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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 8, 2001 as doi:10.1096/fj.01-0008fje. |
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,**2
* Institute of Human Anatomy, University of Parma, Ospedale Maggiore, 14 43100 Parma, Italy;
Institute of Cytomorphology, CNR c/o Research Institute ‘Codivilla-Putti’, 40100 Bologna, Italy;
Department of Anatomical Sciences, Cellular Signalling Laboratory, University of Bologna, 40126 Bologna, Italy;
Department of Biomedical Sciences and Biotechnologies, Human Anatomy Division, University of Brescia, 25123 Brescia, Italy;
¶ Institute of Human Morphology, University of Chieti, 66100 Chieti, Italy;
** Department of Anatomical Sciences, School of Pharmacy, University of Bologna, 40126 Bologna, Italy; and
Liggins Institute, School of Medicine, University of Auckland, Private Bag 92019, Auckland, New Zealand
2Correspondence: Department of Anatomical Sciences, Cellular Signaling Laboratory (AIRC and PFBiotec), University of Bologna, Via Irnerio 48 I-40126 Bologna, Italy. E-mail: lcocco{at}biocfarm.unibo.it
SPECIFIC AIM
Analyzing the nuclear phospholipase C (PLC) signaling in primary human natural killer (NK) cells and its role in their proliferation induced by interleukin 2 (IL-2), we found that 1) IL-2 transiently stimulates nuclear PLCß1b activity within 60 min of treatment; 2) IL-2 induces nuclear translocation of mitogen-activated protein kinase (MAPK), namely, extracellular signal-related kinase 2 (ERK-2) or p42-MAPK and, to a lesser extent, of ERK-1 or p44-MAPK, whose specific inhibition prevents the IL-2-driven nuclear PLCß1 activation; 3) PLCß1b is serine-phosphorylated after IL-2 treatment and the phosphorylation is abolished after MAPK inhibition; 4) inhibition of nuclear PLC activation leads to the inhibition of the IL-2-induced proliferation of NK cells.
PRINCIPAL FINDINGS
1. Nuclear PLC activity in NK cells is up-regulated by IL-2
The PLC assay has been paralleled by the measurement of the actual
mass of nuclear diacylglycerol (DAG) after IL-2 treatment of living NK
cells showed an increase of this pool of DAG in the same time frame of
PLC activation indicating that IL-2 specifically stimulated a nuclear
PLC activity (Fig. 1A
, B
).
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2. Members of the PLCß family are the only isoforms expressed in
the nucleus of human NK cells
Three members of the ß family are expressed in the nucleus of NK
cells: PLCß1b (the nuclear splice variant),
PLCß2, and PLCß3. Both
PLC
and PLC
are found only in the cytoplasmic fraction of these
cells (Fig. 1C
). This hints at the PLCß family as a
target for the signal originated from the plasma membrane.
3. IL-2 induces translocation to the nucleus of MAP kinase and
serine phosphorylation of nuclear PLC-ß1
The kinetics of MAPK enzyme activity (Fig. 2A
) and immunochemical analysis by anti-ERK-1 and ERK-2
antibody (Fig. 2B
) showed that IL-2 was capable of inducing
a marked nuclear translocation of activated ERK-2 (p42) and to a lesser
extent of activated ERK-1 (p44) up to 60 min of stimulation. Using
anti-phosphorylated MAPK antibody, we detected that active ERKs
translocated as well (Fig. 2B
). This was accompanied by an
increase in nuclear kinase activity and a decrease in cytoplasmic
kinase activity. A comparison of the effect of PD 98059 (MEK-1
inhibitor) vs. the PLC inhibitor ET-18-OCH3 on
nuclear PLC activity in vitro and on the actual mass of nuclear DAG
showed that ET-18-OCH3 induced an almost complete
inhibition of nuclear PLC activity both in control and IL-2-treated NK
cells, whereas PD 98059 induced inhibition of nuclear PLC only in NK
cells stimulated with IL-2. These findings hinted at a regulatory role
of ERKs on nuclear PLC activity upon IL-2 stimulation. Moreover, we
have established that the stimulation of nuclear activity is due only
to the activation of nuclear PLCß1, since in
the presence of neutralizing anti-PLCß1 mAb,
the activation induced by IL-2 treatment is completely abolished.
We therefore immunoprecipitated nuclear
PLCß1 and, after checking the blot for the
presence of this isoform, reprobed it with an antibody against
phosphoserine (Fig. 2C
). The anti-phosphoserine
antibody did not stain the nuclear PLCß1b from
control cells, whereas in response to a 60 min stimulation with IL-2,
we saw a high level of immunoreactivity. Phosphorylation of nuclear
PLCß1b was abolished if the cells had been
incubated for 1 h with PD 98059 before IL-2 stimulation (Fig. 2C
). The in vitro phosphorylation assay in the
presence of [32P-]-ATP (Fig. 2D
) of immunoprecipitated phospho-ERK1/2 from nuclei of both
unstimulated and IL-2-stimulated (60 min) NK cells combined with
recombinant PLCß1 either wild-type or mutated
for serine 982, substituted with glycine (S982G), with surrounding
motif PSSP (i.e., the MAPK consensus sequence) showed that wild-type
PLCß1 was phosphorylated only by phospho-ERK1/2
immunoprecipitated from IL-2-stimulated cells, whereas S982G mutant was
not phosphorylated at all. Moreover, the increase of PLC activity was
observed only in phosphorylated PLCß1 (Fig. 2D
).
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4. Inhibition of the ERK2/nuclear PLCß pathway inhibits the
IL-2-dependent proliferation of NK cells
To determine whether the IL-2-driven NK cell proliferation was
dependent on the nuclear PLCß1 activation by
MAPK, NK cells were treated with ET-18-OCH3, PD
98059, and the DNA polymerase 
inhibitor aphidicolin as independent
positive control. The proliferation of IL-2-stimulated purified NK
cells was significantly (P<0.01) inhibited by both
ET-18-OCH3 and PD 98059 in the absence of
cytopathic effects.
CONCLUSIONS AND SIGNIFICANCE
Our data demonstrate the involvement of nuclear
PLCß1, in the response of primary human NK
cells to IL-2. NK cells have a prominent cytoplasmic expression of
PLC1 and 2. The
PLCß family, on the contrary, is localized predominantly in the
nucleus of NK cells, although also present in the cytoplasmic fraction.
IL-2 up-regulates the nuclear PLCß1 activity in
primary NK cells. Inhibition of PLC activity by
ET-18-OCH3 in IL-2-stimulated NK cells blocks
their IL-2-driven proliferation, suggesting that PLC is involved in the
onset of NK cell proliferation. Primary NK cells stimulated with IL-2
apparently respond to PD 98059 differently from the NK cell line YT,
whose proliferative activity was reported not to be inhibited by PD
98059. Our results on the antiproliferative effect of PD 98059 have
been obtained using a 10-fold lower concentration of the inhibitor,
which is not toxic at all, than that capable of blocking NK cell
cytotoxicity. This could account for the high degree of specificity of
the inhibitor in the cell cycle progression of NK cells after IL-2
stimulation.
Upon treatment of NK cells with IL-2, MAPK translocated to the nucleus and PLCß1 was phosphorylated on serine residues. At amino acids 980–983, PLCß1 (both 1a and 1b) displays a typical MAPK consensus sequence, Pro-Ser-Ser-Pro. Such a sequence does not exist in other isoforms of the ß family of PLC (i.e., ß2, ß2, and ß4), which agrees with the fact that IL-2 activates only nuclear PLCß1. A direct confirmation comes from the in vitro phosphorylation experiment, which shows that serine 982 (with surrounding PSSP motif) is the phosphorylation site of activated MAPK and that the phosphorylation is responsible for the increase of PLCß1 activity. Taken together, these findings demonstrate that nuclear PLC activity in NK cells is downstream from the IL-2R and that nuclear PLCß1 is activated upon its phosphorylation by MAP kinase. The proposed mechanism of regulation of nuclear PLCß by ERK-2 constitutes a link between the previously reported evidence of the role of MAPK in several NK functions and the downstream target in the nucleus. In addition, the stimulation of PLC activity upon treatment of primary human NK cells takes place only in the nucleus whereas cytoplasmic PLC is unaffected. The finding that a specific PLC (PLCß1b) is localized to the human NK cell nucleus and linked to the IL-2 response paves the way to the further understanding of what signaling mechanism takes place.
It is possible that nuclear PLCß1b is
responsible for the maintenance of the optimum amount of
PIP2 inside the nucleus itself, since a
PIP2-dependent mechanism has been shown to exist
in chromatin remodeling after T lymphocyte receptor stimulation. Given
the data showing a role for DAG-mediated PKC stimulation in
nuclei during the G2/M phase transition as well
as a selective nuclear translocation of PKC after nuclear PLC
activation, we cannot exclude that PLCß1b could
control IL-2-driven NK cell proliferation by DAG-mediated PKC
activation. This is supported by the fact that nuclear DAG generation,
elicited by IL-2, is inhibited by PD 98059 at the same time when this
compound inhibits nuclear PLC activity and that thereafter NK cells
cease to proliferate. A specific role for nuclear
PLCß1 in cell cycle control has been assigned
in that its overexpression resulted in increased expression of cyclin
D3 and its related cyclin-dependent kinase 4, promoted phosphorylation
of retinoblastoma protein, increasing the binding activity of E2F
transcription factor, and accelerated progression through the
G1 phase and entering in S phase of Friend
erythroleukemia cells. A similar mechanism could also occur in human NK
cells. Our data demonstrate for the first time in human living cells
that nuclear PLCß1 signaling is a downstream
target of the MAPK pathway stimulated by IL-2 and is a key step in the
proliferative response of the human NK cell to IL-2.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0008fje ; to cite this article, use FASEB J. (June 8, 2001) 10.1096/fj.01-0008fje 
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