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Originally published In Press as doi:10.1074/jbc.M004996200 on August 16, 2000
J. Biol. Chem., Vol. 275, Issue 44, 34772-34779, November 3, 2000
Calcium-dependent Threonine Phosphorylation of
Nonmuscle Myosin in Stimulated RBL-2H3 Mast Cells*
Denis B.
Buxton and
Robert S.
Adelstein
From the Laboratory of Molecular Cardiology, NHLBI, National
Institutes of Health, Bethesda, Maryland 20892
Received for publication, June 8, 2000, and in revised form, July 24, 2000
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ABSTRACT |
Stimulation of RBL-2H3 m1 mast cells through the
IgE receptor with antigen, or through a G protein-coupled receptor with
carbachol, leads to the rapid appearance of phosphothreonine in
nonmuscle myosin heavy chain II-A (NMHC-IIA). We demonstrate that this
results from phosphorylation of Thr-1940 by
calcium/calmodulin-dependent protein kinase II (CaM kinase II),
activated by increased intracellular calcium. The phosphorylation site
in rodent NMHC-IIA was localized to the carboxyl terminus of NMHC-IIA
distal to the coiled-coil region, and identified as Thr-1940 by
site-directed mutagenesis. A fusion protein containing the NMHC-IIA
carboxyl terminus was phosphorylated by CaM kinase II in
vitro, while mutation of Thr-1940 to Ala eliminated
phosphorylation. In contrast to rodents, in humans Thr-1940 is replaced
by Ala, and human NMHC-IIA fusion protein was not phosphorylated by CaM
kinase II unless Ala-1940 was mutated to Thr. Similarly, co-transfected
Ala  Thr-1940 human NMHC-IIA was phosphorylated by activated CaM
kinase II in HeLa cells, while wild type was not. In RBL-2H3 m1 cells,
inhibition of CaM kinase II decreased Thr-1940 phosphorylation, and
inhibited release of the secretory granule marker hexosaminidase in
response to carbachol but not to antigen. These data indicate a role
for CaM kinase stimulation and resultant threonine phosphorylation of
NMHC-IIA in RBL-2H3 m1 cell activation.
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INTRODUCTION |
Stimulation of sensitized mast cells by antigen leads to the
activation of signal transduction pathways including signals generated
through the mobilization of calcium and
PKC1 (1-3). One of the
responses to these signals is the fusion of secretory granules with the
plasma membrane, allowing release of preformed mediators such as
histamine and serotonin from the cell. Calcium and PKC are necessary
and sufficient for secretion in permeabilized cells (4). Fusion of the
secretory granules with the plasma membrane is accompanied by
cytoskeletal rearrangements, which may facilitate fusion by removing a
barrier of actomyosin which physically separates the two entities.
Previous work has demonstrated that activation of the rat mast cell
line RBL-2H3 with antigen or with calcium ionophore leads to the rapid
PKC-dependent serine phosphorylation of nonmuscle myosin on
both light chains and heavy chains (5). The phosphorylation of
nonmuscle myosin heavy chain (NMHC)-II, which occurs close to the
carboxyl terminus at the end of the coiled-coil region, has been
proposed to contribute to rearrangement of the actomyosin cytoskeleton
and thus facilitate secretory granule fusion at the plasma membrane and
resultant mediator release (5).
Recent studies in a variety of cell lines have demonstrated that
NMHC-II can also be threonine phosphorylated in response to stimuli. In
rat PC12 cells, NMHC-II phosphorylation in response to bradykinin was
shown to be calcium-dependent, and was regulated by
activity of the small GTPase Rac. NMHC-II phosphorylation was accompanied by loss of cortical myosin (6).
Calcium-dependent phosphorylation of NMHC-II was also shown
in response to nutrient stimulation in the rat insulinoma cell line
RINm5F, and was proposed to play a role in insulin secretion (7).
However, in neither study was the site of threonine phosphorylation of
the myosin heavy chain determined. In light of these findings, it was
of interest to determine whether threonine phosphorylation of NMHC-II also occurs in mast cells. Here we demonstrate that stimulation of
RBL-2H3 m1 cells, an RBL-2H3 cell line made to express the muscarinic
m1 receptor (8), with antigen or carbachol leads to
calcium-dependent threonine phosphorylation of NMHC-IIA. We provide evidence that Thr-1940, situated distal to the coiled-coil region within a CaM kinase consensus sequence, is the residue phosphorylated. We also demonstrate that CaM kinase II is activated by
antigen and carbachol with time courses similar to those obtained for
NMHC-IIA threonine phoshorylation. Finally, a specific inhibitor of CaM
kinase attenuates threonine phosphorylation of NMHC-IIA, and also
inhibits secretion in response to carbachol.
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MATERIALS AND METHODS |
RBL-2H3 m1 cells and Y79 cells were generously provided by Drs.
Michael Beaven and Sachiyo Kawamoto (NHLBI), respectively, and were
cultured in DMEM supplemented with 10% fetal bovine serum and
penicillin/streptomycin. PMA, GF109203X, BAPTA-AM, dimethylsphingosine, autocamtide-2, KN-92, and KN-93 were obtained from Calbiochem (San
Diego CA). Carbachol, BSA-DNP conjugate and phosphoamino acids were
obtained from Sigma. Mouse anti-dinitrophenol IgE was obtained from M. Beaven or from Sigma. Anti-phosphothreonine antibody (rabbit
polyclonal) was obtained from Zymed Laboratories Inc. (South San Francisco CA). Anti-phospho-ERK and protein A/G-agarose were
from Santa Cruz Biotech (Santa Cruz CA). Chymotrypsin-TLCK was from
Worthington Biochemicals (Lakewood, NJ). CaM kinase II was from New
England Biolabs (Beverly, MA). Rabbit polyclonal antibodies to platelet
myosin, which recognizes epitopes primarily in the rod region of
NMHC-IIA, and to the carboxyl terminus of human NMHC-IIA (anti-HA),
have been described (9). A rabbit polyclonal antibody raised against 13 amino acid residues at the amino terminus of human NMHC-IIA was kindly
provided by Dr. Mary Anne Conti (NHLBI). Anti-GFP was obtained from
CLONTECH (Palo Alto, CA). 4-20%
SDS-polyacrylamide gels were obtained from FMC (Rockland, ME), while
4-12% NuPAGE gels were obtained from Novex (San Diego, CA). A
synthetic peptide corresponding to amino acids 1933-1951 of rat
NMHC-IIA was obtained from Research Genetics (Huntsville, AL).
Antigen Stimulation of Mast Cells--
RBL-2H3 cells were
incubated overnight with anti-dinitrophenol IgE (0.5 µg/ml), washed
with phosphate-buffered saline, and the medium replaced with DMEM
containing 0.1% BSA. Cells were then stimulated with DNP-BSA (20 ng/ml), washed twice with ice-cold phosphate-buffered saline containing
5 mM EDTA and 1 mM sodium vanadate, lysis
buffer added, and the cells scraped from the plate with a plastic
scraper. Lysis buffer consisted of 25 mM Hepes, pH 7.5, 0.3 M NaCl, 1.5 mM MgCl2, 0.1% Triton
X-100, 1 mM sodium orthovanadate, 20 mM
 -glycerophosphate, 0.2 mM EDTA, 0.5 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 20 µM leupeptin, 0.15 units/ml aprotinin at 4 °C. Cell
lysates were transferred to a microcentrifuge tube, and
Triton-insoluble material pelleted at 14,000 × g for 10 min. After taking an aliquot for protein determination (10) to
permit equal protein loading of gel lanes, samples were heated in
sample buffer and subjected to SDS-PAGE. Samples were transferred to
polyvinylidene difluoride membranes (Immobilon-P, Millipore) and
subjected to immunoblotting using standard methods. Bound secondary
antibody was detected using luminol blotting reagents (Santa Cruz
Biotech), and the signal captured on Biomax MR film (Kodak). Films were
scanned using a laser densitometer (Molecular Dynamics) and bands
quantified using ImageQuant software.
Immunoprecipitation--
Cleared cell lysates prepared as above
were rocked at 4 °C with the appropriate antibody for 1 h.
Protein A/G-agarose was then added, and rocking continued for a further
2 h. The protein A/G-agarose beads were washed 4 times with
ice-cold phosphate-buffered saline, and then incubated with sample
buffer at 75 °C for 10 min to remove bound protein from the beads.
The supernatant was subjected to SDS-PAGE and immunoblotting as
described above.
Proteolytic Localization of the Threonine Phosphorylation
Site--
For chymotryptic digestion, cell lysate (2 mg/ml) was
incubated with TLCK-treated chymotrypsin, 20 µg/ml at 25 °C.
Aliquots were taken at time points, heated with sample buffer, and
subjected to SDS-PAGE and immunoblotting. For hydroxylamine cleavage,
cell lysate was diluted 1:10 with 2 M hydroxylamine
hydrochloride, 6 M guanidine hydrochloride which had been
adjusted to pH 9.0 with LiOH. The mixture was incubated at 45 °C for
3 h, dialyzed against 6 M urea, 40 mM Hepes, pH 7.5, and concentrated by centrifugation in a
Microcon 10 centrifugal filter (Millipore). An aliquot of the
concentrated digested lysate was then subjected to SDS-PAGE and immunoblotting.
Fusion Protein Expression and Mutation--
A 1906-base pair
fragment (including 3'-untranslated residues), obtained from a clone of
mouse NMHC-IIA provided by M. Conti, was subcloned into pGEX-5T2 to
give a glutathione S-transferase fusion protein including
the carboxyl-terminal 199 amino acids of NMHC-IIA. The threonine
residue located 22 amino acids from the carboxyl terminus,
corresponding to rat NMHC-IIA Thr-1940, was mutated to Ala using the
Quikchange system (Stratagene, La Jolla, CA). The primers used were
5'-TGTTCGGAAAGGCGCCGGCGACTGCTCAG-3' and the complementary
antisense primer; the mutated residue, shown in bold, converts Thr to
Ala and introduces an SfoI site for rapid clone selection. A
450-base pair fragment was subcloned from a clone encoding human
NMHC-IIA (Dr. Qize Wei, NHLBI) into pGEX-4T1 to give a glutathione
S-transferase fusion protein encoding the carboxyl-terminal
150 amino acids of NMHC-IIA. Ala-1940 was mutated to Thr using the
primers 5'-CCGGAAAGGCACCGGGATGGCTCC-3' and the complementary
antisense primer; the mutated residue, shown in bold, converts Ala to
Thr and removes an SfoI site. SDS-PAGE-purified primers were
obtained from Life Technologies, Inc. (Gaithersburg MD). Fusion
proteins were expressed in Escherichia coli and purified by
standard methods (11).
Mammalian Expression Experiments--
Mammalian expression
vector encoding constitutively active CaM kinases I, II, and IV (12)
were generously provided by Dr. Richard Maurer (Oregon Health Sciences
University). An expression vector encoding human NMHC-IIA with GFP
fused to the amino terminus was kindly provided by Q. Wei. Ala-1940
was mutated to Thr as described above for the human pGEX-4T1 construct.
Transfection was performed using Effectene (Qiagen, Valencia, CA)
following the manufacturer's standard protocol. pAdVantage (Promega,
Madison, WI) was co-transfected with the expression vectors to improve expression levels by inhibiting dsRNA-activated inhibitor.
In Vitro Phosphorylation with CaM Kinase II and CaM Kinase
Assay--
Protein or peptide substrates were incubated with CaM
kinase II, and where indicated [ -32P]ATP, 250 µCi/mmol, in a reaction buffer consisting of 20 mM Tris,
pH 7.5, 10 mM MgCl2, 2 mM
CaCl2, 2.4 mM calmodulin, 0.5 mM
dithiothreitol, 0.1 mM EDTA, 50 µM ATP.
Phosphorylated proteins and peptides were then separated on NuPAGE
4-12% gels run in MES buffer. CaM kinase II activity in cell lysates
was determined by phosphorylation of autocamtide-2 and capture of the
phosphorylated peptide on Whatman P81 phosphocellulose as described
previously (13).
Phosphoamino Acid Determination--
After in vitro
labeling of peptide, the reaction mixture was lyophilized and taken up
in 6 M HCl, 50 µl. The mixture was incubated at 110 °C
for 1 h, and lyophilized after addition of 400 µl of water. The
mixture was dissolved in electrophoresis buffer (glacial acetic
acid/pyridine/water, 10:1:189, pH 3.5) (14), marker phosphoamino acids
added, and an aliquot spotted on to a 20 × 20-cm glass-backed cellulose TLC plate. Electrophoresis was carried out for 30 min at 1.3 kV, the plate dried, and the phosphoamino acids visualized with
ninhydrin. The plate was then subjected to autoradiography.
Measurement of Secretion by Hexosaminidase Assay--
The
release of the secretory granule marker hexosaminidase was measured as
described previously (15). Released hexosaminidase was expressed as a
percentage of total hexosaminidase.
Statistical Analysis--
Results are presented as mean ± S.E. Comparisons between 2 groups were performed by paired or unpaired
Student's t test as appropriate. Comparisons between more
than 2 groups were made by repeated measure analysis of variance
followed by post-hoc Tukey HSD test to assess significance of
differences between individual groups.
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RESULTS |
Stimulation of RBL-2H3 Cells Results in Threonine Phosphorylation
of NMHC II-A--
RBL-2H3 m1 cells, sensitized by overnight incubation
with anti-DNP IgE, were stimulated with DNP-BSA, and threonine
phosphorylation of lysate proteins detected by immunoblotting using a
specific antibody to phosphothreonine. Fig.
1A shows that DNP-BSA caused the rapid appearance of phosphothreonine in a band of 220 kDa, with
maximal threonine phosphorylation at 10-15 min. To determine the
specificity of the response to antigen stimulation, cells were
stimulated with carbachol, which acts via the stably transfected m1
muscarinic receptors. Carbachol also caused threonine phosphorylation of the 220-kDa band (Fig. 1B), but in this case the response
was more rapid, detectable within 15 s and maximal at 2 min. The
responses thus parallel the calcium responses to the agents, which are
more rapid for carbachol than for antigen (16). A comparison of the time courses for the 2 agents is shown in Fig. 1C. The
maximal threonine phosphorylation was also greater for carbachol; the mean response to antigen was 59 ± 8% of that to carbachol
(n = 3; p < 0.01).
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Fig. 1.
Stimulation of threonine phosphorylation of
220-kDa protein in RBL-2H3 m1 cells. Threonine phosphorylation of
220-kDa protein in response to: A, antigen stimulation in
sensitized RBL-2H3 m1 cells; B, carbachol stimulation of
non-sensitized cells; C, comparison of time courses. For
antigen stimulation, cells were sensitized overnight with DNP-IgE
before stimulation with 20 ng/ml DNP-BSA. After stimulation with
antigen or with 250 µM carbachol, cells were lysed at the
appropriate time point, and the lysates were then subjected to SDS-PAGE
and immunoblotting with an antibody specific for phosphothreonine.
Squares, carbachol; circles, antigen. Results are
the means of two experiments.
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To confirm the identity of the 220-kDa band, NMHC-IIA was
immunoprecipitated from RBL-2H3 lysates using anti-HA antibody which recognizes the carboxyl terminus of NMHC-IIA, and the
immunoprecipitates immunoblotted with anti-PT antibody. Fig.
2 (left) shows that the
antibody recognized a 220-kDa band in the anti-HA immunocomplexes from
carbachol-treated cells, while in untreated control cells the 220-kDa
band was barely detectable. Omitting anti-HA from the
immunoprecipitation resulted in loss of the phosphothreonine band,
indicating specificity of the immunoprecipitation. Conversely, NMHC-IIA
was increased in anti-PT immunocomplexes obtained from carbachol-treated but not control cells (Fig. 2, right).
Since RBL-2H3 cells contain only NMHC-IIA and are devoid of the
NMHC-IIB isoform (17), the NMHC phosphorylated in these
experiments can be identified unequivocally as NMHC-IIA.
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Fig. 2.
Identification of the 220-kDa
phosphothreonine acceptor as NMHC-IIA. Left panel, proteins
were immunoprecipitated from control or carbachol-treated cells with
antibody to the carboxyl terminus of NMHC-IIA (anti-HA), and
immunoblotted with anti-phosphothreonine (anti-PT) antibody.
Phosphothreonine was greatly increased in the 220-kDa band in
stimulated cells. Omission of anti-HA prevented immunoprecipitation of
the 220-kDa phosphoprotein from stimulated cells, ruling out
nonspecific binding of phosphothreonine to the protein A/G-agarose.
Right panel, proteins were immunoprecipitated with anti-PT
and immunoblotted with anti-HA.
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Threonine Phosphorylation of NMHC Is
Calcium-dependent--
Threonine phosphorylation of NMHC
in RINmF5 and PC12 cells is calcium-dependent (6, 7).
RBL-2H3 cell degranulation in response to antigen requires an increase
in intracellular calcium, and is also dependent on influx of
extracellular calcium. To assess the role of calcium in the threonine
phosphorylation of NMHC in RBL-2H3 cells, the cells were incubated with
the calcium ionophore ionomycin. Antigen-activated threonine
phosphorylation of NMHC-IIA was mimicked by the calcium ionophore
ionomycin (Fig. 3A),
demonstrating that influx of extracellular calcium is sufficient to
cause threonine phosphorylation of NMHC-IIA. Responses to both antigen
stimulation and ionomycin were inhibited by preloading cells with the
calcium chelator BAPTA, indicating that a rise in intracellular calcium is necessary for stimulation of threonine phosphorylation. Incubation of cells with EGTA to chelate extracellular calcium and prevent calcium
influx also inhibited threonine phosphorylation of NMHC in response to
antigen and ionomycin (Fig. 3A), demonstrating that influx
of extracellular calcium is also required. Since the incubation time
with EGTA was brief (2 min), depletion of intracellular calcium stores
is an unlikely explanation for the inhibitory effects of this
chelator.
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Fig. 3.
Role of calcium in NMHC threonine
phosphorylation. A, calcium dependence of NMHC threonine
phosphorylation. Cells were incubated with or without anti-DNP IgE
overnight, washed, and incubated in DMEM containing 0.1% BSA. For
BAPTA loading, cells were incubated with 50 µM BAPTA-AM
for 30 min, washed with phosphate-buffered saline, and incubated with
fresh DMEM, 0.1% BSA. For EGTA-treated cells, EGTA (4 mM)
was added 2 min prior to stimulation. Anti-DNP IgE-sensitized cells
were then stimulated for 15 min with DNP-BSA (lanes 2-4),
while non-sensitized cells were stimulated with 1 µM
ionomycin (lanes 5-7) or 0.1 µM ionomycin
(lanes 8-10) for 5 min. B, effects of
dimethylsphingosine on threonine phosphorylation of NMHC.
DNP-IgE-sensitized cells were washed, and incubated in the presence or
absence of DMS for 30 min. Cells were then stimulated with DNP-BSA (20 ng/ml) for 15 min. Non-sensitized cells were treated identically before
stimulation with carbachol (100 µM) or ionomycin (1 µM) for 5 min. Lysates were prepared and subjected to
SDS-PAGE and immunoblotting with anti-phosphothreonine.
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Antigen-mediated mobilization of calcium in RBL-2H3 cells has been
reported to be mediated primarily by the action of sphingosine kinase
to yield sphingosine 1-phosphate, which releases calcium from
intracellular stores leading in turn to the uptake of extracellular calcium (16). To assess the involvement of sphingosine kinase in the
threonine phosphorylation of NMHC, cells were incubated with the
specific sphingosine kinase inhibitor dimethylsphingosine (DMS) (18).
Threonine phosphorylation of NMHC in response to antigen was completely
abrogated by DMS, indicating that production of sphingosine 1-phosphate
is a necessary step in the activation of the threonine kinase (Fig.
3B). In contrast, DMS had no effect on the response to
carbachol, consistent with the primary route of calcium mobilization
being via the PLC-mediated production of inositol 1,4,5-trisphosphate
(16). DMS also had no effect on ionomycin-mediated NMHC threonine
phosphorylation, consistent with the action of DMS being upstream of
ionomycin at the level of blocking calcium release.
Threonine Phosphorylation of NMHC Involves CaM Kinase and Not
PKC--
PKC is activated in antigen-stimulated RBL-2H3 cells,
resulting in phosphorylation of NMHC at a serine residue in the
carboxyl terminus (5). PKC was not involved in threonine
phosphorylation of NMHC, however, as treatment of RBL-2H3 cells with
the phorbol ester PMA had no effect on threonine phosphorylation of
NMHC (Fig. 4A, top). However,
it did increase levels of phosphorylated ERK (Fig. 4A,
bottom), indicating that PKC was activated. The PKC independence
of NMHC-IIA threonine phosphorylation was confirmed by pretreating
IgE-sensitized cells with the specific PKC inhibitor GF109203X; the
inhibitor had no effect on subsequent threonine phosphorylation of
NMHC-IIA in response to antigen. However, GF109203X did inhibit the
increases in pERK in response to both antigen and PMA (Fig.
4A).
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Fig. 4.
Effect of kinase inhibitors on NMHC threonine
phosphorylation. A, threonine phosphorylation of NMHC is not
mediated by PKC. DNP-IgE-sensitized cells were washed and
incubated ± GF109203X, 200 nM for 30 min before
stimulation with with DNP-BSA (20 ng/ml) for 15 min. Non-sensitized
cells were stimulated with 1 µM PMA for 5 min. Lysates
were prepared and subjected to SDS-PAGE and immunoblotting with
anti-phosphothreonine. Blots were then stripped and probed again with
anti-pERK. B and C, threonine phosphorylation of
NMHC is attenuated by inhibitors of CaM kinase. Control cells
(carbachol, B) or DNP-IgE-sensitized cells (antigen,
C) were washed and incubated for 30 min without addition,
with the CaM kinase inhibitor KN-93 (10 µM), or with the
inactive analog KN-92 (10 µM), before stimulation with
carbachol (100 µM) for 2 min or DNP-BSA (20 ng/ml) for 10 min. Results are expressed as a percentage of the response to stimulus
in the absence of inhibitor, and are mean ± S.E. for four to six
experiments. *, p < 0.01 versus stimulation
without inhibitor.
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The other major class of calcium-activated serine/threonine kinases is
the calcium/calmodulin-dependent protein kinases. To test
the role of CaM kinase in phosphorylation of NMHC-IIA, cells were
preincubated with the CaM kinase inhibitor KN-93, and with the inactive
analog KN-92. KN-93, which has no effect on myosin light chain kinase
or PKC activity (19), caused significant inhibition of NMHC-IIA
phosphothreonine responses to carbachol (Fig. 4B) and
antigen (Fig. 4C). However, the inactive homolog KN-92
was without effect, suggesting that the inhibition in response to KN-93
is a specific effect.
Ionomycin Causes Threonine Phosphorylation of NMHC in Rodent but
Not Human Cells--
Ionomycin has previously been shown to stimulate
threonine phosphorylation of NMHC in PC12, N1E-115, and RINm5F cells
(6, 7), which are all derived from rodent sources. Treatment of mouse
embryonic fibroblasts also resulted in threonine phosphorylation of
NMHC (Fig. 5). Similar results were
obtained both in wild type cells and in cells obtained from embryos in
which NMHC-IIB had been ablated (20), consistent with NMHC-IIA being
the isoform phosphorylated (results not shown). However, stimulation of
human-derived HeLa cells with ionomycin did not lead to detectable
threonine phosphorylation of NMHC (Fig. 5, upper panel),
despite the presence of NMHC-IIA in these cells (Fig. 5, lower
panel). Similarly Y79 cells, a human retinoblastoma cell line, did
not respond to ionomycin by threonine phosphorylation (results not
shown).
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Fig. 5.
Ionomycin does not stimulate threonine
phosphorylation of NMHC in human cells. Lysates from RBL-2H3, HeLa
cells, and mouse embryonic fibroblasts stimulated with ionomycin, 1 µM for 5 min (+) or untreated ( ) were subjected to
SDS-PAGE and immunoblotting with anti-phosphothreonine (upper
panel). Blots were then stripped and probed with anti-HA
(lower panel).
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The Phosphorylated Threonine Is Close to the Carboxyl Terminus of
NMHC-IIA--
Hydroxylamine cleaves specifically between asparagine
and glycine residues, and thus cleaves rat NMHC-IIA into only 6 fragments. Arranged from the amino terminus to the carboxyl terminus,
the sizes are 4.4, 2.4, 21, 7, 44, and 147 kDa. Immunoblotting of hydroxylamine-digested antigen-stimulated RBL-2H3 lysate showed phosphothreonine in 2 bands, at approximately 150 and 190 kDa (Fig.
6). The 150-kDa band is consistent with
the carboxyl-terminal 147-kDa fragment (amino acids 696-1961), while
the 190-kDa band is consistent with incomplete cleavage of the 147-kDa
fragment and the adjacent 44-kDa fragment (amino acids 306-695). To
confirm this, the immunoblot was stripped and reprobed with anti-HA;
the antibody recognized the 150- and 190-kDa bands, showing that they both contain the carboxyl terminus of NMHC-IIA. No other
phosphothreonine bands were detected apart from the band at 35 kDa,
which is found in untreated lysates and is apparently resistant to
hydroxylamine treatment.
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Fig. 6.
Cleavage of NMHC with hydroxylamine.
Lysate from antigen-stimulated RBL-2H3 cells was incubated with
hydroxylamine, and subjected to SDS-PAGE on NuPAGE 4-12% gels using a
MES buffer system and immunoblotting with anti-pThr (left
panel). NMHC-IIA was cleaved to 2 bands (lane 3), at
approximately 150 and 190 kDa. After stripping the blot was reprobed
with anti-HA which recognized the same 2 bands (right
panel), demonstrating them to be the 147-kDa carboxyl-terminal
frament and the 147-kDa fragment uncleaved from the adjacent 44-kDa
fragment. Lanes 1 and 4, control lysate;
lanes 2 and 5, lysate from antigen-stimulated
cells; lanes 3 and 6, hydroxylamine-treated
lysate from antigen-stimulated cells.
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Treatment of antigen-activated RBL-2H3 cell lysate with
TLCK-chymotrypsin led to the rapid disappearance of phosphothreonine from NMHC without the appearance of any major bands at lower molecular weight (results not shown). The disappearance of phosphothreonine paralleled the disappearance of signal obtained after stripping the
blot and reprobing with anti-HA, a rabbit polyclonal antibody raised
against the 12 amino acids at the extreme carboxyl terminus of the
human NMHC-IIA. However, no significant loss of myosin was detected
with anti-platelet myosin antibody, which recognizes epitopes
throughout myosin but primarily in the rod region. These results
suggested that the disappearance of phosphothreonine was the result of
clipping of a small peptide from the carboxyl terminus of NMHC-IIA, so
that the phosphothreonine was removed without significantly affecting
the migration of the remaining myosin molecule. It has previously been
shown that the carboxyl terminus of NMHC distal to the coiled-coil
region is clipped rapidly by chymotrypsin; phosphoserine phosphorylated
by casein kinase II disappears rapidly when NMHC is treated with
chymotrypsin, while the nearby phosphoserine in the coiled-coil region
labeled by PKC is much more resistant to chymotrypsin proteolysis
(21).
CaM Kinase II Phosphorylates the Carboxyl Terminus of Mouse
NMHC-IIA in Vitro--
To explore further the difference between
rodent and human NMHC-IIA in susceptibility to threonine
phosphorylation, glutathione S-transferase fusion proteins
containing the carboxyl termini of mouse and human NMHC-IIA were
expressed in E. coli. Rat (17) and
mouse2 NMHC-IIA contain a
threonine residue (Thr-1940) between the coiled-coil region and the
carboxyl terminus, which is part of a CaM kinase consensus sequence. In
the human NMHC-IIA sequence this threonine is replaced by alanine (22).
Incubation of the mouse fusion protein with CaM kinase II and ATP
resulted in phosphorylation of the fusion protein (Fig.
7A). In contrast, a fusion
protein containing the carboxyl terminus of human NMHC-IIA was not
phosphorylated by CaM kinase II. Immunoblotting confirmed the presence
of phosphothreonine in the mouse but not the human fusion protein
(results not shown). Mutation of Thr-1940 to alanine in the mouse
fusion protein prevented phosphorylation by CaM kinase II (Fig.
7B), while the reciprocal mutation converting Ala to Thr in
the human fusion protein led to phosphorylation by CaM kinase II (Fig.
7C).
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Fig. 7.
Phosphorylation of NMHC-IIA carboxyl terminus
in vitro by CaM kinase II. A, CaM kinase II
phosphorylates mouse but not human NMHC-IIA glutathione
S-transferase carboxyl-terminal fusion protein. Fusion
proteins were incubated with CaM kinase II and
[-32P]ATP. Assay mixtures were then subjected to
SDS-PAGE, and phosphorylation detected by autoradiography. The mouse
fusion protein has an apparent molecular mass of 48 kDa, the human
fusion protein an apparent molecular mass of 45 kDa. B,
mutation of Thr-1940 to Ala prevents phosphorylation of mouse fusion
protein. C, mutation of Ala-1940 to Thr allows
phosphorylation of the human fusion protein. D, CaM kinase
II phosphorylates rat NMHC-IIA peptide. The CaM kinase II substrate
autocamtide-2, and a peptide corresponding to residues 1933-1951 of
rat NMHC-IIA were phosphorylated in vitro by CaM kinase II,
and the assay mixtures subjected to SDS-PAGE. E,
phosphoamino acid analysis of the phosphorylated NMHC-IIA peptide
demonstrated that the phosphorylated amino acid was threonine.
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To confirm that Thr-1940 is phosphorylated in vitro by CaM
kinase II, a peptide corresponding to residues 1933-1951 of rat NMHC-IIA (RRIVRKGTGDCSDEEVDGK) was phosphorylated by CaM kinase II. The peptide was readily phosphorylated (Fig. 7D),
and phosphoamino acid analysis of the phosphorylated peptide confirmed that the phosphate was incorporated only into Thr-1940 (Fig.
7E).
Constitutively Active CaM Kinases II and IV Phosphorylate Thr-1940
in Situ--
To determine whether CaM kinases can phosphorylate
NMHC-IIA in situ, HeLa cells were transfected with plasmids
encoding constitutively active CaM kinases I, II, or IV. Cells were
also co-transfected with a plasmid encoding mutant human NMHC-IIA with
Ala-1940 converted to Thr, and with GFP fused to the amino terminus.
Threonine phosphorylation of cell proteins was analyzed 48 h later
by immunoblotting. Ala Thr-1940 human NMHC-IIA was threonine
phosphorylated in situ by activated CaM kinases II (Fig.
8A, lane 3) or IV (lane
4), but not CaM kinase I (lane 2).
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Fig. 8.
Phosphorylation of human NMHC-IIA
(Ala-1940 Thr) in situ by CaM
kinases. A, HeLa cells were transfected with a plasmid
expressing Ala Thr-1940 human NMHC-IIA GFP fusion protein
(lanes 2-5), and constitutively active CaM kinase I, II,
and IV were co-transfected (lanes 2, 3, and 4,
respectively). After 48 h, cells were lysed, and soluble proteins
immunoblotted with anti-phosphothreonine. B, constitutively
active CaM kinase II was transfected into HeLa cells alone (lane
1) or coexpressed with GFP-fusion proteins of wild-type
(lane 2) or Ala-1940 Thr (lane 3) human
NMHC-IIA. After 48 h, cells were lysed, and soluble proteins
immunoblotted with anti-phosphothreonine (top panel). The
blot was then stripped and blotted again with anti-GFP (lower
panel).
|
|
Co-transfection of wild-type and Ala Thr-1940 NMHC-IIA with CaM
kinase II showed that only the mutant protein was threonine phosphorylated (Fig. 8B, lane 3). Endogenous NMHC-IIA was
also not phosphorylated by transfected CaM kinase II (lanes
1 and 2). Measurement of CaM kinase II activity in the
lysates showed that the activity was similar in all cases, increased
approximately 10-fold over basal levels, excluding differences in CaM
kinase transfection efficiency as a factor. Similar results were
obtained when CaM kinase IV was co-transfected in place of CaM kinase
II (results not shown).
CaM Kinase II Is Activated by Carbachol and Antigen in
RBL-2H3 Cells--
To assess whether CaM kinase II is activated in
RBL-2H3 cells in response to stimuli which cause NMHC phosphorylation,
CaM kinase II activity was determined in cell lysates following cell stimulation (Fig. 9). Carbachol
stimulation of RBL-2H3 m1 cells led to a rapid increase in autonomous
calcium/calmodulin-independent CaM kinase activity which was detectable
within 15 s, peaked at 2 min, and returned toward baseline by 30 min. Stimulation of IgE-sensitized cells with DNP-BSA led to a slower
increase in activity which was maximal at 10-20 min. The increases in
CaM kinase II activity thus parallel the increase in NMHC-IIA threonine phosphorylation.
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Fig. 9.
Time courses of CaM kinase II activation in
response to carbachol and antigen. RBL-2H3 m1 cells were lysed
following stimulation with antigen or carbachol for the times shown,
and CaM kinase II activity was determined by phosphorylation of
autocamtide-2 in the presence of EGTA (autonomous activity) or
calcium/calmodulin (total activity). Autonomous activity is expressed
as a percentage of total activity. Squares, carbachol;
circles, antigen. Total activity did not change (results not
shown).
|
|
Inhibitors of CaM Kinase Inhibit Secretion--
To determine
whether CaM kinase has any functional role in the secretory process in
RBL-2H3 cells, cells were stimulated with antigen or with carbachol in
the presence or absence of inhibitors of CaM kinases, and secretion
monitored by the release of hexosaminidase. KN-93, inhibited
hexosaminidase release in response to carbachol, while KN-92, an
inactive analogue of KN-93, had no effect on secretion (Fig.
10A). In contrast, KN-93 had
only a small inhibitory effect on secretion in response to antigen
(Fig. 10B). Moreover, there was no significant difference
between the inhibitory effects of the active and inactive homologs,
suggesting that this may represent a nonspecific effect.
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Fig. 10.
CaM kinase inhibitors reduce secretion in
response to carbachol but not to antigen. RBL-2H3 m1 cells were
incubated with or without anti-DNP IgE (0.5 mg/ml) overnight. Following
incubation with KN-92 or -93 (10 µM) for 30 min, the
cells were stimulated with carbachol (250 µM) or DNP-BSA
(20 ng/ml). Hexosaminidase released into the medium was determined and
expressed as a percentage of total hexosaminidase, measured by lysing
the cells. Results are mean ± S.E. for six to eight experiments.
*, p < 0.05 versus antigen alone; **,
p < 0.01 versus carbachol alone.
|
|
| |
DISCUSSION |
The activation of secretory cells and release of mediators from
secretory granules requires the granules to penetrate a cortical cytoskeletal barrier of actomyosin and other components in order to
fuse with the plasma membrane and release their contents into the
extracellular space (23). Previous work has shown that stimulation of
sensitized mast cells with antigen leads to the rapid
PKC-dependent phosphorylation of nonmuscle myosin on serine
residues in both the light and heavy chains (5). The kinetics of early
shape change and formation of myosin-deficient lamellopodia in RBL-2H3 cells correlate temporally, consistent with a role for myosin phosphorylation in the disruption of the cortical actomyosin rim that
occurs during mast cell activation (24). This disruption may in turn
facilitate interaction between the secretory granule and the plasma
membrane. Here we demonstrate for the first time that CaM kinase II is
activated in RBL-2H3 cells by stimuli which increase cytoplasmic
calcium. The activation of CaM kinase II results in the phosphorylation
of NMHC-IIA at Thr-1940, close to the carboxyl terminus and distal to
the coiled-coil region.
The essential role of increases in intracellular calcium and PKC
activity in mast cell activation have been recognized for many years
(1-4). However, the potential role of the other main calcium-dependent kinase family, the CaM kinases, in mast
cell activation has not been studied in depth. The CaM kinases are a
family of three kinases, CaM kinases I, II, and IV. CaM kinases I and
IV are monomeric, while CaM kinase II is a large multimer. CaM kinase I
and IV, but not CaM kinase II, are regulated by an upstream kinase, CaM
kinase kinase (25). CaM kinase II is activated in RBL-2H3 m1 cells
stimulated with antigen or with carbachol, and activation of CaM kinase
II is accompanied by the threonine phosphorylation of NMHC-IIA with a
similar time course. The phosphorylation is
calcium-dependent, requiring both an elevation of
intracellular calcium, blocked by BAPTA, and influx of extracellular
calcium, blocked by brief incubation with EGTA. Carbachol stimulation
led to a larger increase in NMHC-IIA phosphothreonine than antigen, paralleling the larger increase in intracellular calcium (16) and the
greater extent of secretion (8) found with carbachol relative to
antigen. Phosphorylation appears to be mediated via a CaM
kinase-dependent pathway rather than by PKC, since
threonine phosphorylation of NMHC is inhibited by the CaM kinase
inhibitor KN-93 but not by the PKC inhibitor GF109203X. In addition,
treatment of the cells with PMA did not result in threonine
phosphorylation of NMHC-IIA. KN-93 inhibits CaM kinase II with an
IC50 of 0.37 µM (19), but also inhibits CaM
kinase I and IV (IC50 2.7 and 50 µM,
respectively) (26). While the time course of activation of CaM kinase
II and effects of constitutively active CaM kinase II are consistent
with a role for this enzyme in NMHC-IIA threonine phosphorylation,
involvement of CaM kinase IV cannot be excluded. Since KN-93 is less
effective against CaM kinase IV, a contribution from CaM kinase IV
could explain the incomplete inhibition of NMHC-IIA threonine
phosphorylation by KN-93.
There is increasing evidence that converging
calcium/calmodulin-dependent pathways are important for
secretion. Calmodulin-dependent activation of myosin light
chain kinase and phosphorylation of myosin light chain is required for
calcium-induced cortical F-actin disassembly (27). CaM kinase
inhibitors also inhibit insulin secretion in pancreatic cells (28) and
gonadotropin-releasing hormone secretion from infundibular explants
(29), suggesting that there may be a more general role for CaM kinases
in secretory processes. Threonine phosphorylation of NMHC-IIA is also
found in pancreatic cells undergoing secretion (7, 30). The effect of
CaM kinase phosphorylation of Thr-1940 in NMHC-IIA on the interactions between actin and myosin (and possibly other cytoskeletal proteins) may
give further insight into the secretory process. The ability of the CaM
kinase inhibitor KN-93, but not the inactive analogue KN-92, to inhibit
secretion in RBL-2H3 cells in response to carbachol lends further
credence for a functional role for CaM kinase in mast cell secretion.
The lack of effect of KN-93 on antigen-mediated secretion, and the
absence of CaM kinase phosphorylation of NMHC-IIA in human cells,
implies that NMHC-IIA threonine phosphorylation may have a facilitatory
role rather than being essential for secretion. This may reflect
redundancy in the IgE-mediated pathway, perhaps between PKC and CaM
kinase-mediated NMHC-IIA phosphorylation. Alternatively, CaM kinase may
be having additional upstream effects in the carbachol-mediated pathway
which are responsible for the inhibition of carbachol-mediated
secretion by KN-93.
The non-helical carboxyl-terminal region of the myosin rod modulates
the assembly of myosin filaments (31-33), and it has been suggested
that phosphorylation of the NMHC carboxyl terminus affects filament
formation in an isoform-specific manner; phosphorylation by either PKC
or casein kinase inhibits assembly of NMHC-IIB isoforms, but not of
NMHC-IIA (21). An alternative mechanism by which threonine
phosphorylation of NMHC-IIA could affect filament assembly is through
interactions with other proteins, and in particular with Mts 1. Mts 1 is a 9-kDa calcium-binding protein of the S100 family, which is
up-regulated in cancer cells and highly motile cells (34). Mts 1 binds
to the non-helical region of NMHC and destabilizes filaments (35).
Binding occurs at residues 1909-1937 of human platelet NMHC-IIA, and
inhibits phosphorylation of Ser-1917 by PKC (36). It will therefore be
of interest to determine whether phosphorylation of NMHC-IIA at
Thr-1940 has any effect on filament assembly or Mts 1 binding.
Preliminary experiments have shown that Mts 1 also inhibits
phosphorylation of NMHC-IIA by CaM kinase II in
vitro.3
Calcium-dependent threonine phosphorylation of NMHC-IIA has
now been shown in a range of rodent cells, including PC12 rat pheochromacytoma cells and mouse NIE-115 neuroblastoma cells (6), rat
islets and rat RINm5F insulinoma cells (7), in the mouse pancreatic
cell line TC6-F7 (30), and in mouse fibroblasts and rat
RBL-2H3 mast cells. However, increasing intracellular calcium with
ionomycin did not result in threonine phosphorylation of NMHC-IIA in
HeLa cells or the Y79 retinoblastoma cell line, both of which are
derived from humans. While the absence of some component of the signal
transduction cascade leading to myosin phosphorylation cannot be
excluded, a more likely explanation is a sequence difference between
rodent and human NMHC-IIA, resulting in the absence of the
phosphorylated threonine or targeting residues in human cells. The
carboxyl terminus of mouse NMHC-IIA was phosphorylated in
vitro by CaM kinase, while human NMHC-IIA carboxyl-terminal was
not. Inspection of the sequences for rat and mouse NMHC-IIA distal to
the coiled-coil region showed 2 conserved threonines which are absent
in human NMHC-IIA; in particular, comparison of the conserved rat (17)
and mouse2 sequence surrounding Thr-1940
(Ile-Val-Arg-Lys-Gly-Thr-Gly) with the optimal CaM kinase
consensus sequences for CaM kinase IV
(Hyd-X-Arg-X-X-Ser/Thr) and
CaM kinase IIa
(Hyd-X-Arg-X-NB-Ser/Thr-Hyd) (37) show
a good correspondence, while the sequence is less well conserved in
human NMHC-IIA (Met-Ala-Arg-Lys-Gly-Ala-Gly) (22).
This high degree of sequence conservation in rat and mouse made it a
good candidate as the kinase target, which was confirmed with mutants
of the NMHC-IIA fusion proteins. Mutation of Thr-1940 to Ala in mouse
NMHC-IIA abrogated in vitro phosphorylation, while mutation
of Ala-1940 to Thr in human NMHC-IIA restored phosphorylation by CaM
kinase II. A peptide corresponding to rat NMHC-IIA residues 1933-1951 was phosphorylated in vitro by CaM kinase
II on Thr-1940, indicating that the effects of mutation of residue 1940 on fusion protein phosphorylation were not due to indirect
conformational changes. Co-expression experiments in HeLa cells showed
that constitutively active CaM kinase II also phosphorylated mutant
full-length NMHC-IIA containing Thr-1940, but not wild type NMHC-IIA.
Taken together these data confirm that Thr-1940 of rodent NMHC-IIA is
phosphorylated by CaM kinase II, and that the absence of this site in
human NMHC-IIA explains the lack of threonine phosphorylation in
stimulated human cells.
The absence of threonine phosphorylation in human cells is interesting,
and may imply that they are able to rearrange their actomyosin
cytoskeleton with only PKC-dependent serine phosphorylation of NMHC-II, in addition to serine phosphorylation of the nonmuscle myosin light chain. Conversely, human cells could have developed an
alternative phosphoacceptor pathway, as has been demonstrated for the
transcription factor C/EBP. The highly conserved threonine phosphoacceptor (Thr-217) in the transcription factor C/EBP required for transforming growth factor  -mediated hepatocyte proliferation in
mouse, is replaced by alanine in rat, while mouse/human Ala-105 has
been replaced by Ser-105 in rat (38). The two phosphoacceptors have
analogous functions, since mutation of mouse Thr-217 or rat Ser-105
with Ala blocks proliferation while replacement with Asp promotes
proliferation in both cases. It will be of interest to determine by
peptide mapping whether any additional phosphoacceptors have evolved in
human NMHC-IIA to substitute for the phosphothreonine.
The activation of CaM kinase II that we have demonstrated also raises
the possibility that CaM kinase could contribute to the changes in gene
expression found following mast cell activation (39). All three CaM
kinases are found in the nucleus as well as the cytosol; for CaM kinase
I and IV, nuclear localization is probably by diffusion through nuclear
pores, while for CaM kinase II certain isoforms are targeted to the
nucleus as a result of an alternatively spliced nuclear localization
signal (40). The nuclear CaM kinases can phosphorylate transcription
factors such as cAMP-response element-binding protein, leading to
changes in gene expression (40).
In summary, stimulation of RBL-2H3 m1 cells with mediators which
increase intracellular calcium leads to activation of CaM kinase II and
phosphorylation of NMHC-IIA on Thr-1940. Inhibition of CaM kinase II
decreases myosin phosphorylation in response to antigen and carbachol,
and also inhibits secretion in response to carbachol. Threonine
phosphorylation of NMHC-IIA thus appears to be obligatory for some but
not all stimulants, and is species-dependent, which may
indicate the existence of alternative pathways to achieve the
appropriate functional response.
| |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Richard Maurer (Oregon
Health Sciences University) for the CaM kinase expression vectors; Dr.
Michael A. Beaven for RBL-2H3 m1 cells, and helpful discussions; Dr.
Sachiyo Kawamoto for Y79 cells; Dr. Mary Anne Conti for the
COOH-terminal mouse NMHC-IIA clone; and Dr. Qize Wei for the human
NMHC-IIA-GFP expression vector.
| |
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. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Laboratory of
Molecular Cardiology, NHLBI, Bldg. 10, Rm. 8N202, 10 Center Dr., Bethesda, MD 20892-1762. Tel.: 301-496-5639; Fax: 301-402-1542; E-mail:
db225a@nih.gov.
Published, JBC Papers in Press, August 16, 2000, DOI 10.1074/jbc.M004996200
2
M. A. Conti and R. S. Adelstein,
unpublished results.
3
D. B. Buxton and R. S. Adelstein,
unpublished results.
| |
ABBREVIATIONS |
The abbreviations used are:
PKC, protein kinase
C;
NMHC, nonmuscle myosin heavy chain;
CaM kinase, calcium/calmodulin-dependent protein kinase;
PMA, phorbol
12-myristate 13-acetate;
GF109203X, 2-[1-(3-dimethylaminopropyl)1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide;
BAPTA-AM, 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid tetra(acetoxymethyl)ester;
KN-92, 2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzyl-amine
phosphate;
KN-93, N-(2-[N-{4-chlorocinnamyl]-N-methylaminomethyl]phenyl)-N-(2-hydroxyethyl)-4-methoxybenezenesulfonamide;
anti-HA, antibody to human NMHC-IIA carboxyl terminus;
ERK, extracellular regulated kinase;
TLCK, N-tosyl-L-lysine chloromethyl ketone;
PAGE, polyacrylamide gel electrophoresis;
GFP, green fluorescent protein;
Hyd, hydrophobic;
NB, non-basic;
MES, 4-morpholineethanesulfonic acid;
DMS, dimethylsphingosine;
BSA, bovine serum albumin;
DNP, dinitrophenol;
DMEM, Dulbecco's modified Eagle's medium.
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A. Verma and A. T. Maurelli
Identification of Two Eukaryote-Like Serine/Threonine Kinases Encoded by Chlamydia trachomatis Serovar L2 and Characterization of Interacting Partners of Pkn1
Infect. Immun.,
October 1, 2003;
71(10):
5772 - 5784.
[Abstract]
[Full Text]
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J. Holst, A. T.R. Sim, and R. I. Ludowyke
Protein Phosphatases 1 and 2A Transiently Associate with Myosin during the Peak Rate of Secretion from Mast Cells
Mol. Biol. Cell,
March 1, 2002;
13(3):
1083 - 1098.
[Abstract]
[Full Text]
[PDF]
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Copyright © 2000 by the American Society for Biochemistry and Molecular Biology.
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