©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification by Targeted Differential Display of an Immediate Early Gene Encoding a Putative Serine/Threonine Kinase(*)

Patrick J. Donohue (§) , Gregory F. Alberts , Yan Guo , Jeffrey A. Winkles (¶)

From the (1) Department of Molecular Biology, Holland Laboratory, American Red Cross, Rockville, Maryland 20855

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Fibroblast growth factor (FGF)() -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) .

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 10high 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 Gstage of the mammalian cell cycle.


MATERIALS AND METHODS

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 10phage were plated at a density of 2 10plaque-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 10phage were plated at a density of 2.5 10plaque-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).


RESULTS

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.

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-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.




DISCUSSION

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 CHzinc 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.

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 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.

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.


FOOTNOTES

*
This study was supported in part by National Institutes of Health Grant HL-39727 (J. A. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U21392 and U22434.

§
This work was performed in partial fulfillment of the requirements for the degree of Doctor of Philosophy from the Graduate Genetics Program, George Washington University, Washington, D.C. 20037. Present address: Laboratory of Biological Chemistry, National Cancer Inst., National Institutes of Health, Bethesda, MD 20892.

To whom reprint requests and correspondence should be addressed: Dept. of Molecular Biology, Holland Laboratory, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. Tel.: 301-738-0655; Fax: 301-738-0465.

The abbreviations used are: FGF, fibroblast growth factor; bp, base pair(s); EGF, epidermal growth factor; Fnk and fnk, FGF-inducible kinase; IGF, insulin-like growth factor; kb, kilobase(s); PCR, polymerase chain reaction; PDGF, platelet-derived growth factor; Plk and plk, polo-like kinase; PMA, phorbol myristate acetate; Sak, Snk/Plk-akin kinase; sgk, serum- and glucocorticoid-regulated kinase; Snk and snk, serum-inducible kinase; TGF, transforming growth factor.

P. J. Donohue and J. A. Winkles, manuscript in preparation.


ACKNOWLEDGEMENTS

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.


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