From the Department of Physiology and Pharmacology, Sackler School
of Medicine, Tel-Aviv University, 69978 Ramat Aviv, Israel and the
Department of Physiology and Biophysics, Mount Sinai
School of Medicine, The Mount Sinai Hospital,
New York, New York 10029-6574
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ABSTRACT |
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Various brain K+ channels,
which may normally exist as complexes of (pore-forming) and
(auxiliary) subunits, were subjected to regulation by metabotropic
glutamate receptors. Kv1.1/Kv
1.1 is a voltage-dependent
K+ channel composed of
and
proteins that are widely
expressed in the brain. Expression of this channel in
Xenopus oocytes resulted in a current that had fast
inactivating and noninactivating components. Previously we showed that
basal and protein kinase A-induced phosphorylation of the
subunit
at Ser-446 decreases the fraction of the noninactivating component. In
this study we investigated the effect of protein kinase C (PKC) on the
channel. We showed that a PKC-activating phorbol ester (phorbol
12-myristate 13-acetate (PMA)) increased the noninactivating fraction
via activation of a PKC subtype that was inhibited by staurosporine and
bisindolylmaleimide but not by calphostin C. However, it was not a
PKC-induced phosphorylation but rather a dephosphorylation that
mediated the effect. PMA reduced the basal phosphorylation of Ser-446
significantly in plasma membrane channels and failed to affect the
inactivation of channels having an
subunit that was mutated at
Ser-446. Also, the activation of coexpressed mGluR1a known to activate
phospholipase C mimicked the effect of PMA on the inactivation via
induction of dephosphorylation at Ser-446. Thus, this study identified
a potential neuronal pathway initiated by activation of metabotropic
glutamate receptor 1a coupled to a signaling cascade that possibly
utilized PKC to induce dephosphorylation and thereby to decrease the
extent of inactivation of a K+ channel.
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INTRODUCTION |
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It is now well accepted that modulation of activity of
voltage-gated K+ channel by protein kinases and
phosphatases can regulate neuronal excitability (for recent reviews see
Refs. 1 and 2). Previously, we showed that basal activity of an
unidentified protein kinase endogenous to Xenopus oocytes as
well as activation of protein kinase A
(PKA)1 and PKC modulate the
delayed rectifier-type of current through a Kv1.1 ( subunit)
homomultimeric channel (3-5). We could correlate the modulation of the
channel activity by the unidentified kinase and by PKA with
phosphorylation of Ser-446 on the cytoplasmic C terminus of the
subunit. In the case of PKC, however, mutations of the numerous
putative phosphorylation sites on the
subunit did not eliminate the
modulation by PKC (5).
Mammalian Shaker family homologues such as Kv1.1 may
normally exist as heteromultimers of with
subunits that supply
the pore-occluding domain that confers fast inactivation upon
coexpression of the two subunits in heterologous systems (6, 7).
Recently we showed (8) that the extent of fast inactivation of the
heteromultimeric Kv1.1/Kv
1.1 (
) channel expressed in
Xenopus oocytes is regulated by the basal and PKA-induced
phosphorylations of the
subunits that affect the interaction of the
channel with microfilaments. Part of the interaction is probably
mediated by a native post-synaptic density-95-like protein of the
oocyte that recognizes the C-terminal end of the
subunit (9).
In this work we studied the effect of PKC on the extent of fast
inactivation of the Kv1.1/Kv1.1 channel. We showed that the PKC
effect is opposite that of PKA, and it is mediated by dephosphorylation of Ser-446 that is phosphorylated by PKA.
Glutamate is a major excitatory neurotransmitter in the brain. mGluRs
participate in synaptic plasticity, both in long term potentiation and
long term depression, as well as in neurotoxicity and neuroprotection
(for reviews see Refs. 11-16). mGluRs were shown to inhibit several
types of K+ currents like the M-type current, the
Ca2+-activated current (IKAHP), a
voltage-dependent K+ current
(IK,slow), and resting K+ currents. mGluRs were
shown to activate K+ currents in cerebellar granule cells
(17). They belong to the superfamily of G protein-coupled seven
transmembrane receptors (14, 18) and comprise eight members encoded by
distinct genes, mGluR1-mGluR8. Group I of mGluRs comprises mGluR1 (the
longer splice variant is mGluR1a) and mGluR5 that activate
phospholipase C (probably via coupling to Gq class of G
proteins) and thereby activate a large endogenous
Ca2+-activated chloride current (19, 20) and a PKC subtype,
possibly PKC-µ (21), when expressed in oocytes. In this study we
identify a potential physiological pathway initiated by activation of
mGluR1a coupled to a signaling cascade that possibly utilizes PKC to
modulate the extent of inactivation of Kv1.1/Kv1.1 channel.
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EXPERIMENTAL PROCEDURES |
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Materials--
Chemicals were from Sigma unless stated
otherwise. Vanadate (sodium orthovanadate) and okadaic acid were from
Alomone Labs (Jerusalem); bisindolylmaleimide (BIS) and calyculin A
were from Calbiochem; cyclosporin A was a gift from Sandoz
Pharmaceutical, Basel, Switzerland.
[35S]Methionine/cysteine mix and
[-32P]ATP were from Amersham Corp. Kv1.1 antiserum was
generated against a 23-amino acid peptide that corresponds to the N
terminus of Kv1.1 (SGENADEASAAPGHPQDGSYPRQ), as described (3). Kv1.1,
S446A and Kv
1.1 cDNAs and mRNA preparation were described
previously (8). Kv
1.1 cDNA was a gift of Dr. Pongs (Hamburg,
Germany). mGluR1a cDNA was kindly provided by Dr. Nakanishi (Kyoto,
Japan).
Oocytes, Drug Treatments, and Electrophysiological
Recording--
Frogs (Xenopus laevis) were maintained and
dissected, and their oocytes were prepared as described (22).
Previously (8) we showed that coinjection of Kv1.1 () and Kv
1.1
(
) mRNAs in a 1 to 30 ratio is enough to saturate
with
and to render over 90% of the channels in the form of
in the
plasma membrane. Thus, for biochemical studies oocytes were injected
(50 nl/oocyte) with 100-200 ng/µl Kv1.1 and 3-7
µg/µl Kv
1.1 mRNAs. For electrophysiological studies a ratio of 1 to 100 was used as done previously (8) to ensure
saturation of
with
beyond any doubt; thus, 2-5 ng/µl Kv1.1 and 200-500 ng/µl Kv
1.1 mRNAs
were injected. 5 ng/µl mGluR1a mRNA were injected for both
electrophysiological and biochemical studies. The concentrations of the
injected S446A mRNA deviated slightly from the above concentrations
as it was adjusted to give current amplitudes similar to wild type.
Injected oocytes were incubated at 22 °C for 1-4 days in ND96
solution (8) and then assayed either electrophysiologically or
biochemically. Oocytes injected with mGluR1a were injected with 20 nl
of 50 mM K+-EGTA (pH 7.6) 2-6 h before the
electrophysiological experiment; this corresponds to ~1
mM EGTA in the oocyte. Staurosporine (3 mM in
Me2SO stock, 3 µM final), calphostin C (5 mM in Me2SO stock, 5 µM final),
BIS (2.5 mM in water stock, 5 µM final),
okadaic acid (2 mM in Me2SO stock, 2 µM final), calyculin A (2 mM in
Me2SO stock, 0.3 µM final), and cyclosporin A
(33 mM in ethanol/Tween 80 1:2 stock, 250 µM
final) were added to the standard NDE solution in which the oocytes
were incubated for at least 2 h before the experiment; all the
stock solutions were kept at
20 °C except for that of cyclosporin
A which was prepared daily (the solution of calphostin C was exposed to
daylight for its activation). Control solutions always included the
same concentration of the vehicle as in the corresponding solutions
containing the drug of choice. 10 nM
-phorbol
12-myristate 13-acetate (
-PMA) and
-PMA were applied by constant
bath perfusion after stability of the current had been verified for at
least 5 min. 100 µM glutamate was bath perfused for 1 min
and then washed away.
Metabolic Labeling with [35S]Methionine/Cysteine ([35S]Met/Cys) in Vivo-- This was done essentially as described (4). Following injection of mRNA(s), six to eight oocytes were incubated at 22 °C for 2-4 days in NDE containing 0.2 mCi/ml [35S]Met/Cys. Before homogenization oocytes were incubated with either 100 or 10 nM PMA for 25 min in a dark environment or with 100 µM glutamate for 1 min, as required by the experiment. Homogenization was done in 150-300 µl of medium consisting of 20 mM Tris (pH 7.4), 5 mM EDTA, 5 mM EGTA, 100 mM NaCl, 50 µg/ml phenylmethylsulfonyl fluoride, 1 mM iodoacetamide, 1 µM pepstatin, 1 mM 1,10-phenanthroline supplemented with protein phosphatase inhibitors as follows: 50 nM okadaic acid, 0.5 mM vanadate, and 50 mM KF. Yolk was removed by centrifugation at 1000 × g for 10 min at 4 °C. After addition of Triton X-100 to a final concentration of 4%, followed by centrifugation for 15 min at 4 °C, antiserum was added to the supernatant for 16 h. After 1 h incubation with protein A-Sepharose, immunoprecipitates were washed four times with immunowash buffer (150 mM NaCl, 6 mM EDTA, 50 mM Tris (pH 7.5), 0.1% Triton X-100); the final wash contained no Triton. Samples were boiled in SDS-gel loading buffer and electrophoresed on 8% polyacrylamide-SDS gel (SDS-PAGE).
Plasma membranes were separated mechanically, as described (3). Defolliculated oocytes were placed for 10 min in ice-cold hypotonic solution (5 mM NaCl, 5 mM Hepes, 1 mM phenylmethylsulfonyl fluoride, 1 µM pepstatin, and 1 mM 1,10-phenanthroline) supplemented with phosphatase inhibitors, as specified above. Plasma membranes together with the vitelline membranes (extracellular collagen-like matrix) were removed manually with watchmaker's forceps. Ten internal fractions or 20-30 plasma membranes were processed as described for whole oocytes.PKC Stimulation in Oocyte Homogenates--
This was done
essentially as described (23). Yolk-free homogenates of 10 oocytes that
were injected with mRNA(s) 2-3 days before homogenization were
incubated in 20 mM Tris-HCl (pH 7.4), 5 mM
MgCl2, 5 mM NaH2PO4, 1 mM EDTA, 1 mM dithiothreitol supplemented with
protease inhibitors (as above) in a final volume of 100 µl at
25 °C for 40 min in the presence of 100 nM PMA, 1.5 mM CaCl2 with either 3 mM ATP or 50 µM [-32P]ATP (3000 Ci/mmol). Phosphatase
inhibitors (as above) were added, and the following steps were as
described above for [35S]Met/Cys labeling.
Quantification of Labeling Intensities and Generation of Digitized PhosphorImager Scans-- Gels were dried and placed in a PhosphorImager (Molecular Dynamics) cassette for about 1 day. Using the software ImageQuant, a digitized scan was derived, and relative intensities of protein bands were estimated quantitatively by the software ImageQuant as described (4).
Statistical Analysis-- Data are presented as means ± S.E.; n denotes the number of oocytes assayed. Student's t test was used to calculate the statistical significance of differences between two populations.
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RESULTS |
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PMA Decreases the Extent of Inactivation of the
Kv1.1/Kv1.1 Current--
Previously (5) we showed that oocytes
injected with Kv1.1 (
) RNA and assayed by the two-electrode voltage
clamp technique express a delayed rectifier type of K+
current (
current) that is modulated by PMA. Incubation with 10 nM PMA causes gradual reduction of the
current
amplitude (Ref. 5; e.g. Fig.
1D) that is mediated by
activation of the oocyte's PKC.
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PMA Brings about Dephosphorylation of the Protein--
Previously (3, 4), we showed that the
protein is
extensively phosphorylated in its basal state in the oocyte and that this phosphorylation is manifested as a shift in its migration in
SDS-PAGE; the nonphosphorylated form migrates as a 54-kDa protein, whereas the phosphorylated form migrates as a 57-kDa protein. This
phosphorylation was totally abolished when Ser-446 was replaced by
alanine (S446A mutant). In this study we looked for PKC-induced phosphorylation of the channel proteins first in homogenates of oocytes
and then under in vivo conditions. Fig.
2 shows an experiment in which the
oocyte's PKC was stimulated in homogenates of oocytes expressing
or
channels by stimulating with added 100 nM PMA and
1.5 mM CaCl2 in the presence of added
-ATP,
as was described by Beguin et al. (23) for the Na,K-ATPase.
To our surprise, PKC activation brought about dephosphorylation, rather
than phosphorylation, of the
protein both in
and in
channels. Dephosphorylation of the basally phosphorylated
protein
at Ser-446 was evident from SDS-PAGE analyses of
32P-labeled or [35S]Met/Cys-labeled channel
proteins that were coimmunoprecipitated by a specific anti-
antibody. Thus, the 32P- labeled 57-kDa band that
corresponds to the phosphorylated
protein could be precipitated
from homogenates incubated with [
-32P]ATP (the low
intensity band at 57 kDa in control oocytes that do not express the
channel may represent an endogenous K+ channel; Ref. 3) but
could not be detected after the addition of CaCl2 and PMA
(Fig. 2, right panel). Correspondingly, in homogenates of
oocytes that were metabolically labeled with [35S]Met/Cys
and incubated with cold ATP, the addition of CaCl2 and PMA
induced dephosphorylation of
that was quantified as the reduction
in the extent of its phosphorylation. Thus, following addition of
CaCl2 and PMA, the extent of phosphorylation of
(calculated as the ratio of labeling intensities of the 57-kDa over the
54-kDa bands; see Ref. 4) was reduced to 0.33 and 0.4 of that of
control (without PMA) in
(compare lanes 5 and 2) and
(compare lanes 4 and 1)
channels, respectively (Fig. 2, left panel). The
protein
was not phosphorylated (Fig. 2, right panel). The total
amount of
did not change significantly by the treatment (compare
lanes 1 with 4 (27% increase) and lanes 2 with 5 (4% increase)). This experiment shows that
phosphorylation of the
protein by an enzyme constitutively active
in the oocyte's homogenate was reduced upon stimulation of the
oocyte's PKC. Interestingly, a massive dephosphorylation of oocyte's
proteins probably occurred upon PKC stimulation, as judged from the
extensive reduction of the 32P labeling of the other
proteins nonspecifically pulled into the immune complex (right
panel of Fig. 2), whereas their total amount (intensity of their
[35S]Met/Cys labeling; left panel of Fig. 2)
was not reduced.
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Dephosphorylation of Ser-446 of the Protein Underlies the
Modulation of Extent of Inactivation by PMA--
Next we tested the
possibility that dephosphorylation of Ser-446 underlies the decrease in
the extent of inactivation by PMA. We performed two experiments in
which the modulation by PMA of wild-type (
WT
)
channels was compared with that of the mutant (
S446A
)
channels where Ser-446 in
was replaced by alanine and thereby
rendered the channel totally non-phosphorylated in its basal state (4).
Although the peak amplitude of
S446A
was decreased by
PMA to the same extent as that of
WT
(Fig. 4A), the extent of
inactivation of the mutant was unaffected compared with WT where the
extent of inactivation decreased (as shown in the increased
Is fraction; Fig. 4C). The inactivation of two
other phosphorylation-irrelevant serine mutants S489I and S322A (first in the C terminus and second in the loop between S4 and S5
transmembrane segments, respectively; Ref. 5) was not reduced by PMA
(Fig. 4D). It is noteworthy that the Is current
component of
S446A
did not exhibit biphasic
modulation by PMA; rather, PMA caused an apparent decrease of
Is that was larger than that of
WT
(Fig. 4B). This substantiates the notion that a PMA-induced
increase of Is that overlaps a PMA-induced reduction of
total current (including Ii and Is) underlies
the decrease in the extent of inactivation of the WT channel caused by
PMA.
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mGluR1a Mimics the Effect by PMA--
Since mGluR1a activates
phospholipase C and lately was shown to activate PKC in oocytes (21),
we tested whether activation of mGluR1a, coexpressed with the channel
in oocytes, will mimic the modulation by PMA. Coinjection of ,
,
and mGluR1a RNAs into oocytes gave rise to an
current with an
average amplitude of 53 ± 0.04% of the current elicited in
oocytes that were injected with the same RNA amounts of
and
without mGluR1a (51 and 61 oocytes tested, respectively; 4 frogs;
p < 0.001). The reduced amplitude could be due to
lower expression of the
proteins in oocytes injected with
mGluR1a; however, no evidence for that was obtained in concomitant
biochemical analyses of the level of expression of the proteins in six
experiments (see below). The other alternative would be that the
reduced amplitudes were intrinsic to the receptor modulation of the
current (see below) due to enough receptor molecules being
spontaneously active without agonist, thereby continually activating at
a significant level a signaling cascade that utilizes PKC
(cf. Ref. 30). This notion is supported by the observation
that in two experiments the currents elicited in oocytes injected with
mGluR1a had significantly larger (~140%) Is fractions as
compared with those in oocytes not injected with the receptor.
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The Reduction in Inactivation by mGluR1a Is Due to
Dephosphorylation of Ser-446--
Biochemical analysis of mGluR1a
expressing oocytes in three experiments out of six done (see Fig.
6 for a representative example in oocytes
of one frog) showed that coexpression of the receptor already caused
some reduction in the extent of basal phosphorylation of the protein; the intensity ratio of the 57-kDa over the 54-kDa bands was
reduced to 0.82 ± 0.04 that in oocytes that were not expressing
the receptor. This could correspond to the large Is fractions observed in electrophysiologically assayed oocytes injected with the receptor (see above), substantiating the notion that in
oocytes of some frogs there are enough receptor molecules that are
spontaneously active. Biochemical analysis of all six experiments showed that incubation of the oocytes for 1-5 min in 100 µM glutamate before homogenization resulted in extent of
phosphorylation of the
protein that was 0.72 ± 0.05 that in
oocytes expressing the receptor but were not exposed to the agonist.
Thus, also in this respect mGluR1a mimicked the PMA modulation and
induced dephosphorylation of the
protein, suggesting that its
effect on the extent of inactivation may result from the
dephosphorylation.
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DISCUSSION |
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This study shows that in Xenopus oocytes activation of
PKC by PMA decreases the extent of inactivation (increases the
Is fraction) of rat brain Kv1.1/Kv1.1 (
) current.
It further suggests that the metabotropic glutamate receptor mGluR1a
that is coupled to phospholipase C is a plausible candidate to initiate
such a cellular process and to modulate the inactivation of the
channel in a physiologically relevant environment. Several interesting
aspects to this modulation are as follows: (i) it does not involve
PKC-mediated phosphorylation but rather a PKC-mediated
dephosphorylation of the channel; (ii) it is opposite to the modulation
by PKA that increases the extent of inactivation of the channel (8);
(iii) PKA and PKC signaling pathways converge onto the same site
bringing about phosphorylation and dephosphorylation of Ser-446,
respectively.
PKC-induced Dephosphorylation of Ser-446 Decreases the Extent of
Inactivation of the Channel--
An intriguing mode of
modulation of an ion channel is described here that involves induction
of a dephosphorylating activity by activation of PKC. Involvement of
PKC is evident from the inhibition of the PMA effect on inactivation by
two potent blockers, staurosporine (a wide specificity kinase blocker)
and BIS (a specific PKC blocker). The fact that calphostin C (a
specific PKC blocker) did not inhibit the effect may be interpreted as
an indication of the PKC subtype involved, as detailed in the
following. PKC isoenzymes have been classified into three groups with
different structure and cofactor regulation (31, 32). Activation by PMA
makes the involvement of "atypical" PKC isoenzymes unlikely.
Ca2+ chelation that did not impair the effect by mGluR1a
leaves out the possibility of "conventional" PKCs. Of the "new"
PKCs that are left as candidates, PKC-µ is the most plausible
isoenzyme to mediate the decrease in inactivation as it is insensitive
to calphostin C (33).
mGluR1a-induced Dephosphorylation of Ser-446 Decreases the Extent
of Inactivation of the Channel--
In this study we show that
activation of mGluR1a by glutamate mimics the effect of PMA on
inactivation, the onset being faster (4 min to reach 40% of response
as compared with 10 min for PMA) and the response saturating. The fast
onset of the response could be due to colocalization of the receptor,
the channel, and the oocyte's signaling molecules involved in the
response in sub-membranous sites targeted by protein(s) which serve as
a scaffold (for review see Ref. 44). Notably, a post-synaptic
density-95-like endogenous protein was shown by us to interact with the
channel and to affect its extent of inactivation (9). The effect of
glutamate on the current amplitudes was small compared with that of
PMA, possibly because it was somewhat occluded by the effect exerted by
spontaneous coupling of the receptor without glutamate to the signaling
machinery, since coexpression of the receptor with the channel reduced
the current amplitudes significantly even in the absence of
agonist.
Modulation of the Extent of Inactivation by Dephosphorylation,
Possible Mechanisms--
It is evident from this study that
stimulation of PKC causes a decrease in the extent of inactivation of
the current by dephosphorylating Ser-446. This effect is
opposite that shown by us for a constitutively active kinase (yet
unidentified) or for a stimulated PKA that causes an increase in the
extent of inactivation by phosphorylating Ser-446 that impairs
interaction between the channel and the microfilaments (8). Part of the interaction with the microfilaments was shown to be mediated via a
post-synaptic density-95-like protein that interacts with the C-terminal end of Kv1.1 (9). A similar phenomenon was described in
mammalian cells for the K+ channel Kir 2.3 that dissociates
from post-synaptic density-95 upon PKA phosphorylation of a serine
residue at its C terminus (47). We proposed and have now
confirmed2 a kinetic model
that assumes two modes of gating of the
channel, inactivating
and a noninactivating. In the noninactivating mode the channel's
interaction with microfilaments results in impaired inactivation and
gives rise to the sustained current component (Is), whereas
in the inactivating mode the channel does not interact with the
microfilaments and gives rise to the inactivating current component
(Ii); the equilibrium between the modes is influenced by
the extent of phosphorylation of Ser-446. In this context it is
expected that the PKC-induced dephosphorylation shifts the equilibrium
toward the noninactivating mode which is manifested in the increase in
the sustained fraction of the current.
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ACKNOWLEDGEMENTS |
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We thank Dr. Gal Levin for helpful discussions and help during the first stages of the project and Dr. Nathan Dascal for the critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by a grant from the United States-Israel Binational Science Foundation (to I. L.).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: Dept. of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, 69978 Ramat Aviv, Israel. Tel.: 972-3-6409863; Fax: 972-3-6409113; E-mail: ilotan{at}post.tau.ac.il.
1 The abbreviations used are: PKA, protein kinase A; PKC, protein kinase C; mGluR, metabotropic glutamate receptors; PMA, phorbol 12-myristate 13-acetate; WT, wild type; PAGE, polyacrylamide gel electrophoresis; BIS, bisindolylmaleimide; PP, protein phosphatase.
2 D. Zinger Lahat, D. Chikvashvili, N. Dascal, and I. Lotan, manuscript in preparation.
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REFERENCES |
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