From the Basic Research Laboratory, NCI, National
Institutes of Health, Frederick, Maryland and ¶ Oral and
Pharyngeal Cancer Branch, NIDCR, National Institutes of Health,
Bethesda, Maryland 20892
Received for publication, August 22, 2002, and in revised form, January 8, 2003
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ABSTRACT |
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A novel protein kinase, polyploidy-associated
protein kinase (PAPK), was isolated using a subtraction cDNA
library approach from a mouse erythroleukemia cell line that had been
induced to polyploidy after serum withdrawal. PAPK shares homology with
members of the Ste20/germinal center kinase family of protein kinases and is ubiquitously expressed as two spliced forms, PAPK-A and PAPK-B,
that encode for proteins of 418 and 189 amino acids, respectively. The
expression of endogenous PAPK-A protein increased after growth factor
withdrawal in murine hematopoietic and fibroblast cells. When tested in
an in vitro kinase assay, PAPK-A was activated in response
to the stress-inducing agent hydrogen peroxide and slightly by fetal
calf serum. Biochemical characterization of the PAPK-A-initiated
pathway revealed that this novel kinase does not affect MAP kinase
activity but can stimulate both c-Jun N-terminal kinase 1 (JNK1) and
ERK6/p38 The sterile-20 (Ste20) family of serine-threonine kinases was
first discovered, and extensively studied, as an essential component for the pheromone-response pathway in Saccharomyces
cerevisiae (1, 2). It has also been recognized that this family of kinases plays a key role in several other known yeast signaling pathways. They include the control of morphological changes,
cytokinesis, response to nutrient starvation, and localizing cell
growth with respect to the division plate (3-5).
Over the past few years, there has been a tremendous increase in the
number of mammalian homologues of Ste20 kinases identified. Based on
their structure, these mammalian Ste20-like kinases can be divided into
two subfamilies (6). The first class, p21-activated kinases
(PAKs),1 is activated upon
binding to the guanosine triphosphatases Cdc42 and/or Rac1. Upon
binding to Cdc42/Rac1-GTP, PAKs undergo a conformational change, which
enables autophosphorylation and subsequent activation of the kinase to
occur (7). PAKs have been shown to be involved in changes in cell
motility, morphology (8), apoptosis (9-12), and transformation (13,
14). PAKs have also been implicated in activating MAP kinases,
including ERK, JNK/SAPK, and p38 (8).
The second class of Ste20-related kinases is the germinal center kinase
(GCK), which contains an N-terminal catalytic domain and lacks the
p21-binding domain. A large number of serine/threonine kinases
belonging to the GCK family have been implicated as upstream regulators
of MAP kinase signaling pathways (15, 16). The GCK family kinases
include GCK (17, 18), hematopoietic progenitor kinase-1 (HPK1) (19,
20), NCK-interacting kinase (NIK; also referred to as HGK) (21, 22),
GCK-like kinase (23), kinase homologous to SPS1/STE20 (KHS1; also
referred to as GCKR) (24, 25), STE20/oxidant stress-response kinase-1
(SOK-1; also referred to as YSK1, stk25) (26, 27), mammalian STE20-like
kinase-1 (MST1; also referred to as Krs-2) (28, 29), mammalian
STE20-like kinase-2 (MST2; also referred to as Krs-1) (28, 29),
mammalian STE20-like kinase-3 (MST3) (30, 31), MST4 (also referred to as MASK) (32-34), misshapen/NIK-related kinase (35), Traf2 and NCK interacting kinase (TNIK) (36), NIK-related kinase (also referred
to as NESK) (37, 38), lymphocyte-oriented kinase (39), SLK (40, 41),
PASK/SPAK (42, 43), TAO1 (44), PSK1/TAO2 (45, 46), and
JNK/SAPK-inhibitory kinase (JIK; also referred to as DPK) (47, 48). All
GCKs contain N-terminal Ste20-like kinase domains and long C-terminal
regulatory domains. Several members of the GCK group have been shown to
be potent and selective activators of SAPK/JNK or p38.
In the present study, using a subtractive screen for polyploidy
induction, we cloned and characterized a widely expressed molecule
termed polyploidy-associated protein kinase (PAP kinase; PAPK), a novel
member of the mammalian Ste20/GCK kinase family. We demonstrate in this
report that PAPK, like many other GCK family members, is able to
specifically activate the JNK and ERK6/p38 Plasmids--
Expression vectors of pcDNAIII-HA-ERK2,
pcDNAIIIHA-JNK1, pcDNA-HA-p38 Cell Culture and Transfection--
293T, NIH3T3, HeLa, EL4, and
Friend erythroleukemia (C19, DS19 (53)) cells were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum (FCS). HCD-57 cells, an erythropoietin
(Epo)-dependent erythroleukemia cell line, were maintained
as described previously (54). Interleukin 3-dependent BaF3,
FDCP2, and 32D cells were maintained in RPMI 1640 medium with 10% FCS
and 10% WEHI-conditioned medium (as an IL-3 source). NIH3T3 cells
stably expressing the regulatory GAL4-DBD/hPR-LBD/p65 AD fusion protein
from the pSwitch plasmid (GeneSwitch-3T3) were obtained from
Invitrogen. To obtain clones with mifepristone-inducible expression of
PAPK-A and PAPK-A(K89M), GeneSwitch-3T3 cells were transfected with 2 µg of the pGene/V5-His plasmid (Invitrogen) carrying the coding
region of Myc-tagged PAPK-A, PAPK-A(K89M) cDNA, and empty vector by
using LipofectAMINE 2000 (Invitrogen) and subjected to selection for
stable transfectants with 0.4 mg of Zeocin per ml. Two to 3 weeks
later, colonies were picked, expanded, and tested for induction of the
transgene by mifepristone (1 × 10 cDNA Subtraction--
cDNA subtraction was performed
using a PCR-selected cDNA Subtraction kit
(Clontech). The mRNA from C19 cells cultured
for 24 h in serum-free medium was used as the "driver." The
mRNA from polyploid C19 cells cultured for 5 days in serum-free
medium was used as the "tester." The subtractive screening was
performed according to the manufacturer's instructions. A partial
fragment of PAPK was obtained as a candidate-positive cDNA clone.
Cloning of PAPK and DNA Construct--
5'- and 3'-RACE analyses
for the cDNA fragment obtained were performed using C19
erythroleukemia cDNA as a template with the Marathon cDNA
amplification kit (Clontech) in accordance with the
manufacturer's instructions. The internal primers used were 5'-CATCTTGCACGGCACACTCCTACAGGAACAC-3' (sense for 3'-RACE) and 5'-CACTGTTGGCAGCTGGCTTTGGGTTATTTCTCC-3' (antisense for 5'). Each RACE
product was cloned into the pCR2.1-TOPO vector (Invitrogen) and
sequenced. Full-length mouse PAPK-A and PAPK-B were obtained by PCR
using cDNA from C19 cells as a template with the Marathon cDNA
amplification kit (Clontech).
EcoRI-SalI fragments of full-length PAPK-A and
PAPK-B were cloned into pCMV-Myc (Clontech).
A kinase-inactive form of PAPK-A (PAPK-A-K89M) was created by
substituting lysine 89 with a methionine using a QuickChange site-directed mutagenesis kit (Stratagene) with the mutagenic primers 5'-GAACACTGGTAACTGTAATGATTACAAACCTGGAAAG-3
(sense) and 5'-CTTTCCAGGTTTGTAATCATTACAGTTACCAGTGTTC-3'
(antisense) (the mutated bases are underlined) by PCR and cloned into
pCMV-myc.
Generation of Polyclonal Antibodies against PAPK--
An
antiserum against PAPK was raised in rabbits by immunization with a
keyhole limpet hemocyanin-conjugated synthetic polypeptide corresponding to the C-terminal 20 amino acids (amino acids 399-418, SPWSELEFQFPDDKDPVWEF). Anti-PAPK antibodies were typically used at a
1:1000 dilution.
RNA and Protein Analysis--
Poly(A+) RNA was
prepared with the FastTrac Kit (Invitrogen). Mouse Multiple Tissue
Northern blots were purchased from Origene. Cells were lysed in lysis
buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl,
10% glycerol, 1% Nonidet P-40, 2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM sodium
orthovanadate, and aprotinin and leupeptin at 1 µg/ml each). Cells
were lysed on ice for 20 min, and supernatant was collected at 14,000 rpm at 4 °C. Cell lysates were subjected to immunoblotting, as
described previously (54), using mouse monoclonal anti-HA antibody
(12CA5, Roche Molecular Biochemicals), mouse monoclonal anti-Myc
antibody (7E10, Clontech), mouse monoclonal anti-FLAG antibody (M2, Stratagene), mouse monoclonal anti-GST antibody
(Cell Signaling), rabbit anti-JNK antibody (Cell Signaling), and rabbit
anti-phospho-JNK antibody (Cell Signaling). For the PAPK and MAP kinase
assays, protein was immunoprecipitated with anti-Myc monoclonal
antibody (7E10) or anti-HA monoclonal antibody (12CA5) at 4 °C for
5 h or overnight, respectively. Immunocomplexes were recovered
with protein G-agarose (Upstate Biotechnology, Inc.) for 1 h.
Beads were washed twice with lysis buffer and twice with kinase
reaction buffer (25 mM Tris-HCl, pH 7.5, 10 mM
MgCl2, 2 mM dithiothreitol, 0.1 mM
orthovanadate for the MAP kinase assay or 20 mM HEPES, pH
7.6, 10 mM MgCl2, 2 mM
MnCl2, 2 mM dithiothreitol, 0.1 mM
orthovanadate for the PAPK assay). Samples were then resuspended in 30 µl of kinase reaction buffer containing 5 or 10 µCi of
[ Cell Death Assay--
In order to induce apoptosis in NIH3T3
cells stably expressing vectors, cells were plated at 1 × 105 cells per well in 12-well plates for 1 day and then
washed in DMEM. Cells were cultured with serum-free DMEM in
the presence or absence of mifepristone (1 × 10 Morphological Analyses--
1 × 105 cells
grown in 60-mm dishes were cultured for 1 day in serum containing
medium and then transferred to DMEM with 10% FCS in the
presence mifepristone (1 × 10 cDNA Cloning and Identification of PAPK, a Novel
Ste20/GCK-like Kinase--
In the course of studying
erythroleukemia (MEL) cell lines derived from mice infected with the
Friend spleen focus-forming virus, we observed that growing these
cells in serum-free medium induced a dramatic change in their
morphology. Within 3-5 days, the cells changed from erythroblastic to
giant polyploid cells (Fig.
1A). Although the altered
cells resemble megakaryocytes, they do not express typical
megakaryocyte markers such as acetylcholinesterase and CD41 (data not
shown). In order to isolate genes in MEL cells whose expression is
altered after induction of this morphological change, we performed a
subtractive cDNA screening using mRNAs from the MEL cell line
C19 (53) before and after induction. Among the isolated cDNA
clones, we found a cDNA fragment corresponding to a gene that is
up-regulated during induction of polyploidy. Northern blot analysis
using this cDNA revealed that the corresponding mRNA for this
gene is normally present in C19 cells but is dramatically up-regulated
5 days after induction of the cells to polyploidy (Fig.
1B).
3'- and 5'-RACE analyses showed that at least two mRNA variants
with different 3'-sequences were isolated, and their cDNAs were
sequenced (Fig. 2A). Sequence
analysis revealed that the larger of the two should encode a
polypeptide of 418 amino acids that contains a kinase domain (amino
acids 58-369) in the center of the coding region. The smaller message
appears to be encoded by a gene containing a stop codon within the
kinase-encoding domain and should encode for a 189-amino acid
polypeptide lacking 172 amino acids from the C terminus. We named these
proteins polyploidy-associated protein kinase (PAPK)-A and PAPK-B. The
calculated molecular mass of PAPK-A is 46.8 kDa, whereas that of PAPK-B
is 21.4 kDa.
Sequence analysis indicates that PAPK-A is a novel protein kinase
belonging to the Ste20/GCK family of serine/threonine kinases. When the
kinase domain of PAPK-A is compared with those of other protein kinases
in this family (Fig. 2B), it shows the highest homology to
that of NY-BR-96 (52% homology) and SPAK (30% homology). The
phylogenetic tree for the PAPK-A kinase domain is shown in Fig.
2C. Within the sequence of PAPK-A there are two consensus Src homology 3-binding sites (PXXP) (55) at amino acids 382 and 397, which may be involved in protein-protein interactions, and a
consensus AKT phosphorylation site (RXRXXS) (56)
at amino acid position 16 (residues 11-16). When the sequence for
PAPK-A was BLASTed against the assembled mouse genomic sequence in the NCBI data base, it was found on mouse chromosome 1.
Expression of PAPK--
The expression pattern of PAPK was
examined using a mouse multitissue Northern blot (Fig.
3). When PAPK-A cDNA was used as a
probe, a 2.4-kb transcript was shown to be expressed ubiquitously, with
higher expression levels in brain, heart, kidney, liver, and testis. A
similar pattern was observed using a probe corresponding to nucleotides
692-863 of PAPK-A, which are missing in PAPK-B.
To identify and characterize the PAPK-A protein, we produced
anti-PAPK-A antibody by immunizing rabbits with a peptide corresponding to the C terminus of the predicted protein. The specificity of the
anti-PAPK antibody was confirmed using 293T cells transfected with a
Myc-tagged PAPK-A cDNA expression plasmid. Both the anti-PAPK antibody and the anti-Myc antibody recognized the same protein (data
not shown). Because the antibody is directed against the C terminus of
PAPK-A, which is missing in PAPK-B, it recognizes only PAPK-A and
detects a 48-kDa protein in the C19 and DS19 (53, 54) MEL cell lines
(Fig. 4A). When C19 and DS19
cells are induced to polyploidy by growth in serum-free medium, the
level of PAPK-A protein is significantly increased (Fig.
4A). However, when serum-grown MEL cells are induced to
differentiate with the chemical hexamethylenebisacetamide, PAPK-A levels decrease (Fig. 4B). In addition to MEL cells,
a large amount of PAPK-A protein can also be detected in the mouse T-cell line EL4 grown in serum but not in other mouse cell lines examined, including the Epo-dependent erythroleukemia cell
line HCD-57, the IL-3-dependent myeloid cell lines FDCP2
and 32D, the IL-3-dependent proB cell line BaF3, and the
embryonic mouse fibroblast cell line NIH3T3 (Fig. 4A). Of
interest, however, is the observation that the levels of PAPK-A can be
increased by withdrawing Epo from HCD-57 cells (Fig. 4C) or
removing IL-3 from FDCP2, 32D, or BaF3 cells (data not shown), each of
which results in apoptosis of the cells. These data suggest that PAPK-A
is up-regulated during apoptosis. Furthermore, when NIH3T3 cells were
starved for 24 h in DMEM with 0.5% bovine serum albumin, PAPK-A
expression increased, and when cells were re-fed with DMEM plus 10%
FCS, PAPK-A levels gradually decreased over 24 h to the steady
state level (Fig. 4D). Collectively, these data suggest that
PAPK-A expression levels are regulated by growth factor deprivation and
that this novel kinase may play a role in the apoptotic response.
PAPK Functions as a Protein Kinase--
To determine whether PAPK
has protein kinase activity, we transfected 293T cells with Myc-tagged
PAPK-A, isolated the protein from the cell lysate by
immunoprecipitation with an anti-myc monoclonal antibody, and carried
out an immune complex kinase assay using myelin basic protein (MBP) as
an exogenous substrate. Cells transfected with empty vector or with
vector expressing Myc-tagged PAPK-B, which is missing the majority of
the kinase domain of PAPK-A and is not expected to have any kinase
activity, were used as controls. Although the immunoprecipitates from
cell lysates prepared from 293T cells transfected with vector alone or
with PAPK-B phosphorylated MBP to a limited extent, the kinase activity
was markedly increased in immunoprecipitates from cells transfected
with PAPK-A (Fig. 5). These data indicate
that PAPK-A, but not PAPK-B, is a functional protein kinase. To
eliminate the possibility that an associated kinase might be
co-precipitating with PAPK-A, we generated a kinase-inactive form of
PAPK-A, designated K89M-PAPK-A, by replacing lysine 89 in the
ATP-binding domain with a methionine. When tested in the same immune
complex kinase assay, the level of phosphorylation of MBP by the
kinase-inactive mutant was less when compared with that of wild-type
PAPK-A (Fig. 5). Thus, in addition to its own kinase activity, PAPK-A
may also function as a scaffold for other protein kinases as this
kinase-inactive mutant of PAPK-A still resulted in some MBP
phosphorylation.
Activation of PAPK-A by Cellular Stimuli--
To determine whether
PAPK-A is activated by any of the cellular stimuli that are known to
activate MAP kinases, we transfected HeLa cells with Myc-tagged PAPK-A
and carried out the PAPK-A kinase assay after exposing the cells to
various cellular stresses or cytokines (Fig.
6). PAPK-A kinase activity markedly
increased (greater than 5-fold) after treatment of the cells with
H2O2 and slightly (almost 2-fold) after
exposure to fetal calf serum. Treatment with
12-O-tetradecanoylphorbol-13-acetate, tumor necrosis
factor- PAPK Activates MAP Kinase Pathways--
To determine whether PAPK
is a component of any of the known MAP kinase pathways, we tested the
effect of overexpression of PAPK-A and PAPK-B on the activation of
various MAP kinases (Fig. 7). 293T cells
were transiently transfected with HA-tagged ERK2, JNK1/SAPK, p38 JNK Activation by PAPK--
MKK7 is one of the upstream activators
of JNK1. To determine whether PAPK-A activates JNK1 through MKK7, 293T
cells were transiently co-transfected with HA-tagged JNK1 with or
without Myc-tagged PAPK-A, FLAG-tagged MKK7, or a combination. After
transfection, HA-tagged JNK1 was immunoprecipitated from cell lysates,
and a kinase assay was carried out using the ATF2 fusion protein as a
substrate. Although MKK7 activates JNK1 in 293T cells, the kinase activity is enhanced when MKK7 is co-expressed with PAPK-A (Fig. 8A, left panel).
When 293T cells expressing HA-tagged JNK1 are co-transfected with a
kinase-inactive mutant of PAPK-A (K89M-PAPK-A), JNK1 is activated, and
the kinase activity is slightly enhanced when the mutant is
co-expressed with MKK7 (Fig. 8A, right panel). When 293T cells expressing HA-tagged JNK1 were transiently
co-transfected with Myc-tagged PAPK-A and a FLAG-tagged
dominant-negative MKK7 and starved of serum for 12 h,
PAPK-A-induced JNK1 activation was inhibited (Fig. 8B,
left panel). However, K89M-PAPK-A induced JNK1 activation
was not inhibited by the dominant-negative MKK7 (Fig. 8B,
right panel). These results suggest that although PAPK-A functions upstream of MKK7, different mechanisms for JNK1 activation by
PAPK-A might exist.
Overexpression of PAPK-A in NIH3T3 Cells Induces Morphological
Changes--
To determine whether overexpression of PAPK-A had any
effect on cell morphology or viability, we generated inducible NIH3T3 cell lines that expressed either wild-type PAPK-A or kinase-inactive K89M-PAPK-A under the control of a mifepristone-responsive element. In
this system, addition of mifepristone induced expression of the
recombinant proteins (Fig.
9A). Cells expressing an empty vector were used as a control.
Before mifepristone treatment, all of the cell lines looked
morphologically similar when they were examined by phase contrast microscopy (Fig. 9B). However, when mifepristone was added
to the cultures, only those expressing PAPK-A showed a large increase in the number of cells exhibiting a distinct stretching-like morphology associated with extensions in a stellate shape (Fig. 9B). To
quantitate the number of cells exhibiting this distinct morphology, we
counted 300 cells from each uninduced or induced cell line in
triplicate, and we determined the frequency of cells that exhibited
extensions that were longer than the body length. By using these
criteria, <10% of the cells in each culture exhibited this morphology
before mifepristone treatment (Fig. 9C), and when the
control line or the line expressing the kinase-inactive mutant of
PAPK-A (K89M) was treated with the drug, the frequency remained at
<10% 24 or 48 h later. However, 24 h after mifepristone
treatment of the line expressing PAPK-A, 20% of the cells exhibited
this distinct morphology, and the frequency rose to 29% by 48 h
(Fig. 9C). These data indicate that PAPK-A induces
morphological changes in NIH3T3 cells and that this function requires
its kinase activity.
Overexpression of PAPK-A in NIH3T3 Cells Blocks Cell
Death--
The fact that withdrawal of cytokines or FCS consistently
results in increased expression of PAPK-A suggested that PAPK might have a function that promotes or inhibits apoptosis. When PAPK-A is
overexpressed in NIH3T3 cells growing in DMEM with 10% FCS, the cells
did not undergo apoptosis, indicating that PAPK-A is not pro-apoptotic.
Because serum withdrawal induces apoptosis in NIH3T3 cells, we tested
whether expression of PAPK in these cells made them resistant or more
sensitive to apoptosis. Our results indicate that NIH3T3 cells
overexpressing PAPK-A are resistant to cell death induced by serum
withdrawal. Fig. 10A shows a
typical picture of uninduced and induced cells after growth in
serum-free medium for 30 h. All of the uninduced cells show
condensed nuclei typical of apoptotic cells. After induction with
mifepristone, NIH3T3 cells stably expressing empty vector or the
kinase-inactive K89M-PAPK-A mutant are apoptotic, whereas those
expressing PAPK-A appear healthy. To determine the kinetics for
apoptotic cell death, we stained nuclei with Hoechst 33342 at various
times after serum withdrawal (Fig. 10B). Although 26.7% ± 1.6 of NIH3T3 cells expressing empty vector (closed squares)
and 40.8% ± 3.2 of NIH3T3 cells expressing kinase-inactive PAPK-A
(closed triangles) show apoptosis 48 h after
serum withdrawal, only 10.9% ± 2.4 of those expressing PAPK-A show
apoptosis (closed diamonds). Interestingly, NIH3T3 cells
overexpressing the kinase-inactive K89M-PAPK-A mutant (closed triangles) are even more sensitive than uninduced or
vector-induced NIH3T3 cells for cell death at 24, 48, and 72 h
after serum withdrawal, suggesting that this kinase-inactive mutant of
PAPK-A may be acting as a dominant-negative mutant for endogenous
PAPK-A. Furthermore, to monitor a marker of cells undergoing apoptosis,
we examined cells for expression of cleaved PARP, one of the main
targets of caspase 3. As shown in Fig. 10C, whereas cleaved
PARP could be detected 24 and 48 h after serum withdrawal in
control cells or cells expressing the kinase-inactive mutant of PAPK-A,
cells expressing wild-type of PAPK-A did not show significant
expression of cleaved PARP. To determine whether PAPK-A-induced
protection against cell death involved JNK1 activation, we carried out
Western blot analysis using anti-phospho-JNK antibody. As shown in Fig. 10D, JNK phosphorylation was detected in cells expressing
wild-type PAPK-A after serum withdrawal, but we failed to detect JNK
phosphorylation in control cells (vector). Interestingly, even higher
levels of JNK phosphorylation were detected after induction of the
kinase-inactive mutant of PAPK-A, which does not protect the cells from
apoptosis induced by serum withdrawal. These data suggest that PAPK-A
kinase activity is not required for phosphorylation of JNK and that JNK activation by PAPK-A is not sufficient to protect cells from death under these conditions. Unfortunately, it is not technically possible to detect phosphorylation of endogenous p38 We have cloned and characterized a murine protein kinase, PAPK,
which when overexpressed promotes cell survival as well as cytoskeletal
changes. From our phylogenetic tree analysis of the kinase domain, we
determined that PAPK is a Ste20-like kinase that is most closely
related to the GCK subfamily of these serine/threonine kinases. Unlike
GCK kinase family members that have an N-terminal kinase domain and a
C-terminal regulatory domain (15, 16), PAPK has a kinase domain in the
center of the protein and lacks the long C-terminal regulatory domain.
Among known GCK kinase family members, PAPK is most highly related to
NY-BR-96 (GenBankTM accession number AF308302), with
52% identity within the kinase domain. While this work was in
progress, we also identified the human orthologue of PAPK-A, which is
similar to ALS2CR2 (GenBankTM accession number,
AB038950), an uncharacterized gene cloned in a search for genes
involved in juvenile amyotrophic lateral sclerosis on chromosome
2q33-q34 (57). We also isolated an inactive mutant of PAPK, termed
PAPK-B. Analysis of the genomic sequence of PAPK-B indicated that it is
an alternatively spliced isoform of PAPK. PAPK-B is missing a 172-bp
region of putative exon 9, causing a frameshift within the kinase
domain and premature termination. PAPK-B does not exhibit
kinase activity toward MBP using exogenous substrate.
In this study, we have focused on elucidating the function of
PAPK-A. Like other GCK family members, PAPK-A exhibits kinase activity
toward MBP. In addition, PAPK-A resembles other GCK family kinases in
that overexpression activates JNK. When a putative kinase-inactive form
of PAPK-A, K89M-PAPK-A, was generated by replacing lysine 89 in the
ATP-binding site with a methionine and tested in an in vitro
kinase assay, phosphorylation of MBP was reduced but not to background
levels. Interestingly, we observed that PAPK-B and the kinase-inactive
K89M-PAPK-A also activated JNK as determined by the in vitro
kinase assay and by Western blot analysis using anti-phospho-JNK
antibodies. These data suggest that PAPK-A, in addition to harboring
protein kinase activity, may also bind other signal tranducing
molecules, including kinases that can phosphorylate MBP and/or activate
JNK. Other GCK kinases have been shown to activate the JNK pathway in
the absence of kinase activity (18). For example, a mutant of TNIK that
lacks a kinase domain was shown to activate JNK by binding to TRAF2 (36, 58), which potently activates JNK (59). The nature of the
PAPK-associated proteins is still unknown and under current investigation.
MKK7 is an upstream activator of JNK (71). We found that PAP
kinase-mediated JNK activation was inhibited by dominant-negative MKK7
and enhanced by expression of MKK7. These observations suggest that
PAPK-A is upstream of MKK7. Unlike PAPK-A-induced activation of JNK,
activation of JNK by the kinase-inactive mutant of PAPK-A is not
inhibited by dominant-negative MKK7, suggesting that different mechanisms for JNK activation by PAP kinase exist involving the aforementioned putative PAPK-associated proteins. Because SEK1/MKK4 is
also an upstream activator of JNK1 (71), PAP kinase may also activate
JNK1 through SEK1/MKK4. Our preliminary data demonstrate that PAP
kinase-mediated JNK activation is enhanced by expression of SEK1/MKK4
(data not shown). However, we are unable to conclude that the SEK1/MKK4
pathway plays a role in PAPK-A-induced activation of JNK1 because
dominant-negative SEK1/MKK4 directly inhibits the basal activity of
JNK. Further detailed analysis will be required to determine whether
PAPK-A-induced activation of JNK involves SEK1/MKK4.
Although we did not detect ERK2, p38 We found that PAPK-A is activated to a greater extent by oxidative
stress induced by H2O2 than by other cellular
stimuli such as cytokines, growth factors, and other stressors,
suggesting that PAP kinase may represent a unique redox-sensitive
kinase. Oxidative stress has been linked to both cell death (apoptosis) and cell survival (64). Thus, PAPK may be involved in redox-sensitive signal transduction pathways for cell survival. Although PAPK is
ubiquitously expressed in adult tissues, PAPK-A expression levels
increase when cells undergo apoptosis.
We isolated PAP kinase from an MEL cell line that was derived from a
mouse infected with the Friend spleen focus-forming virus. The
erythroid progenitors that serve as targets for spleen focus-forming virus were recently shown to be bipotent (erythroid and megakaryocytic) (65), suggesting that MEL cells may also be bipotent cells. When MEL
cells are grown in serum-free medium, PAPK levels are greatly increased
and the cells become polyploid, resembling megakaryocytes. When the
cells are grown in serum and hexamethylenebisacetamide, which
induces their differentiation into red blood cells, PAPK levels
decrease. These observations suggest that PAPK may regulate the
erythroid/megakaryocyte commitment pathway. We are currently carrying
out studies to test this possibility.
Unfortunately, we were unable to make stable lines of either MEL cells
or fibroblasts constitutively expressing PAPK-A. Thus, in this study,
we used an inducible system for expression of PAPK in NIH3T3 cells.
Cells overexpressing PAPK-A exhibited a stretching morphology
associated with membrane extensions in a stellate shape, as described
previously for H-Ras- and RIT-transformed NIH3T3 cells (49). Our
preliminary data using immunofluorescence and confocal microscopy to
identify filamentous actin-containing structures suggest that actin
stress fibers have disappeared in cells overexpressing PAPK-A,
suggesting that PAPK-A may induce actin rearrangement. Such
morphological changes might be associated with cell motility. The
morphological changes induced by PAP kinase-A appear to require its
kinase activity. Ste20 family members such as PAKs (66) and GCK family
members such as SLK (67), PASK (68), PSK (46), and TNIK (36) have all
been implicated in the regulation of cytoskeletal reorganization. It
has been reported that the low molecular weight GTPase Rho regulates
the formation of actin stress fibers and the assembly of focal
adhesions and that the inhibition of Rho function by C3 toxin blocks
stress fiber formation (69, 70). It is possible that PAPK-A modulates
the activity of Rho family proteins. Further experiments are necessary
to determine this possibility.
Our studies demonstrate that PAPK-A expression in NIH3T3 cells confers
resistance to cell death induced by serum withdrawal, a function that
requires kinase activity. Activation of the JNK pathway has been shown
to play a role in pro-apoptotic pathways (71). However, it is unlikely
that activation of JNK by PAPK-A has an anti-apoptotic function, due to
the fact that the kinase-dead form of this protein is unable to protect
cells from death despite its ability to activate JNK. Activation of
p38 In this study, we found PAPK-A expression levels increased during
apoptosis in murine hematopoietic and fibroblast cells. Although we do
not know whether the kinase activity of PAPK-A is also increased in
these intact cells, the fact that PAPK-A has intrinsic kinase activity
even without stimulation suggests that increased protein levels likely
reflect enhanced activity. Interestingly, NIH3T3 cells overexpressing
the kinase-inactive mutant of PAPK-A are more sensitive to cell death
than uninduced or vector-induced NIH3T3 cells after serum withdrawal,
suggesting that endogenous PAPK-A plays a role in protecting cells
against death induced by serum withdrawal. Activation of a death signal transduction pathway may result in the stimulation of the PAPK-A promoter, leading to increased levels of PAPK-A expression and providing a negative feedback signal to prevent cell death.
. The kinase activity of PAPK appears to be required for the
activation of ERK6/p38
but not JNK1. When an inducible construct of
PAPK-A was expressed in stably transfected NIH3T3 cells, the cells
exhibited distinct cytoskeletal changes and became resistant to
apoptotic cell death induced by serum withdrawal, effects of PAPK that
require its kinase activity. These data suggest that PAPK is a new
member of the Ste20/germinal center kinase family that modulates
cytoskeletal organization and cell survival.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
pathway when
overexpressed in 293T cells. In addition, overexpression of PAPK in
NIH3T3 cells resulted in resistance to cell death and led to distinct
changes in cell morphology. Our results suggest that PAPK represents a
novel molecule regulating cytoskeleton structures and cell survival.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, pCEFL-HA-p38
,
pCEFL-HA-ERK6/p38
, and pCEFL-HA-ERK5 were described previously (49,
50). The expression plasmids for GST-SEK1 (pEBG-SEK1) and GST-SEK1-KR
(pEBG-SEK1-KR) encoding a dominant-negative SEK1 (51) were a generous
gift of John M. Kyriakis (Massachusetts General Hospital). The
expression plasmids MKK7 and MKK7-K76E encoding a dominant-negative
MKK7 (52) were a generous gift of Tse-Hua Tan (Baylor College of
Medicine). pFC-MEKK1, pFC-MEK1, and pFC-MKK3 were purchased from Stratagene.
8 M)
(Invitrogen). 293T and HeLa cells in 60-mm culture dishes (2 × 106) were transfected with various expression plasmids by
LipofectAMINE 2000 in accordance with the manufacturer's instructions.
-32P]ATP and 200 µM cold ATP. After 30 min at 30 °C, the reactions were terminated by addition of 10 µl
of 4× Laemmli buffer. In vitro kinase assays were performed
using as substrates myelin basic protein (MBP; Upstate Biotechnology,
Inc.) for ERK6/p38
, ERK5, and PAPK; GST-Elk1 (residues
307-428; Cell Signaling) for ERK2; or GST-ATF2 (residues
19-96; Cell Signaling) for JNK, p38
, and p38
. Samples were
separated by SDS-14% PAGE, transferred to nitrocellulose filters, and
subjected to autoradiography.
8
M). At the indicated times, cells were stained with Hoechst
33342 (Molecular Probes) and visualized by using a fluorescence
microscope. Abnormal nuclei were scored for apoptosis in a blind
fashion. To analyze a marker for cells undergoing apoptosis, we carried out Western blot analysis by using a mouse monoclonal antibody to
cleaved PARP (7C9; Cell Signaling).
8 M). At
the indicated times, the cultures were examined by phase contrast
microscopy, and the percentages of cells undergoing morphological change were counted. Three separate experiments were carried out, and
for each experiment, a total of 300 cells per culture were examined for
each time point. The percentage of cells exhibiting extensions that
were longer than the length of the cell body was determined.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Serum withdrawal from mouse
erythroleukemia cells induces polyploidy and up-regulation of a 2.4-kb
transcript. A, the Friend MEL cell line C19 was cultured in
DMEM containing 10% FCS (left panel) (steady state) or
grown for 5 days in serum-free medium (right panel), which
induces polyploidy. The cells were stained with HEMA 3 stain (Fisher)
and photographed at ×40 magnification. B,
poly(A+) RNA was prepared from C19 cells grown in
DMEM with 10% FCS (left lane) (steady state) or
for 5 days in serum-free medium (right lane) (induced to
polyploidy). Northern blotting was then carried out using a cDNA
probe (PAPK-A) from a gene that was shown by subtractive cDNA
screening to be differentially expressed under the two
conditions.
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Fig. 2.
Primary structure of PAPK and homologies with
known MAP kinases. A, nucleotide and deduced protein
sequence of PAPK-A cDNA and its spliced variant PAPK-B. The
GenBankTM accession numbers for PAPK-A and PAPK-B are
AB057666 and AB057667. B, sequence alignment of the kinase
domain of PAPK-A with other GCK family kinases. C,
phylogenetic tree of GCK family kinases. Generation of the phylogenetic
tree was carried out using the Phylip program.
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Fig. 3.
Tissue distribution pattern of PAPK.
Poly(A+) RNA of various mouse tissues (Origene) was probed
with PAPK-A cDNA labeled by random priming.
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Fig. 4.
Expression of PAPK-A protein.
A, various cell lines were examined for PAPK-A protein
expression by Western blotting using anti-PAPK antiserum. The MEL cell
lines C19 and DS19 were examined after growth in DMEM plus
10% FCS (steady state) as well as after being cultured for 5 days in
serum-free medium (induced to polyploidy). The other cell lines
examined were all steady state cultures. Western blotting with
anti- -tubulin was used as a loading control. B, C19 cells
were induced to differentiate by exposure to 3 mM
hexamethylenebisacetamide, and at various times after induction
were examined for expression of PAPK-A by Western blotting. Fold change
in expression from steady state levels (day 0) is indicated
below each lane. Antiserum to Erk2 was used as a loading
control. C, the Epo-dependent HCD-57 cell line
was induced to apoptosis by withdrawal of Epo; and at various times
after Epo withdrawal, lysates were examined for expression of PAPK-A by
Western blotting. D, NIH3T3 cells were starved for 24 h
in DMEM with 0.5% bovine serum albumin and then cultured in DMEM with
10% FCS. Lysates were then examined for expression of PAPK-A by
Western blotting.
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Fig. 5.
Catalytic activity of PAPK. 293T cells
(2 × 106/60-mm dish) were transfected with 3 µg of
an empty vector (pCMV), myc-PAPK-A, myc-K89M-PAPK-A, or myc-PAPK-B. The
cells were collected 32 h after transfection. Immunocomplex kinase
assays were then performed with anti-Myc antibody using MBP as an
exogenous substrate (upper panel). [32P]ATP
incorporated into the phosphorylated substrate was quantified, and fold
change compared with cells transfected with vector alone (background)
is indicated below each lane. Independent experiments were
performed three times with similar results. The bottom
panels show immunoblots with anti-Myc antibody.
, anisomycin, NaCl, or IL-1 (Fig. 6), as well as with
tunicamycin, thapsigargin, or sorbitol (data not shown), did not
increase PAPK-A activity. The level of overexpressed PAPK-A was
verified by immunoblotting with anti-myc antibody (bottom
panel). Similar data were obtained using 293T cells (data not
shown). Thus, PAPK-A may be stimulated by a limited number of
extracellular stimuli.
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Fig. 6.
PAPK-A activation in HeLa cells after various
stimuli. HeLa cells were transfected with 3 µg of Myc-tagged
PAPK-A plasmid. After incubation for 24 h, cells were starved for
5 h and then stimulated in DMEM with either 300 ng/ml
12-O-tetradecanoylphorbol-13-acetate (TPA) for 30 min, 1 mM H2O2 for 10 min, 100 ng/ml tumor necrosis factor- (TNF) for 30 min, 10 µg/ml
anisomycin for 30 min, 0.7 M NaCl for 30 min, 30% FCS for
30 min, or 20 ng/ml IL-1 for 30 min. Following stimulation, Myc-tagged
PAPK-A was immunoprecipitated (IP) from cell lysates using
the monoclonal antibody 9E10, and the kinase activity was measured
using MBP as the substrate. Independent experiments were performed
three times with similar results.
,
p38
, ERK5, or ERK6/p38
with or without mammalian expression
vectors encoding Myc-tagged PAPK-A or PAPK-B. The HA-tagged kinases
were then immunoprecipitated from the cell lysates and analyzed for
kinase activity using the appropriate substrate. As shown in Fig. 7,
co-expression of PAPK-A with ERK2 (A), p38
(C), p38
(D), or ERK5 (F) failed to
activate their kinase activity even though each kinase could be
activated by previously described upstream activating kinases. In
contrast, both JNK1 (B) and ERK6/p38
(E) were
markedly activated (2.45- and 2.57-fold, respectively) by
overexpression of PAPK-A. Interestingly, JNK1, but not ERK6/p38
, was
also slightly activated (1.5-fold) by overexpression of PAPK-B. By
using anti-phospho-JNK antibodies, we were also able to detect phosphorylated JNK1 in cells overexpressing PAPK-B (data not shown). This suggests that although the kinase activity of PAPK is required for
maximal activation of JNK1, PAPK might also have a function as a
scaffold or adaptor protein.
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Fig. 7.
Effect of PAPK on the MAP kinase
pathway. 293T cells (2 × 10 6/60-mm dish)
were transfected with 2 µg of the following HA-tagged MAP kinase
plasmids: ERK2 (A), JNK1 (B), p38
(C), p38
(D), ERK6 (E), or ERK5
(F) with or without 2 µg of Myc-tagged PAPK-A or PAPK-B.
MEK1 was used as a positive control for activation of ERK2; MEKK served
as a positive control for JNK1 activation, and MKK3 was used as a
positive control for activation of p38
, p38
, and ERK6. Cells
stimulated with 1 mM H2O2 for 10 min served as a positive control for activation of ERK5. Empty vector
(2 µg) was used to normalize the amount of transfected DNA. After
incubation for 32 h, the lysates were immunoprecipitated with
anti-HA antibody, and then in vitro kinase
assays were carried out using the appropriate substrates as indicated.
[32P]ATP incorporated into the phosphorylated substrates
was quantified, and the values were expressed as fold change over the
MAP kinase alone. Results shown are representative of at least three
independent experiments. IB, immunoblot.
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Fig. 8.
Activation of JNK by PAPK-A. A,
293T cells were transfected with 2 µg of HA-tagged JNK1 plus either 4 µg of empty vector, 2 µg of Myc-tagged PAPK-A (left
panel), or 2 µg of Myc-tagged K89M-PAPK-A (right
panel) with or without 2 µg of FLAG-tagged MKK7. Empty vector
was used to normalize the amount of transfected DNA. After incubation
for 32 h, the lysates were immunoprecipitated with anti-HA
antibody, and then an in vitro kinase assay was carried out
using GST-ATF2 as the substrate. Three independent experiments were
performed. [32P]ATP incorporated into the phosphorylated
substrate was quantified, and fold change compared with cells
transfected with JNK1 alone is indicated. B, 293T cells were
transfected with 1 µg of HA-tagged JNK1 and either 2 µg of
Myc-tagged PAPK-A (left panel) or Myc-tagged K89M-PAPK-A
(right panel) with or without 2 µg of a FLAG-tagged
dominant-negative MKK7. 293 cells transfected with HA-tagged JNK1 and
FLAG-tagged dominant-negative MKK7 was included as a control in both
panels. Empty vector was used to normalize the amount of transfected
DNA. After incubation for 24 h, cells were starved in DMEM without
FCS for 12 h. The lysates were immunoprecipitated with anti-HA
antibody, and then an in vitro kinase assay was carried out
using GST-ATF2 as the substrate. Three independent experiments were
performed. [32P]ATP incorporated into the phosphorylated
substrate was quantified, and fold change compared with cells
transfected with JNK1 alone is indicated. IB,
immunoblot.
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Fig. 9.
Overexpression of PAPK-A in NIH3T3 cells
induces morphological changes. A, NIH3T3 cells were
stably transfected with mifepristone-dependent and
Myc-tagged constructs of PAPK-A and the kinase-inactive mutant
K89M-PAPK-A. At various times after addition of mifepristone (1 × 10 8 M), the cells were examined for
expression of PAPK-A by Western blotting with an anti-Myc antibody.
B, phase contrast microscopy of PAPK-A and PAPK-A (K89M)
stably transfected NIH3T3 cells cultured in 10% FCS before and after
induction of PAPK-A by treatment with mifepristone for 48 h. Phase
contrast microscopy of representative cells is shown. C,
NIH3T3 cells (1 × 105/60-mm dish) stably expressing
the inducible PAPK-A, K89M PAPK-A, or empty vector were cultured in
DMEM with 10% FCS. The next day they were incubated with
or without mifepristone (1 × l0
8 M).
After 24 and 48 h, 300 cells each in three separate cultures for
each cell line were examined using a phase contrast microscope. Data
show the average % of cells exhibiting extensions (see "Experimental
Procedures") ± S.E. for three cultures.
in NIH3T3 cells, so we
were unable to determine whether PAPK-A activation of this kinase plays
a role in protecting the cells from apoptosis induced by serum
withdrawal.
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Fig. 10.
Overexpression of PAPK-A in NIH3T3 cells
confers protection against stress-induced cell death. A,
NIH3T3 cells were examined by phase contrast microscopy for dead cells
in culture without FCS before and after induction of vector, wild-type
of PAPK-A, and kinase-inactive mutant of PAPK-A (K89M-PAPK-A) at 30 h.
B, NIH3T3 cells (1 × 105) stably
expressing inducible PAPK-A (diamonds), K89M-PAPK-A
(circles), or empty vector (squares) were
cultured in 12-well plates in DMEM plus 10% FCS. The next day, the
cells were washed and transferred to DMEM without FCS in
the presence (closed symbols) or absence (open
symbols) of mifepristone (1 × 10 8
M) for the indicated times. Apoptotic nuclei were
determined by staining with Hoechst 33342 by using a fluorescence
microscope. About 500 cells were counted at the indicated times under
the fluorescence microscope. Data shown represent the averages for
three cultures. S.E. was less than 3.2. C, cells were
induced to apoptosis by FCS withdrawal. As a marker for apoptosis,
the expression of cleaved PARP was determined by Western blot analysis
using mouse monoclonal anti-cleaved PARP(7C9) antibody at the indicated
times after treatment with mifepristone in serum-free media.
D, cells were induced to apoptosis by FCS withdrawal. The
phosphorylation of JNK was determined by Western blot analysis at the
indicated times after treatment with mifepristone in serum-free medium.
Asterisk represents a nonspecific band.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, p38
, or ERK5 activation by
PAP kinase, PAPK-A, but not PAPK-B, preferentially activated ERK6/p38
. This suggests that unlike JNK activation by PAP kinase, activation of ERK6/p38
requires PAPK-A kinase activity. It is unknown whether other GCKs activate ERK6/p38
. It was shown
previously (49, 60) that ERK6/p38
is activated by MKK4, MKK3, and
MKK6. ERK6/p38
is preferentially expressed in heart, skeletal
muscle, lung, thymus, and testes (61-63). Because PAPK-A expression is elevated in some of these tissues, it will be interesting to explore the possibility that PAPK-A, via the ERK6/p38
pathway, plays a role
in the physiology of these tissues.
may also be required for the anti-apoptotic function of PAPK-A.
Alternatively, the quality or quantity of JNK activation by PAPK-A may
contribute to cell survival. Of interest, among Ste20 kinase family
members, only PAKs have so far been shown to have an anti-apoptotic
function. For example, PAKs have been shown to induce BAD
phosphorylation, resulting in a reduction in the interaction between
BAD and Bcl-2 or Bcl-XL and an increase in the association
of BAD with 14-3-3 or an inhibition of caspase activation (10, 11,
72-74). It is, therefore, possible that PAPK-A modulates Bcl-2 family proteins.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. John M. Kyriakis for providing the expression constructs of SEK1 and dominant-negative SEK1, Dr. Tse-Hua Tan for providing the expression constructs of MKK7 and NKK7-K76E, and Dr. Atsushi Oue for helpful discussions.
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FOOTNOTES |
---|
* 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 may be addressed: Basic Research Laboratory, Bldg. 469, Rm. 205, NCI-Frederick, Frederick, MD 21702-1201. Tel.: 301-846-5740; Fax: 301-846-6164; E-mail: nishigakik@mail.ncifcrf.gov.
To whom correspondence may be addressed: Basic Research
Laboratory, Bldg. 469, Rm. 205, NCI-Frederick, Frederick, MD
21702-1201. Tel.: 301-846-5740; Fax: 301-846-6164; E-mail:
ruscetti@ncifcrf.gov.
Published, JBC Papers in Press, February 6, 2003, DOI 10.1074/jbc.M208601200
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ABBREVIATIONS |
---|
The abbreviations used are: PAKs, p21-activated kinases; PAPK, polyploidy-associated protein kinase; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; MAP, mitogen-activated protein; IL, interleukin; HA, hemagglutinin; JNK, c-Jun N-terminal kinase; SAPK, stress-activated protein kinase; MEL, mouse erythroleukemia; GCK, germinal center kinase; RACE, rapid amplification of cDNA ends; NIK, NCK-interacting kinase; TNIK, Traf2 and NCK interacting kinase; Epo, erythropoietin; GST, glutathione S-transferase; MBP, myelin basic protein.
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