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J. Biol. Chem., Vol. 278, Issue 26, 23773-23785, June 27, 2003
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/Protein Kinase D3 Nuclear Localization, Catalytic Activation, and Intracellular Redistribution in Response to G Protein-coupled Receptor Agonists*
From the Unit of Signal Transduction and Gastrointestinal Cancer, Division of Digestive Diseases, Department of Medicine, UCLA-CURE Digestive Diseases Research Center and Molecular Biology Institute, David Geffen School of Medicine, University of California, Los Angeles, California 90095-1786
Received for publication, January 8, 2003 , and in revised form, March 24, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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/PKD3. Whereas PKD has been the focus
of most studies, virtually nothing is known about the effect of G
protein-coupled receptor agonists (GPCR) on the regulatory properties and
intracellular distribution of PKD3. Consequently, we examined the mechanism
that mediates its activation and intracellular distribution. GPCR agonists
induced a rapid activation of PKD3 by a protein kinase C (PKC)-dependent
pathway that leads to the phosphorylation of the activation loop of PKD3.
Comparison of the steady-state distribution of endogenous or tagged PKD3
versus PKD and PKD2 in unstimulated cells indicated that whereas PKD
and PKD2 are predominantly cytoplasmic, PKD3 is present both in the nucleus
and cytoplasm. This distribution of PKD3 results from its continuous shuttling
between both compartments by a mechanism that requires a nuclear import
receptor and a competent CRM1-nuclear export pathway. Cell stimulation with
the GPCR agonist neurotensin induced a rapid and reversible plasma membrane
translocation of PKD3 that is PKC-dependent. Interestingly, the nuclear
accumulation of PKD3 can be dramatically enhanced in response to its
activation. Thus, this study demonstrates that the intracellular distribution
of PKD isoenzymes are distinct, and suggests that their signaling properties
are regulated by differential localization. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, 
I,
II, and 
), which are regulated by calcium, DAG, and
phospholipids; the novel PKCs (
, 
, 
, and 
), which are
regulated by diacylglycerol (DAG) and phospholipids; and the atypical PKCs
(
and 
), whose regulation is less characterized, but that have
been proposed to be regulated by D-3 phosphoinositides
(6). The DAG-regulated PKC
isoforms all bind phorbol esters and are major cellular targets for this class
of tumor promoter (7). It is
increasingly recognized that each isoform has specific functions in
vivo (8). However, the
mechanisms by which PKC-mediated signals in the plasma membrane are propagated
to critical downstream targets remain largely undefined.
Protein kinase D (PKD)/protein kinase Cµ
(9,
10) and two recently
identified serine protein kinases termed PKD2 and PKC/PKD3, which are
similar in overall structure and primary amino acid sequence to PKD
(11,
12), constitute a new protein
kinase subfamily separate from the previously identified PKCs. The salient
features of the PKD structure include the presence of a catalytic domain
distantly related to Ca2+-regulated kinases, a
pleckstrin homology (PH) domain that regulates PKD activity, and a highly
hydrophobic stretch of amino acids in its NH2-terminal region
(9,
10,
13,
14). The
NH2-terminal region of PKD contains in addition to the PH domain, a
cysteine-rich domain (CRD) that confers high affinity binding to phorbol
esters and DAG (9,
14–16).
Neuropeptides such as neurotensin (NT) and mammalian bombesin-like peptides
act as potent cellular mitogens for a variety of cell types
(17–20)
and have been implicated as autocrine/paracrine growth factors for some human
cancer cells (19,
21) including pancreatic
cancer cells (22).
Neuropeptides bind to G protein-coupled receptors (GPCR) that promote
Gq-mediated activation of isoforms of phospholipase C
(23) to produce two second
messengers: inositol 1,4,5-trisphosphate that mobilizes
Ca2+ from internal stores and DAG that activates PKC
(24,
25). PKD can be activated in
intact cells by tumor promoting phorbol esters, GPCR agonists, growth factors,
and antigen-receptor engagement
(26–33)
via PKC-mediated phosphorylation of Ser744 and Ser748
within the activation loop of the catalytic domain
(27,
31,
33–36).
PKD has been localized in the cytosol and in several intracellular
compartments including Golgi, plasma membrane, and mitochondria
(29,
37–40)
and undergoes rapid translocation from the cytosol to the plasma membrane
(41). PKD has been implicated
in the regulation of a variety of cellular functions, including DNA synthesis
and cell proliferation (42),
Golgi organization and function
(43,
44), epidermal growth factor
receptor and c-Jun signaling
(45–47),
NF
B-mediated gene expression
(48) and cell migration
(49).
Much less information is available on the regulation, biological roles, and intracellular distribution of other members of the PKD family. PKD2 can be activated by phorbol esters and the GPCR agonist gastrin (12, 50) but the precise mechanism mediating its activation is still under investigation (50). As for its intracellular distribution, we recently showed that PKD2 is present only in the cytosol of unstimulated or NT-stimulated cells (51). Regarding PKD3, virtually nothing is known about its regulatory properties or intracellular distribution in response to GPCR agonists, although its cDNA sequence obtained from expressed sequence tag deduction and rapid amplification of cDNA ends was published before the cloning of PKD2 (11).
The experiments presented in this paper were designed to examine the
mechanisms regulating the intracellular distribution and catalytic activity of
PKD3. In striking contrast to the predominantly cytoplasmic localization of
PKD and PKD2 shown in our previous studies, we demonstrate here that PKD3 is
present both in the cytoplasm and in the nucleus of non-stimulated cell.
Several lines of evidence, including studies using fluorescence loss in
photobleaching (FLIP), indicate that PKD3 continuously shuttles between the
nucleus and the cytoplasm by a mechanism that requires a nuclear import
receptor and a competent CRM1-nuclear export pathway. Our results also show
that GPCR agonists induce a striking translocation of PKD3 from the cytosol to
the plasma membrane that leads to its catalytic activation and rapid plasma
membrane dissociation through PKC-mediated phosphorylation of the activation
loop of PKD3 (Ser731 and Ser735). Although PKD and PKD3
can be activated by PKC, we found that only PKD forms a stable molecular
complex with this novel PKC isoform. Upon its activation, PKD3 rapidly
dissociates from the plasma membrane, moves into the cytosol, and is
subsequently imported into the nucleus at a faster rate than in non-stimulate
cells, which results in its further nuclear accumulation. These findings
reveal a connection between PKD3 and PKC and support a novel mechanism by
which GPCR-mediated activation of the closely related serine/threonine kinases
of the PKD family can potentially generate diverse physiological responses
based on their unique subcellular distribution.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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mRNA Amplification
To detect the expression of sequences encoding PKD, PKD2, and PKD3 in
Panc-1 cells we employed reverse transcriptase-PCR (RT-PCR) with specific
oligonucleotide primers designed using the public domain Primer3 program
(52).2
The cDNA sequences used to design the primers were based on the published PKD
(10) and PKD2
(12) sequences. For PKD3, we
designed specific primers with the Primer3 program based on the cDNA sequence
obtained from expressed sequence tag deduction and rapid amplification of cDNA
ends (11). The primers
sequences for PKD were, sense primer 5'-GAGATGGCTTGCTCCATTGT-3'
corresponding to nucleotide positions 235–254 and the antisense primer
5'-TCGCCTTCCTGGATATCACT-3' corresponding to nucleotide positions
361–380, considering for both primers as nucleotide number 1 the first
nucleotide of the ATG initiation codon. The primers sequences for PKD2 were,
sense primer 5'-CACCTTCGAGGACTTCCAGA-3' corresponding to
nucleotide positions 404–421 and the antisense primer
5'-CAGCGCTTGTGGTAGTTCAG-3' corresponding to nucleotide positions
537–556, considering for both primers as nucleotide number 1 the first
nucleotide of the ATG initiation codon. The primers sequences for PKD3 were,
sense primer 5'-TCAGCCCAGAAGTCTGTATTACCC-3' corresponding to
nucleotide positions 25–48 and the antisense primer
5'-TGGAGTTGGTGAGTGATGGTGC-3' corresponding to nucleotide positions
139–160, considering for both primers as nucleotide number 1 the first
nucleotide of the ATG initiation codon. RT-PCR was performed on 1 µg of
total RNA extracted from Panc-1 cells using TRIzol reagent (Invitrogen).
First-strand cDNA was synthesized at 45 °C by using the designed PKD,
PKD2, or PKD3 antisense oligonucleotides described above and ThermoScript
reverse transcriptase (Invitrogen) under the conditions suggested by the
manufacturer. A fraction of the obtained cDNAs were amplified by PCR using
Platinum TaqDNA polymerase high fidelity (Invitrogen), as suggested
by the manufacturer, and the sense and antisense primers specific for PKD,
PKD2, or PKD3 are indicated above. As controls, in a set of reactions the
sense primers for PKD, PKD2, or PKD3 were not added to the obtained cDNAs
during the PCR amplification step. The products of PCR were resolved in 2.5%
NuSieve GTG-agarose (FMC BioProducts, Rockland, ME), 1x TBE buffer (100
mM Tris, 90 mM boric acid, 1 mM EDTA, pH
8.4). The gel was stained for 60 min with ethidium bromide (0.5 µg/ml) in
1x TBE, follow with two 15-min washes with distilled water. Gel was
viewed and images captured using a Gel Doc 1000 system (Bio-Rad). The
predicted sizes of the RT-PCR products for PKD, PKD2, and PKD3 were 145, 152,
and 135 bp, respectively.
PKD3 Cloning and cDNA Constructs
Primers for the synthesis of the cDNA encoding PKD3 were designed with the
Primer3 program using the cDNA sequence obtained from expressed sequence tag
deduction and rapid amplification of cDNA ends
(11). The sense primer was
5'-CGGGGTACCTCAGATGTCTGCAAATAATTCCCCT-3' corresponding to
nucleotide positions –3 to 21 and the antisense primer was
5'-CGGGGTACCGAAGAACAAGTTACACAGCA-3' corresponding to nucleotide
positions 2725–2746, considering for both primers as nucleotide number 1
the first nucleotide of the ATG initiation codon. Total RNA (3 µg)
extracted from Panc-1 cells using TRIzol reagent was employed as template for
the synthesis at 55 °C of the cDNA encoding PKD3 using the ThermoScript
reverse transcriptase and the antisense primers indicated above. A fraction of
the obtained cDNAs were amplified by PCR using Platinum TaqDNA
polymerase high fidelity, as suggested by the manufacturer, and the sense and
antisense primers indicated above. The PCR products were digested with
KpnI and resolved in a 1% agarose gel, 1x TBE buffer. The band
with the predicted size for PKD3 was eluted from the gel and cloned into a
vector encoding the green fluorescent protein, pEGFP-C3 (BD Biosciences and
Clontech, Palo Alto, CA), previously digested with KpnI. The obtained
construct, pGFP-PKD3, was verified by DNA sequence analysis and the products
of expression analyzed by Western blot using antibodies against GFP. The
deduced amino acid sequence for the PKD3 cDNA clone obtained from Panc-1 cells
was identical to the published one
(11). The product of
expression of pGFP-PKD3 matched the predicted protein size and did not show
any degradation. The cDNA encoding PKD3 was isolated from pGFP-PKD3 using
KpnI and subcloned into pFLAG-CMV-4 (Sigma), previously digested with
KpnI. The obtained construct, pFLAG-PKD3, was verified by DNA
sequence analysis and the product of expression analyzed by Western blot using
the murine monoclonal antibodies M2 and M5 (Sigma) against the FLAG epitope.
The vectors encoding the chimeric fusion protein between GFP and PKD or PKD2
were previously described (29,
51). A schematic
representation of the employed constructs is shown in
Fig. 1B.
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The constitutively active PKC clones , , and were
obtained from Peter Parker, Protein Phosphorylation Laboratory, Imperial
Cancer Research Fund (53,
54). Dr. J. Battey (Laboratory
of Molecular Biology, NIDCD, National Institutes of Health) kindly provided
the vector bNR-pCD2 containing the cDNA encoding the bombesin/gastrin
releasing peptide receptor.
Immunoprecipitations
Transfected cells were washed twice with Dulbecco's modified Eagle's medium
and equilibrated in 5 ml of the same medium at 37 °C for 1–2 h
before treating the cultures with NT (100 nM), bombesin (10
nM), or phorbol 12,13-dibutyrate (PDBu) (200 nM) for 10
min. The cells were then lysed in buffer A (50 mM Tris-HCl, pH 7.6,
2 mM EGTA, 2 mM EDTA, 1 mM dithiothreitol,
100 µg/ml leupeptin, 1 mM 4-(2-aminoethyl)benzenesulfonyl
fluoride, hydrochloride (Pefabloc), 1% Triton X-100) and the fusion or
endogenous PKD or PKD3 proteins were immunoprecipitated at 4 °C for 3 h
with rabbit polyclonal anti-GFP, murine monoclonal (M2 and M5) anti-FLAG,
rabbit anti-PKD (PA-1), or rabbit anti-PKD3 (N17) antibodies. The immune
complexes were recovered using protein A or protein G coupled to agarose for
the rabbit polyclonal or murine monoclonal antibodies, respectively.
In Vitro Kinase Assays
Immune complexes were washed twice with lysis buffer, then twice with
kinase buffer consisting of 30 mM Tris-HCl, pH 7.4, 10
mM MgCl2, 1 mM dithiothreitol.
Autophosphorylation reactions were initiated by combining 20 µl of immune
complexes with 5 µl of a phosphorylation mixture containing 100
µM [-32P]ATP (specific activity, 400–600
cpm/pmol) in kinase buffer. Following incubation at 30 °C for 10 min, the
reactions were terminated by addition of 1 ml of ice-cold kinase buffer and
placed on ice. Immune complexes were recovered by centrifugation, and the
proteins were extracted for SDS-PAGE analysis by addition of 2x SDS-PAGE
sample buffer (200 mM Tris-HCl, pH 6.8, 0.1 mM sodium
orthovanadate, 1 mM EDTA, 6% SDS, 2 mM EDTA, 4%
2-mercaptoethanol, 10% glycerol). Dried SDS-PAGE gels were subjected to
autoradiography to visualize radiolabeled protein bands.
Western Blot Analysis
Samples of cell lysates were directly solubilized by boiling in SDS-PAGE
sample buffer. Following SDS-PAGE on 8% gels, proteins were transferred to
Immobilon-P membranes (Millipore Corp., Bedford, MA) and blocked by overnight
incubation with 5% nonfat dried milk in phosphate-buffered saline (PBS), pH
7.2. Membranes were incubated at room temperature for 3 h with rabbit
antiserum specifically recognizing GFP, PKD3 (N17), or the murine monoclonal
antibodies recognizing the FLAG epitope diluted in PBS containing 3% nonfat
dried milk. To determine the phosphorylation/activation state of the different
PKD isoforms, the membranes were incubated with the Aloop antibody that
specifically recognizes the phosphorylated state of the equivalent serines
within the activation loop of the three PKD isoforms (PKD:
Ser744-Ser748; PKD2:
Ser706-Ser710; PKD3:
Ser731-Ser735) (Refs.
36,
50, and this article).
Cell Imaging
Live Cell Imaging—To maintain a constant temperature of 37
°C during the experimental procedures, cells were grown on the 15-mm glass
coverslips and subsequently mounted in a perfusion chamber (RC-25 Warner
Instrument Corp., Hamden, CT) and perfused with medium preheated at 37 °C
by a SH-27B solution in-line heater (Warner Instrument Corp.). The medium was
supplemented with 10 mM HEPES, pH 7.2. The perfusion chamber was
mounted on an epifluorescence microscope (Zeiss Axioskop) and cells were
imaged with a Zeiss water immersion objective (Achroplan x40/0.75w, Carl
Zeiss Inc., Jena, Germany). Images were captured as uncompressed 24-bit TIFF
files with a SPOT cooled (–12 °C) single CCD color digital camera
(three pass method) driven by SPOT version 2.1 software (Diagnostic
Instruments, Inc., Sterling Heights, MI). GFP fluorescence was observed with a
HI Q filter set for fluorescein isothiocyanate (Chroma Technology,
Brattleboro, VT). Fifty cells were analyzed per experiment and each experiment
was performed in triplicate. The selected cells displayed in the appropriate
figures were representative of 90% of the population of positive cells.
Indirect Immunofluorescence—Indirect immunofluorescence was performed in 10% buffered formalin phosphate fixed, 0.3% Triton X-100/PBS permeabilized cultures. After extensive PBS washing, fixed cells were incubated for 18 h at 25 °C in blocking buffer (PBS, 1% gelatin-0.05% Tween 20) (BB) and then stained at 25 °C for 60 min with a mixture of murine monoclonal antibodies M2 and M5, which are directed against the FLAG epitope. Subsequently, the cells were washed with PBS, 0.05% Tween 20 at 25 °C and stained at 25 °C for 60 min with fluorescein-conjugated goat anti-mouse diluted in BB and washed again with PBS, 0.05% Tween 20. The samples were then stained at 25 °C for 10 min with DAPI (4',6 diamidino-2-phenylindole) (300 nM) diluted in PBS. After several PBS washes, the samples were mounted with a gelvatol-glycerol solution containing 2.5% 1,4-diazobicyclo[2.2.2]octane (41). The samples were examined and imaged with an epifluorescence microscope (Zeiss Axioskop) and a Zeiss oil immersion objective (Plan-Apochromat x40/1.0, Carl Zeiss Inc., Jena, Germany). Images were captured as uncompressed 24-bit TIFF files with a SPOT cooled (–12 °C) single CCD color digital camera (three pass method) driven by SPOT version 2.1 software (Diagnostic Instruments, Inc.). Fluorescein or DAPI signals were observed with HI Q filter sets for fluorescein isothiocyanate or DAPI, respectively (Chroma Technology). The selected cells displayed in the appropriate figures were representative of 80% of the population of positive cells.
FLIP—FLIP experiments were performed on a Zeiss LSM310 confocal microscope (Carl Zeiss Inc.) using a x100/1.0 water immersion lens. First, a control image was obtained at focal plane at a depth approximately midway through the cell using the 488-nm line of the Argon laser for excitation of GFP with an attenuation of 90% and stored as 8-bit TIFF file. Then a small rectangular region of interest was defined within the cytoplasm or nuclei, the laser attenuation was completely removed and the region of interest (noted by white outlined areas in the Fig. 6) was continuously scanned at 488 and 514 nm at full laser power for 2 min (cytoplasm) or 30 s (nuclei) by using the LSM software provided with the Zeiss 310 microscope, resulting in continuous photo-bleaching (loss of fluorescence) of any molecule within that region. In time, freely moving molecules located previously outside of the region of interest will diffuse into that region and become photobleached, resulting in a time-dependent loss of fluorescence within the larger area (cytoplasm or nucleus). At the end of the bleaching period, laser attenuation and excitation wavelengths were restored to those of the control image, and the fluorescence throughout the cell was monitored at various time intervals. During experiments, a constant temperature of 37 °C was maintained following the methodology described under "Live Cell Imaging."
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Materials
[-32P]ATP (370 MBq/ml) and horseradish
peroxidase-conjugated donkey anti-rabbit IgG were from Amersham Biosciences.
The rabbit anti-PKC, anti-PKC, anti-PKC, and anti-GFP
antibodies were obtained from Santa Cruz Biotechnology, Inc., Santa Cruz, CA.
The Aloop antibody (phospho-PKD/PKCµ Ser744-Ser748
antibody), which specifically recognizes the phosphorylated state of the
equivalent serines within the activation loop of the three PKD isoforms (PKD:
Ser744-Ser748; PKD2:
Ser706-Ser710; PKD3:
Ser731-Ser735), was obtained from Cell Signaling
Technology, Beverly, MA. The polyclonal anti-PKD antibody PA-1 was previously
described (13). Fluorescein or
Texas Redconjugated goat anti-mouse or anti-rabbit immunoglobulins were
obtained from Molecular Probes, Eugene, OR. The polyclonal anti-PKD3 N17
antibody was raised by immunizing rabbits with a peptide corresponding to the
human PKD3 17 amino-terminal residues SANNSPPSAQKSVLPTA using standard
procedures. Neurotensin, DAPI, and the murine monoclonal anti-FLAG antibodies
M2 and M5 were obtained from Sigma. Leptomycin B (LMB) was a generous gift
from Dr. Minoru Yoshida, Department of Biotechnology, University of Tokyo. All
the other reagents were the highest grade commercially available.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Once established that putative transcripts encoding for PKD3 were present in total RNA isolated from Panc-1 cells, we used that same total RNA to synthesize a full-length cDNA for PKD3 by RT-PCR. The deduced amino acid sequence of the obtained PKD3 cDNA (890 amino acids with a predicted molecular mass of 100.5 kDa) matched 100% the one obtained from expressed sequence tag deduction and rapid amplification of cDNA ends for PKD3 (11). PKD3 has 71 and 66% overall homology to PKD and PKD2, respectively. The catalytic domain is the most conserved structural domain between the different members of the PKD family. In contrast, the regulatory domain is the most divergent region of this family (Table I). In particular, the PH domain of PKD3 has the lowest level of homology to the PH domains of PKD and PKD2 (Table I). The PH domain of PKD mediates regulation of its catalytic activity, complex formation with other signaling molecules, and its nuclear export (13, 62, 63). Therefore, it is possible that these sequence differences are important for the regulation and intracellular distribution of PKD3. In addition, hydropathy analysis revealed that PKD3 does not have a hydrophobic domain in its NH2 terminus as the one present in PKD and PKD2. Overall, PKD3 is the most distinct member of the PKD family.
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To facilitate the unambiguous detection of PKD3, we constructed a plasmid
encoding a chimeric PKD3 protein by fusing the GFP of Aequorea
victoria to 5' of the cDNA encoding PKD3
(Fig. 1B). The
obtained construct, pGFP-PKD3, was transfected into Panc-1 cells and the
product of its expression analyzed by Western blot using antibodies against
GFP (Fig. 1C, left
panel). The product of expression of pGFP-PKD3 matched the predicted size
for the encoded protein (
130 kDa) and did not show any degradation.
Differential Intracellular Distribution of PKD Isoforms—In view of the differences between PKD and PKD3 in their regulatory region (Table I), we consider the possibility that PKD3 is a kinase with an intracellular distribution distinct from PKD. To test this hypothesis, we transiently transfected pGFP-PKD3 into Panc-1 cells and its intracellular distribution was examined in living cells. A construct encoding a fusion protein between GFP and PKD was also used for comparison because the distribution of the encoded GFP-PKD protein has already been described in several cell types (40, 41, 63). In addition, we also compared the distribution of GFP-PKD3 to GFP-PKD2, because we recently demonstrated that PKD2 is principally localized in the cytoplasm in Panc-1 cells (51).
In agreement with our recent results (41, 51), we found that GFP-PKD and GFP-PKD2 expressed in Panc-1 cells are predominantly in the cytoplasm (Fig. 1C). In striking contrast, GFP-PKD3 accumulates in the nucleus, in addition to its cytosolic localization (Fig. 1C). The morphology of GFP-, GFP-PKD-, GFP-PKD2-, or GFP-PKD3-transfected cells was indistinguishable from that of non-transfected Panc-1 cells (data not shown). A similar nuclear accumulation of GFP-PKD3 was observed when this fusion protein was transiently expressed in other cell types, including COS-7 or MDCK epithelial cells (Fig. 1C), NIH and Swiss 3T3 fibroblasts, and in the intestinal epithelial cell line IEC-18 (data not shown).
The molecular weight of GFP-PKD3 (
130,000) far exceeds the size limit
for passive nuclear diffusion (45,000–60,000)
(64–67).
Nevertheless, its fluorescent tag could interfere with the normal distribution
of PKD3. Therefore, we constructed a plasmid encoding a different chimeric
PKD3 protein by fusing the small FLAG epitope (8 amino acid residues) to the
5' of the cDNA encoding PKD3 (Fig.
1B). The obtained construct, pFLAG-PKD3, was transfected
into Panc-1 cells and its expression was analyzed by Western blot using
monoclonal antibodies against the FLAG epitope
(Fig. 1D, left
panel). The product of expression of pFLAG-PKD3 matched the predicted
size for the encoded protein (100 kDa) and did not show any
degradation.
We then analyzed the localization of FLAG-PKD3 by indirect immunofluorescence of fixed cells. No difference was detected between the distributions of FLAG-PKD3 and GFP-PKD3 in different cell types, including Panc-1 or MDCK cells (Fig. 1D, right panels), demonstrating that the unique intracellular distribution of GFP-PKD3 was not because of the fluorescent tag. The localization of FLAG-PKD3 in the nucleus of Panc-1 and MDCK cells was corroborated by DAPI costaining of those cells (Fig. 1D).
Intracellular Distribution of Endogenous PKD3—Our results indicated that PKD3 displayed an intracellular distribution that was unique within the PKD family. However, this conclusion was based on the observed distribution of ectopically expressed PKD3 (GFP-PKD3 and FLAG-PKD3). To corroborate this conclusion, we examined the intracellular distribution of endogenous PKD3 using a specific antibody that recognizes PKD3.
A polyclonal anti-PKD3 antibody (N17) was raised by immunizing rabbits with the peptide SANNSPPSAQKSVLPTA corresponding to 17 amino acids in the NH2 terminus of human PKD3. The N17 antibody specificity was tested against different members of the PKD family using Western blot analysis. Panc-1 cells transiently transfected with pGFP-PKD, pGFP-PKD2, or pGFP-PKD3 were lysed 18 h post-transfection and the solubilized proteins were resolved by SDS-PAGE and Western blotted using the N17 antibody. As shown in Fig. 2A, top panel, the N17 antibody only recognized GFP-PKD3. The reactivity of N17 against GFP-PKD3 was eliminated by preincubating the N17 antiserum with the immunizing peptide (Fig. 2A, top panel). Parallel controls were stained with an anti-GFP antibody to verify the expression and equal loading of the three PKD isoforms (Fig. 2A, bottom panel).
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Next, we examined the intracellular distribution of endogenous PKD3 by indirect immunofluorescence in unstimulated fixed Panc-1 cells using the N17 antibody. Contrasting with the cytoplasmic localization of endogenous PKD (Fig. 2B, left panel), endogenous PKD3, in addition to its cytoplasmic localization, showed a very prominent nuclear localization (Fig. 2B, center panel). The same distribution of endogenous PKD3 was detected in MiaPaca-2 cells, another human pancreatic cancer cell line (data not shown). No signal was detected when the cells were stained with the N17 antibody previously preincubated with the immunizing peptide (Fig. 2B, right panel). A similar intracellular distribution of endogenous PKD3 was observed in Panc-1 cells using a polyclonal antibody recognizing the carboxyl-terminal 16 residues (HFIMAPNPDDMEEDP) of human PKD3 (data not shown). Thus, the intracellular distribution of endogenous PKD3 is identical to that of ectopically expressed FLAG-PKD3 or GFP-PKD3. Our results indicate that in contrast to PKD (41) or PKD2 (51), PKD3 localizes not only to the cytosol but also to the nucleus in unstimulated cells.
Phorbol Esters and GPCR Agonists Activate PKD3—The distinct
intracellular distribution of PKD3 prompted us to examine the pathways and
mechanisms leading to its catalytic activation. Panc-1 cultures transiently
transfected with pGFP-PKD, pGFP-PKD3, or pEGFP-C3 (empty vector) (see
Fig. 3A) were
stimulated with 200 nM PDBu for 10 min, lysed, and the different
chimeric proteins immunoprecipitated from the extracts with an antibody
against GFP. The immune complexes were incubated with
[-32P]ATP, subjected to SDS-PAGE, and analyzed by
autoradiography to detect the prominent 140–130-kDa bands corresponding
to the autophosphorylated PKD moieties of GFP-PKD and GFP-PKD3, respectively.
GFP-PKD3 isolated from unstimulated Panc-1 cells had low catalytic activity
that was markedly activated by PDBu stimulation of intact cells. This rapid
and striking increase in PKD3 kinase activity was maintained during cell lysis
and immunoprecipitation. PDBu treatment of Panc-1 cells expressing GFP-PKD
also induced a rapid and striking increase in PKD kinase activity shown for
comparison (Fig.
3A).
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Recently we reported that NT binding to its heptahelical GPCR induces PKC-dependent PKD activation and DNA synthesis in Panc-1 cells (22). Interestingly, the expression of the NT receptor is markedly increased in pancreatic cancer samples as compared with normal controls (68–70). It is therefore important to characterize NT-mediated signal transduction pathways in human pancreatic cancers and in this study we examined whether NT induces PKD3 activation in Panc-1 cells. Cultures of these cells transiently transfected with pGFP-PKD, pGFP-PKD3, or pEGFP-C3 (empty vector) were stimulated for 10 min with 50 nM NT, lysed, and the catalytic activity of the different chimeric proteins was assayed as described above. The results presented in Fig. 3A show that stimulation of Panc-1 cells with NT induced a marked increase in the catalytic activity of the PKD3 moiety of GFP-PKD3 as well as in the PKD moiety of GFP-PKD. No catalytic basal activity was detected in the control Panc-1 cells expressing GFP in the absence or presence of NT or PDBu.
Similarly, we also analyzed the catalytic activation of FLAG-PKD3 in Panc-1
cells. Cultures of these cells transiently transfected with pFLAG-CMV-4 (empty
vector) or pFLAG-PKD3 (see Fig.
1B) were stimulated for 10 min with 50 nM NT
or 200 nM PDBu, lysed, and FLAG-PKD3 was immunoprecipitated from
the cell extracts with a mixture of murine monoclonal antibodies against the
FLAG epitope. The immune complexes were incubated with
[-32P]ATP, subjected to SDS-PAGE, and analyzed by
autoradiography to detect a prominent 100-kDa band corresponding to the
autophosphorylated PKD3. The results presented in
Fig. 3B show that
treatment of Panc-1 cells with either PDBu or NT also induced a striking
increase in the catalytic activity of FLAG-PKD3 that was comparable with the
one detected, under the same conditions, with GFP-tagged PKD3. No catalytic
basal activity was detected in control Panc-1 cells transfected with
pFLAG-CMV-4 in the absence or presence of NT or PDBu.
To substantiate the results shown in
Fig. 3A with tagged
PKD3, we also examined the catalytic activation of endogenous PKD3 in Panc-1
cells stimulated with NT or PDBu. Panc-1 cells were treated for 10 min with 50
nM NT or 200 nM PDBu, lysed, and PKD3 was
immunoprecipitated from the cell extracts with the N17 antibody. The immune
complexes were incubated with [-32P]ATP, subjected to
SDS-PAGE, and analyzed by autoradiography. A prominent 100-kDa band
corresponding to the autophosphorylated PKD3 was detected in the Panc-1 cells
treated with either PDBu or NT (Fig.
3C). No catalytic basal activity was detected in control
Panc-1 cells in the absence of NT or PDBu.
Next, we examined whether an increase in PKD3 catalytic activity can be demonstrated in another cell system by stimulation with phorbol ester or through another GPCR-mediated pathway. COS-7 cells transiently cotransfected with the bombesin GPCR and pGFP-PKD, pGFP-PKD3, or pEGFP-C3 (empty vector) were stimulated for 10 min with 10 nM bombesin or 200 nM PDBu, lysed, and the catalytic activity of the different chimeric proteins assayed by in vitro kinase assays as described above. As shown in Fig. 3D, bombesin or PDBu stimulation of COS-7 cells transiently cotransfected with GFP-PKD3 together with the bombesin GPCR induced a rapid increase in the catalytic activity of PKD3 as well as in its control expressing the bombesin GPCR and GFP-PKD. No catalytic basal activity was detected in COS-7 cells expressing GFP and the bombesin GPCR in the absence or presence of bombesin or PDBu (data not shown). The results presented in Fig. 3 demonstrate, for the first time, that PKD3 can be converted from inactive to an active state by cell treatment with either a biologically active phorbol ester or the GPCR agonists NT or bombesin.
PKD3 Activation Correlates with the Phosphorylation of Its Activation Loop—The signal-dependent phosphorylation of amino acid residues within the activation loop of many protein kinases plays a critical role in their activation. Here we examined whether the activation of PKD3 by treatment of intact cells with PDBu, NT, or bombesin also induces the phosphorylation of Ser731 and Ser735 located in the activation loop of PKD3. These residues in PKD3 are equivalent to Ser744 and Ser748 within the activation loop of PKD.
Panc-1 cells transiently transfected with pEGFP-C3 (empty vector), pGFP-PKD, or pGFP-PKD3 were treated for 10 min with NT (50 nM) or PDBu (200 nM) and lysed. The solubilized proteins were resolved by SDS-PAGE and Western blotted using the Aloop antibody that recognizes the phosphorylated serines within the PKDs activation loop sequence IGEKS-FRRSVVGT. As shown in Fig. 4A, top panel, NT or PDBu induced the activation loop phosphorylation of the PKD3 moiety of GFP-PKD3, which correspond to Ser731 and Ser735, as well as the activation loop phosphorylation of the PKD moiety of GFP-PKD, which correspond to Ser744 and Ser748. Several bands in the vicinity of the 100-kDa marker were detected in PDBu and NT challenged control Panc-1 cells expressing GFP (Fig. 4A, top panel). These bands, which were also detectable in Panc-1 cells expressing GFP-PKD and GFP-PKD3, correspond to the endogenously expressed PKDs upon their activation mediated by PDBu or NT (Fig. 4A, top panel). The same membrane was stripped and subsequently probed with an antibody against GFP to verify the expression and equal loading of GFP-PKDs (Fig. 4A, bottom panel). Similar results were also obtained with COS-7 cells expressing the bombesin receptor and GFP-PKD3 when they were challenged with PDBu or bombesin (Fig. 4B) or in Panc-1 cells expressing FLAG-PKD3 when the cultures were challenged with NT or PDBu (Fig. 4C). The results displayed in Figs. 3 and 4 demonstrate, for the first time, that PDBu and GPCR agonists induce a rapid and persistent increase in the catalytic activity of PKD3 as measured by autokinase activity and direct activation loop phosphorylation.
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Activation of Neurotensin-GPCR Induces Transient Translocation of GFP-PKD3 from the Cytosol to the Plasma Membrane—Taking into consideration the similarity in the activation but the differential intracellular distribution of PKD and PKD3, we decided to compare their spatial and temporal distribution in Panc-1 cells in response to NT. As illustrated by the image presented in Fig. 4D, GFP-PKD expressed in unstimulated Panc-1 cells was distributed throughout the cytosol, whereas GFP-PKD3 was present in both cytosol and nucleus in agreement with the results presented in Fig. 1C. In each case, very little fluorescent signal was localized to the plasma membrane. Real time imaging of living cells revealed that NT stimulation of Panc-1 cells induced a rapid translocation of GFP-PKD3 to the plasma membrane (Fig. 4D, arrows). Translocation of GFP-PKD3 occurred within 30 s and reached a maximum between 2 and 3 min. The association of this chimeric protein with the plasma membrane was transient. The reverse translocation of GFP-PKD3 from the plasma membrane to the cytosol was complete within 5 min of NT stimulation (Fig. 4D). The rapid and transient translocation of GFP-PKD3 to the plasma membrane is comparable with that of GFP-PKD (Fig. 4D). The distribution of GFP, detected in the nuclei as well as throughout the cytosol, was not affected by NT stimulation (data not shown).
Inhibition of PKC Prevents the Activation and Rapid Plasma Membrane Dissociation of PKD3—To determine whether GPCR agonists induce PKD3 activity and activation loop phosphorylation through a PKC-mediated pathway, we used the inhibitors of phorbol ester-sensitive isoforms of PKC, GF I (also known as GF 109203X or bisindolylmaleimide I), or Ro 31-8220. Panc-1 cells transiently transfected with pGFP-PKD3 were treated for 1 h with GF I or Ro 31-8220 prior to stimulation with 50 nM NT or 200 nM PDBu. As shown in Fig. 5A, exposure to either GF I or Ro 31-8220 prevented the activation of PKD3 as well as the phosphorylation of its activation loop induced by NT or PDBu. In contrast, the compound GF V, which is related to GF I but biologically inactive, did not prevent PKD3 phosphorylation in response to NT or PDBu in Panc-1 cells. Inhibition of the activation loop phosphorylation of PKD3 as well as inhibition of its catalytic activity was also observed in GF I or Ro 31-8220 pre-treated COS-7 cells expressing the bombesin receptor and GFP-PKD3 when these cells were challenged with either PDBu or bombesin (Fig. 5B).
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We then examined whether PKC inhibitors affect the rapid translocation of PKD3 from the plasma membrane to the cytosol. Treatment of cultures expressing GFP-PKD3 with GF I prior to stimulation with NT did not prevent their translocation from the cytosol to the plasma membrane, indicating that classical and novel PKCs are not necessary for this step (Fig. 5 C, bottom panel). However, addition of GF I dramatically delayed the plasma membrane dissociation of GFP-PKD3 (Fig. 5C, bottom panel, arrows) that we detected in cells untreated with PKC inhibitors (Fig. 5C, top panel; see also Fig. 4D). Similar results were obtained using Ro 31-8220, another PKC inhibitor (data not shown). Therefore, these results demonstrate that agonist stimulation of the NT GPCR induces a striking and transient translocation of GFP-PKD3 to the plasma membrane of Panc-1 cells and that its rapid reverse translocation can be delayed by PKC catalytic activity inhibitors.
As demonstrated above, treatment of intact cells with the inhibitors GF I or Ro 31-8220 inhibited PKD3 activation induced by subsequent addition of PDBu or NT. In striking contrast, GF I or Ro 31-8220 added directly to the in vitro kinase assays at identical or higher concentrations to those used previously in intact cells did not inhibit PKD3 activity stimulated by PDBu in intact cells (Fig. 5D). Thus, the observed in vivo inhibition of PKD3 catalytic activity in response to GF I and Ro 31-8220 was not direct but rather because of the inhibition of the catalytic activity of PKC.
PKD3 Activation by Novel PKCs—Next, we examined whether
novel PKC isoforms activate PKD3 by contransfecting COS-7 cells with plasmids
encoding GFP-PKD3 and the constitutively active PKC* or PKC*. The
cells were lysed 48 h post-transfection and GFP-PKD3 was immunoprecipitated
from the lysates using the GFP antiserum. The immunocomplexes were subjected
to in vitro kinase assays to measure PKD3 activity by
autophosphorylation. As shown in Fig.
6A, upper panel, the constitutively active
PKC*orPKC* induced the activation of PKD3 as measured by its
autokinase activity. This activation is comparable with the one induced by
PDBu alone. The addition of PDBu to the cultures expressing GFP-PKD3 and the
constitutively active PKC* or PKC* further enhanced the catalytic
activity of PKD3. In contrast, no PKD3 activation was induced in the cells
cotransfected with GFP-PKD3 and constitutively active PKC* (see
Fig. 6A, upper
panel). No basal kinase activity was detected in control COS-7 cells
expressing GFP and constitutively active PKC* or PKC* (data not
shown).
We also found that in COS-7 cells, constitutively active PKC* or
PKC* mediated the activation loop phosphorylation of PKD3 as revealed by
Western blot analysis using the Aloop antibody
(Fig. 6A, center
panel). The same membrane was stripped and subsequently probed with an
antibody against GFP to verify the expression and equal loading of GFP-PKD3
(Fig. 6A, bottom
panel).
Previous results from our laboratory demonstrated that PKC, in
addition to mediating the activation of PKD, forms a complex with PKD via the
regulatory region of PKD (13).
In view of the differences between the PH domains of PKD and PKD3
(Table I), we examined whether
PKD3 interacts with PKC. Panc-1 cells were cotransfected with pGFP-PKD3
and pCO2 (empty vector) or the plasmid encoding the constitutively active form
of the novel PKC*. Panc-1 cells were also cotransfected with pGFP-PKD and
pCO2 (empty vector) or the plasmid encoding the constitutively active
PKC* for comparison because the interaction between PKD and PKC has
been previously characterized. The transfected cells were lysed 48 h
post-transfection and GFP-PKD or GFP-PKD3 in the extracts were
immunoprecipitated using antiserum specific for GFP, PKD, or PKD3. The
immunocomplexes were subjected to in vitro kinase assays to measure
PKD and PKD3 activity by autophosphorylation
(Fig. 6B). GFP or PKD
immunoprecipitates from GFP-PKD/PKC*-cotransfected cells showed two
prominent phosphorylated bands: one corresponding to GFP-PKD (140 kDa) and
another corresponding to PKC* (80 kDa), in agreement with previous
results (13). In striking
contrast, GFP or PKD3 immunoprecipitates from PKD3/PKC*-cotransfected
cells did not show any other band in addition to that corresponding to
phosphorylated GFP-PKD3 (130 kDa) (Fig.
6B). Western blot analysis was used to corroborate that
the 80-kDa band corresponded to PKC* and that the levels of expression of
this protein and the GFP-tagged PKDs were similar in all conditions (data not
shown). These results identify an important difference between PKD and PKD3 in
their ability to form a molecular complex with PKC*.
PKD3 Continuously Shuttles between the Cytoplasm and the Nucleus—The observed intracellular distribution of PKD3 could result from its continuous transport between the nucleus and the cytoplasm. If this interpretation is correct, interference with the nuclear export machinery should further increase the nuclear accumulation of GFP-PKD3.
LMB is an antifungal antibiotic (71) that inhibits the formation of complexes consisting of CRM1, RanGTP, and proteins containing a leucine-rich nuclear export signal, thereby blocking CRM1-dependent nuclear export (72–75). Consequently, we decided to use LMB to determine whether the nuclear localization of GFP-PKD3 was CRM1-dependent and therefore the result of its continuous shuttling between the cytoplasm and nucleus.
Panc-1 cells transiently transfected with pGFP-PKD3 were incubated with LMB (10 ng/ml) for 1 h and the distribution of GFP-PKD3 analyzed by imaging live cells. As shown in Fig. 7A, an increase in the nuclear accumulation of GFP-PKD3 was detected in response to LMB. This result strongly supports the conclusion that GFP-PKD3 is continuously shuttling between the nucleus and the cytoplasm and that this shuttling requires a competent CRM1 nuclear export pathway. LMB did not induce any detectable nuclear accumulation of GFP (Fig. 7A).
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Effect of PKD3 Activation in Its Nuclear Transport—To determine whether NT-mediated activation of PKD3 increases the rate of its nuclear import, we blocked the CRM1-dependent nuclear export pathway with LMB and compared the relative nuclear accumulation of PKD3 in non-stimulated versus NT-stimulated Panc-1 cells. The addition of NT to LMB-treated Panc-1 cells did not induce any significant change in the intracellular distribution of GFP (Fig. 7A). However, NT induced an increase in the rate of nuclear import of GFP-PKD3 as indicated by its more rapid nuclear accumulation when compared with non-stimulated cells in the presence of LMB (Fig. 7A). The results imply that the nuclear transport of PKD3 is a regulated process associated to its activation. Therefore, we hypothesized that constitutively active PKCs that activate PKD3 should also modify its nuclear transport without cell stimulation.
Panc-1 cells were cotransfected with plasmids encoding GFP-PKD3 and active
PKC* or PKC* and the distribution of GFP-PKD3 was analyzed in fixed
cells. As our results shown, the constitutively active PKC* or PKC*
induced a dramatic nuclear accumulation of GFP-PKD3
(Fig. 7B). No further
nuclear accumulation of GFP-PKD3 than the one detected in cells cotransfected
with the empty vector pCO2 (data not shown) was observed in the cells
expressing the constitutively active atypical PKC*, despite its nuclear
presence (Fig. 7B).
The expression of GFP-PKD3 did not affect the intracellular distribution of
any of the constitutively active PKCs (compare left and center
panels in Fig.
7B). Similar results were obtained in living or fixed
COS-7 cells coexpressing GFP-PKD3 and PKC* or PKC* (data not
shown). No change in the intracellular distribution of GFP was detected in
control Panc-1 cells expressing GFP and PKC* or PKC* (data not
shown). These results demonstrated that the rate of nuclear transport for PKD3
is dramatically modified as a result of its activation via NT or
constitutively active novel PKCs.
A Nuclear Transport Receptor Mediates the Nuclear Import of PKD3—One of the functional characteristics of nuclear transport receptors is the saturability of signal recognition (76). We previously employed this criterion to block the nuclear import of PKD via its CRD, because this PKD domain is responsible for its nuclear import (63). Taking into consideration that the CRD of PKD and PKD3 are highly homologous (see Table I), we hypothesized that the CRD of PKD should block the nuclear import of PKD3 by interfering with its nuclear import receptor.
Consequently, a protein consisting of GFP fused to the NH2 terminus of the CRD of PKD (GFP-CRD) (63) was coexpressed with FLAG-PKD3 to block the nuclear import of PKD3. GFP-CRD was distributed throughout the cytosol and the nuclei of Panc-1 cells with a distinct accumulation in the plasma membrane and Golgi, in agreement with our previous observations (63), whereas FLAG-PKD3 was predominantly located in the nuclear compartment (Fig. 7C, arrows). The coexpression of GFP-CRD and FLAG-PKD3 (ratio 10:1) did not produce any major changes in the distribution of GFP-CRD. However, the nuclear import of FLAG-PKD3 was prevented, as revealed by its absence in the nuclear compartment (Fig. 7C). These results suggested that the CRD of PKD successfully competed with FLAG-PKD3 for the nuclear import receptor that delivers PKD3 to the nucleus and they support the conclusion that the nuclear import of PKD3 utilizes the same import machinery as PKD. Coexpression of GFP, which uniformly distributes in the cytoplasm and nuclei of Panc-1 cells did not interfere with the nuclear accumulation of Flag-PKD3 (data not shown).
Intracellular Dynamic Properties of PKD3—Our preceding results strongly suggested that PKD3 shuttles between the cytoplasm and the nucleus. To further support this conclusion and to study the dynamic behavior of PKD3 in the nucleus and in the cytoplasm we used FLIP.
Panc-1 cells were transiently transfected with the plasmid encoding GFP-PKD3 and analyzed by FLIP in live cells as indicated under "Experimental Procedures." As illustrated by the images presented in Fig. 8, GFP-PKD3 was distributed throughout the cytosol and the nucleus before photobleaching. FLIP was performed either in the cytoplasmic or nuclear compartments, within the restricted areas indicated by rectangles. After FLIP, the total cell fluorescence was followed over time. Bleaching the GFP-PKD3 present in the cytoplasm (Fig. 8, top panel) resulted in almost complete disappearance of fluorescent signal from this compartment by 2 min. This result showed that most of the GFP-PKD3 cytoplasmic molecules passed through the area being bleached within 2 min suggesting a rapid and random movement. The recovery of fluorescent signal over the cytoplasm was relatively slow, very likely because of the free diffusion barrier imposed by the nuclear envelope to the GFP-PKD3 molecules inside the nuclear compartment. By 12 min a significant amount of GFP-PKD3 was detectable in the cytoplasm, which coincided with a drop in the intensity of the fluorescent signal over the non-bleached nuclear compartment, indicating transfer of GFP-PKD3 from nuclei into the cytoplasm.
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Bleaching GFP-PKD3 present in the nucleus resulted in the complete disappearance of fluorescent signal from this compartment by 30 s, in accordance with the smaller volume of the nucleus, followed by partial recovery of fluorescent signal over the bleached area by 9 min (Fig. 8, bottom panels). The spontaneous recovery after bleaching of the whole cell was less than 3% of total initial fluorescence (data not shown), which coincides with previous reports indicating that spontaneous recovery of bleached GFP did not contribute to signals (77–79). Unbleached control cells, adjacent to the photobleached ones, showed no fluorescence changes during photobleaching, indicating that there was no generalized bleaching effect during imaging (see Fig. 8). Thus, any fluorescent signal increase detected after the restricted photobleaching within a compartment was the result of non-bleached GFP-PKD3 molecules entering the other non-bleached compartment. These results demonstrate that in non-stimulated cells GFP-tagged PKD3 continuously shuttles between the cytoplasm and nucleus and indicates that PKD3 moves rapidly within each compartment.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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