(Received for publication, February 22, 1995; and in revised form, June 20, 1995)
From the
The LAMMER subfamily of kinases has been conserved throughout
evolution, and its members are thought to play important roles in the
regulation of cellular growth and differentiation programs. STY is a
murine LAMMER kinase which has been implicated in the control of PC12
cell differentiation. Multiple transcripts are derived from the Sty gene, and their relative abundance is developmentally regulated.
Alternative splicing of the primary Sty transcript generates
mRNAs encoding full-length catalytically active (STY) and truncated,
kinase-deficient polypeptides. Both STY and its truncated isoform,
STY, are localized in the nucleus and are capable of
heterodimerizing. We also demonstrate that STY functions as a dual
specificity kinase in mammalian cells.
The Sty gene encodes a member of the recently
discovered family of dual specificity kinases(1, 2) .
To date, at least 18 distinct genes encoding dual specificity kinases
have been identified in the genomes of yeast and mammals (for reviews,
see Douville et al.(3) and Lindberg et
al.(4) ). These kinases, expressed either as bacterial
products or isolated from mammalian cells, have the ability to
autophosphorylate on serine, threonine, and tyrosine residues. Two of
the best studied of these enzymes are the yeast wee1 kinase, a regulator of progression through the cell
cycle(5) , and MEK (MAPK/ERK kinase), believed to be a key
molecule in mitogen-stimulated signaling pathways (6) . We have
previously reported the cloning of the murine Sty cDNA which
when expressed in bacteria encodes a protein capable of phosphorylating
serine, threonine, and tyrosine residues in vitro(1) .
The STY kinase contains an amino acid motif, LAMMER, found in kinase
subdomain X(7) , a feature which is shared with at least eight
other dual specificity kinases expressed in humans, mice, plants, and
insects(7, 8, 9) . The LAMMER motif
containing protein kinases appears to be conserved throughout evolution
suggesting that these enzymes may play important roles in the control
of cellular growth and differentiation. Indeed, the Doa kinase, a Drosophila gene product, is critical to the
development of the fly embryo and affects eye differentiation in the
adult(7, 10) . The AFC1 kinase gene of Arabidopsis
thaliana can complement yeast signal transduction mutants via
activation of the transcription factor STE12(8) . In mammalian
cells, the physiological function of the LAMMER kinases is largely
unknown although overexpression of the STY kinase in PC12 cells appears
to initiate their differentiation into neural derivatives possibly
through activation of a protein kinase cascade(11) . As has
been observed with other members of the LAMMER kinase family, the Sty gene appears to express several differentially processed
transcripts. To gain a full understanding of the physiological role of
the STY kinase, we felt it important to identify and biochemically
characterize all its gene products. We report here the cloning and
sequencing of three novel Sty cDNAs. Two of these cDNAs are
derived from incompletely processed transcripts which accumulate in the
nucleus in a developmentally regulated fashion. The third cDNA encodes
a truncated polypeptide (STY
), which like the full-length
STY kinase is found in the nucleus and contains domains which
facilitate dimerization. Furthermore, we present evidence that the STY
kinase is able to phosphorylate serine, threonine, and tyrosine
residues when expressed in mammalian cells.
The STY
polypeptide was tagged at the amino terminus with a six-repeat human
Myc epitope by subcloning the Myc epitope isolated from Bluescript
KS MTG (17) (SalI/PstI-blunted fragment) into the
pECE/Sty plasmid (KpnI-blunted). The Myc epitope in
pECE/M-Sty was confirmed to be in-frame by sequencing.
pECE/M-Sty and
pECE/M-Sty
were generated by PCR-directed
mutagenesis using the pECE/M-Sty plasmid as template and the
following oligonucleotides:
gcatagttaaaaatgtggatag
and
gcactgctacacgtctac
for
pECE/M-Sty
or
cccgtgtgaatggtgctg
and
atgaaattgttgatactttaggt
for
pECE/M-Sty
. 50-µl reactions were
performed containing 20 ng of plasmid, 25 pmol of each oligonucleotide,
0.2 mM concentration of each dNTP, 2 units of Vent
(exo
) DNA Polymerase (New England Biolabs) in buffer
supplied by the manufacturer. Amplification conditions were 95 °C,
1 min; 56 °C, 1 min; 72 °C, 5 min for 20 cycles. The reaction
product was phosphorylated with polynucleotide kinase and
recircularized with T4 DNA ligase.
An amino-terminal deletion
mutant,
pECE/M-Sty,
lacking the first 60 amino acids of STY, was created by substitution of
the BamHI/BamHI Sty fragment from
pECE/M-Sty with the XhoII/BamHI Sty fragment generated by subsequent digestion of the BamHI/BamHI fragment with XhoII. All
mutations were confirmed by sequencing.
The Myc epitope in these constructs is immunoreactive with the monoclonal antibody (mAb) 9E10 in immunoprecipitation/immunoblot analysis(18) .
Sty was also subcloned into the bacterial expression vector pGEX-3X (Pharmacia) to generate the fusion protein GST-STY. The 1.7-kb Sty fragment was isolated from pTZ19R/Sty by PCR amplification as described above. This blunt/EcoRI fragment was subcloned into pGEX-3X (BamHI, blunted, followed by EcoRI digestion). Sty was confirmed to be in-frame with GST by sequencing.
For immunoblots, membranes were blocked in 5% Blotto in TBST (150 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.05% Tween 20). Anti-Myc immunoblotting was performed using ammonium sulfate-precipitated 9E10 hybridoma culture supernatant at 1:50-1:100. Immunoblots were visualized with horseradish peroxidase-conjugated goat anti-mouse antibody followed by enhanced chemiluminescence (Amersham) and exposure to Kodak XAR-5 x-ray film. Phosphoamino acid analysis was performed as described previously(19) .
Figure 2: Schematic representation of Sty transcripts and analysis of their subcellular localization. Total RNA from NIH 3T3 cells was separated into cytoplasmic and nuclear fractions and subjected to Northern blot analysis. Each lane represents the hybridization of the Sty1.8 cDNA to 20 µg of total RNA. The approximate sizes of the Sty mRNAs are (from top to bottom) 5.6, 3.2, 1.8, and 1.7 kb. Positions of 18 S and 28 S RNA were determined by ethidium bromide staining of the agarose gel. Exons (EA-ED) are depicted by thick lines and introns (IA-ID) as thin lines. The alternatively spliced exon (EB) is hatched.
Figure 1:
Nucleotide and predicted amino acid
sequences of the Sty5.6 partial cDNA and StycDNA. The amino acid
sequence is derived from that predicted from the Sty1.8
cDNA(1) . A, Sty5.6 partial cDNA. Predicted
intron/exon boundaries are indicated in boldface. The
alternatively spliced exon is underlined, and consensus amino
acids found in kinase subdomains are highlighted and indicated
by Roman numerals. Amino acids indicated by an asterisk are encoded by codons split between two adjacent exons. Exons (E)
and introns (I) are labeled as follows: EA, nucleotides 1-456;
IA, nucleotides 457-1326; EB, nucleotides 1327-1417; IB,
nucleotides 1418-1811; EC, nucleotides 1812-1995; IC,
nucleotides 1996-2082; ED, nucleotides 2083-2249; ID,
nucleotides 2250-2572. B, Sty1.7 (Sty
) cDNA.
To determine whether Sty5.6 represented the primary
transcript from the Sty gene, fragments of the Sty locus were isolated from a D3 embryonic stem cell genomic
DASHII library (see ``Materials and Methods''). A
fragment surrounding the predicted initiating methionine was cloned
into the sequencing vector pGEM-4. Portions of this genomic fragment
were sequenced using oligonucleotides residing in the Sty cDNA. Two introns were identified in addition to those found in Sty5.6, demonstrating that Sty5.6 is not the primary
transcript but is an incompletely spliced RNA. The intron/exon
boundaries and relative positions of these introns are indicated in Table 1.
Figure 3:
Schematic representation of the
alternative splicing of exon B. A, comparison of the amino
acid sequence surrounding exon B of STY (mouse) with that of the human
homologue (hCLK). The sequence encoded by exon B in mouse which is
absent in the human cDNA is denoted by the dashed line. Amino
acid positions indicated are relative to those predicted for STY (1) and hCLK(17) . Identical residues are identified by bars, and conservative substitutions by dots. B, the nucleotide sequence of the intron/exon boundaries
surrounding the alternatively spliced exon is shown between the
predicted splicing products. Splicing in of exon B would result in
production of full-length STY. Splicing out of exon B results in a
frameshift which introduces a premature stop codon (tag, indicated by a period). The resulting protein (STY) is truncated
prior to the catalytic domain.
To investigate this possibility, we performed reverse transcriptase coupled with PCR amplification (RT-PCR) to amplify the entire coding sequence of Sty (nucleotide position 7-1539, data not shown) from P19 cell-derived cDNA. Two DNA species were amplified, the sizes of which were consistent with alternative slicing of exon B (data not shown). To verify the identity of the PCR products, the DNA was isolated, reamplified, and subcloned into the pCRII vector. Sequencing revealed that the larger amplification product corresponded to full-length Sty, whereas the smaller lacked exon B (Fig. 1). This result was observed with cDNA derived from P19 cells (see P19 lane, Fig. 4) suggesting that the alternatively spliced transcript lacking exon B (Sty1.7) arises from a mRNA that co-migrates with the 1.8-kb transcript.
Figure 4:
RT-PCR analysis of Sty transcripts demonstrates alternative splicing. Oligonucleotide
primers flanking exon B were used to amplify cDNA generated from adult
mouse tissues or P19 EC cells as indicated above each lane or to
amplify the Sty1.8 cDNA. PCR products were resolved on a 1.5%
agarose gel and stained with ethidium bromide. The predicted product
sizes of 543 bp (Sty) and 453 bp (Sty)
are indicated on the left, and DNA marker sizes are shown on
the right.
Expression of Sty1.7 and Sty1.8 was assayed by RT-PCR amplification of the region encompassing exon B from cDNA of several mouse tissues (Fig. 4). All tissues tested contained both Sty1.7 and Sty1.8 transcripts. A PCR product of approximately 1 kb in size was also reproducibly amplified from all tissues, the size of which is consistent with that of a splicing intermediate derived from Sty5.6 (data not shown).
The Sty1.7 mRNA, generated by alternative splicing of exon B,
should encode a protein homologous to the hCLK protein product. The
protein product from the murine Sty1.7 transcript is predicted
to be truncated and catalytically inactive (we have termed this product
STY for truncated STY) (see Fig. 3B).
However, the protein product of hClk, which lacks exon B, is
predicted to be catalytically active. This apparent contradiction
suggested that either the sequence encompassing exon B is absent in the
human genome and that in its absence the gene has evolved to maintain a
catalytically active molecule or, alternatively, the hClk cDNA
is the homologue of Sty1.7 and should encode a truncated
molecule. To investigate this further, a human liver cDNA library (gift
of Dr. D. A. Gray) was screened with a full-length Sty cDNA
probe. From an initial screening of 200,000 recombinants, 30 clones
were identified. Of these, 10 were picked at random and analyzed by PCR
to determine if they contained the exon A-exon C region. One positive
clone was analyzed further, and the sequence of this boundary indicated
that it was the human homologue of Sty1.7 and would encode a
truncated protein product (data not shown). The human cDNA, as
published by Johnson and Smith(21) , contains an additional G
residue at this boundary which would maintain the open reading frame
through the catalytic domain. The absence of this residue in our mouse
and human cDNAs causes a frameshift to occur which introduces a stop
codon (Fig. 3B).
Figure 5:
Expression and catalytic activity of
wild-type and mutant STY proteins in COS-1 cells. COS-1 cells were
transfected with either pECE expression vector alone (lane 1)
or vector encoding Myc epitope-tagged STY (M-STY), catalytic mutant STY
(K190R), truncated STY (T) or an amino-terminal deletion of STY
(XhoII) (lanes 2-5, respectively).
Myc-tagged proteins were immunoprecipitated with the anti-Myc
monoclonal antibody and subjected to an in vitro kinase assay.
Proteins were subjected to SDS-PAGE and transfer to nitrocellulose.
Expression levels were determined by immunoblotting with the anti-Myc
antibody and enhanced chemiluminescence (A). Autoradiogram of in vitro catalytic activities of wild-type and mutant STY
proteins (B). Phosphoamino acid analysis by two-dimensional
thin layer electrophoresis of wild-type M-STY labeled in vitro (C). Positions of molecular mass markers (kDa) are
indicated to the left of each panel. The positions of
phosphoserine (pSer), phosphothreonine (pThr), and
phosphotyrosine (pTyr) are also
indicated.
Figure 6:
STY protein is phosphorylated on tyrosine in vivo. COS-1 cells were transfected with the pECE vector
encoding Myc epitope-tagged STY (M-STY) and grown overnight in the
absence (lanes 1 and 3) or presence (lanes 2 and 4) of 50 µM sodium vanadate
(VO). M-STY was immunoprecipitated (IP) with
either the anti-Myc monoclonal antibody (myc, lanes 1 and 2) or an antiphosphotyrosine monoclonal antibody (pTyr, lanes 3 and 4). Immunoprecipitated
Myc-tagged proteins were visualized by immunoblot analysis and enhanced
chemiluminescence with the anti-Myc antibody. Positions of molecular
mass markers (kDa) are indicated on the left.
Figure 7:
Subcellular localization of wild-type and
mutant STY proteins. COS-1 cells were transfected with the pECE vector
alone (a) or vector encoding Myc epitope-tagged STY (b), an amino-terminal deletion of STY
(STY, c)
or truncated STY (STY
, d). Myc-tagged STY proteins
were visualized by indirect immunofluorescence with the anti-Myc
monoclonal antibody followed by fluorescein isothiocyanate-conjugated
secondary antibody. Cells were photographed at a magnification of
40 with exposure times of 30 s (b-d) and 60 s (a).
Figure 8:
STY can dimerize in vitro. COS-1
cells were transfected with the pECE expression vector encoding Myc
epitope-tagged STY (M-STY, lanes 1-4), Tik (M-Tik, lanes 5-8) or truncated STY (M-STY, lanes 9-11). Protein from equivalent fractions of cell
lysates was subjected to immunoprecipitation with the anti-Myc
monoclonal antibody (lanes 1, 5, and 10) or affinity
purification with glutathione-Sepharose 4B (Seph, lanes 2 and 6), GST coupled to Sepharose (GST, lanes
3, 7, and 9), or GST-STY coupled to Sepharose (GST-STY, lanes 4, 8, and 11). Purified Myc-tagged proteins
were visualized by immunoblot analysis and enhanced chemiluminescence
with the anti-Myc antibody. Positions of molecular mass markers (kDa)
are indicated on the left and right.
Truncated STY
(M-STY) also formed heterodimers with full-length STY (Fig. 8, lanes 9-11) demonstrating that the STY
dimerization domain is contained in the amino-terminal portion of the
molecule and is not dependent upon catalytic activity of the kinase.
These results, however, do not differentiate between a direct
association of STY with itself or an association mediated through an
intermediary molecule.
Isoforms of several protein kinases have been predicted and
identified based on cDNA cloning of multiple RNA transcripts (23, 24, 25) . For instance, six transcripts,
coding for at least two protein isoforms, are derived from the gene
encoding the trkB neurogenic tyrosine kinase
receptor(23) . These trkB proteins,
gp145 and gp95
,
have identical extracellular and transmembrane domains, but only
gp145
contains the cytoplasmic kinase domain.
Similarly, the fibroblast growth factor tyrosine kinase receptor 1 gene
generates several transcripts which encode receptor
variants(24) . One of these, a receptor-like molecule that
lacks the transmembrane and kinase domains, is secreted and
catalytically inactive.
The expression of the Sty gene is developmentally regulated at the level of transcript processing. Embryonic stem cells express two major transcripts (1.7 and 1.8 kb), whereas differentiated cells express two additional partially spliced mRNAs (3.2 and 5.6 kb). While the two larger mRNAs are sequestered in the nucleus and are unavailable for protein translation, the 1.7- and 1.8-kb mRNAs are capable of directing synthesis of a truncated and full-length STY protein, respectively. In general, unspliced primary transcripts are relatively short-lived with half-lives on the order of minutes(26) . However, several examples of developmentally regulated mRNAs exist which reflect developmental changes in expression of factors involved in the splicing process. In Drosophila, differentially expressed splicing factors control the expression of the sex lethal and transformer RNAs during sexual determination(27) . In the developing pituitary, increased transcription of the proopiomelanocortin (POMC) gene results in accumulation of immature transcripts, possibly due to limiting amounts of specific splicing factors(28) . Immature T cell antigen mRNA species appear to accumulate at specific stages of T cell development due to the presence of a cycloheximide-sensitive splicing inhibitor protein(29) . It is unknown at this time which, if any, of these mechanisms accounts for the accumulation of Sty mRNA precursors. It is of interest to note, however, that two leukemic cell lines, P388 (pre-B) and SP10 (myeloma), like embryonic stem cells, do not accumulate these larger transcripts(1) . Thus the 1.7/1.8-kb embryonic transcripts may be favored in rapidly proliferating cells such as embryonic or tumor cells.
Amino acid
comparison of STY with its human homologue, hCLK, revealed a
30-amino-acid insertion in STY. Isolation and sequencing of the Sty5.6 partial cDNA clone revealed that this 30-amino acid
segment was contained within a single exon. Alternative splicing of
this exon would generate either the full-length product, STY
(containing the exon), or a truncated polypeptide (STY,
lacking this exon) due to a frameshift which introduces a stop codon
(see Fig. 3B). Johnson and Smith (21) suggested
that the hClk cDNA, which lacks the exon, coded for an active
kinase. We have isolated a human Clk cDNA and sequenced it in
this region. Our sequence indicated that the G residue at position 546
in the hClk cDNA is not present, and, as a result, an mRNA
with this 91-bp exon absent would encode a truncated protein as
predicted by the mouse sequence. One simple interpretation of these
data is that the original human cDNA contained a sequencing error and
that STY
and STY isoforms are encoded in both the human and
mouse genomes. A similar conclusion has been reached by Hanes et
al.(9) who have recently reported the sequence of two new
human kinase cDNAs with significant sequence homology to both Sty and hClk. As we have shown here for Sty, the
primary transcripts of these two kinases (hClk2 and
hClk3) undergo alternative splicing to generate mRNAs
predicted to encode full-length and truncated, catalytically inactive
kinases(9) . This striking conservation of amino acid sequence
and splicing patterns for three separate kinase genes suggests that
they may represent a subfamily of enzymes with related physiological
targets. It is of interest to note that the Doa and AFC1
LAMMER kinases have been implicated in the control of transcription.
The nuclear localization of STY is consistent with its having a role in
the regulation of transcription and/or RNA processing. With respect to
this latter idea, we and others have recently found that STY and
STY
can interact with members of the SR
(serine-arginine-rich) family of splicing factors.
We are
testing the possibility that hCLK2, hCLK3, and their truncated
derivatives also associate with splicing factors.
One commonly
accepted paradigm of kinase regulation is that dimerization is required
for kinase activation and subsequent signal
transduction(30, 31) . Evidence for this idea includes
the demonstration that catalytically inactive kinase mutants dominantly
suppress wild type kinase activity(32) . STY and STY show identical patterns of expression and subcellular
localization and can form heterodimers in vitro. We suggest
that a dynamic interaction between STY and STY
may be
involved in the regulation of the biological properties of STY and
related kinases.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s) U11054 [GenBank](Sty5.6) and U21209 [GenBank](Sty).