmi
S.
,
Tsutsumi
O.
,
Taketani
Y.
,
Inagaki
M.
(
1996
).
Mitosis-specific phosphorylation of vimentin by protein kinase C coupled with reorganization of intracellular membranes.
J. Cell Biol
133
,
141
149
Wolfson
M.
,
Yang
C. P.
,
Horwitz
S. B.
(
1997
).
Taxol induces tyrosine phosphorylation of Shc and its association with Grb2 in murine RAW 264.
7 cells. Int. J. Cancer
70
,
248
252
Yamashita
K.
,
Yasuda
H.
,
Pines
J.
,
Yasumoto
K.
,
Nishitani
H.
,
Ohtsubo
M.
,
Hunter
T.
,
Sugimura
T.
,
Nishimoto
T.
(
1990
).
Okadaic acid, a potent inhibitor of type 1 and type 2A protein phosphatases, activates cdc2/H1 kinase and transiently induces a premature mitosis-like state in BHK21 cells.
EMBO J
9
,
4331
4338
This content is only available via PDF.