Threonine phosphorylations induced by RX-871024 and insulin
secretagogues in
TC6-F7 cells
Jie
An1,
Genshi
Zhao2,
Lisa M.
Churgay3,
John J.
Osborne1,
John E.
Hale3,
Gerald W.
Becker3,
Gerald
Gold1,
Lawrence E.
Stramm1, and
Yuguang
Shi1
1 Endocrine Research,
2 Infectious Disease, and
3 Research Technology and
Proteins, Lilly Research Laboratories, Indianapolis, Indiana 46285
 |
ABSTRACT |
Treatment of the pancreatic
-cell line
TC6-F7 with an imidazoline compound, RX-871024, KCl, or tolbutamide
resulted in increased threonine phosphorylation of a 220-kDa protein
(p220) concurrent with enhanced insulin secretion, which can be
partially antagonized by diazoxide, an ATP-sensitive potassium
(KATP) channel activator. Although phosphorylation of p220 was regulated by cytoplasmic free
calcium concentration
([Ca2+]i),
membrane depolarization alone was not sufficient to induce phosphorylation. Phosphorylation of p220 also was not directly mediated
by protein kinase A, protein kinase C, or insulin exocytosis. Analysis
of subcellular fractions indicated that p220 is a hydrophilic protein
localized exclusively in the cytosol. Subsequently, p220 was purified
to homogeneity, sequenced, and identified as nonmuscle myosin heavy
chain-A (MHC-A). Stimulation of threonine phosphorylation of nonmuscle
MHC-A by KCl treatment also resulted in increased phosphorylation of a
40-kDa protein, which was coimmunoprecipitated by antibody to MHC-A.
Our results suggest that both nonmuscle MHC-A and the 40-kDa protein
may play roles in regulating signal transduction, leading to insulin secretion.
imidazoline; free calcium concentration; nonmuscle
myosin
 |
INTRODUCTION |
IN PANCREATIC
-CELLS, signal
transduction involved in insulin secretion is a complex process
encompassing the integration and transmission of a number of metabolic,
hormonal, and neuronal signals to the exocytotic machinery (14, 31, 33,
44). Depolarization of the plasma membrane as a result of closure of the ATP-sensitive potassium channel
(KATP) leads to the opening of
the voltage-dependent calcium channel (VDCC) (4) and the increase of
the free cytosolic Ca2+
concentration
([Ca2+]i),
which in turn triggers insulin release (4, 44). Several classes of
antihyperglycemic agents, such as imidazolines and sulfonylureas, have
been shown to affect
[Ca2+]i.
Sulfonylurea compounds, which are widely used to treat type II
diabetes, stimulate insulin secretion primarily by inhibiting the
KATP channels in the plasma
membrane. By comparison, the molecular targets underlying
imidazoline-stimulated insulin secretion have not been clearly
identified. In addition to blocking of
KATP channels and increasing
[Ca2+]i,
imidazolines also have been shown to potentiate glucose-stimulated insulin secretion in depolarized
-cells (9, 46).
Whereas Ca2+ is an important
second messenger that couples membrane depolarization to insulin
secretion, the intervening steps that link
[Ca2+]i
elevation to insulin secretion have not been well characterized. Protein kinase A (PKA), protein kinase C (PKC), and
Ca2+/calmodulin (CaM) kinase are
considered as candidates that convey signals necessary for
stimulus-secretion coupling (8). PKC and PKA have been suggested to
play multiple roles in
Ca2+-dependent and
Ca2+-independent insulin secretion
pathways, both by generating second messengers and by targeting the
exocytotic machinery (1, 2, 22, 44). PKC has also been implicated in
sulfonylurea- and imidazoline-stimulated insulin secretion by
modulating Ca2+-independent
pathways (11, 46).
Microtubule proteins and associated kinases also may play a role in
insulin secretion (25, 32, 38). For instance,
Ca2+-dependent phosphorylation of
a microtubule-associated protein (MAP-2) and phosphorylation of myosin
light chains were reported to be involved in insulin secretion and in
release of other hormones and neurotransmitters (6, 16, 17, 24,
26-28). Whereas phosphorylation of vertebrate nonmuscle myosin
heavy chain (MHC) has been demonstrated both in vivo and in vitro (12,
23, 29, 30, 35, 40, 42, 43), little is known about the regulation and
functional significance of such phosphorylation. In lower eukaryotic
organisms, MHC participates in diverse cellular processes including
cell motility, cytokinesis, morphogenesis, and development (21, 39, 41,
45).
In this study, we identified and characterized a
threonine-phosphorylated protein (p220), which was subsequently
purified and identified as nonmuscle MHC-A. We also showed that
stimulation of
TC6-F7 cells with imidazoline and other insulin
secretagogues resulted in increased threonine phosphorylation of
nonmuscle MHC-A, which occurred distal to the calcium channel
activation and proximal to exocytosis. In addition, we demonstrated a
time-dependent phosphorylation of a 40-kDa protein that associated with
nonmuscle MHC-A in response to KCl stimulation.
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MATERIALS AND METHODS |
Cell culture and chemical treatment.
TC6-F7 cells were maintained in Dulbecco's modified Eagle's medium
(DMEM) containing 25 mM glucose (GIBCO-BRL), 15% heat-inactivated horse serum (Hyclone), and 2.5% fetal bovine serum (Hyclone) at 37°C with 10% CO2-90% air.
Unless mentioned otherwise, 6.5 × 106
TC6-F7 cells (passage
50-55) were plated into 60-mm cell culture dishes. For
experiments, cells were washed twice with phosphate-buffered saline
(PBS) and then exposed to compounds dissolved in DMEM without glucose,
because pilot experiments showed that there was no difference in p220
threonine phosphorylation when cells were treated with compounds in
DMEM with or without glucose (data not shown). After treatment, cells
were washed once with PBS and proteins were extracted in 200 µl of
boiling lysis buffer (1% SDS, 1 mM sodium vanadate, 1 µM okadaic
acid, and 10 mM Tris, pH 7.4). At least two independent experiments
were done with each compound.
SDS-PAGE and Western blot analysis.
Proteins from lysed cells were boiled for 5 min in lysis buffer and
centrifuged to remove insoluble material. The protein concentration of
the supernatant was determined using bicinchoninic acid (Pierce). A
4-20% gradient Tris-glycine polyacrylamide SDS gel (Novex) was
loaded with 25 µg of total protein per well and run at 125 V for 3 h
in running buffer containing 0.1% SDS, 192 mM glycine, and 25 mM Tris
(pH 8.3) (Novex). Proteins were then transferred to a polyvinylidene
difluoride (PVDF) membrane, and the membrane was blocked for 1 h with a
TBST buffer (25 mM Tris, pH 7.5, 137 mM NaCl, 2.6 mM KCl, 0.1%
Tween-20, and 0.2% casein). The blot was then incubated for 1 h with
1-2 µg/ml rabbit anti-phosphotyrosine, anti-phosphoserine, or
anti-phosphothreonine antibody (Zymed) in the TBST. After three 5-min
washes with the TBST buffer, the PVDF blot was incubated for 1 h with
0.3-0.5 µg/ml horseradish peroxidase-conjugated goat anti-rabbit
IgG (Zymed) in TBST. After three 5-min washes with the TBST buffer,
proxidase activity was detected using the enhanced chemiluminescence
system (Amersham Life Science).
Insulin secretion assay.
After
TC6-F7 cells were treated with 3 ml of media containing the
specified compound, 1 ml of the solution was collected and centrifuged
at 10,000 rpm for 3 min. The supernatant was transferred to a new tube
and stored at
20°C for insulin measurement using a
scintillation proximity assay (SPA) procedure (3). Briefly, samples
were diluted in SPA buffer (50 mM
Na2HPO4,
150 mM NaCl, 25 mM EDTA, 0.01% thimerosal, and 0.1% BSA, pH 7.6) at a
ratio of 1:75, and 50 µl of this dilution were added to the wells of a 96-well microtiter plate. Subsequent additions (in order) were 50 µl of 125I-labeled porcine
insulin (NEN Life Sciences, ~10,000
counts · min
1 · well
1),
50 µl of guinea pig anti-rat insulin antibody (Linco Research), and
50 µl of protein A SPA beads (Amersham). The plate was covered with
an adhesive plastic sheet, mixed briefly on a plate shaker, and
incubated overnight at room temperature. Light emitted by the SPA beads
as a result of bound radioactivity was detected using a Wallac 1450 Microbeta Scintillation Counter. Insulin concentrations were determined
from standard curves of rat insulin using the program RIASYS (Lilly
Research Laboratories). Results represent duplicate determinations from
at least two independent studies and are reported as means ± SE.
Data were analyzed using Student's t-test. A probability of
P < 0.05 was considered
statistically significant.
Purification and microsequencing of p220 from
TC6-F7 cells.
Subcellular fractionation was carried out according to Shi et al. (36).
For large-scale purification of p220,
TC6-F7 cells were treated with
KCl (75 mM) for 5 min and homogenized in the hypotonic HEPES buffer (10 mM HEPES, PH 7.4, 1 mM MgCl2, 1 mM aminoethylisothiouronium
bromide hydrobromide, 1 mM Na3VO4,
and 1 mM phenylmethylsulfonyl difluoride). Protein lysate was enriched for high-molecular-weight proteins by a Centricon-100 filter unit (Amicon) and was subjected to HPLC using a DEAE ion exchange column (Pharmacia). The column was washed with buffer
A (25 mM Tris, pH 8.2, 1 mM
MgCl2, and 5% glycerol) and
eluted with a linear gradient of 0-1,000 mM NaCl in
buffer A. Positive fractions were identified by anti-phosphothreonine immunoblot analysis and purified by
fast performance liquid chromatography (FPLC) using a hydroxyapitate (HA) column (Bio-Rad Laboratories). The column was washed with 20 mM
potassium phosphate buffer (pH 7.0) and eluted with a linear gradient
of 20-700 mM of potassium phosphate buffer (pH 7.0). Fractions
containing p220 were pooled and concentrated using a Centricon-100
apparatus (Amicon). The concentrated sample was subjected to 4%
Tris-glycine gel electrophoresis. The gel was then stained with a
colloidal Coomassie G-250 (Novex), and p220 bands were isolated for
microsequencing analysis at the W. M. Keck Foundation Biotechnology
Resource Laboratory at Yale University.
Immunoprecipitation assay.
All immunoprecipitation analyses were performed at 4°C. Briefly,
KCl-stimulated
TC6-F7 cells were washed twice with ice-cold PBS
(GIBCO-BRL), homogenized in 1 ml of hypotonic HEPES buffer, and
centrifuged at 800 g to eliminate
nuclei. Samples containing equal amounts of protein were incubated for
90 min with the rabbit anti-human nonmuscle myosin antibody (Biomedical
Technologies). The immunocomplex was added to 100 µl of a slurry of
protein A-Sepharose (PAS) pretreated with washing buffer (10 mM HEPES,
150 mM NaCl, 10 mM benzamidine · HCl, 1 mM
phenylmethylsulfonyl fluoride, 5 mM EDTA, and 0.5% Triton X-114) and
incubated for an additional 90 min. After four brief washes, the
immunocomplex was eluted from PAS by boiling in 20 µl of 2×
Tris · glycine SDS sample buffer (Novex), followed by
SDS-PAGE on a 4-20% Tris-glycine gradient SDS gel (Novex) and
Western blot analysis.
 |
RESULTS |
Time- and dose-dependent phosphorylation of p220 induced by
imidazoline and other depolarization reagents.
To identify signal molecules involved in imidazoline-stimulated insulin
secretion, we investigated protein phosphorylation in
TC6-F7 cells
treated with an insulin secretagogue, RX-871024, which contains an
imidazoline ring. Protein lysates were separated by SDS-PAGE, and
phosphorylated proteins were analyzed by immunoblot assays by use of
antibodies against phosphoamino acids. Treatment of
TC6-F7 cells
with 100 µM RX-871024 resulted in increased threonine phosphorylation
of a protein with an apparent molecular mass of 220 kDa
(p220, Fig.
1A).
Western blot analysis with anti-phosphotyrosine antibody also detected
increases in tyrosine phosphorylation of a 42-kDa protein, which was
later identified as extracellular signal-regulated mitogen-activated
protein kinase, or ERK2 MAP kinase (data not shown). In contrast,
initial results indicated that antibody against phosphoserine did not
detect any changes in serine phosphorylation. Thus we chose to study
p220 phosphorylation in greater detail because p220 was abundant and
its phosphorylation was markedly increased by exposure of cells to
RX-871024.

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Fig. 1.
Threonine phosphorylation of p220 induced by RX-871024, KCl, and
glibenclamide in TC6-F7 cells. Cells were treated with 100 µM
RX-871024 (A), 75 mM KCl
(B), and 5 µM glibenclamide
(C) for periods of 0, 1, 5, 10, and
20 min in Dulbecco's modified Eagle's medium. At the end of each
incubation period, cells were harvested, and cell lysates were
quantified using the bicinchoninic acid method. Protein lysate (25 µg) from each treatment was analyzed by SDS-PAGE, followed by Western
blot analysis with anti-phosphothreonine antibody.
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One of the reported mechanisms of RX-871024 on insulin secretion
involved closure of ATP-regulated
K+ channels and membrane
depolarization (10). We therefore tested the effects of two other
membrane depolarization reagents, KCl and glibenclamide. Treatment of
TC6-F7 cells with 75 mM KCl or 5 µM glibenclamide caused
time-dependent increases of p220 phosphorylation (Fig. 1,
B and
C). However, there were significant
differences in the time course of phosphorylation of p220 for KCl vs.
RX-871024 and glibenclamide. Threonine phosphorylation of p220 induced
by KCl occurred instantaneously on addition of KCl and peaked in 5 min
after the treatment. In comparison, p220 phosphorylation induced by
RX-871024 (compare Fig. 1, A with
B) or glibenclamide (compare Fig. 1,
B with
C) was slower in onset and peaked at
20 min.
p220 Phosphorylation and insulin secretion.
RX-871024 has been reported to potentiate glucose-stimulated insulin
secretion (46). However, we and others have experienced a low threshold
of glucose-stimulated insulin secretion from
TC6-F7 around 2 mM of
glucose. In agreement with the lack of glucose responsiveness in
TC6-F7 cells, p220 phosphorylation was not affected by changing levels of glucose in the media (data not shown).
To investigate whether p220 threonine phosphorylation was related to
insulin secretion, we analyzed in parallel insulin secretion from
TC6-F7 cells treated with KCl and RX-871024. As shown in Fig.
2A,
stimulation of
TC6-F7 cells with 15 mM, 45 mM, or 75 mM KCl caused
dose-dependent increases in insulin secretion concurrently with
increased threonine phosphorylation of p220 as measured 5 min after the
treatment. Levels of insulin secretion were significantly
(P < 0.05) higher after treatment
with 45 mM and 75 mM KCl vs. the control. Similar results were observed
when Earle's balanced salt solution buffer was adjusted for osmolarity by reducing the NaCl concentration to exclude possible osmotic effects
on insulin secretion (data not shown). p220 Phosphorylation was also
correlated with insulin secretion from
TC6-F7 cells treated with
RX-871024. As demonstrated in Fig. 2B,
100 µM RX-871024 increased both insulin secretion and p220 threonine
phosphorylation measured after 20 min of treatment compared with the
negative control.

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Fig. 2.
Correlation between insulin secretion and p220 phosphorylation.
A: dose-dependent effect of KCl on
both insulin release and threonine phosphorylation of p220 in TC6-F7
cells. Cells were treated with KCl at concentrations of 0, 15, 45, and
75 mM, respectively, for 5 min in Earle's balanced salt solution
(EBSS) buffer. B: effect of RX-871024
on insulin release and threonine phosphorylation of p220 in TC6-F7
cells. Cells were treated with RX-871024 (100 µM) for 0, 5, and 20 min in EBSS buffer. The presence and absence of the compound was shown
as "+" and " ", respectively. At the end of each
incubation period, the supernatant was removed for insulin
quantification using a scintillation proximity assay.
* Significantly different from negative control
(P < 0.05).
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p220 Phosphorylation distal to
Ca2+ channel
activation.
The effect of membrane potential on p220 phosphorylation was further
investigated by treatment of
TC6-F7 cells with diazoxide, a
KATP channel activator. As
indicated by Fig
3A, both
p220 phosphorylation and insulin secretion were inhibited by the
presence of 150 µM diazoxide in the media. Because membrane
depolarization of
-cells often leads to activation of
Ca2+ channels, we next
investigated whether Ca2+ channels
played a role in regulating p220 phosphorylation. As shown in Fig.
3B, pretreatment of
TC6-F7 cells
with 50 µM nifedipine, an L-type
Ca2+ channel blocker, completely
abolished both insulin secretion and p220 phosphorylation induced by
treatment of 75 mM KCl. In the absence of KCl, nifedipine did not have
any effect, either on p220 phosphorylation or on insulin secretion
(Fig. 3B).

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Fig. 3.
Effects of diazoxide, nifedipine, and extracellular
Ca2+ on KCl-induced insulin
release and threonine phosphorylation of p220 in TC6-F7 cells.
A: cells were pretreated with
diazoxide (150 µM) for 5 min and then treated with KCl (75 mM) for 5 min in EBSS buffer containing 10 mM glucose.
B: cells were pretreated with or
without nifedipine (50 µM) for 15 min and then treated with or
without KCl (75 mM) for 5 min in EBSS buffer.
C: cells were treated with or without
KCl (75 mM) for 5 min in EBSS buffer containing glucose (10 mM) in the
presence or absence of calcium (1.8 mM). At the end of each treatment
period, the supernatant was removed for insulin quantification. The
presence or absence of a compound was shown as "+" and
" ", respectively. * Significantly different from
negative controls (P < 0.05).
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RX-871024 stimulates insulin release by both
Ca2+-dependent and
Ca2+-independent mechanisms (46).
To separate the effect of membrane depolarization from the effect of
[Ca2+]i
on p220 phosphorylation, we next compared p220 phosphorylation in
TC6-F7 cells cultured in media with and without
Ca2+. As indicated by Fig.
3C, treatment of
TC6-F7 cells with
75 mM KCl induced strong p220 phosphorylation concurrent with insulin secretion only in the presence of 1.8 mM extracellular
Ca2+, suggesting that membrane
depolarization alone was not sufficient to induce p220 phosphorylation.
Extracellular Ca2+ alone in the
absence of depolarizing concentration of KCl did not stimulate p220
phosphorylation (Fig. 3C).
Regulation of p220 phosphorylation by PKA and PKC.
PKA has been implicated to play a role in insulin secretion stimulated
by RX-871024 (46). To further understand the signal events that
regulate p220 threonine phosphorylation, we tested the effect of IBMX
on p220 phosphorylation. IBMX, an inhibitor of phosphodiesterase,
potentiates glucose-induced insulin secretion by raising cAMP levels
and activating the PKA-mediated signal transduction pathway (15).
Whereas neither glucose nor IBMX alone stimulated insulin secretion
from the cells, treatment of
TC6-F7 cells with IBMX (100 µM) in
the presence of glucose (10 mM) resulted in a sharp increase in insulin
secretion as measured at 5 and 20 min after the stimulation (Fig.
4). In contrast to the treatment by KCl, no
p220 phosphorylation was induced by insulin secretion stimulated by
IBMX either at 5 or 20 min after the treatment. Thus p220
phosphorylation is not directly regulated by PKA. The results also
suggest that p220 phosphorylation is not a signal event obligatory to
insulin exocytosis. Likewise, p220 phosphorylation was not affected by
treatment of
TC6-F7 cells with phorbol 12-myristate 13-acetate
(PMA), an activator of PKC, and H7, an inhibitor of PKC, suggesting
that p220 is not a substrate of PKC (data not shown).

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Fig. 4.
Comparison of effects of IBMX and KCl on insulin release and threonine
phosphorylation of p220 in TC6-F7 cells. Cells were treated with
IBMX (100 µM) or KCl (75 mM) in the presence or absence of glucose
(10 mM) for 0, 5, and 20 min. At the end of the treatment period, the
supernatant was removed for insulin quantification. Cells were
harvested, and cell lysates were analyzed as described in Fig. 1. The
presence or absence of a compound was shown as "+" and
" ", respectively. * and + Significantly different
from negative controls (P < 0.05);
different symbols represent different control groups.
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Phosphorylated p220 is a cytosolic protein.
Subcellular fractionation was carried out to characterize the
biochemical features of p220 and to facilitate purification of p220
from
TC6-F7 cells. After treatment with 75 mM KCl for 1 and 5 min to stimulate p220 phosphorylation,
TC6-F7 cells were homogenized and separated into a cytosol (C, Fig.
5) and a particulate (P, Fig. 5) membrane
fraction. The membrane fraction was washed in 0.5 M NaCl. Triton X-114
extracts of cytosol and membrane fractions were subjected to
temperature-induced Triton X-114 phase transition. The aqueous phase
(A, Fig. 5), which contained hydrophilic proteins, and the detergent
phase (D, Fig. 5), which contained hydrophobic proteins, were separated
by SDS-PAGE and subjected to immunoblotting analysis using
anti-phosphothreonine antibodies. As indicated by Fig. 5,
phosphorylated p220 was a hydrophilic protein and localized exclusively
in the aqueous phase of the cytosolic fraction.

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Fig. 5.
Subcellular localization of p220 in TC6-F7 cells. Cells were treated
with 75 mM of KCl for 1 (lanes
1-4) and 5 min (lanes
5-8) before subcellular fractionation. A
cytosolic fraction (C) and an extract of a washed particulate fraction
(P) were prepared from TC6-F7 cells and subjected to
temperature-induced Triton X-114 phase separation. Aqueous (A) and
detergent (D) phases were analyzed by SDS-PAGE immunoblot analysis with
anti-phosphothreonine antibody. Positions of molecular mass markers (M)
are indicated with numbers on left.
The position of p220 is indicated with an arrow.
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Purification of p220 and identification as nonmuscle MHC.
p220 was purified from
TC6-F7 cells treated with 75 mM KCl.
Cytosolic fractions enriched for high molecular proteins by Centricon filtration were separated by ion exchange chromatography by use of a
DEAE column. Column fractions containing phosphorylated p220 were
identified by Western blot analysis using anti-phosphothreonine antibodies. Combined fractions containing p220 were further purified on
an HA column, which resulted in significant enrichment of p220, as
indicated in Fig. 6. As a final
purification step, p220 was separated from other proteins by SDS-PAGE
on 4% Tris-glycine gels. After Coomassie blue staining of the gel, a
p220 band was isolated and subjected to microsequencing analysis.

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Fig. 6.
Immunoblot analysis of purified fractions from a hydroxyapitate
(HA)-fast performance liquid chromatography (FPLC) column.
Column fractions were separated by 4-20% SDS-PAGE followed by
Western blot analysis using anti-phosphothreonine antibody.
Lane 1, a fraction containing p220
from a DEAE-HPLC column. Lane M,
molecular mass marker. Lanes
7-10, positive fractions containing p220 from
HA-FPLC column purification. Position of p220 is indicated with an
arrow.
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Two peptide sequences were generated from the microsequencing analysis.
The first peptide contained the sequence R-G-D-L-P-F-V-V-T-R, and the
second contained the sequence T-D-L-L-L-E-P-Y-N-K. Each peptide
sequence was compared with proteins in the SwissProt database. Both
peptides shared high sequence homology with vertebrate nonmuscle MHC-A.
The first peptide shares 100% homology with a sequence fragment
located at the COOH terminus of chick nonmuscle MHC-A (Fig.
7) and matched all but one amino acid of a sequence
located in the corresponding region of the human nonmuscle MHC-A (Fig. 7). The second peptide shared 100% sequence identity with a peptide sequence close to the NH2 terminus
of both human and chick nonmuscle MHC-A (Fig. 7). No matches were found
with any other members of the myosin super family, including nonmuscle
MHC-B, which shares high homology with nonmuscle MHC-A, or any other
protein sequences in the data bank. This was also supported by
immunoprecipitation and immunoblot analysis by use of a polyclonal
nonmuscle MHC antibody and partially purified DEAE fractions. Only
p220-positive fractions contained proteins that were immunoreactive
with nonmuscle MHC antibody (data not shown), suggesting that the
threonine-phosphorylated p220 is the nonmuscle MHC-A.

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Fig. 7.
Sequence comparison of p220 peptides with nonmuscle myosin heavy chain
A (MHC-A) from human and chick. Nos. indicate position of peptide in
myosin protein sequence. The only mismatch of p220 peptide sequence
with human nonmuscle MHC-A is underlined.
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Immunoprecipitation analyses were carried out to verify
whether nonmuscle MHC-A becomes phosphorylated on threonine residue(s) in response to insulin secretion by use of human nonmuscle MHC-A antibody and lysates from
TC6-F7 cells treated with KCl (Fig. 8). The immunoprecipitates were
characterized by immunoblot analysis with anti-phosphothreonine
antibody (Fig. 8A) and nonmuscle MHC antibody (Fig 8B). Treatment of
TC6-F7 with 75 mM KCl resulted in time-dependent threonine
phosphorylation of nonmuscle MHC identical to that of p220 (compare
Fig. 8, A with
B). As indicated by Fig. 8B, roughly equal amounts of nonmuscle
MHC-A were immunoprecipitated from the lysates of
TC6-F7 cells
treated with KCl for different lengths of time. In addition to
nonmuscle MHC phosphorylation, a protein with molecular mass of ~40
kDa (p40) was coimmunoprecipitated by the nonmuscle MHC antibody. The
p40 protein also demonstrated time-dependent phosphorylation on
threonine residue(s) in response to the KCl treatment (Fig.
8A). The p40 protein was not
recognized by the nonmuscle MHC antibody from the Western blot analysis
(Fig. 8B), suggesting that the
molecule is not a degraded product of p220.

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Fig. 8.
Nonmuscle MHC threonine phosphorylation and association with the p40
protein induced by KCl in TC6-F7 cells. Cells were treated with 75 mM KCl for 0 (lane 1), 1 (lane 2), 5 (lane
3), 10 (lane 4),
and 20 min (lane 5), respectively.
Immunoprecipitation was carried out using human nonmuscle MHC antibody,
followed by Western blot analyses using rabbit anti-phosphothreonine
antibody (A) and rabbit
anti-nonmuscle MHC antibody (B).
Positions of molecular mass markers are indicated with nos. on
left. Positions of nonmuscle MHC and
p40 proteins are indicated with arrows.
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DISCUSSION |
The signal molecules involved in the insulinotropic effects of
RX-871024 have not been well characterized. In the present study, we
have demonstrated for the first time that stimulation of pancreatic
-cells with RX-871024 leads to increased threonine phosphorylation
of a 220-kDa protein (p220). Whereas imidazolines have been shown to
potentiate glucose-stimulated insulin secretion (46), our data showed
that p220 phosphorylation stimulated by RX-871024 was mainly related to
the membrane depolarization effect of imidazolines. This is supported
by our experiments showing that p220 phosphorylation also occurs in
-cells treated with KCl and glibenclamide. Although these three
secretagogues differ in mechanisms involved in insulin secretion, all
have been shown to cause
-cell depolarization, activation of L-type
VDCC, and increase in
[Ca2+]i.
Indeed, our data indicate that p220 phosphorylation can be blocked by
treatment with diazoxide, a KATP
channel activator, by treatment with nifedipine, an L-type
Ca2+-channel inhibitor, and by
depletion of extracellular calcium. Our data also suggest that
depolarization without affecting
[Ca2+]i
was not sufficient to elicit p220 phosphorylation.
Through protein purification and peptide sequencing analyses, we
identified p220 as nonmuscle MHC-A. Subsequent immunoprecipitation analyses also confirmed that the phosphorylation profile of nonmuscle MHC coincided with that of p220 when cells were tested with KCl. Two
isoforms of MHC, MHC-A and MHC-B, were reported to be present in
mammals (18, 19, 34, 37). Even though both forms were recently found to
be expressed in pancreatic
-cells (43), our sequence data suggest
that the A form is the predominant one that is responsive to
stimulation of acute insulin secretion. This is also consistent with a
recent report (16) that only a single form of MHC was detected in
pancreatic
-cells by immunoblot analysis. However, it is possible
that the B isoform was also responsive to KCl stimulation but was not
detected without enrichment of the protein by immunoprecipitation,
because our immunoprecipitation analysis using nonmuscle MHC antibody
resulted in detection of two isoforms (Fig. 8). Interestingly,
threonine phosphorylation of both forms appeared to be equally
stimulated by KCl treatment, as demonstrated by immunoprecipitation analysis.
One of the suggested mechanisms of RX-871024 involves a direct
stimulation of insulin exocytosis (9). A potential role of MHC
phosphorylation in stimulating exocytosis was tested in our experiments
by treatment of
-cells with nondepolarizing secretagogues, such as
IBMX. It was expected that if MHC phosphorylation were an obligatory
event that led to insulin exocytosis, p220 phosphorylation should also
occur in exocytosis stimulated by different mechanisms. We chose to use
IBMX because the secretagogue stimulates insulin secretion and bypasses
the membrane depolarization step. IBMX potentiates glucose-stimulated
insulin secretion by increasing the level of cAMP, which activates
PKA-mediated signal transduction pathways in
-cells. Whereas IBMX
elicited a sharp increase in insulin release in the presence of high
glucose, p220 phosphorylation was not affected by IBMX. The results
suggest that phosphorylation of nonmuscle MHC is not an obligatory
event in insulin exocytosis. Our data also suggest that MHC
phosphorylation is not mediated by PKA.
A number of kinases, including PKC, CaM kinase, and casein kinase II,
have been suggested to phosphorylate MHC both in lower eukaryotic
systems and in mammalian cells (5, 20, 40). However, the only kinase
with demonstrated specificity for MHC came from biochemical and
molecular characterization of
Dictyostelium myosin heavy chain
kinase, or MHCK (13). Because PKC has been implicated in insulin
release stimulated by imidazoline and glibenclamide (11, 46), we also
tested the involvement of PKC in regulating MHC phosphorylation by
using an inhibitor and an activator of the kinase. Our data showed that
p220 phosphorylation was not affected by either the PKC activator PMA
or the PKC inhibitor H7. The results suggest that MHC threonine
phosphorylation is not directly regulated by the PKC-mediated signal
transduction pathways, a conclusion which is consistent with previous
reports that the PKC-mediated phosphorylation of MHC primarily affects serine residues (7, 40).
One of the interesting observations from our current studies involved
identification of a 40-kDa (p40) molecule associated with p220
phosphorylation. Because p40 was identified only after immunoprecipitation, it is possible that the protein was
nonspecifically immunoprecipitated by the polyclonal MHC antibody.
Alternatively, p40 may be physically associated with MHC and hence
enriched by the immunoprecipitation of nonmuscle MHC. This was partly
supported by the observation that p40 was the only other
threonine-phosphorylated protein immunoprecipitated by the MHC antibody
among all the threonine-phosphorylated proteins in the cell lysate, and
that p40 protein was not recognized by the polyclonal MHC antibody on
Western blot after enrichment with immunoprecipitation. Furthermore,
p40 was also responsive to KCl stimulation and demonstrated
time-dependent phosphorylation on threonine residue(s). Interestingly,
the threonine phosphorylation of p40 appeared to be at a different rate
than the phosphorylation of nonmuscle MHC. In comparison with the
threonine phosphorylation of nonmuscle MHC, which peaked at 5 min after
KCl treatment, p40 phosphorylation became more pronounced after 10 min
of treatment. The molecular identity of p40 remains unknown, and its
identification may help explain how nonmuscle MHC phosphorylation is
regulated in pancreatic
-cells. More importantly, the identification
of p40 also may reveal a missing link between nonmuscle MHC
phosphorylation and signal transduction pathways involved in acute
insulin secretion.
 |
ACKNOWLEDGEMENTS |
We thank Valerie X. Williams, Carol Broderick, and Dr. Xiaodong Liu
for technical assistance, and Drs. Christopher Newgard, Mark L. Heiman,
Michael Statnick, and Scott E. Hayes for critically reading the manuscript.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for correspondence and reprint requests: Y. Shi, Endocrine
Division, DC 0545, Lilly Research Laboratories, Eli Lilly and Company,
Indianapolis, IN 46285 (E-mail: Shi_Yuguang{at}Lilly.com).
Received 19 February 1999; accepted in final form 11 June 1999.
 |
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