Analysis of the role of Ser1/Ser2/Thr9 phosphorylation on myosin II assembly and function in live cells.

Abstract

Phosphorylation of non-muscle myosin II regulatory light chain (RLC) at Thr18/Ser19 is well established as a key regulatory event that controls myosin II assembly and activation, both in vitro and in living cells. RLC can also be phosphorylated at Ser1/Ser2/Thr9 by protein kinase C (PKC). Biophysical studies show that phosphorylation at these sites leads to an increase in the Km of myosin light chain kinase (MLCK) for RLC, thereby indirectly inhibiting myosin II activity. Despite unequivocal evidence that PKC phosphorylation at Ser1/Ser2/Thr9 can regulate myosin II function in vitro, there is little evidence that this mechanism regulates myosin II function in live cells.
The purpose of these studies was to investigate the role of Ser1/Ser2/Thr9 phosphorylation in live cells. To do this we utilized phospho-specific antibodies and created GFP-tagged RLC reporters with phosphomimetic aspartic acid substitutions or unphosphorylatable alanine substitutions at the putative inhibitory sites or the previously characterized activation sites. Cell lines stably expressing the RLC-GFP constructs were assayed for myosin recruitment during cell division, the ability to complete cell division, and myosin assembly levels under resting or spreading conditions. Our data shows that manipulation of the activation sites (Thr18/Ser19) significantly alters myosin II function in a number of these assays while manipulation of the putative inhibitory sites (Ser1/Ser2/Thr9) does not.
These studies suggest that inhibitory phosphorylation of RLC is not a substantial regulatory mechanism, although we cannot rule out its role in other cellular processes or perhaps other types of cells or tissues in vivo.

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RESEARCH ARTICLEOpen Access
Analysis of the role of Ser1/Ser2/Thr9
phosphorylation on myosin II assembly and
function in live cells
Jordan R Beach1,2, Lucila S Licate1, James F Crish1and Thomas T Egelhoff1*
Abstract
Background: Phosphorylation of non-muscle myosin II regulatory light chain (RLC) at Thr18/Ser19 is well
established as a key regulatory event that controls myosin II assembly and activation, both in vitro and in living
cells. RLC can also be phosphorylated at Ser1/Ser2/Thr9 by protein kinase C (PKC). Biophysical studies show that
phosphorylation at these sites leads to an increase in the Km of myosin light chain kinase (MLCK) for RLC, thereby
indirectly inhibiting myosin II activity. Despite unequivocal evidence that PKC phosphorylation at Ser1/Ser2/Thr9
can regulate myosin II function in vitro, there is little evidence that this mechanism regulates myosin II function in
live cells.
Results: The purpose of these studies was to investigate the role of Ser1/Ser2/Thr9 phosphorylation in live cells. To
do this we utilized phospho-specific antibodies and created GFP-tagged RLC reporters with phosphomimetic
aspartic acid substitutions or unphosphorylatable alanine substitutions at the putative inhibitory sites or the
previously characterized activation sites. Cell lines stably expressing the RLC-GFP constructs were assayed for
myosin recruitment during cell division, the ability to complete cell division, and myosin assembly levels under
resting or spreading conditions. Our data shows that manipulation of the activation sites (Thr18/Ser19) significantly
alters myosin II function in a number of these assays while manipulation of the putative inhibitory sites (Ser1/Ser2/
Thr9) does not.
Conclusions: These studies suggest that inhibitory phosphorylation of RLC is not a substantial regulatory
mechanism, although we cannot rule out its role in other cellular processes or perhaps other types of cells or
tissues in vivo.
Background
Non-muscle myosin II is expressed in nearly every
eukaryotic cell, where it plays critical roles in a number
of cellular processes, including cell division and cell
migration. Myosin II molecules are comprised of two
heavy chains (MHC), two essential light chains (ELC)
and two regulatory light chains (RLC). The MHC con-
sists of a globular head domain that contains that actin
binding and ATPase properties, a linker region that con-
tains the binding sites for the ELC and RLC and a
coiled-coil rod domain that allows the MHC to dimerize
and assemble into bipolar filaments. Myosin II is in
constant equilibrium between monomeric and filamen-
tous forms. The cell achieves spatio-temporal control of
myosin II assembly and activation by modulation of this
equilibrium, primarily through phosphorylation events.
There are two groups of residues on the RLC that are
phosphorylated by distinct kinases and have contrasting
effects on myosin II biophysical properties. The first
group is Thr18/Ser19. These residues are phosphory-
lated by myosin light chain kinase, Rho kinase and
others [1]. Phosphorylation at Thr18/Ser19 is a well-
established regulatory mechanisms that increases the
actin-activated ATPase activity of the holoenzyme and
shifts the molecule into a filamentous state [2,3]. There-
fore, Thr18/Ser19 phosphorylation essentially “activates”
the myosin molecule to produce force. The second
group of phosphorylated residues is at the N-terminus
* Correspondence: egelhot@ccf.org
1Department of Cell Biology, Lerner Research Institute NC-10, Cleveland
Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA
Full list of author information is available at the end of the article
Beach et al. BMC Cell Biology 2011, 12:52
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© 2011 Beach et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

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of the RLC at Ser1, Ser2 and Thr9 [4]. These residues
have been shown to be phosphorylated by PKC [5]. Bio-
physical studies showed that PKC phosphorylation leads
to a 9-fold increase in the Km of MLCK for RLC,
thereby indirectly favoring a less active state for the
myosin II itself [6]. Further in vitro studies with Xeno-
pus myosin II using alanine substitution at either Ser1/
Ser2 or Thr9 followed by PKC pre-phosphorylation of
the remaining non-mutated residue identified Thr9 as
the critical inhibitory phosphorylation event [7].
Live cell studies showed that phosphorylation at Ser1/
Ser2 (but not Thr9) is elevated 6-12 fold higher in cells
arrested in mitosis versus non-mitotic cells [8]. Release
of the cells from mitotic arrest results in a decrease in
Ser1/Ser2 phosphorylation over the next hour, as the
cells progress through cell division [8]. These studies
support the hypothesis that “inhibitory” phosphorylation
at Ser1/Ser2, and perhaps Thr9, is a mechanism by
which the contractile machinery for cell division is held
in an inactive form during metaphase then activated
after the metaphase/anaphase transition. One recent
study identified elevated Ser1 phosphorylation in fibro-
blasts following treatment with platelet-derived growth
factor (PDGF) [9], concordant with disassembly of acto-
myosin stress fibers. Based on visual scoring, stress fiber
disassembly was reported to be attenuated with expres-
sion of an un-phosphorylatable RLC at Ser1/Ser2 [9].
However, aside from this single report, no studies have
addressed the importance of Ser1/Ser2/Thr9 phosphory-
lation in live cell settings.
The goal of our studies was to quantify the effect of
RLC inhibitory phosphorylation at Ser1/Ser2/Thr9 on
myosin II activity and assembly in live cells in multiple
settings. Utilizing phospho-specific antibodies and GFP-
tagged RLC constructs with mutations at the putative
activating and inhibitory sites, we find clear modulatory
roles for Thr18/Ser19 phosphorylation. However, our
data is inconsistent with the hypothesis that Ser1/Ser2/
Thr9 phosphorylation significantly regulates myosin II
function in live cells in the settings of cytokinesis or cell
spreading.
Results and Discussion
Using a phospho-specific antibody to RLC Ser1, we
immunostained HeLa and primary human keratinoctyes
under normal growth conditions. In agreement with
published reports [8,10], RLC Ser1 phosphorylation is
dramatically enhanced in mitotic cells (Figure 1A). The
previous study by Yamakita and colleagues examined
RLC phosphorylation following a semi-synchronous
release from mitotic arrest in metaphase. In doing so
they observed that Ser1/Ser2 phosphorylation decreased
as the cells progressed through mitosis. However, it is
not clear if the residual Ser1/Ser2 phosphorylation in
their “released” cells was in cells that had not yet transi-
tioned in to anaphase or in cells that were in anaphase.
To determine if RLC remains phosphorylated at Ser1 in
cells that are actively dividing, we imaged HeLa cells in
the anaphase state. In doing so, we observed that RLC
Ser1 phosphorylation is enhanced specifically in the
contractile ring of anaphase cells (Figure 1B). This was
also true in primary human keratinocytes (Figure 1B,
bottom row). The Ser1-P antibody also strongly localizes
to peri-chromosomal regions in the HeLa cell line but
not the primary keratinocytes. As we never observe
endogenous RLC or MHC localizing to these areas (data
not shown) this peri-chromosomal staining either repre-
sents a very small percentage of the total myosin II in
the cell or it represents spurious antibody reactivity with
something other than the RLC.
It appears contradictory that myosin II is phosphory-
lated at putative inhibitory sites in the contractile ring,
where myosin II activity is proposed to be the dominant
force contributing to furrow ingression. However, inhi-
bitory phosphorylation and activated myosin II in a spe-
cific local, such as the contractile ring, may not be
mutually exclusive. In Dictyostelium discoideum, myosin
II disassembly is necessary for efficient completion of
cytokinesis [11]. Similarly, it may be that in mammalian
cells, RLC inhibitory phosphorylation contributes to dis-
assembly of myosin II in the contractile ring as the fur-
row ingresses.
To determine if modulation of the putative inhibitory
residues would alter myosin II behavior during cell divi-
sion, we created GFP-tagged RLC constructs with non-
phosphorylatable alanine substitutions (RLC-4A) and
phosphomimetic aspartic acid substitutions (RLC-4D) at
Ser1/Ser2/Thr9/Thr10. Thr10 was previously shown to
be a secondary phosphorylation site when Thr9 is
mutated [7], so this residue was also mutated in all con-
structs. As controls, we also created non-phosphorylata-
ble (RLC-T18A/S19A) and phosphomimetic (RLC-
T18D/S19D) mutations at the activation sites. These
constructs, along with the wild type RLC-GFP, were sta-
bly transfected into tetracycline-responsive HeLa cells,
allowing temporally regulated expression of the mutated
RLC-GFP proteins to avoid any chance of compensatory
background changes or adaptation in cells carrying
mutant constructs. When induced, these constructs
expressed at slightly higher levels than the endogenous
RLC and oftentimes appear to suppress expression of
the endogenous RLC (Figure 2A), presumably resulting
in a higher replacement of exogenous RLC on the myo-
sin II molecule. Note that the RLC antibody used
appears to have a lower affinity for the RLC-4A con-
struct. To further quantitate the percent replacement of
RLC-GFP for endogenous RLC we performed immuno-
precipitation of MHC IIA followed by western blotting
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(Figure 2B). Unfortunately, the IgG heavy chain co-
migrates with the RLC-GFP constructs, preventing any
ratio analysis of RLC:RLC-GFP. Use of a mouse primary
antibody for the IP followed by a rabbit antibody for
western blotting or use of immuno-depleted secondary
antibodies did not alleviate this problem. However, we
were able to ratio the endogenous RLC to the amount
of MHC IIA immunoprecipitated. Under the assumption
that the remaining MHC IIA is occupied with RLC-GFP,
we estimate our percent replacement to be ~80-95%.
To determine if mutations at the putative inhibitory
sites altered the ability of myosin II to localize to and
assemble in the furrow during cell division, we stained
HeLa cells for the most abundant MHC isoform, MHC
Figure 1 RLC Ser1 phosphorylation is elevated in mitotic cells and localizes to the contractile ring. (A) HeLa and primary human
keratinocytes (PHK) under normal growth conditions were fixed and immunostained for RLC Ser1-P (green) and stained for actin (red). (B) RLC
Ser1-P is specifically enhanced in the contractile ring of dividing cells.
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IIA. Recruitment of myosin II to the furrow was
assessed by fluorescent microscopy (Figure 2C), and
quantitated by normalizing peak cortical furrow signal
to cytoplasmic signal in a number of cells (Figure 2D).
Relative to expression of wild type RLC, expression of
RLC-T18A/S19A resulted in a decrease in the amount
of cortically assembled myosin II in the furrow (Figure
2C &2D). In contrast, expression of RLC-T18D/S19D
resulted in a relative overassembly of myosin II in the
furrow (Figure 2C &2D). If phosphorylation of RLC at
Ser1/Ser2/Thr9 inhibits myosin II assembly during cyto-
kinesis, then expression of RLC-4D should result in a
decrease in myosin II cortical signal in the furrow. This
does not appear to be the case (Figure 2C &2D). In fact
furrow recruitment was slightly elevated in this setting.
Furthermore, myosin II cortical assembly in RLC-4A
expressing cells was statistically indistinguishable from
wild type RLC expressing cells. This data suggests that
phosphorylation of Ser1/Ser2/Thr9 does not alter myo-
sin II assembly levels in the contractile ring.
Figure 2 Expression, localization and assembly of MHC IIA during cell division. (A) Whole cell lysates of HeLa cells expressing the indicated
RLC construct were probed for total RLC, GFP and MHC IIA. (B) MHC IIA was immunoprecipitated from the indicated cell line and the eluate was
probed for MHC IIA, RLC and GFP. Note that the RLC-GFP and IgG heavy chain migrate too close to differentiate. The number under each
construct indicates the relative amount of RLC present after normalization to MHC IIA based on densitometry. (C) Anaphase cells expressing the
indicated RLC construct (green) were fixed and stained for MHC IIA (red). Representative images are shown. (C) MHC IIA recruitment was
quantitated for a minimum of 13 cells. Data are mean +/- SEM. * indicates p = < 0.003.
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Note that the RLC-GFP constructs often localize to
the spindle independent of the MHC (Figure 2C). We
do not see this spindle localization when we immunos-
tain for endogenous RLC protein (data not shown). This
spindle localization appears to be different than the
peri-chromosomal staining seen with the Ser1-P anti-
body (Figure 1B). Therefore, these appear to be indepen-
dent anomalies from as of yet unknown origins.
It remains possible that phosphorylation at Ser1/Ser2/
Thr9 may alter myosin II activity during cell division,
resulting in an altered rate of furrow ingression. To
investigate this, we measured furrow ingression rates of
dividing cells expressing each of the RLC constructs
(Figure 3A). The only cells that consistently had a statis-
tically significant slower furrow rate relative to wild type
were the cells expressing RLC-T18A/S19A. Surprisingly,
even these cells were able to complete furrow ingression
and divide successfully, similar to previous reports [12].
Furthermore, analysis of nucleation state as an indicator
of multinucleation revealed that none of the RLC
mutant constructs induced a multinucleation phenotype
(Figure 3B). The myosin II inhibitor blebbistatin did
cause a significant shift into a multinucleated state, indi-
cating an absolute necessity for myosin II activity (Fig-
ure 3B). Similar results were obtained in HEK293 cells
(data not shown). In summary, our data suggests that
modulation of the RLC activating sites (Thr18/Ser19)
clearly impacts myosin II recruitment to the furrow, but
has modest effects on furrow rates, and no detectable
effect on completion of furrowing as assessed by
multinucleation. Phosphomimetic substitution of the
putative inhibitory sites (Ser1/Ser2/Thr9) on RLC con-
ferred an effect on assembly opposite of what would be
predicted if these sites are inhibitory and had little or
no effect on furrow rates. Alanine substitutions at the
putative inhibitory sites conferred a phenotype virtually
indistinguishable from wild type.
We next sought to determine if modulation of RLC
Ser1/Ser2/Thr9 phosphorylation could affect myosin II
assembly in non-mitotic cells. HeLa cells under normal
growth conditions that were expressing the RLC con-
structs were immunostained for MHC IIA (Figure 4A).
Relative to cells expressing wild type RLC, cells expres-
sing RLC-T18A/S19A displayed fewer stress fibers and
generally appeared larger, indicating a possible defect in
myosin II assembly and contractility (Figure 4A, column
1&2). In contrast, cells expressing RLC-T18D/S19D
appeared to have enhanced myosin II assembly in stress
fibers (Figure 4A, column 3). MHC IIA assembly in cells
expressing RLC-4D or RLC-4A, which might be pre-
dicted to cause an under-assembly or over-assembly
defect, respectively, was indistinguishable from the wild
type (Figure 4A, columns 4&5).
To quantitate assembly levels of myosin II when dif-
ferent RLC constructs were expressed, we used triton-
insoluble fractionation to separate cytoskeletal and cyto-
solic cellular fractions. Expression of RLC-T18D/S19D
resulted in an increase in assembled myosin II (Figure
4B). Expression of RLC-T18A/S19A, RLC-4A and RLC-
4D did not significantly alter resting assembly levels
Figure 3 Analysis of furrow ingression and multinucleation. (A) HeLa cells expressing the indicated RLC construct were imaged throughout
cell division. Furrow width was measured every 30 seconds for a minimum of 5 cells. Data are mean +/- SEM. * indicates p = <0.05. (B) HeLa
cells expressing the indicated RLC construct or treated with 60 uM blebbistatin for 24 hours were analyzed for nucleation state using propidium
iodide and flow cytometry. Black bars represent cells with DNA content of less than 4n. Grey bars represent cells with DNA content of 4n or
greater.
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