From the
Fibroblast growth factor (FGF)-1 mitogenic signal transduction
is mediated in part by gene products that are specifically expressed in
response to cell surface receptor binding and activation. We have used
a targeted differential display method to identify FGF-1-inducible
genes in murine NIH 3T3 fibroblasts. Here we report that one of these
genes is predicted to encode a novel serine/threonine-specific protein
kinase. This putative kinase has been named Fnk, for FGF-inducible
kinase. The deduced Fnk amino acid sequence has 49, 36, 33, 32, and 22%
overall identity to mouse serum-inducible kinase (Snk), mouse polo-like
kinase (Plk), Drosophila polo, Saccharomyces Cdc5,
and mouse Snk/Plk-akin kinase (Sak), respectively. These proteins are
all members of the polo subfamily of structurally related
serine/threonine kinases. The Plk, polo, Cdc5, and Sak kinases are
required for cell division. FGF-1 induction of Fnk mRNA expression is
first detected at 30 min after mitogen addition, reflects
transcriptional activation, and does not require de novo protein synthesis. FGF-2, platelet-derived growth factor-BB, calf
serum, or phorbol myristate acetate treatment of quiescent cells also
induces fnk gene expression. Fnk mRNA is expressed in vivo in a tissue-specific manner, with relatively high levels detected
in newborn and adult mouse skin. These results indicate that Fnk may be
a transiently ex-pressed protein kinase involved in the early signaling
events required for growth factor-stimulated cell cycle progression.
Fibroblast growth factor (FGF)
We are presently identifying and characterizing
novel FGF-1-inducible genes in NIH 3T3 cells in order to gain further
insight into the FGF-1 intracellular signal transduction pathway. The
NIH 3T3 fibroblast cell line is particularly well suited for studies on
FGF-1 signaling since these cells (i) are relatively easy to maintain
and amplify, (ii) will enter a quiescent growth state when placed in
medium containing a low serum concentration, (iii) express
The effect of whole calf serum, individual serum growth factors, or
phorbol ester treatment on Fnk mRNA levels was then studied.
Serum-starved NIH 3T3 cells were either left untreated or treated for
various lengths of time with calf serum, PMA, or various purified
growth factors. Cells were collected, RNA was isolated, and Fnk mRNA
levels were analyzed by RNA gel blot hybridization. Both serum and the
phorbol ester PMA, a potent activator of protein kinase C
(18) ,
increased Fnk mRNA levels with kinetics similar to those observed after
FGF-1 treatment (Fig. 3, A and B). PDGF-BB
treatment also increased Fnk mRNA levels to the same extent as FGF-1;
in contrast, TGF-
One of the cellular responses that occurs following mitogenic
stimulation of quiescent cells is the transcriptional activation of the
immediate early and delayed early gene families
(30, 31) . Some of these genes are known to encode
proteins required for cell cycle progression, DNA replication, and/or
cytokinesis. Genes regulated by various growth promoting agents (for
example, serum
(32, 33, 34, 35, 36, 37, 38) ,
PDGF
(39) , EGF
(40, 41) , or IGF-1
(42) ), have been successfully identified by subtracted cDNA
probe hybridization or differential screening of cDNA libraries. Our
approach to identify FGF-1-inducible genes, termed targeted
differential display, is based on the reverse transcription-PCR
technique. Recently, Stone and Wharton
(43) described a similar
but more technically complex strategy, which they termed targeted RNA
fingerprinting, to identify cDNAs representing cell cycle-regulated
genes that encode proteins with zinc finger motifs.
The original Fnk
cDNA was amplified using a pair of degenerate oligonucleotide primers
designed to recognize cDNAs encoding proteins containing both a protein
kinase domain and a zinc finger domain. The sense protein kinase
primer, originally described by Wilks
(14) , encodes the peptide
IHRDL, which is a conserved motif located in subdomain VIb of both
serine/threonine-specific and tyrosine-specific protein kinase
catalytic domains
(28, 29) . As expected, this primer
annealed to a region of the Fnk cDNA sequence that encoded a similar
peptide, LHRDL. Interestingly, this LHRDL motif is present twice in the
Fnk deduced sequence, within subdomains III and VIb of the putative
protein kinase catalytic domain. The original PCR-derived Fnk cDNA
clone was amplified as a result of the kinase domain primer annealing
to the DNA sequence encoding the amino-terminal LHRDL motif. The
antisense zinc finger primer was designed by aligning the H/C-link
regions of several mouse C
The fnk gene has
properties similar to those described for many of the previously
identified mitogen-inducible immediate early genes. First, FGF-1
stimulation of quiescent cells rapidly increases Fnk mRNA levels, with
peak expression detected at 1 h. It is likely that this response is
due, at least in part, to transcriptional activation of the fnk gene, since Fnk mRNA accumulation does not occur in the presence
of actinomycin D. Second, Fnk mRNA levels are only transiently elevated
for a period of
The deduced Fnk amino acid sequence is
most closely related to members of the polo subfamily of
serine/threonine kinases. The Drosophila polo cDNA was
isolated by screening a cDNA library with a genomic fragment cloned
from a mutant polo allele tagged with the P-element transposon
(25) . Mutations in polo cause abnormal mitotic and meiotic
divisions
(25) . Polo transcripts are abundant in Drosophila embryos and in larval or adult tissues characterized by extensive
mitotic activity. Fenton and Glover
(48) have demonstrated that
polo immunoprecipitated from Drosophila embryo lysates has
kinase activity in vitro. Four protein kinases containing
significant amino acid sequence identity to the predicted polo protein,
primarily in the catalytic domain, have been reported: Snk
(21) , Plk
(22, 23, 24, 49, 50) , Cdc5
(26) , and Sak
(27) . Analysis of the deduced Fnk
sequence indicates that Fnk is the sixth member of this rapidly
expanding subfamily. The highest degree of amino acid sequence identity
is between Fnk and Snk (49%), Plk (36%), and polo (33%). The Snk cDNA
was isolated from an NIH 3T3 F-2 cDNA library as a sequence
artifactually ligated to another cDNA under investigation
(21) .
Snk mRNA expression is rapidly and transiently induced in NIH 3T3 cells
treated with serum or phorbol ester in the presence of cycloheximide;
therefore, snk is also an immediate early gene. Snk
transcripts are expressed at detectable levels in mouse brain, lung,
and heart. In regard to Plk, both human
(23, 24, 49, 50) and murine
(22, 23, 24) Plk cDNA clones have been isolated
by reverse transcription-PCR using degenerate oligonucleotide primers
corresponding to conserved regions in the catalytic domain of protein
kinases. Plk mRNA levels are also increased following serum stimulation
of quiescent NIH 3T3
(24) or A-431
(49) cells; however,
in comparison to the kinetics of serum-induced Fnk or Snk mRNA
accumulation, Plk mRNA expression peaks much later in the cell cycle,
during S phase
(24) . Furthermore, in contrast to the findings
reported for Snk
(21) and shown here for Fnk (Fig. 2),
the increase in Plk mRNA levels is not due to transcriptional
activation
(24) . The plk gene is expressed in numerous
fetal and newborn mouse tissues, but in adults it is only expressed in
hemopoietic tissues, thymus, placenta, ovaries, and testes
(22, 23, 24, 49, 50) . Plk mRNA
expression has also been detected in tumor cell lines and tumor tissue
(49) . Plk may play a role in cell growth control. Hamanaka
et al. (23) reported that microinjection of sense Plk
mRNA into quiescent NIH 3T3 cells stimulated DNA synthesis, whereas
microinjection of antisense Plk RNA into growing cells inhibited DNA
synthesis.
fnk, snk, and sgk (51, 52) are the only immediate early genes identified to date that
are predicted to encode putative serine/threonine protein kinases. All
three mRNAs, and presumably the respective proteins as well, are
rapidly and transiently expressed in mitogen-treated cells. It is
possible that the enzymatic activity of these kinases is solely
regulated at the transcriptional level, with the level of activity
directly proportional to intracellular concentration. Alternatively,
one or more of these putative kinases may require post-translational
modifications ( e.g. phosphorylation) for maximal catalytic
function. In either case, it is likely that these three proteins
participate in the intracellular signaling cascades that promote growth
factor-stimulated cell cycle progression. Additional studies are under
way to determine whether Fnk is in fact a serine/threonine-specific
kinase required for FGF-1 mitogenic signal transduction.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank/EMBL Data Bank with accession number(s) U21392 and U22434.
We thank Dr. L. Lau for the cDNA library, T. Lanahan
for the mouse genomic library, Dr. W. Burgess for the FGF-1, Dr. R.
Friesel for the protein kinase domain oligonucleotide primer, and Dr.
C. Bieberich for the mouse tissue samples. We are also grateful to S.
Appleby and C. Liu for performing the automated DNA sequence analysis
and B. Hampton for help with data base searches and sequence
alignments. We also thank Dr. R. Friesel and Dr. D. Hsu for critical
review of the manuscript and K. Wawzinski for excellent secretarial
assistance.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
-1 and
FGF-2, two members of the FGF family of structurally related proteins,
are multifunctional regulators of cell proliferation, migration, and
differentiation
(1) . Both proteins are potent vascular cell
mitogens and angiogenic factors; thus, they may play an important role
in the pathogenesis of various diseases including atherosclerosis,
diabetic retinopathy, rheumatoid arthritis, and cancer. The biological
effects of FGF-1 and FGF-2 are mediated via binding to a family of
receptor tyrosine kinases
(2) and to heparan sulfate
proteoglycans
(3) present on the surface of most cell types.
Although it is known that ligand binding stimulates FGF receptor
tyrosine autophosphorylation and dimerization
(4) , the
subsequent biochemical pathway responsible for FGF mitogenic signal
transduction has not been elucidated. However, several studies have
demonstrated the FGF-1-dependent phosphorylation of numerous
cytoplasmic proteins, including phospholipase C-
(5) , Shc
(6) , Raf-1
(7) , extracellular signal-regulated kinases
1 and 2
(6) , focal adhesion kinase
(8) , and cortactin
(9) . Furthermore, FGF-1 stimulation of quiescent NIH 3T3 cells
promotes the transcriptional activation of various genes, including the
protooncogenes c- fos, c- jun, and c- myc (10) .
10
high affinity FGF receptors per cell
(11) ,
and (iv) display a strong mitogenic response to FGF-1 stimulation
(10) . We have identified numerous FGF-1-regulated genes using a
PCR-based differential display technique. Four of these genes
(phosphofructokinase, fatty acid synthase, Ca
-ATPase,
and FR-1) have been described to date
(12, 13) . Here we report that FGF-1 stimulation of NIH
3T3 cells induces the expression of an immediate early gene encoding a
novel putative serine/threonine kinase, which we have named Fnk, for
FGF-inducible kinase. The deduced Fnk sequence is most closely related
to members of the polo subfamily of serine/threonine protein kinases.
fnk represents the third immediate early gene described to
date that is predicted to encode a protein kinase likely to function
primarily during the early G
stage of the mammalian cell
cycle.
Cell Culture
Murine NIH 3T3 cells (American Type
Culture Collection) were grown at 37 °C in Dulbecco's
modified Eagle's medium (Mediatech) supplemented with 10% (v/v)
heat-inactivated bovine calf serum (Hyclone Laboratories) and a 1:100
dilution of a penicillin-streptomycin-fungizone solution (JRH
Biosciences). The cells were expanded by trypsin-EDTA (JRH Biosciences)
treatment and subculturing at a split ratio of 1:7 every 2-3
days. Subconfluent cells were incubated for 72 h in the above
medium containing a reduced serum concentration (0.5%) to induce a
relatively quiescent cell population. Cells were then either left
untreated or treated for various times with either 10% calf serum or
0.5% calf serum supplemented with 10 ng/ml recombinant human FGF-1
(gift of W. Burgess, American Red Cross) and 5 units/ml heparin
(Upjohn), 10% calf serum, 10 ng/ml PDGF-BB (Genzyme Corp.), 20 ng/ml
EGF (Genzyme Corp.), 2 ng/ml TGF-
1 (R & D Systems), 20 ng/ml
IGF-1 (Bachem), or 30 ng/ml PMA (Sigma). In some experiments, cells
were treated with 10 µg/ml cycloheximide (Sigma) or 2 µg/ml
actinomycin D (Calbiochem).
RNA Isolation
Cells were harvested by trypsin-EDTA
treatment, and total RNA was isolated using RNazol B (Tel-Test) or RNA
Stat-60 (Tel-Test) according to the manufacturer's instructions.
Tissues from newborn (1-5 days old) or adult FVB/N mice (Taconic
Farms) were homogenized in RNA Stat-60 (3 ml of reagent/500 µg of
tissue) using a Tissumizer (Tekmar). RNA concentrations were calculated
from the absorbance at 260 nM.
Targeted Differential Display
Total RNA (1 µg)
isolated from serum-starved or FGF-1-stimulated cells was converted to
cDNA using random hexamer primers (Boehringer Mannheim) as described
(13) . The PCR conditions were identical to those we described
previously
(13) . The degenerate sense protein kinase domain
primer was described previously by Wilks
(14) and is
5`-CGGATCCAC MG NGA YYT-3`, where M denotes A or C, N denotes all four bases, and Y denotes C or T. The degenerate antisense zinc finger domain primer
has been described
(12) . An aliquot of each amplification
mixture was subjected to electrophoresis in a 1.8% agarose gel, and DNA
was visualized by ethidium bromide staining.
/ HaeIII restriction fragments (Clontech
Laboratories) were used as size standards. The appropriate DNA fragment
was excised, recovered using the freeze-squeeze method
(15) ,
reamplified, and ligated into the cloning vector pCR1000 (Invitrogen
Corp.).
cDNA and Genomic Library Screening
A mouse Balb/c
3T3 cell gt10 cDNA library (gift of L. Lau, University of Illinois
College of Medicine) was screened with the subcloned PCR-derived DNA
fragment to obtain a larger cDNA clone. Briefly, the DNA fragment was
labeled with [
P]dCTP (3000 Ci/mmol, DuPont NEN)
using a random primer labeling kit (Boehringer Mannheim). Approximately
1.8
10
phage were plated at a density of 2
10
plaque-forming units/150-mm dish using Escherichia
coli C600 Hfl as host. Duplicate plaque lifts (Colony/Plaque
Screen, DuPont) were hybridized and washed as described
(13) .
Five positive phage were purified by two additional rounds of
screening. Purified
clones were amplified on E. coli C600 Hfl and
DNA isolated by polyethylene glycol/NaCl
precipitation. The cDNA inserts were released from the
gt10 vector
by EcoRI digestion and subcloned into the plasmid
pGEM3Zf+ (Promega Corp.). A mouse BALB/c liver
Fix II
genomic library (gift of T. Lanahan, Johns Hopkins University) was
screened with a 461-bp fragment derived from the 5` end of the
2.2-kb cDNA to obtain additional 5` sequence not present in the
cDNA clones. Approximately 5
10
phage were plated
at a density of 2.5
10
plaque-forming units/150-mm
dish using E. coli C600 Hfl as host. Filter hybridization and
washing was performed as described above, and two positive phage were
plaque-purified. DNA was isolated, and a
1.2-kb fragment released
by BglII digestion of one genomic clone was subcloned into the
BamHI site of pGEM3Zf+.
DNA Sequence Analysis
Plasmid DNA was purified
using a Magic Miniprep Kit (Promega Corp.), and both strands of the
entire 2.2-kb cDNA insert and the 5` 329 bp of the BglII
genomic DNA restriction fragment were sequenced by the
dideoxynucleotide chain termination method. Sequencing was either done
automatically using an Applied Biosystems model 373A DNA sequencer or
manually using a Sequenase 2.0 kit (U. S. Biochemical Corp.). The
nucleic acid and deduced protein sequences were compared with sequences
in the data base server at the National Center for Biotechnology using
the tBlastn and B10-357 programs. Protein sequences were aligned using
a Genetics Computer Group software package.
RNA Gel Blot Hybridization
Ten µg of each RNA
sample was denatured and subjected to electrophoresis in 1.2% agarose
gels containing 2.2 M formaldehyde. The gels were routinely
stained with ethidium bromide to verify that each lane contained
similar amounts of undegraded rRNA. RNA was electroblotted onto
Zetabind nylon membranes (Cuno, Inc.) and cross-linked by UV
irradiation using a Stratalinker (Stratagene). Radiolabeling of the
cDNA insert as well as membrane hybridization and washing were
performed as described above for library screening. In some
experiments, we verified that similar amounts of RNA were applied to
each lane by rehybridizing the membranes with a human
glyceraldehyde-3-phosphate dehydrogenase cDNA insert. This insert was
an 800-bp PstI/ XbaI fragment of pHcGAP (American Type
Culture Collection).
Identification of an FGF-1-inducible Gene by Targeted
Differential Display
RNA isolated from serum-starved or
FGF-1-stimulated NIH 3T3 cells was converted to cDNA using reverse
transcriptase and random primers. PCR was then performed using a
degenerate sense protein kinase domain primer and a degenerate
antisense zinc finger domain primer. Amplification products were
displayed using agarose gel electrophoresis and ethidium bromide
staining. The pattern of amplified cDNAs obtained from quiescent and
FGF-1-stimulated cellular RNA were, for the most part, similar
(Fig. 1 A). However, two DNA fragments, 200 bp and
800 bp in size, were amplified to a greater degree when cDNA
representing the RNA isolated from cells treated with FGF-1 was used as
template. Characterization of the larger DNA fragment will be the
subject of another report.
(
)
The
200-bp
fragment was excised from the gel, reamplified, and subcloned. Initial
RNA gel blot hybridization experiments indicated that this DNA fragment
hybridized to an FGF-1- and serum-inducible
2.4-kb transcript.
Therefore, to isolate larger cDNA clones, the DNA fragment was
radiolabeled and used to screen a
gt10 cDNA library prepared by
Lau and Nathans
(16) using RNA isolated from serum-treated
Balb/c 3T3 cells. Five positive phage were isolated, and their cDNA
inserts were subcloned. All inserts were
2.2 kb in size; one of
these inserts was sequenced in its entirety and also used as a probe
for RNA gel blot hybridization experiments. Based on these results,
described in detail below, the gene and the corresponding protein
represented by the cloned cDNA have been named fnk and Fnk,
for FGF-inducible kinase.
Figure 1:
Identification of an FGF-1-inducible
mRNA in NIH 3T3 fibroblasts by targeted differential display.
A, serum-starved cells were either left untreated or treated
with FGF-1 for 2 or 12 h. RNA was isolated, cDNA was synthesized, and
the PCR was performed using protein kinase and zinc finger
oligonucleotide primers. Amplification products were separated by
agarose gel electrophoresis and visualized by ethidium bromide
staining. The DNA size markers ( lane M; in bp) are
/ HaeIII restriction fragments. The arrow denotes the DNA fragment that was recovered and cloned.
B, serum-starved cells were either left untreated or treated
with FGF-1 for the indicated time periods. RNA was isolated, and
equivalent amounts of each sample were analyzed by RNA gel blot
hybridization. The upper and lower bars on
the left represent the positions of 28 S and 18 S rRNA,
respectively. The bottom panel is a photograph of the
28 S rRNA band.
Regulation of Fnk mRNA Expression in NIH 3T3
Cells
RNA gel blot hybridization analysis using RNA isolated
from serum-starved or FGF-1-treated NIH 3T3 cells was then performed to
confirm the differential display results indicating that fnk was an FGF-1-inducible gene. A single Fnk transcript of 2.4
kb was rapidly and transiently expressed following FGF-1 stimulation;
maximum levels were detected at 1 h, and expression returned to basal
levels by 8 h (Fig. 1 B). The structurally and
functionally related mitogen FGF-2 increased Fnk mRNA levels to the
same degree with identical kinetics (data not shown). It should be
noted that in this time course experiment, as well as in others
( e.g. Fig. 3
, A and B), a slight
decrease in the apparent size of Fnk mRNA is detected between 0.5 and 1
h after cellular stimulation. The mechanism responsible for this
decrease is unknown. However, Fnk mRNA is only transiently expressed,
and its 3`-untranslated region contains AU-rich motifs (see below);
consequently, it may have a relatively short half-life
(17) . It
is possible that the decrease in Fnk mRNA size reflects deadenylation
prior to transcript degradation
(17) .
Figure 3:
Effect of calf serum, PMA, or individual
growth factors on Fnk mRNA levels. Serum-starved cells were either left
untreated or treated with 10% calf serum ( CS) ( panel A), PMA ( panel B), FGF-1, PDGF-BB,
TGF-1, EGF, or IGF-1 ( panel C) for the indicated
time periods. RNA was isolated, and equivalent amounts of each sample
were analyzed by RNA gel blot hybridization.
The effect of the RNA
synthesis inhibitor actinomycin D on FGF-1 induction of Fnk mRNA levels
was then examined. Serum-starved NIH 3T3 cells were left untreated or
treated with FGF-1 alone, both FGF-1 and actinomycin D, or actinomycin
D alone for 0.5, 1, 2, or 4 h. Cells were collected, RNA was isolated,
and Fnk mRNA levels were analyzed by RNA gel blot hybridization.
Actinomycin D treatment prevented the FGF-1 induction of Fnk mRNA
(Fig. 2 A); thus, the increase in Fnk mRNA expression
after FGF-1 addition is due, at least in part, to transcriptional
activation of the fnk gene.
Figure 2:
Effect of actinomycin D or cycloheximide
on FGF-1 induction of Fnk mRNA. A, serum-starved cells were
either left untreated or treated with FGF-1, FGF-1 and actinomycin D
( Act.D), or actinomycin D alone for the times indicated. RNA
was isolated, and equivalent amounts of each sample were analyzed by
RNA gel blot hybridization. In this and the subsequent RNA gel blot
hybridization figures, only the region of the autoradiogram that
contained a Fnk mRNA hybridization signal is shown. Also, in some
cases, the blots were rehybridized to a glyceraldehyde-3-phosphate
dehydrogenase cDNA probe. Similarly, only the region of the
autoradiogram that contained glyceraldehyde-3-phosphate dehydrogenase
mRNA hybridization is shown. B, serum-starved cells were
either left untreated or treated with FGF-1, FGF-1 and cycloheximide
( CHX), or cycloheximide alone for the times indicated. RNA was
isolated, and equivalent amounts of each sample were analyzed by RNA
gel blot hybridization.
We next used the protein
synthesis inhibitor cycloheximide to determine whether FGF-1 induction
of Fnk mRNA levels was dependent on de novo protein synthesis.
Serum-starved cells were either left untreated or treated with FGF-1
alone, both FGF-1 and cycloheximide, or cycloheximide alone for 0.5, 1,
2, or 4 h. RNA was isolated, and RNA gel blot hybridization analysis
was performed. The addition of cycloheximide to FGF-1-stimulated cells
did not prevent fnk gene induction but instead
``superinduced'' Fnk mRNA levels (Fig. 2 B).
This indicates that the fnk gene does not require protein
synthesis for transcriptional activation; thus, by this criterion, it
can be classified as a growth factor-regulated immediate early gene.
1, EGF, or IGF-1 treatment increased Fnk mRNA
expression only slightly above basal levels (Fig. 3 C).
Fnk cDNA and Gene Sequence Analysis
Both strands
of the 2.2-kb Fnk cDNA insert were sequenced by the
dideoxynucleotide chain termination method. The nucleotide sequence
contained a long open reading frame encoding a protein of 609 amino
acids, but an initiating ATG methionine codon was not present. Partial
DNA sequence analysis of the other four Fnk cDNA clones isolated in the
original library screen indicated that they did not contain additional
5` coding sequence. Furthermore, full-length Fnk cDNA clones could not
be obtained by screening additional murine cDNA libraries. Therefore, a
mouse liver genomic DNA library was screened using a 461-bp 5`
restriction fragment of the Fnk cDNA. Positive phage were
plaque-purified, and DNA was isolated. Southern blot hybridization
analysis identified a
1.2-kb BglII fragment that was
likely to encode additional amino-terminal protein sequence. DNA
sequence analysis indicated that 302-bp of this fragment represented
new 5` sequence not present in the cDNA. The composite nucleotide and
deduced protein sequences of Fnk are shown in Fig. 4. The
nucleotide sequence contains a long open reading frame that encodes a
protein of 631 amino acids with a predicted molecular mass of 69,995
daltons. The presumed initiating ATG is flanked by a favorable sequence
for translation initiation
(19, 20) . There is an
in-frame TAG termination codon located 84 nucleotides upstream of this
ATG. The cDNA clone contained a 335-nucleotide 3`-untranslated region
with a consensus polyadenylation signal and three copies of a TTATTTAT
sequence motif. AU-rich sequence elements are found in the
3`-untranslated regions of many immediate early mRNAs and may be
responsible for their rapid decay rate
(17) . Comparison of the
PCR primer sequences with the
2.2-kb Fnk cDNA sequence identified
the two regions that flanked the original PCR-derived
200-bp cDNA
clone. The sense protein kinase and antisense zinc finger
oligonucleotides had
76 and
65% nucleotide sequence identity,
respectively, to sequences within the Fnk coding region. Analysis of
the Fnk predicted amino acid sequence indicates that this protein does
in fact have a protein kinase catalytic domain but not a zinc finger
motif (see below).
Figure 4:
Nucleotide sequence and deduced amino
acid sequence for Fnk. Numbers to the left refer to
the first amino acids on the lines, and the numbers to the right refer to the last nucleotides on the lines. The nucleotide
sequence obtained from a genomic clone is in boldface type; the remaining sequence is from a cDNA clone. The
solid line above nucleotides 574-590 indicates
the sequence that hybridized to the sense protein kinase domain primer,
and the solid line above nucleotides 786-805
indicates the sequence that hybridized to the antisense zinc finger
primer. The TAG stop codon is denoted by an asterisk, the
putative mRNA destabilizing sequence motifs are underlined,
and the polyadenylation signal is
boxed.
Fnk Amino Acid Sequence Comparisons
A search of
the sequence data bases revealed that the deduced amino acid sequence
of Fnk was most similar to members of the polo subfamily of
serine/threonine protein kinases. Specifically, there is 49, 36, 33,
32, and 22% overall sequence identity to mouse Snk
(21) , mouse
Plk
(22, 23, 24) , Drosophila polo
(25) , Saccharomyces Cdc5
(26) and mouse Sak-a
(27) , respectively. An alignment of the deduced Fnk amino acid
sequence and the three most structurally similar polo subfamily members
is shown in Fig. 5. Two regions of relatively high amino acid
sequence identity are apparent, the protein kinase catalytic domain
located in the amino-terminal half of the proteins and a region in the
carboxyl-terminal half termed the polo homology 2 domain by Hamanaka
et al. (23) . This latter domain is present in all
members of the polo subfamily except for Sak.
Figure 5:
Sequence identity between the predicted
Fnk amino acid sequence and three members of the polo subfamily of
serine/threonine kinases. The aligned sequences are murine Fnk, murine
Snk, murine Plk, and Drosophila polo. Numbers to the right refer to the last Fnk amino acid in the
numbered lines. Columns that are boxed indicate
identical residues at that position. Gaps represented by
dashes were inserted to maximize the alignment. The boundaries
of the Fnk putative kinase domain, residues 63 and 315, are marked by
asterisks. Roman numerals above the kinase
domain sequence are the 12 conserved subdomains identified by Hanks and
Quinn (29). A region of sequence identity in the carboxyl-terminal
region of the four proteins, the polo homology 2 (PH2) domain (23), is
also noted.
The catalytic domain
of Fnk consists of 253 amino acids and includes features characteristic
of protein kinases in general and serine/threonine-specific kinases in
particular
(28, 29) . Within this domain, the amino acid
sequence identity between Fnk and Snk, Plk, polo, Cdc5, or Sak-a is 67,
52, 50, 45, and 41%, respectively. Fnk contains the amino acid residues
that are most highly conserved in all known protein kinases; for
example, a K residue in subdomain II (amino acid 92), an E residue in
subdomain III (amino acid 111), and the DFG sequence in subdomain VII
(amino acids 204 to 206). However, the consensus
G XG XXG XV sequence present in many
nucleotide-binding proteins is present as
G XG XXA XC (amino acids 69-76) in Fnk.
The G A and V
C substitutions are found in all polo
subfamily members except for Sak as well as in several other protein
kinases
(28, 29) . Finally, Fnk contains two sequence
motifs that structurally distinguish serine/threonine-specific kinases
from tyrosine-specific kinases
(28, 29) . The DLKLGN
sequence present in subdomain VIb and the GTPNYVAPE sequence present in
subdomain VIII are similar to the DLKP XN and
GTP XYL XPE consensus sequences commonly found in
serine/threonine kinases.
Fnk mRNA Expression Levels in Mouse Tissues
We
next used RNA gel blot hybridization analysis to examine the tissue
distribution of Fnk mRNA. Six different tissues were obtained from
newborn animals, and 12 different tissues were obtained from adult
animals. In the newborn animals, Fnk transcripts were expressed at a
low level in the heart but at moderate or high levels in intestine,
kidney, liver, lung, and skin (Fig. 6 A). In the adult
animals, Fnk mRNA was expressed at a high level in skin but was
undetectable or expressed at a low level in all of the other tissues
examined (Fig. 6 B). These results indicate that fnk gene expression is regulated in vivo in both an age- and
tissue-specific manner.
Figure 6:
Fnk mRNA expression levels in various
mouse tissues. Total RNA was isolated from the indicated tissues, and
equivalent amounts of each sample were analyzed by RNA gel blot
hybridization. A, newborn mouse tissues; B, adult
mouse tissues.
H
zinc finger cDNA
sequences
(44, 45, 46, 47) . The
complementary sense primer to this region encodes the peptide HQRIHTG.
The Fnk cDNA sequence recognized by the primer encoded the peptide
HRDLKLG, which has only 29% sequence identity to the targeted zinc
finger motif. Coincidentally, the first four residues of this sequence
are part of the LHRDL motif mentioned above. Additional amino acid
residues characteristic of H/C-link regions were not found in the
deduced Fnk sequence. Therefore, in the case of Fnk, the targeting
approach for amplifying particular types of cDNAs was only partially
successful since only one of the two targeted domains was actually
present in the deduced sequence of the cloned cDNA. Some of the other
FGF-1-inducible genes that we have identified using this approach also
do not encode proteins with the targeted structural domains
(12, 13) . This is likely to reflect the necessity to
use degenerate oligonucleotide primers and thus a relatively low
annealing temperature in the PCR assays.
8 h. This implies that Fnk mRNA has a relatively
short half-life, consistent with the presence of three UAUUUAU motifs
in its 3`-untranslated region
(17) . Third, FGF-1 induces Fnk
mRNA expression in the presence of cycloheximide; thus, gene activation
does not require the de novo synthesis of intermediary
proteins. The simultaneous addition of FGF-1 and cycloheximide actually
elevated Fnk mRNA levels to a greater degree than FGF-1 alone, and the
kinetics of accumulation were prolonged. It is likely that
cycloheximide is preventing the synthesis of labile proteins required
for Fnk transcriptional repression and/or Fnk mRNA decay.
Alternatively, Fnk mRNA degradation may be coupled to translation.
Fourth, fnk gene expression can be induced by numerous
growth-promoting agents (FGF-1, FGF-2, serum, PDGF-BB) as well as by
PMA, a tumor-promoting phorbol ester than can bind to and activate the
intracellular signaling molecule protein kinase C. Fifth, Fnk mRNA is
expressed in a tissue-specific manner, with maximal levels detected in
newborn and adult mouse skin.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.