(Received for publication, October 28, 1994)
From the
To gain insight into the signal transduction pathways utilized by the Wnt-1-responsive mammary epithelial cell line C57MG, we screened for non-src family member tyrosine kinases expressed in these cells using a polymerase chain reaction-based technique. We identified five cDNA clones encoding receptor tyrosine kinases for which the ligand is known (fibroblast growth factor receptor, platelet-derived growth factor receptor, epithelial growth factor receptor, insulin receptor, and insulin-like growth factor receptor), two putative receptor tyrosine kinases for which the ligand remains to be identified (the products of ryk and the mouse klg homolog), and a novel tyrosine kinase. We cloned cDNAs encoding both the murine and human homologs of this kinase, the sequences of which were subsequently published under the names sky (Ohashi, K., Mizuno, K., Kuma, K., Miyata, T., and Nakamura, T.(1994) Oncogene 9, 699-705) and rse (Mark, M. R., Scadden, D. T., Wang, Z., Gu, Q., Goddard, A., and Godowski, P. J.(1994) J. Biol. Chem. 269, 10720-10728). Mouse sky RNA levels are abundant in mammary tumors derived from transgenic mice that express wnt-1, fgf-3, or both oncogenes in their mammary glands. However, little or no expression of sky is detected in mammary glands from virgin animals or in preneoplastic mammary glands from wnt-1 transgenic mice. Moreover, we find that the human homolog of sky is expressed at elevated levels when normal human mammary epithelial cells are rendered tumorigenic by the introduction of two viral oncogenes. Transient transfection of the human SKY cDNA into the quail fibrosarcoma cell line QT6 reveals that SKY is an active tyrosine kinase that augments the level of cellular phosphotyrosine. Introduction of murine Sky into RatB1a fibroblasts by retrovirus-mediated gene transfer results in morphological transformation, growth in soft agar, and the formation of tumors in nude mice. These data raise the possibility that the Sky tyrosine kinase is involved in the development and/or progression of mammary tumors.
Protein tyrosine kinases (PTKs) ()are key players in
many of the signal transduction pathways used to control cell growth,
differentiation, and division. PTKs can be divided into two general
categories based on sequence similarities: receptor tyrosine kinases,
transmembrane proteins stimulated by cognate ligands (1) , and
cytoplasmic tyrosine kinases, including members of the src, fps, and abl gene families(2) . Genes
encoding several PTKs from each category have been transduced to
generate oncogenic retroviruses(3) , and mutations of others
have been implicated in human malignancies and cell
transformation(4, 5) .
Receptor tyrosine kinases possess an extracellular ligand-binding domain, a transmembrane region, and a cytoplasmic catalytic domain. In the presence of the appropriate ligand, many receptor PTKs form dimers, an event which triggers a receptor's intrinsic tyrosine kinase activity and leads to autophosphorylation, thereby transducing an external signal to the inside of the cell(6) . In many cases, these autophosphorylated tyrosine residues are targets for binding by cytoplasmic proteins, including PTKs, via SH2 (src homology region 2) domains. These steps initiate an array of signal transduction pathways that ultimately lead to the nucleus, eliciting a variety of cellular responses and activities.
We are interested in studying the signal transduction pathways involved in mammary carcinogenesis, particularly as results from the expression of the wnt-1 gene. It is well established that wnt-1 is a mammary oncogene. wnt-1 was first identified in murine mammary tumors as a locus at which a mouse mammary tumor virus provirus is frequently integrated, causing ectopic expression of wnt-1(7) . Furthermore, expression of a mouse mammary tumor virus-wnt-1 transgene in mammary glands results in hyperplasia and development of mammary tumors(8) . The mouse mammary epithelial cell line C57MG (9) has been useful for studying the effects of the wnt-1 gene. Introduction of wnt-1 into C57MGs results in a marked morphological transformation, extended growth past confluence, and focus formation(10) . However, many aspects of C57MG cells have not received much attention, especially the identity of the signaling molecules used by these cells.
Toward this end, we have used a polymerase chain reaction (PCR)-based approach to identify receptor PTKs expressed in C57MG cells. This method takes advantage of the high degree of conservation of amino acid residues within the approximately 270-amino acid catalytic domain of all tyrosine kinases(2) . Our analysis identified the known tyrosine kinase receptors for insulin, fibroblast growth factor, epithelial growth factor, platelet-derived growth factor, and insulin-like growth factor as well as the receptor-type tyrosine kinases Klg and Ryk, the ligands for which are not yet known. In addition, we cloned a cDNA for a receptor-like tyrosine kinase that at the time was unreported.
Recently, the sequence of this cDNA, cloned from both murine and human sources, has been published under the names sky(11) and rse(12) . These papers have stated that the sky mRNA is expressed most abundantly in the brains of humans and rats and that this message encodes a putative receptor tyrosine kinase possessing two immunoglobulin-like and two fibronectin type III domains in the extracellular region(11, 12) . These latter characteristics place Sky in the ufo/axl family of receptor tyrosine kinases. The amino acid sequence of the human (SKY) and murine (Sky) homologs are 90% identical, although the human protein contains an additional 10 amino acid residues in the putative signal peptide sequence(12) . Furthermore, Mark et al.(12) have reported that a version of SKY engineered with a heterologous signal sequence and amino-terminal epitope tag is located at the cell surface and exhibits tyrosine kinase activity.
In this report, we show that sky mRNA is significantly more abundant in mammary tumors isolated from transgenic mice than in hyperplastic mammary glands and mammary glands of virgin females. In addition, SKY mRNA levels are up-regulated in human mammary epithelial cells but not in fibroblasts after transformation with SV40 large T antigen and v-Ha-ras. We also demonstrate that the native human SKY gene product is an active tyrosine kinase that undergoes glycosylation and oligomerization, like other transmembrane PTKs. Furthermore, RatB1a fibroblasts overexpressing murine Sky exhibit a transformed morphology, grow as colonies in soft agar, and form tumors when injected into nude mice. These data raise the intriguing possibility that this signaling protein, the Sky tyrosine kinase, may be involved in the development and/or progression of certain mammary tumors.
Figure 1:
Expression of sky during
murine mammary gland postnatal development. Mammary glands from female
mice at various stages of postnatal development were isolated, and
total RNA was harvested as described under ``Materials and
Methods.'' For each sample (lanes1-11),
poly(A) RNA from 50 µg of total RNA was recovered
with oligo(dT)-cellulose and analyzed by Northern blot.
Poly(A)
RNA (0.7 µg) isolated from C57MG cells
also was analyzed (lane12). The blot was hybridized
and exposed separately with random-primed cDNA probes for murine sky, rat GAPDH, and rat
-casein. The blot was stripped
between hybridizations and exposed to ensure that the previous probe
was removed completely. The sky RNA is 3.8 kb, and GAPDH and
-casein RNA both are approximately 1.1 kb. The sample from 10 days
of pregnancy (lane3) appeared to be partially
degraded as visualized by ethidium bromide staining of the agarose gel
(data not shown).
Figure 4:
A,
human SKY is overexpressed in a tumorigenic human breast cell
line. 10 µg of total RNA from various HMECs was analyzed by
Northern blot. The parental strain (184, lane1) was
chemically mutagenized to give rise to an immortal line (184A1, lane2), which was subsequently passaged in growth
factor-depleted media (184A1N4, lane3) and then made
tumorigenic by the introduction of SV40 large T antigen and Ha-ras (184A1N4-TH, lane4). The blot was hybridized
with a random-primed human SKY cDNA probe and subsequently
with a rat GAPDH cDNA probe. B, sky expression is not
increased in MEF cell lines transformed by activated oncogenes.
Poly(A) RNA isolated from primary (lane 1)
and immortalized (lane 2) MEF cells, as well as from
immortalized MEF cells expressing v-Ha-ras (lane3), v-erbB (lane4), and an
activated allele of c-src (SrcA, lane5)
were analyzed by Northern blot. The blot was hybridized with a
random-primed murine sky cDNA probe and subsequently a rat
GAPDH probe.
Figure 9:
The SKY
tyrosine kinase functions as a dimer. QT6 cells were transfected with
10 µg of the HA-tagged wild-type (lane1) or
Kinase (lane4) SKY expression
construct alone or in combination with 10 µg of the untagged
versions of the
Kinase (lane2) or wild-type (lane3) SKY expression construct. The
pcDNA3 vector plasmid (10 µg) was included in the transfections of lanes1 and 4. Cell lysates were prepared 2
days after transfection and immunoprecipitated with the 12CA5E antibody
directed against the HA epitope. Immunocomplexes were assayed for in vitro kinase activity as described in Fig. 6.
K, deletion of kinase subdomains V to
IX.
Figure 6:
The SKY gene product is an active tyrosine kinase. QT6 cells were
transfected with 20 µg of the HA-tagged wild-type (SKY-HA)
and Kinase (SKY
K-HA) expression constructs. A vector
control was transfected as well. Lysates prepared 2 days after
transfection were immunoprecipitated with the 12CA5E antibody directed
against the HA epitope, and the immunocomplexes were divided into three
equal portions. A, immunocomplexes were assayed for in
vitro kinase activity as described under ``Materials and
Methods'' in the absence (lanes1-3) or
presence (lanes4-6) of the synthetic substrate
poly(Glu,Tyr). Radiolabeled complexes were resolved on a 10% SDS-PAGE
gel followed by autoradiography. B, the third portion of each
immunocomplex was analyzed by immunoblot using the 12CA5E antibody as
described in Fig. 5. Vector, pcDNA3;
K,
deletion of kinase subdomains V to IX; Ig,
immunoglobulin.
Figure 5:
Expression of the human SKY protein
increases the level of cellular phosphotyrosine. QT6 cells transfected
with 20 µg of the various SKY expression constructs were harvested
2 days after transfection, and the lysates were then prepared. A, 50 µg of total cell lysate was electrophoresed through
a 7.5% SDS-PAGE gel, immunoblotted, and probed with an
anti-phosphotyrosine antibody (4G10). B, the remainder of the
lysate was immunoprecipitated with an antibody (12CA5E) recognizing the
HA epitope and protein A-Sepharose. The immunocomplexes were resolved
on a 7.5% SDS-PAGE gel, immunoblotted, and probed with the 12CA5E
antibody. The position of the SKY proteins is indicated, as is the
heavy chain of the 12CA5E immunoglobulin (Ig).
Chemiluminescence was used for detection in both A and B. Vector, pcDNA3; -HA, HA epitope
engineered at the extreme carboxyl terminus of the protein; K, deletion of kinase subdomains V to
IX.
We also measured sky RNA in a variety of mouse tissues. Northern blot hybridization revealed a 3.8-kb transcript that is relatively abundant in the brain, testes, ovaries, and in a 9.5-day-old whole embryo and is detectable in the lung, thymus, and adrenal glands (Fig. 2). The integrity of the RNA was verified by hybridization with the GAPDH probe. Our estimates of sky RNA levels in the lung and spleen may be low since relative levels of GAPDH were lower in these samples, even though equal amounts of total RNA were loaded in each lane (as judged by ethidium bromide staining) (data not shown).
Figure 2: Murine sky RNA is detectable in brain, testes, and ovaries. 30 µg of total RNA isolated from the indicated adult mouse organs (lanes1-12) and from a 9.5-day-old mouse embryo (lane13) was analyzed by Northern blot using a random-primed murine sky cDNA probe. The blot was subsequently hybridized with a rat GAPDH cDNA probe. The sample derived from salivary gland (lane12) appeared to be mostly degraded as visualized by ethidium bromide staining of the agarose gel (data not shown).
Northern blot hybridization with a mouse sky probe showed that, as described above, sky RNA is almost undetectable in normal mammary glands (Fig. 3, lane1). Similarly low levels were observed in hyperplastic glands isolated from the wnt-1 transgenic mice (lanes2-4), suggesting that sky expression is not strictly correlated with a proliferative state. In contrast, sky transcripts were abundant in mammary tumors isolated from the wnt-1 and fgf-3 transgenic and bitransgenic animals (lanes5-10). Analysis of RNAs from 12 tumors ( Fig. 3and data not shown) revealed that, on average, sky mRNA is over 10-fold more abundant in these tumors than in normal mammary glands.
Figure 3:
sky RNA is abundant in mammary
tumors from wnt-1 and fgf-3 transgenic and
bitransgenic mice. Poly(A) RNA isolated from virgin
mammary glands (MG, lane1), hyperplastic
mammary glands (HG) from wnt-1 transgenic mice (lanes2-4), or mammary tumors (MT)
from wnt-1 (lanes5 and 6) and fgf-3 (lanes7 and 8) transgenic
and bitransgenic (lanes9 and 10) mice was
analyzed by Northern blot. The blot was hybridized with a random-primed
murine sky cDNA probe and subsequently with a rat GAPDH cDNA
probe. The blot was quantitated using a PhosphorImager (Molecular
Dynamics). Relative expression is indicated and was calculated relative
to virgin mammary gland and standardized against GAPDH
levels.
To determine if a gene amplification or rearrangement correlated with the increased expression of the sky gene in the tumors, we isolated genomic DNA from the identical tumor samples used in the Northern blot of Fig. 3, digested the genomic DNA separately with several restriction enzymes, and analyzed it by Southern blot. No amplification or rearrangement of the sky gene was evident in DNA from any of the tumors (data not shown).
The fact that 184A1N4-TH cells
overexpress sky prompted us to examine whether, in general,
the introduction of an oncogene into a cell line would lead to an
induction of sky transcripts. We isolated poly(A) RNA from primary MEF, immortalized MEF, and immortalized MEF in
which the oncogenes v-Ha-ras, v-erbB, and an
activated allele of c-src (SrcA) had been introduced
separately. These latter cells were morphologically transformed and
exhibited an elevated level of cellular phosphotyrosine relative to
both the parental cell line and primary MEF cells (data not shown). In
contrast to the HMEC cell lines, however, the introduction of oncogenes
into immortalized MEF did not increase sky transcript levels
when normalized to GAPDH RNA levels (Fig. 4B, compare lanes3-5 with lanes1 and 2). These data suggest that oncogenic transformation is not
sufficient to induce the level of sky expression in all cell
types.
To detect the SKY protein synthesized in the transfected cells,
we engineered an HA epitope tag onto the extreme carboxyl terminus of
the protein. As a control, we created an internal deletion within the
conserved kinase domain of the protein, removing subdomains V to IX
almost entirely, generating a protein, which we term Kinase, that
should be enzymatically inactive. The
Kinase version also was
engineered to contain the HA tag.
Our preliminary experiments were hindered by low SKY protein levels, even though SKY transcripts were detected easily by Northern blot (data not shown). We reasoned that the high GC content of the 5`-end of the SKY cDNA might reduce the translation efficiency of the SKY mRNA, thereby affecting the protein yield. Using synthetic oligonucleotides, we altered the base composition of the first 19 codons of SKY so as to reduce the GC content from approximately 82 to 42% while maintaining the native amino acid sequence. The putative translation initiation sequence (26) upstream of the initial methionine residue was left unchanged. In side-by-side comparisons, these alterations significantly and reproducibly enhanced SKY protein levels after transfection (data not shown).
In our initial assays, we examined the effect of
expression of several SKY constructs on total cellular phosphotyrosine
levels in QT6 cells. An immunoblot of total cell lysates from these
transfectants was probed with an antibody that recognizes
phosphotyrosine residues (Fig. 5A). Expression of the
full-length SKY protein with or without the HA epitope resulted in a
dramatic increase in the overall level of cellular phosphotyrosine
relative to the vector control (Fig. 5A, compare lanes2 and 3 to lane1).
The major phosphorylated species in the lysate appeared to be the SKY
protein itself, since it comigrated at 110,000 daltons with SKY-HA
immunoprecipitated with an antibody against the HA epitope (12CA5E; Fig. 5B, lane3). Cells transfected
with the Kinase version of SKY did not exhibit an increase in
phosphotyrosine levels (Fig. 5A, lanes4 and 5), although this truncated protein was produced, as
judged by immunoprecipitation (Fig. 5B, lane5). Note that the recovery of the
Kinase form of SKY
was more efficient than recovery of the full-length version (Fig. 5B, compare lane3 with 5). We believe this discrepancy was due to a difference in the
accessibility of the HA epitope tag, since the difference in recovery
was not reflected by transcript levels and was not observed when these
proteins were precipitated by concanavalin A-Sepharose (data not
shown). In any event, taken together, these data strongly suggest that
the SKY gene product is a bona fide tyrosine kinase and that, as
predicted, an internal deletion in the kinase domain abrogates kinase
activity.
To test more directly the kinase activity of the SKY
protein, we performed in vitro kinase assays with
immunoprecipitated lysates from transfected QT6 cells. We detected a P-labeled protein of approximately 110,000 daltons in
immunocomplexes from cells transfected with the SKY-HA cDNA (Fig. 6A, lane2) but not from cells
transfected with the
Kinase construct; (lane3)
or with the vector alone (lane1). Therefore, we
conclude that SKY exhibited autophosphorylation activity, and this
activity was dependent on the integrity of the kinase domain. We also
tested for phosphorylation of the synthetic substrate poly(Glu, Tyr).
This exogenous substrate became phosphorylated only in reactions with
SKY (lane5), suggesting that SKY kinase activity is
not limited to autophosphorylation. The
Kinase protein was unable
to phosphorylate poly(Glu,Tyr) (lane6), confirming
that it is enzymatically nonfunctional. Both the full-length and
Kinase form of the SKY protein were synthesized, as examined by
immunoblot analysis (Fig. 6B, lanes2 and 3). We conclude that the SKY gene product possesses
intrinsic tyrosine kinase activity.
Figure 7:
Sky overexpression can transform
fibroblasts. RatB1a fibroblasts were infected with recombinant
retroviruses expressing murine Sky-HA, SkyK-HA, or activated
c-src (SrcA). Uninfected cells were used as a control. 2 days
after infection, cells were passaged and observed for morphology (A), cultured in soft agar and assayed for colony formation
after 12 days (B), and lysed and analyzed for Sky protein
expression by concanavalin A precipitation followed by anti-HA
immunoblot (C). Solidarrows denote the two
Sky-HA species, while brokenarrows highlight the two
Sky
K-HA bands. Molecular weight markers are shown at the left of the figure. Cells also were injected into both rear hind flanks
of two nude mice and assayed for tumor formation, as described in the
text.
K, deletion of kinase subdomains V to
IX.
Infected cells from the same experiment were cultured in soft agar
and assayed for colony formation. RatB1a cells expressing Sky-HA formed
large, multicellular colonies (Fig. 7B, panel2) as did cells expressing SrcA (panel4). Uninfected cells (panel1) and
cells expressing the inactive SkyK-HA (panel3)
formed colonies containing only few cells.
Nude mice inoculated with
Sky-HA-expressing RatB1a cells developed tumors at the site of
injection with a latency of 17 days. Uninfected and SkyK-HA cells
did not form tumors in this time, while SrcA-expressing cells had
visible tumors in 9 days. Expression of the Sky constructs in the
infected RatB1a cells was confirmed by concanavalin A precipitation
(see below) followed by anti-HA immunoblot analysis (Fig. 7C, lanes1-3). Thus,
overexpression of HA-tagged Sky is sufficient to cause cell
transformation and tumor formation.
Figure 8:
SKY is a
glycoprotein. HA-tagged wild-type (SKY-HA) and Kinase (SKY
K-HA) expression constructs (20 µg each) were
transfected into QT6 cells. 2 days after transfection, cells were first
treated for 30 min with the indicated amounts of tunicamycin and then
metabolically labeled with [
S]cysteine in the
presence of tunicamycin. Cell lysates were prepared 4 h after metabolic
labeling and immunoprecipitated with the 12CA5E antibody directed
against the HA epitope. Immunocomplexes were resolved on a 7.5%
SDS-PAGE gel followed by fluorography. Solidarrows indicate positions of wild-type and mutant SKY proteins in the
absence of tunicamycin, and brokenarrows indicate
positions of wild-type and
Kinase SKY proteins after tunicamycin
treatment.
K, deletion of kinase subdomains V to
IX.
These data lead to
several conclusions. First, the presence of both species within the
same immunocomplex strongly suggested that the full-length and
Kinase SKY proteins were part of a heterodimer. Second, this
heterodimer appeared to be functional for autophosphorylation. Both
points were particularly evident in the case in which the
Kinase
subunit was HA-tagged and the full-length subunit was not (lane3). Under these conditions, only heterodimers and
inactive
Kinase homodimers could be immunoprecipitated with the HA
antibody (lane4). Homodimers of the full-length SKY
protein cannot be immunoprecipitated. Therefore, an active heterodimer
must have been responsible for the phosphorylation observed in lane3. We have ruled out the possibility that our results
were due to nonspecific aggregation of full-length and
Kinase SKY
proteins during immunoprecipitation since phosphorylation of both
species was not observed when lysates from cells transfected separately
with SKY-HA and SKY
K-HA were mixed, immunoprecipitated, and
assayed for in vitro kinase activity (data not shown). Taken
together with the data of Fig. 8, these results suggest that SKY
is a dimeric receptor tyrosine kinase. However, our data are also
consistent with the possibility that SKY can exist as a higher order
multimer such as a trimer or tetramer.
In this report, we have shown that the sky gene encodes a glycosylated active tyrosine kinase that most likely functions as a dimer and has several biological features that imply a role for this gene in oncogenesis, especially in the mammary gland. sky is expressed at least 10-fold more abundantly in murine mammary tumors than in preneoplastic and normal mammary glands. Similarly, SKY RNA levels are elevated in a tumorigenic human mammary epithelial cell line but not in its non-tumorigenic progenitors. Furthermore, RatB1a cells overexpressing HA-tagged murine Sky become morphologically transformed, grow as colonies in soft agar, and form tumors when injected into nude mice.
The expression and activity of a number of tyrosine kinases, including both the growth factor receptor and cytoplasmic classes, have been found to be elevated in mammary tumors (cf. (27, 28, 29, 30, 31) ). For example, in approximately 30% of human breast tumors, the HER-2/neu receptor tyrosine kinase gene is amplified and overexpressed(32) , and a large number of human mammary cancers exhibit an increase in the specific kinase activity of c-SRC(33, 34, 35) . It is possible that sky overexpression may override normal growth regulatory controls in the mammary gland and participate directly or indirectly in the formation or development of mammary tumors. The fact that sky transcripts cannot be detected in the preneoplastic glands of wnt-1 transgenic mice suggests that sky expression is induced in the latter stages of the multi-step pathway leading to mammary tumorigenesis. Therefore, the observed increase in sky transcript levels in the transgenic mouse mammary tumors (Fig. 3) most likely is a secondary event, with the primary event being the expression of the introduced oncogene (wnt-1 and/or fgf-3). This supposition is supported by the expression pattern of human SKY in the HMECs (Fig. 4A), in which only tumorigenic, and not simply immortalized, human mammary cells exhibit high levels of SKY RNA. However, our examination of oncogene-transformed MEF cells shows that the introduction of an oncogene is not sufficient to induce expression of sky (Fig. 4B). Nonetheless, overexpression of sky may be sufficient to lead to tumorigenesis since we have observed that RatB1a fibroblasts overexpressing the murine Sky protein are morphologically transformed and form large colonies in soft agar (Fig. 7). It is noteworthy that AXL, the prototype of the ufo/axl family to which Sky belongs, also has been shown to transform cells when overexpressed(36) .
sky expression may be regulated in the mouse mammary gland during pregnancy, lactation, and involution (Fig. 1), suggesting that sky could be involved in normal mammary gland development. However, we have not yet determined the specific mammary cell types in which sky is transcribed; the abundance of such cell types may influence the amount of sky mRNA recovered from glands at a particular stage. The fact that murine sky is expressed in the C57MG cell line predicts that sky will be present in at least some mammary epithelial cells, although expression in other cell types is also possible. In situ hybridization and immunohistochemical studies should clarify this issue.
The SKY protein is a dimeric tyrosine kinase that seems to be constitutively active in transfected cells. There are several possible explanations for this observation. First, since the SKY kinase appears to be a receptor tyrosine kinase, it is possible that the SKY ligand, as yet unidentified, is present in our culture system. This ligand might then induce dimerization and activate the SKY kinase. Alternatively, it is conceivable that the SKY gene product normally is present as a monomer but that when expressed to a high level in our transient system, a regulatory system is bypassed and the kinase is able to dimerize. Interestingly, our in vitro evidence shows that a SKY dimer with only one functional catalytic domain remains active (Fig. 9). These observations raise the question as to whether dimerization of the SKY kinase is required for it to attain full kinase activity. However, these data must be interpreted cautiously, since, in vitro, a comparable epithelial growth factor receptor heterodimer exhibits kinase activity, while in vivo this heterodimer appears non-functional(37) .
Mark et al.(12) also observed constitutive tyrosine kinase activity with an epitope-tagged version of human SKY (RSE). Although these authors did not show formally that SKY was present as a dimer, they did demonstrate that the addition of a monoclonal antibody against the epitope induced tyrosine phosphorylation of the SKY protein, presumably by promoting the formation of activated dimers. Furthermore, in contrast to our HA-tagged version of SKY, which migrates at approximately 110 kDa, these investigators observed both 120- and 140-kDa species of their epitope-tagged SKY protein in 3T3 cells. Mark et al.(12) speculate that the multiple species they observe may represent either proteolytically processed or differentially glycosylated forms of the protein. Interestingly, although we could only detect a single form of SKY in QT6, COS, or 293T cells, we did observe multiple species in infected RatB1a (Fig. 7C) and NIH 3T3 cells (data not shown). These observations suggest that different cell lines may differentially modify the SKY protein.
The expression pattern and transforming capacity of the Sky tyrosine kinase suggest that Sky may play an important role in the development and/or progression of mammary carcinogenesis. The identification of Sky's ligand and downstream substrates will be required to understand the mechanisms by which Sky may promote tumorigenesis.
Note Added in Proof-Lai et al. (Lai, C., Gore, M., and Lemke, G.(1994) Oncogene9, 2567-2578) recently have reported the cloning and characterization of tyro3, which is identical to sky.