Nuclear Translocation of the N-terminal Prodomain of
Interleukin-16*
Yujun
Zhang
,
Hardy
Kornfeld,
William W.
Cruikshank,
Sue
Kim,
Christine C.
Reardon, and
David M.
Center
From the Pulmonary Center, Boston University School of Medicine,
Boston, Massachusetts 02118
Received for publication, September 18, 2000, and in revised form, October 12, 2000
 |
ABSTRACT |
Interleukin-16 (IL-16) is a pleiotropic
cytokine that functions as a chemoattractant factor, a modulator of T
cell activation, and an inhibitor of human immunodeficiency virus (HIV)
replication. These diverse functions are exclusively attributed to the
secreted C-terminal peptide of 121 amino acids (mature IL-16), which is cleaved from the precursor protein (pro-IL-16) by caspase-3. Human pro-IL-16 is comprised of 631 amino acids with three PDZ
domains, one of which is present in secreted mature IL-16. No cellular localization or biologic functions have been ascribed to the unusually large and highly conserved N-terminal prodomain formed as a result of
proteolytic release of the third PDZ domain of pro-IL-16. Here we show
that the N-terminal prodomain of pro-IL-16 translocates into the
nucleus following cleavage of the C-terminal segment. The nuclear
localization signal of pro-IL-16 consists of a classical bipartite
nuclear targeting motif. We also show that the nuclear targeting of the
IL-16 prodomain induces a G0/G1 arrest in
the cell cycle. Taken together, the high degree of conservation of the
prodomain among species, the presence of two PDZ motifs, and the
nuclear localization and subsequent inhibitory effect on cell cycle
progression suggest that pro-IL-16 is cleaved into two functional proteins, a C-terminal-secreted cytokine and an N-terminal product, which affects the cell cycle.
 |
INTRODUCTION |
Interleukin-16 (IL-16)1
was originally identified from mitogen-stimulated T cell culture medium
as a T cell-specific chemoattractant factor (1, 2). IL-16 functions
also as a modulator of T cell activation (3) and as an inhibitor of
HIV-1 replication (4-6). Control of IL-16 expression occurs at both
transcriptional and translational levels (7). In particular, secreted
IL-16 comprises the 121 C-terminal amino acids of the larger
intracellular precursor protein following cleavage by caspase-3 (8).
Mature IL-16 is unusual in that it contains one of the three PDZ
domains present in the C-terminal half of pro-IL-16. There are few, if any, examples of secreted PDZ-containing proteins. There is very high
cross species homology of the entire pro-IL-16, including the
N-terminal prodomain, which results from caspase-3 cleavage of the
mature secreted cytokine (9). Along with the high interspecies homology, the presence of two additional PDZ domains in the prodomain suggests that pro-IL-16 might have intracellular functions in addition
to serving as a precursor of the secreted cytokine.
The distribution pattern of a protein within the cell can provide
important clues to its function. As the first step toward understanding
the function of pro-IL-16, we used fluorescence immunochemistry to
study the intracellular distribution of this protein. COS cell
expression systems were used to provide detailed characterization of
subcellular localization of pro-IL-16 and its N-terminal prodomain
after proteolytic processing. Previously published data have shown that
COS cells do not express endogenous IL-16 but are able to express and
process pro-IL-16 via endogenous caspase-3 following cDNA
transfection (8). This processing results in the extracellular release
of biologically active mature IL-16, thus mimicking the natural
secretory processes previously noted in T cells. Our data demonstrate
that in transfected COS cells, pro-IL-16 is distributed in the
cytoplasm and is concentrated in the perinuclear region. Following
caspase-3 cleavage the remaining N-terminal prodomain translocates to
the nucleus. Nuclear localization of IL-16 prodomain in transfected COS
cells has a negative effect on cell growth.
 |
EXPERIMENTAL PROCEDURES |
cDNA Constructs--
Pro-IL-16 cDNA was subcloned into
vector pXM (Genetics Institute). An N-terminal GFP pro-IL-16 fusion
construct was produced by inserting the GFP coding sequence from
pEGFP-1 (CLONTECH) into pXM-pro-IL-16 using the
5'-KpnI site in the vector and a 3'-BsrGI site
introduced after the first methionine of pro-IL-16 by polymerase chain
reaction. For the FLAG-tagged constructs, we used polymerase chain
reaction to add the FLAG sequence to either the N- or C-terminal of
pro-IL-16 either after the first methionine or right before the stop
codon, respectively. Truncated pro-IL-16 constructs were created using
recombination polymerase chain reaction to delete the selected portion
of pro-IL-16 as listed in Fig. 3A and cloned back into the
pXM vector. All constructs were confirmed by sequence analysis.
Transfections--
For the immunoblotting experiments,
transfections were performed, and cells were collected as described
previously (8). For the immunocytochemistry experiments, cells were
plated on chamber slides 24 h before transfection. The
transfections were performed on the slides with SuperFect reagent
(Qiagen) according to the manufacturer's protocol.
Immunoblotting and Immunocytochemistry--
Anti-IL-16
(monoclonal antibody: clone 14.1) (10) and anti-FLAG antibodies (Sigma)
were used for immunoblotting. Immunoblots were performed as described
previously (8). For immunohistochemistry, only the anti-FLAG antibody
was used. Briefly, 48 h after transfection, cells growing on the
chamber slides were washed twice with PBS and fixed in 4%
paraformaldehyde for 10 min. After washing twice with PBS, fixed cells
were permeabilized with 0.5% Triton X-100 for 5 min, washed twice with
PBS, and were then incubated with 5% milk and 50 µg/ml normal goat
serum for 1 h at room temperature. After removing the blocking
milk, the cells were incubated with anti-FLAG M2 antibody (Sigma) for
1 h at room temperature. The slides were washed three times (5 min
each) with PBS/Tween (0.05%) before incubation with
rhodamine-conjugated goat-anti-mouse IgG (1:500) in 1% milk for 1 h. The slides were then washed two times with PBS/Tween, the chamber
and gasket were disassembled, the slides were washed once more with
PBS/Tween, and were finally rinsed with distilled water. After removal
of excess water the slides were mounted with Pro Long antifade solution
(Molecular Probes) covered with coverslips and sealed with nail polish.
Slides were examined either with confocal microscopy or an Axioplan
microscope (Zeiss) with a × 40 objective. Images of cells were
captured using 400 ASA slide film (Eastman Kodak Co.).
Cell Fractionation--
Forty-eight hours after transfection,
COS cells grown in P100 plates were harvested by trypsin treatment and
resuspended in ice-cold PBS. Cells were rinsed two times with cold PBS
prior to incubation with Buffer I (20 mM Tris-HCl, 0.5 mM dithiothreitol, 10 mM
-glycerol
phosphate, 300 mM sucrose, 0.2 mM EGTA, 5 mM MgCl2, and 10 mM KCl and the
protease inhibitors aprotinin, chymostatin, antipain, and pepstatin at
10 µg/ml) on ice for 15 min. 20 µl of 10% Nonidet P-40 were added
into the 400-µl cell suspension and hand-mixed for 10 s. The
sample was then centrifuged at 2000 rpm for 5 min at 4 °C using a
desk-top microcentrifuge to pellet the nuclei. The supernatant was
further fractionated by centrifugation at 14,000 rpm for 20 min at
4 °C. The supernatant fraction of this centrifugation was collected
and classified as the cytoplasmic fraction. The nuclear pellet was
washed twice with ice-cold PBS before adding 50 µl of Buffer II (10 mM Tris-HCl, 0.5 mM dithiothreitol, 10 mM
-glycerol phosphate, 0.2 mM EGTA, 5 mM MgCl2, 350 mM KCl, 25% glycerol
and the protease inhibitors aprotinin, chymostatin, antipain, and
pepstatin at 10 µg/ml) to lyse the nuclei. After a 15-min incubation
on ice, nuclear lysates were centrifuged at 14,000 rpm for 5 min at
4 °C, and this supernatant fraction was classified as nuclear. The
protein concentration in each fraction was determined using Bio-Rad
bovine gamma globulin as standard protein and Bio-Rad protein assay reagent.
Cell Cycle Analysis--
5 × 106 COS cells
were washed with PBS buffer and fixed in 80% ethanol on ice for 1 h. After fixation, cells were washed with PBS twice before resuspension
in RNase A (100 µg/ml)/PIB (propidium iodide staining buffer: PBS
buffer with the addition of 0.12% Triton X-100 and 0.12 mM
EDTA) for 45 min at 37 °C. PI solution (5 mg/ml propidium iodide
dissolved in PIB) was added at 50 µg/ml, and the reaction was
incubated at room temperature in the dark for at least 1 h. The
analysis was performed on the day of the assay using a flow cytometer
(Becton Dickinson).
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RESULTS |
COS Cell Expression and Processing of Pro-IL-16 and Its
Derivative--
Schematic representations of the wild-type pro-IL-16
and a construct of the full-length 80-kDa pro-IL-16 with a
GFP-fused N-terminal and a FLAG-tagged C-terminal (GFP·80·FLAG) are
shown in Fig. 1A. Expression
and natural processing of the GFP·80·FLAG protein in the
transfected COS cells were compared with wild-type pro-IL-16 by Western
blot of the transfected COS cell lysate probed with anti-IL-16 antibody
(Fig. 1B). The Western analysis demonstrates that pro-IL-16
is processed normally in COS cells, justifying the use of this system
to explore the fate of pro-IL-16 after cleavage.

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Fig. 1.
Both wild-type and modified pro-IL-16 undergo
normal processing in transfected COS cells. A, schematic
representations of wild-type pro-IL-16 protein as well as N-terminal
GFP-fused and C-terminal FLAG-tagged pro-IL-16 protein
(GFP·80·FLAG). B, Western blot of COS cell lysates
following transfection either with wild-type pro-IL-16 cDNA
(lane 1) or with (GFP)-pro-IL-16-(FLAG) (lane 2)
constructs. The Western blot was probed with monoclonal anti-IL-16
antibody. This antibody recognizes a region in the C-terminal third PDZ
domain (i.e. the mature secreted IL-16) of pro-IL-16.
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Subcellular Localization of IL-16--
Double immunofluorescence
confocal microscopic analysis of COS cells transfected with the
GFP·80·FLAG construct revealed that the GFP green fluorescence
localized to both cytoplasm and nucleus (Fig.
2A). In contrast, staining
with a rhodamine-labeled anti-FLAG antibody localized red fluorescence
in the cytoplasm only (Fig. 2B). An overlay of green and red
fluorescent images is shown in Fig. 2C, where the double
exposure shows the coincidence of green N-terminal signal and red
C-terminal signal as yellow. Specific immunofluorescent staining
of pro-IL-16 was confirmed by phase contrast imaging of untransfected
COS cells (Fig. 2D, arrowhead). The differential
distribution of red versus green fluorescence of pro-IL-16
in the GFP·80·FLAG-transfected COS cells suggests that the
full-length pro-IL-16 protein is localized in the COS cell cytoplasm,
particularly concentrated in the endoplasmic reticulum and perinuclear
region (the reticular pattern of yellow fluorescence resulted from the
overlay of Fig. 2, A and B). After cleavage, the
N-terminal prodomain of pro-IL-16 appears to translocate into the
nucleus (the nuclear staining of green fluorescence) and C-terminal fragment of active IL-16 appears to remain in the cytoplasm (the cytoplasmic distribution of red fluorescence) prior to secretion.

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Fig. 2.
Confocal images of autofluorescence
(green) and immunofluorescence (red)
of GFP·80·FLAG-transfected COS cells. A,
green fluorescence represents both full-length as well as
N-terminal-cleaved pro-IL-16 proteins and shows minimal nuclear and
predominant perinuclear localization. B, red
fluorescence is limited to cytoplasmic staining, which represents
either full-length pro-IL-16 or C-terminal mature IL-16. C,
an overlay image of green and red fluorescence
shows that the full-length pro-IL-16 protein is located in perinuclear
and reticular regions (yellow); the N-terminal profragment
of IL-16 targets into the nucleus, and the C-terminal IL-16 remains in
the cytoplasm. D, phase contrast image of COS cells shown in
A-C. Arrows indicate COS cells that do not
express pro-IL-16 protein.
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To confirm the hypothesis suggested by the experiments depicted in Fig.
2, as well as to identify the region responsible for nuclear targeting,
we made three selectively truncated pro-IL-16 constructs with
N-terminal FLAG tags (Fig.
3A). These constructs produce:
1) a protein truncated at the C terminus after amino acid 510 (60 kDa),
which constitutes the N-terminal prodomain of IL-16 following caspase-3
cleavage of pro-IL-16; 2) a protein truncated at the N terminus before
residue 511 (20 kDa), which constitutes secreted IL-16; and 3) a
protein that is truncated at both N and C termini before residue 258 and after residue 510 (30 kDa) (Fig. 3A). The authenticity
of the protein products of the above constructs was confirmed by
immunoblot analysis of the transfected COS cell lysates with anti-FLAG
antibody. As shown in Fig. 3B, the sizes of these truncated
proteins of pro-IL-16 were in agreement with the predicted molecular
masses. Immunofluorescence analyses of COS cells transfected with 60- and 20-kDa constructs confirmed the double-stained confocal images of
pro-IL-16. Specifically, the N-terminal portion of pro-IL-16 (60-kDa
prodomain) translocated into the nucleus (Fig.
4A), and the C-terminal mature
IL-16 (20 kDa) remained in the cytoplasm (Fig. 4B). Taken
together, these data suggest that in the transfected COS cells, the
prodomain of IL-16 translocates to the nucleus following cleavage of
C-terminal bioactive IL-16. The cytoplasmic fluorescent staining of COS
cells transfected with the 30-kDa construct (Fig. 4C)
implies that the nuclear localization signal of pro-IL-16 is located in
the N-terminal first 257 amino acid residues of pro-IL-16.

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Fig. 3.
Schematic diagram of three pro-IL-16
deletions and their expression in transfected COS cells.
A, structural alignment of deletions. B, Western
blot of lysates of COS cells transfected with the N-terminal
FLAG-tagged deletion constructs noted on the top of each
lane and designated by the kDa. The blot was probed with
anti-FLAG M2 antibody.
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Fig. 4.
Differential distribution of the selectively
truncated Pro-IL-16 proteins in transfected COS cells.
A1 and A2, N-terminal prodomain of IL-16 (60 kDa)
is localized in the nucleus. B1 and B2,
C-terminal mature IL-16 (20 kDa) locates in the cytoplasm.
C1 and C2, the 30-kDa protein of pro-IL-16
remains in the cytoplasm.
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Identification of a Nuclear Localization Sequence
(NLS)--
Knowing the N-terminal region of pro-IL-16 between amino
acid 1 and 257 is responsible for nuclear targeting, we next searched the sequence of pro-IL-16 to see whether there were any consensus nuclear-targeting motifs. One basic region that could function as a
bipartite nuclear localization sequence (NLS) was found between amino
acids 79 and 101 (79KKGPPVAPKPAWFRQSLKGLRNR101).
Similar bipartite motifs have been found in many nuclear proteins and
serve as consensus sequences for nuclear targeting (11). To evaluate
the nuclear localization properties of this NLS, we performed
mutagenesis to change all five basic amino acids into alanines
(indicated as underlined) at the both N and C termini of this NLS
(79AAGPPVAPKPAWFRQSLAGLANA101).
As shown in Fig. 5, the NLS-mutated
prodomain of pro-IL-16 shows reduced nuclear-targeting activity (60 kDa
NLSM), when compared with nonmutated protein (60 kDa NLSW).

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Fig. 5.
Mutation of the NLS significantly reduced
nuclear targeting activity of the N-terminal prodomain of IL-16.
A, with nonmutated NLS, the C-terminal-deleted pro-IL-16
locates predominantly in the nucleus (60 kDa NLSW). B, after
NLS mutation (60 kDa NLSM, as shown in Fig. 6), the nuclear
localization of C-terminal-deleted pro-IL-16 is dramatically
reduced.
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To determine whether the prodomain of pro-IL-16 localized to the
nucleus has an effect on cell growth, we performed cell cycle analysis
on transfected COS cells. To facilitate flow cytometric cell cycle
analysis in transiently transfected cells, we made new pro-IL-16
wild-type and NLS mutant constructs with an N-terminal-fused GFP
reporter (Fig. 6A). As a
control for the effects of nuclear GFP, we made a construct that fused
an NLS sequence from human T cell leukemia virus type 1 (HTLV-1) tax
protein to GFP (NLS·GFP). To investigate nuclear targeting of the NLS
mutant of pro-IL-16 and confirm the distribution of expressed proteins,
subcellular fractionation was performed following transfection of COS
cells with plasmid GFP constructs; NLS·GFP, GFP·60, or
GFP·60-NLSM. Immunoblots of the transfected COS cells with anti-GFP
antibody demonstrate that the IL-16 prodomain with mutated NLS
(GFP·60-NLSM) shows greatly reduced nuclear targeting activity (Table
I and Fig. 6B, upper
panel, lanes 3 and 4). The integrity of the
cytoplasmic and nuclear fractions were confirmed by stripping and
reprobing the blot with an anti-tubulin antibody as an indication of
nuclear fraction free of the contamination of cytoplasmic proteins
(Fig. 6B, bottom panel). As shown in Table I and
Fig. 6B, in the transfected COS cells GFP is exclusively
located in the cytoplasm; NLS·GFP is exclusively located in the
nucleus; and the wild-type prodomain of IL-16 (GFP·60) distribute
equally both in the cytoplasm and nucleus.

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Fig. 6.
The NLS of pro-IL-16 regulates the
translocation of the IL-16 prodomain. A, schematic
representation of the NLS mutation in the prodomain of IL-16
(GFP60 NLSM). B, immunoblot of cytoplasmic
(C) and nuclear (N) fractions of COS cells
transfected with constructs of either GFP·60 (lanes 1 and
2, GFP60), NLS-mutated GFP·60 (lanes 3 and
4, GFP60 NLSM), nuclear-localized GFP (lanes 5 and 6, NLS GFP), or GFP (lanes 7 and
8), respectively. The blot was probed first with anti-GFP
polyclonal antibody (upper) and then stripped and reprobed
with anti-tubulin monoclonal antibody (bottom). Subcellular
fractionation confirms the nuclear targeting motif of pro-IL-16.
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Effects of Nuclear Targeting of IL-16 Prodomain on the Cell
Cycle--
Forty-eight hours after transfecting COS cells with the
above constructs, the cells were collected and stained with propidium iodide and then analyzed by flow cytometry to identify transfected cells (GFP-positive). The cell cycle profile of the transfected COS
cells (gated on green fluorescent-positive cells) revealed that
overexpression of the wild-type prodomain of IL-16 (Fig. 7, GFP60), but not NLS mutant
(Fig. 7, GFP60 NLSM), induced an accumulation of the cells
in G0/G1 phase (Table
II). Cell cycle analysis of cells
transfected with an independent NLS linked to GFP (Fig. 7, NLS
GFP) had a similar G0/G1 profile to GFP
alone transfected cells (Fig. 7, GFP) as well as
untransfected cells (not shown). Fluorescence activated cell sorting
(FACS) analysis revealed that ~40% of GFP or NLS·GFP-transfected
cells were in G0/G1, whereas ~53% of
GFP·60-transfected cells remained in G0/G1 phase (Table II). There is ~13% G0/G1 arrest
in COS cells overexpressing GFP·60 compared with GFP or NLS·GFP. By
contrast, only 41% of GFP·60-NLSM transfected COS cells were in the
G0/G1 phase, and COS cells that expressed this
mutant protein had a similar cell cycle profile as those cells that
expressed GFP or NLS·GFP (Table II). We confirmed these studies by
transfection of the same expression vectors in 3T3 cells (data not
shown).

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Fig. 7.
FACS analysis of IL-16 prodomain-induced
growth arrest. COS cells were transiently transfected with
indicated constructs. Forty-eight hours after transfection, cells were
collected, stained with propidium iodide, and sorted by flow cytometry
to identify transfected cells (GFP positive). The gated green
fluorescent-positive cells were analyzed for cell cycle profile by
relative DNA content according to the red fluorescence of
propidium iodide.
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Table II
Nuclear targeting of IL-16 prodomain effects on cell cycle
COS cells were transfected with constructs GFP, NLS·GFP, GFP·60, or
GFP·60-NLSM. Forty-eight hours after transfection, cell cycle
analysis was performed by flow cytometry. Mean % values from three
experiments are shown.
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DISCUSSION |
The autofluorescent (GFP fusion) and immunofluorescent (FLAG tag)
localization studies suggest that in transfected COS cells, the
N-terminal prodomain of IL-16 translocates into the nucleus following
cleavage and release of the C-terminal bioactive IL-16. Using
site-directed mutagenesis we were able to identify a putative NLS of
pro-IL-16 between amino acids 79 and 101. However, the current studies
did not demonstrate whether other sequences in pro-IL-16 might also
contribute to the nuclear import. Our cell fractionation results of COS
cells transfected with wild-type or NLS-mutated pro-IL-16 constructs
correlated with the fluorescent immunohistochemistry results and
confirmed the nuclear localization properties of the prodomain of
IL-16. The function of pro-IL-16 in the nucleus appears to be cell
growth-related as nuclear translocation of the prodomain results in
G0/G1 arrest.
Our data suggest that the 80 kDa primary pro-IL-16 translation product
could have two distinct functions after caspase-3 processing. The
C-terminal segment of pro-IL-16 is a secreted protein that acts as a
typical cytokine. The remaining N-terminal prodomain of IL-16
translocates to the nucleus after release of C-terminal polypeptide
where it may influence cell cycle regulation. A similar phenomenon has
been reported for IL-1
. Pro-IL-1
is released by caspase-1
cleavage and appears to affect cell growth (12). IL-16 and IL-1
share some general similarities. They are both processed by caspase
family enzymes. Neither secreted mature cytokine has a secretory leader
sequence, and following caspase enzyme processing their remaining
prodomains translocate to the nucleus. However, unlike the negative
effect we observed with N-terminal prodomain of IL-16, the N-terminal
prodomain of IL-1
has been reported to function as a transforming
nuclear oncoprotein (12).
Pro-IL-16 contains multiple PDZ domains. Proteins with PDZ motifs often
function in intracellular signal transduction (13) and in structures at
plasma membrane (14, 15). Pro-IL-16 of lymphocyte origin contains one
putative cdc2 kinase substrate site (56TPPK59),
which lies N-terminal to the nuclear localization sequence. The
sequence 56TPPK59 is similar to the consensus
sequence that is phosphorylated by the mitotic cdc2 kinase or H1 kinase
(16). The cdc2 kinase motif suggests a way that nuclear pro-IL-16 might
participate in cell cycle control. Along these lines, several PDZ
domain-containing proteins have been found to localize in the nucleus
(17, 18) although their functions are not completely understood.
It is unclear how full-length unprocessed pro-IL-16 remains
predominantly in the transfected COS cells cytoplasm, whereas the
prodomain translocates to the nucleus. Extranuclear sequestration mechanisms for regulating entry of proteins into the nucleus have been
reported for many transcription factors, such as NF-kB (19), SREBP-1
(sterol regulatory element-binding protein 1, Ref. 20), and certain
steroid hormone receptors. In each of these cases the entry of protein
into nucleus is a regulated event. In the case of SREBP-1 and -2, nuclear entry is controlled by the proteolytic processing of the
C-terminal segment bound to the endoplasmic reticular membrane (20).
However, overcoming an existing nuclear exporting signal controls entry
of other nuclear proteins. LIMK-1, a protein serine/threonine kinase
concentrates in the nucleus after deletion of a C-terminal portion of
PDZ domain, which contains two nuclear exporting signals (21, 22). The
perinuclear localization of pro-IL-16 prior to cleavage suggests an
element in the C terminus (perhaps the PDZ domain) may bind to a
perinuclear protein. Once cleaved from the third PDZ domain, the
released N-terminal prodomain is free to translocate into the nucleus.
However, any of the previously noted mechanisms might regulate
translocation of the prodomain of IL-16 into the nucleus. The cellular
localization and the biological function of pro-IL-16 in human T
lymphocytes are currently under investigation.
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ACKNOWLEDGEMENTS |
We thank Dr. Matthew J. Fenton and Dr.
Zhixiong Xiao for many helpful discussions and Terry K. Means and
William F. Brazer for technical assistance.
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FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant HL-32802.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: Pulmonary Center,
R-304, Boston University School of Medicine, 80 E. Concord St., Boston,
MA 02118. Tel.: 617-638-4860; Fax: 617-536-8093; E-mail: yzhang@bupula.bu.edu.
Published, JBC Papers in Press, October 13, 2000, DOI 10.1074/jbc.M008513200
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ABBREVIATIONS |
The abbreviations used are:
IL-16, interleukin-16;
GFP, green fluorescent protein;
EGFP, enhanced GFP;
PBS, phosphate-buffered saline;
PI, propidium iodide;
PIB, PI buffer;
NLS, nuclear localization signal;
FACS, fluorescence-activated cell
sorter;
HIV, human immunodeficiency virus.
 |
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