1Department of Physiology, Joan C. Edwards School of Medicine, and 2Department of Chemistry, Marshall University, Huntington, West Virginia 25704
Submitted 7 November 2002 ; accepted in final form 4 March 2003
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ABSTRACT |
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cytoskeleton; protein kinase C-; translocation
There is evidence that the translocation of an individual PKC isoform may
be varied in a single cell type through use of different stimulatory agents
(18), whether highly
differentiated (7,
10,
14) or less highly
differentiated passaged (1)
cells are used and depending on cell culture conditions
(8). In addition, our
laboratory reported (16) that
PKC- may be selectively relocated at the plasmalemma or the perinucleus
of A7r5 smooth muscle cells, depending on the concentration of phorbol ester.
The A7r5 clonal cell line was chosen for these studies because it exhibits an
adult smooth muscle phenotype
(5). This cell line was
originally derived from embryonic rat aortic smooth muscle
(15) and shows expression and
promoter activity of several highly restricted smooth muscle cell markers
(5). In addition to these
indications of smooth muscle phenotype, A7r5 cells retain the ability to
contract by both Ca2+-dependent and -independent
mechanisms (6,
17,
20), which is lost in passaged
smooth muscle cells. Interestingly, our studies
(16) with A7r5 cells indicated
that translocation of PKC-
to the perinucleus but not the
subplasmalemma was blocked by the use of colchicine to disrupt cell
microtubules. These studies indicated that concentrations of phorbol
12,13-dibutyrate (PDBu)
107 M resulted in
colchicine-insensitive translocation of PKC-
to the subplasmalemma,
whereas PDBu concentrations >107 M caused
colchicine-sensitive relocation of the enzyme in the perinuclear region. By
comparison, concentrations of PDBu ranging from
109 to 105 M
resulted only in colchicine-sensitive translocation of PKC-
to the
perinuclear region of multipassaged cells derived from rat aortic smooth
muscle (1). These observations
suggest that in highly proliferative, dedifferentiated passaged smooth muscle
cells, only the colchicine-sensitive mechanism for perinuclear translocation
of PKC-
is operative or sensitive to PDBu activation, whereas high
concentrations of PDBu are required to unmask this mechanism in A7r5 cells.
Hence, it was concluded that multiple pathways are available for the
redistribution of active PKC-
in A7r5 cells that could utilize
different mechanisms for the movement or docking of the isoform at specific
target sites. However, primarily because of interference from the intensity of
PKC-
immunostaining, this early study was unable to show clear evidence
for a direct association of PKC-
with microtubules. Hence, the role of
the microtubular cytoskeleton in PKC-
localization at the perinucleus
after phorbol ester stimulation remained unclear.
In the present study, we investigated the association of PKC- with
microtubular structure in A7r5 smooth muscle cells stimulated with
106 M PDBu, the concentration previously
demonstrated to cause colchicine-sensitive PKC-
translocation to the
perinucleus. The results confirm earlier observations that perinuclear
translocation of PKC-
requires an intact microtubular cytoskeleton. The
results further indicate at least a partial association of PKC-
with
microtubules in the unstimulated and PDBu-stimulated cell.
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MATERIALS AND METHODS |
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Cell plating and treatment. A7r5 cells were plated onto 22 mm x 22 mm no. 1 glass coverslips placed in six-well culture plates and returned to the incubator for a minimum of 24 h to allow cell attachment and spreading. Cells were treated before fixation according to one of five protocols: 1) control cells received vehicle but no treatment before fixation; 2) PDBu cells were treated with 106 M PDBu (Sigma, St. Louis, MO) for up to 30 min; 3) control-colchicine cells were treated with 40 µg/ml colchicine (Sigma) for 30 min; 4) PDBu-colchicine cells were treated with 106 M PDBu for 30 min followed by treatment with 40 µg/ml colchicine for 30 min; and 5) colchicine-PDBu cells were treated with 40 µg/ml colchicine for 30 min followed by treatment with 106 M PDBu for 30 min. Cells were fixed and permeabilized by the addition of ice-cold acetone for 1 min. Fixed cells were preserved in 1x PBS buffer.
Confocal and fluorescence resonance energy transfer microscopy. In
preparation for experiments using laser scanning confocal microscopy, A7r5
cells were plated and fixed as described in Cell plating and
treatment. After fixation the cells were washed three times with PBS
containing 0.5% Tween 20 (PBS-T), pH 7.5, and incubated for 30 min in a
blocking solution (5% nonfat dry milk in PBS). Cells were stained for
-tubulin by incubation in a 1:500 dilution of monoclonal
anti-
-tubulin clone TUB 2.1 FITC-labeled antibody (Sigma) for a minimum
of 2 h at room temperature. For
-actin staining, fixed cells were
incubated with a 1:500 dilution of monoclonal anti-
-smooth muscle actin
clone 1A4 FITC-labeled antibody (Sigma) for 30 min at room temperature. For
total F-actin staining, cells were incubated with 2.5 x
106 M tetramethylrhodamine isothiocyanate
(TRITC)-labeled phalloidin (Sigma) for 30 min at room temperature. Cells were
stained for vimentin via incubation with a 1:300 dilution of monoclonal
anti-vimentin clone V9 (Sigma) at room temperature for 2 h. Fixed cells were
stained for PKC-
with a 1:500 dilution of anti-PKC-
-clone M4
(Upstate Biotechnology, Lake Placid, NY). Treatment with anti-vimentin and
anti-PKC-
solutions was followed by the addition of Alexa Fluor
488-labeled secondary antibody (Molecular Probes, Eugene, OR). In studies
using fluorescence resonance energy transfer (FRET) microscopy, Alexa Fluor
546 (Molecular Probes) was the secondary antibody to anti-PKC-
.
Stained cells were surveyed by mounting on a Nikon Diaphot microscope. Confocal microscopy was performed with a Bio-Rad model 1024 scanning system equipped with a krypton/argon laser. Cells stained with Alex Fluor 488 or FITC conjugates were excited with the 488-nm laser line at 10% power. The resulting emission was visualized with a 522 DF 32 band-pass filter. For colocalization studies, the Bio-Rad scanning system's multichannel capacity was engaged. When both an FITC conjugate and Alexa Fluor 594 were present, a HQ 598/40 band-pass filter was used to observe the secondary fluorophore simultaneously on a second channel. Similarly, multiple channels were necessary in FRET microscopy. Within a FRET system two fluorophores with overlapping emission and excitation spectra are implemented. Here FITC (excitation, 488 nm; emission, 520 nm) and Alexa Fluor 546 (excitation, 546 nm; emission, 580 nm) were the donor-acceptor pair used. The donor molecule (FITC) was directly excited, and the resulting emission was visualized with the 522 DF 32 band-pass filter. However, a portion of energy given off by the fluorophore is not released as light but is transferred to a neighboring fluorophore that can be excited at that wavelength. Thus the FITC emission induced the Alexa Fluor 546 molecule to release energy as light at its characteristic emission wavelength, which was visualized on a second channel with an HQ 598/40 band-pass filter. The resonance energy transfer can only occur if the donor and acceptor molecules are close enough to each other for the transfer to occur efficiently. The Forster distance (R0) is the distance at which fluorescence resonance energy transfer from the donor dye to the acceptor dye is 50%. Distances >R0 will result in less intense emission, whereas distances <R0 will yield higher intensities. The R0 calculated for this pair is 64 Å. Resultant images were analyzed with Lasersharp and Confocal Assistant software (Bio-Rad, Hercules, CA).
To calculate the distance between PKC- and
-tubulin, images of
cells were acquired with laser scanning confocal microscopy with excitation at
the 488-nm laser line. To obtain an internal control, the imaging area was
then excited at the 568-nm laser line at 100% power to photobleach the
acceptor molecule (PKC-
). Subsequently, a second set of images was
acquired again with 488-nm laser line excitation and the appropriate
multichannel filter was set to measure
-tubulin FITC emission (522 DF
32) and to verify the absence of anti-PKC-
secondary antibody Alexa
Fluor 546 emission (HQ598/40). The images were analyzed by two methods, the
determination of whole cell emission intensity and a single line scan emission
intensity measurement. By selecting the entire scan area, an intensity profile
was generated for each whole cell image [Image J Software, National Institutes
of Health (NIH)] and the resulting plot was analyzed with Peakfit v4.11
software (SPSS Science, Chicago, IL) to obtain the area under the curve. These
values were then used in calculating the efficiency of energy transfer
(E) and the FRET distance between the donor and acceptor pair
(r) with the following equations
(13)
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The resulting distances were analyzed by Sigma Stat 2.03 (SPSS Science) in
comparisons of values obtained for control cells, those stimulated with PDBu,
and colchicine-treated cells. Images analyzed by line scan emission intensity
were also evaluated with Image J. For each set of images
(ID, before photobleaching; IDNA,
after photobleaching), a line was arbitrarily drawn to bisect the cell
nucleus, extending to the visible peripheral borders of the cell. The
resulting plot profile was then used to evaluate two aspects of the
intracellular regional distribution of the association between -tubulin
and PKC-
. Initially, we compared the FRET distance in the perinuclear
vs. the peripheral region of the cell. This was accomplished by averaging the
two highest values within the 20-pixel distance of the nuclear mass and the
cell peripheral border. Second, we identified the point of maximum efficiency
of energy transfer (E) on the line scan as a focal area in which
PKC-
interaction with tubulin might approach saturating conditions.
Atomic force microscopy. In experiments using atomic force microscopy (AFM), fixed cells were rinsed with deionized water and dried with compressed air before study. Coverslips were mounted to 20-mm AFM metal specimen disks (TMMicroscopes, Sunnyvale, CA) with 12-mm carbon conductive tabs (Ted Pella, Redding, CA). Cells were imaged in contact mode with a TMMicroscopes Explorer atomic force microscope (2). All images were acquired with either TMMicroscopes model 1520-00 or model 1930-00 cantilevers. Subsequent analysis of images was performed with SPMLab version 5.01 analysis software (TMMicroscopes). On the basis of the whole cell scan of each cell, a line scan was performed bisecting the nucleus in an axis arbitrarily chosen to provide measurement outside the visible perimeter of the cell. The reference point for each cell height measurement was the default point assigned as zero by the SPMLab analysis software. Because the height of the nuclear sleeve was the object of study, and to avoid sampling bias, the measurement of cell height (untreated) or ground substance (colchicine treated) was obtained by using the whole cell scan to visually set measurement points on the sleeve rim or immediately adjacent to the nuclear mass, which were then identified for analysis on the line scan. The two measurements on opposite sides of the nucleus were then averaged to give a single value for height.
AFM measurements showed a well-defined, elevated structure surrounding the
nucleus (see Figs. 9 and
11) that was absent in
colchicine-treated cells (see Figs.
10 and
12). In light of these
observations it was concluded that the central network of microtubules formed
a sleevelike structure surrounding the nucleus. The sleeve diameter and
thickness were directly measured, and sleeve height was obtained by
subtracting the average cell height of colchicine-treated cells (ground
substance) from the cell height of individual untreated cells (see
Fig. 13). With these
measurements the average volume (V) of the microtubular sleeve was then
determined with the formula for calculation of the volume of a hollow cylinder
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Statistics. Differences in FRET distances and AFM-obtained topographic measurements were analyzed by one- or two-way ANOVA followed by Student's t-test (Sigma Stat 2.03, SPSS Science). Differences were considered significant if P < 0.05 in all cases. Data are presented as means ± SE throughout the text.
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RESULTS |
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Figure 2 shows the
structural distribution of the microtubular (-tubulin), actin
(
-actin, phalloidin), and intermediate filament (vimentin) components
of the cytoskeleton in unstimulated (control) and PDBu-activated A7r5 cells.
Unlike the distribution of actin filaments, the dense network of microtubular
and intermediate filament structure surrounding the nucleus provided an early
suggestion of these two filament types as candidates for a role in PKC-
localization. Dual immunostaining suggested a significant degree of
colocalization of microtubules and PKC-
in both control and
PDBu-stimulated cells but particularly in the perinuclear region of the
PDBu-treated cells (Fig. 3).
The conclusion of tubulin-PKC-
interaction was strengthened by FRET
microscopic evaluation of whole cell scans that indicated an association
between microtubules and PKC-
in both stimulated and unstimulated cells
as well as colchicine-treated control and PDBu-stimulated cells
(Fig. 4). These images further
suggested that the tubulin-PKC-
association was largely confined to the
central region of cells having an intact microtubular system.
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Consistent with results from whole cell emission scans, FRET analysis of
line scans from control (Fig.
5) and PDBu-stimulated (Fig.
6) cells indicated a heterogeneous distribution of
tubulin-PKC- association with evidence of high levels of efficiency of
energy transfer (E) in the perinuclear region compared with that at
the periphery of the cell.
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The calculation of FRET distances from whole cell emission scans yielded
similar values among control, PDBu-stimulated, and colchicine-treated groups
(Table 1), supporting the
conclusion of significant tubulin-PKC- association in cells both before
and after disruption of microtubular structure. Furthermore, FRET distances
were significantly increased in the periphery compared with the perinucleus in
both control and PDBu-stimulated cells, verifying visual observations of
differences in the regional distribution of tubulin-PKC-
interaction
within the cell. Interestingly, the FRET distances calculated for points of
peak E obtained from each line scan
(Table 1) were very similar
among the different groups (60.1 ± 0.4 Å) and fell at the upper
end of the range of measurements reported between known loci on actin and
myosin (22). These latter
findings further suggest the occurrence of discrete "hot spots" of
tubulin-PKC-
interaction within the cell.
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Because colchicine caused dispersal of PKC- from the perinuclear
region in cells preactivated with PDBu
(Fig. 1), it was of interest to
evaluate the effect of the drug on the major cytoskeletal elements. Colchicine
treatment had no detectable effect on actin microfilaments or on intermediate
filaments (Fig. 7), greatly
reducing the likelihood of their involvement in PKC-
localization. By
comparison, the drug caused the complete disruption of all but the very
peripheral microtubular structure within 20 min after addition to the medium
(Fig. 8). Interestingly, the
colchicine-induced dissolution of microtubules was clearly observed to occur
at variable rates in different regions of the cell. The organization of
microtubules was consistent among the great majority of A7r5 cells imaged,
forming a dense network about the nucleus from which cables of microtubules
radiated to a second dense network at the subplasmalemma. On treatment with
colchicine, we invariably noted initial dissolution of the central network,
which was generally complete before loss in structural integrity of radiating
cables or peripheral structure was noticeable.
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Topographic imaging of fixed cells by AFM revealed a well-defined, elevated
structure surrounding the nucleus (Fig.
9), which was absent after treatment with colchicine
(Fig. 10). A similar analysis
of the PDBu-activated cell (Figs.
11 and
12) indicated that the
contraction of the cell significantly altered the dimensions and increased the
volume of this structure by roughly fivefold
(Fig. 13). The results suggest
that the dense network of microtubules at the center of the cell forms a
sleevelike structure that, given the enhanced sensitivity to colchicine and
the regional association with PKC-, may represent a more dynamic and
specialized subgroup of microtubules. The significant change in the dimensions
and volume of the microtubular sleeve could reflect compression of the
structure during cell contraction or an artifact of sample preparation.
Alternatively, these results could be suggesting active polymerization and an
increase of microtubular structure in the central region of the activated A7r5
cell.
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DISCUSSION |
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It is noteworthy that both confocal imaging
(Fig. 3) and FRET analysis
(Fig. 4) indicate a
heterogeneous distribution of PKC- in association with microtubules.
Visual inspection of whole cell images suggests that the interaction is
primarily confined to the central region, whereas the microtubular structure
at the cell periphery appears devoid of PKC-
. FRET measurements
(Table 1) verified a
predominance of tubulin-PKC-
interaction in the region immediately
adjacent to the nucleus and further indicated the existence of discrete focal
points of intense resonance energy transfer. The FRET distances calculated for
these hot spots of maximal tubulin-PKC-
interaction are within the
range of values reported for other intermolecular interactions
(22) and could be indicating
the direct binding of PKC-
with tubulin. However, the magnitude of the
FRET distance recorded could also be consistent with the association of
-tubulin and PKC-
via a linker protein. In view of the highly
restricted compartmentalization of the tubulin-PKC-
interaction, we
consider it unlikely that PKC-
is constitutively bound to tubulin. It
must be emphasized that the results do not allow quantitation of
PKC-
-microtubule association, and the possibility remains that a
portion of PKC-
is associated with other structures in the perinuclear
region. Together, however, the results suggest that the microtubules could act
as a scaffold for PKC-
binding. This function is primarily confined to
the perinuclear microtubular structure and occurs through an interaction of
PKC-
with microtubule-associated compounds.
In comparison with other cytoskeletal components, the microtubules of the A7r5 cell form a unique structural configuration, as seen with confocal microscopy (Fig. 2). A dense network about the nucleus is connected by thick cables radiating across the cell body to a dense network of microtubules lying under the plasmalemma. Interestingly, a highly reproducible sequence of colchicine-induced dissolution of microtubular structure was observed in these various regions of the cell (Fig. 8). There was initial loss of the central perinuclear network with complete disassembly realized by 5 min after colchicine addition. By comparison, significant loss of radiating cables occurred at 1020 min, whereas the subplasmalemma network of microtubules was often at least partially intact after 20 min of colchicine treatment. Because colchicine is thought to prevent polymerization by binding tubulin subunits, the high level of colchicine sensitivity suggests that microtubular structure in the perinuclear region is more dynamically robust than in other regions of the cell.
Topographic analysis of fixed cells by AFM indicated that the perinuclear
network of microtubules formed an elevated sleevelike structure enveloping the
nucleus (Figs. 9 and
10). A similar evaluation of
PDBu-activated cells indicated that the dimensions of the perinuclear sleeve
were significantly altered during cell shortening (Figs.
11 and
12). By using measurements of
height, thickness, and diameter (Fig.
13), we calculated the volume of the sleeve assuming a form
roughly the shape of a hollow cylinder. This estimate indicated that the
volume of the microtubular sleeve was 4.9 µm3 in control
cells and 25.5 µm3 in cells treated with PDBu. Differences in
microtubular volume between control and PDBu-stimulated cells could reflect an
artifact of colchicine treatment or sample preparation for imaging.
Alternatively, the fivefold increase observed in PDBu-treated cells could
represent an increase in microtubular mass in the activated cells. As noted in
RESULTS, the interaction between PKC-
and
-tubulin was
retained after treatment with colchicine, suggesting that PKC-
may
associate with monomeric or small aggregates of tubulin in the cytosol. The
possibility occurs that such a tubulin-PKC-
complex is translocated to
the perinucleus during expansion of the microtubular structure in this region
during cell activation.
Activation of PKCs has been shown to result in changes in cytoskeletal
structure in association with the phosphorylation of an array of cytoskeletal
proteins (12). It is well
documented that PKCs may interact with each of the cytoskeletal components,
with some evidence to suggest increased activity in the activated cell
(9). Hence, there is a large
literature on the role of PKCs in cytoskeletal regulation. However, the great
bulk of research has focused on regulation of actin microfilaments
(12) and relatively little is
known of the role of PKC in microtubule dynamics. Kabir et al.
(11) reported the interesting
finding that activation of PKC by phorbol esters in Aplysia bag cell
neurons in primary cell culture resulted in a doubling in the length of a
typical microtubule growth episode while increasing and decreasing rescue and
catastrophe frequencies, respectively. They concluded that PKC may play an
important role in regulating cellular processes involving directed
microtubular growth. This group subsequently showed
(21) phorbol ester-induced
translocation of PKCs to microtubules, suggesting a direct role of these
kinases in the regulation of distal microtubular advance in neural growth
cones. Their findings open the possibility that the translocation of
PKC- to the perinuclear microtubules in A7r5 cells could serve a
regulatory function directed toward microtubular growth and stability in this
region of the cell. This function, in turn, would appear consistent with
increased dynamic activity evidenced by colchicine sensitivity and the
increase in volume of perinuclear microtubular structure in the present study.
Alternatively, the translocation of PKC-
could reflect a specialized
role of perinuclear microtubules as a scaffold for interaction of regulatory
molecules influencing a variety of cell functions in the central region of the
cell. There is evidence from a variety of cell types suggesting a link between
PKC-mediated function and the capacity for microtubule reorganization. For
example, the prevention of microtubular depolymerization with taxol blocked
PKC (phorbol ester)-induced NF-
B activation in murine NIH/3T3 cells
(24) while causing
hyperphosphorylation of vimentin and reorganization of the intermediate
filament cytoskeleton via a PKC signaling pathway in 9L rat brain tumor cells
(3). Others have proposed that
microtubule catastrophe may be obligatory for PKC (phorbol ester)-mediated
MCL1 gene expression in the MCL1 human myeloblastic leukemia cell line
(25). As a matter of
speculation, a dynamic microtubular scaffold could serve to compartmentalize
regulatory interactions while providing for rapid and potentially directional
expansion during growth of structure and reduction of interaction or
compartmentalized release of compounds during catastrophe and loss of
structure.
In summary, the results confirm earlier conclusions that the microtubular
cytoskeleton is required for PDBu-induced localization of PKC- at the
perinuclear region in A7r5 smooth muscle cells. The results suggest that this
is due to an association of activated PKC-
with microtubule-associated
compounds confined to the microtubules in this region of the cell. In addition
to the regional limitation of PKC-
localization, the distribution of
microtubular structure in the cell and differences in colchicine sensitivity
suggest that microtubules in the perinuclear region represent a distinct
subpopulation that may differ in their dynamic growth characteristics from
those in other regions. PKC could be an important factor in determining these
characteristics. Alternatively, the perinuclear microtubules could serve as a
scaffold for interaction of regulatory proteins at the cell center.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Binnig G, Quate CF, and Gerber C. Atomic force microscope. Phys Rev Lett 56: 930933, 1986.[ISI][Medline]
3. Chu JJ, Chen KD, Lin YL, Fei CY, Chiang AS, Chiang CD, and Lai YK. Taxol induces concomitant hyperphosphorylation and reorganization of vimentin intermediate filaments in 9L rat brain tumor cells. J Cell Biochem 68: 472483, 1998.[ISI][Medline]
4. Dekker LV and Parker P. Protein kinase Ca question of specificity. Trends Biochem Sci 19: 7377, 1994.[ISI][Medline]
5. Firulli AB, Han D, Kelley-Roloff L, Kateliansky VE, Schwartz SM, Olsen EN, and Miano JM. Comparative molecular analysis of four rat smooth muscle cell lines. In Vitro Cell Dev Biol Anim 34: 217226, 1998.[ISI][Medline]
6. Fultz ME, Li C, Geng W, and Wright GL. Remodeling in the contracting A7r5 smooth muscle cell. J Muscle Res Cell Motil 21: 775787, 2000.[ISI][Medline]
7. Haller H,
Lindschau C, Maasch C, Olthoff H, Kurschied D, and Luft FC.
Integrin-induced protein kinase C translocation to focal adhesions mediates
vascular smooth muscle spreading. Circ Res
82: 157165,
1998.
8. Haller H, Smallwood JI, and Rasmussen H. Protein kinase C translocation in intact vascular smooth muscle strips. Biochem J 270: 375381, 1990.[ISI][Medline]
9. Jaken S. PKC interacts with intracellular components. In: Protein Kinase C. Current Concepts and Future Perspectives, edited by Lester DS and Epand RM. New York: Ellis Horwood, 1992, p. 237254.
10. Jensen PE, Gong MC, Somlyo AV, and Somlyo AP. Separate upstream and convergent downstream pathways of G-protein- and phorbol ester-mediated Ca2+ sensitization of myosin light chain phosphorylation in smooth muscle. Biochem J 318: 469475, 1996.[ISI][Medline]
11. Kabir N,
Schaefer AW, Nakhost A, Sossin WS, and Forscher P. Protein kinase C
activation promotes microtubule advance in neuronal growth cones by increasing
average microtubule growth life times. J Cell Biol
152: 10331044,
2001.
12. Keenan C and Kelleher D. Protein kinase C and the cytoskeleton. Cell Signal 10: 225232, 1998.[ISI][Medline]
13. Kenworthy AK. Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy. Methods 24: 289296, 2001.[ISI][Medline]
14. Khalil RA,
Lajoie C, and Morgan KG. In situ determination of
[Ca2+]i threshold for translocation of the
alpha protein kinase C isoform. Am J Physiol Cell
Physiol 266:
C1544C1551, 1994.
15. Kimes BW and Brandt BL. Characterization of two putative smooth muscle cell lines from rat thoracic aorta. Exp Cell Res 98: 349366, 1976.[ISI][Medline]
16. Li C, Fultz ME,
Geng W, Ohno S, Norton M, and Wright GL. Concentration-dependent phorbol
stimulation of PKC at the nucleus or subplasmalemma in A7r5 cells.
Pflügers Arch 443:
3847, 2001.[ISI][Medline]
17. Li C, Fultz ME, Parkash J, Rhoten WB, and Wright GL. Ca2+-dependent remodeling in the contracting A7r5 cell. J Muscle Res Cell Motil 22: 521534, 2001.[ISI][Medline]
18. Li C, Fultz ME,
and Wright GL. PKC shows variable patterns of translocation in
response to different stimulatory agents. Acta Physiol
Scand 174:
237246, 2002.[ISI][Medline]
19. Mochly-Rosen D. Localization of protein kinases by anchoring proteins: a theme in signal transduction. Science 268: 247251, 1995.[ISI][Medline]
20. Nakajima S,
Fujmoto M, and Veda M. Spatial changes of
[Ca2+]i and contraction caused by phorbol
esters in vascular smooth muscle. Am J Physiol Cell
Physiol 265:
C1138C1145, 1993.
21. Nakhost A,
Kabir N, Forscher P, and Sossin WS. Protein kinase C isoforms are
translocated to microtubules in neurons. J Biol Chem
277: 4063340639,
2002.
22. Dos Remedios CG, Miki M, and Barden JA. Fluorescence resonance energy transfer measurements of distances in actin and myosin. A critical evaluation. J Muscle Res Cell Motil 8: 97117, 1987.[ISI][Medline]
23. Schmaltz D,
Kalkbrenner F, Hucho F, and Buchner K. Transport of protein kinase C alpha
to the nucleus requires an intact cytoskeleton while the transport of a
protein containing a canonical nuclear localization signal does not.
J Cell Sci 109:
24012406, 1996.
24. Spencer W, Kwon H, Crepieux P, Leclerc N, Lin R, and Hiscott J. Taxol selectively blocks microtubule dependent NF-kappa B activation by phorbol ester via inhibition of I kappa B alpha phosphorylation and degradation. Oncogene 18: 495505, 1999.[ISI][Medline]
25. Townsend KJ, Trusty JL, Traupman MA, Eastman A, and Craig RW. Expression of the antiapoptotic MCL1 gene product is regulated by a mitogen activated protein kinase-mediated pathway triggered through microtubule disruption and protein kinase C. Oncogene 17: 12231234, 1998.[ISI][Medline]