©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mouse Mammary Tumors Express Elevated Levels of RNA Encoding the Murine Homolog of SKY, a Putative Receptor Tyrosine Kinase (*)

(Received for publication, October 28, 1994)

Ian C. A. Taylor (1) (2)(§)(¶) Sophie Roy (1) (2)(¶)(**) Paul Yaswen (3)(§§) Martha R. Stampfer (3)(§§) Harold E. Varmus (1) (2)(¶¶)

From the  (1)Department of Microbiology and Immunology, University of California, San Francisco, California 94143, (2)NCI, National Institutes of Health, Bethesda, Maryland 20892, and the (3)Lawrence Berkeley Laboratory, University of California, Berkeley, California 94720

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Protein tyrosine kinases (PTKs) (^1)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.


MATERIALS AND METHODS

Cell Lines and Transfections

QT6 quail fibrosarcoma cells were grown in M-199 media supplemented with 5% fetal bovine serum (FBS), 1% chicken serum, 10% tryptose phosphate broth, and antibiotics. QT6 cells were transfected at approximately 40% confluence in a 100-mm dish by the calcium phosphate precipitation method(13) , with the amount of plasmid DNA stated in the figure legends. Plasmid DNA used in transfections was purified either by banding twice on CsCl density gradients or using Qiagen column chromatography (Qiagen). C57MG mammary epithelial cells were grown in Dulbecco's minimal essential (DME H-21) media supplemented with 10% FBS, 10 µg/ml bovine insulin (Sigma), and antibiotics. Primary and oncogene-transformed mouse embryo fibroblasts (MEF) and 293T and NIH 3T3 cells were grown in DME H-21 media supplemented with 10% FBS and antibiotics. RatB1a fibroblasts were grown in DME H-21 media supplemented with 7.5% calf serum, 2.5% FBS, and antibiotics.

RNA Isolation

Poly(A) RNA was isolated from C57MG cells either by the SDS/proteinase K method (14) or from total RNA prepared by the guanidinium/CsCl method(13) . Total RNA from human mammary epithelial cells (HMEC) (reviewed in (15) ; see also ``Results'') was isolated from subconfluent cultures by the guanidinium/CsCl method(13) . Total RNA from mouse organs and mammary tumors was prepared with RNazol (Cinna/Biotecx Laboratories International) following the manufacturer's instructions. In all cases, oligo(dT)-cellulose (Collaborative Research) was used to select poly(A) RNA. For the experiment depicted in Fig. 1, mammary gland numbers 2 and 4 were dissected from virgin (age 4 weeks), pregnant, lactating, and post-lactating FVB/N female mice. The day of plug was considered day 0 of gestation, the day of birth was considered day 0 of lactation, and the day the pups were weaned was considered day 0 of mammary gland involution.


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 beta-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 beta-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).



cDNA Library Construction

4 µg of C57MG poly(A) RNA was primed with a dT(18)-NotI oligonucleotide (Invitrogen) and converted into cDNA using 800 units of Superscript II reverse transcriptase (Life Technologies, Inc.). Second-strand cDNA was synthesized with 25 units of Escherichia coli DNA polymerase I and 5 units of RNase H (Life Technologies, Inc.). EcoRI adaptors were ligated onto the double-stranded cDNA, and the cDNA subsequently was digested with NotI. Digested cDNA was size selected (geq1 kb) by agarose gel electrophoresis and ligated into the vector pcDNAI (Invitrogen), which was digested with NotI and EcoRI and gel purified twice. The cDNA library was electroporated into strain MC1061/P3. A second C57MG cDNA library was constructed essentially as described above except that BstXI adaptors were used, the vector was pcDNA3 (Invitrogen) digested with NotI and BstXI, and the electroporated strain was JM109. A human ovary cDNA library was constructed as above using commercially available poly(A) RNA (Clontech). The cDNA was blunt-end cloned into pcDNAI that was digested with EcoRV, and the library was electroporated into MC1061/P3.

Identification and Cloning of sky

sky was identified in a PCR-based screen for protein tyrosine kinase genes expressed in the C57MG mammary epithelial cell line. 1 µg of the pcDNAI-based C57MG library was amplified with degenerate primers corresponding to conserved regions of the kinase domain of protein tyrosine kinases. The primers used were 5`-CGGGATCCAC(A/C)G(G/A/T/C)GA(C/T)(C/T)T(G/A/T/C)GC(G/A/T/C)GC(G/A/T/C)(A/C)G-3` and 5`-GGAATTCA(C/T)(G/A/T/C)CC(A/G)(A/ T)A(G/A/T/C)(G/C)(A/T)CCA(G/A/T/C)AC(A/G)TC-3`. The restriction enzyme sites, BamHI and EcoRI, respectively, that were included in these primers are underlined. Amplification conditions were 30 cycles of 1 min, 95 °C; 5 min, 37 °C; and 5 min, 63 °C. The resulting 210-base pair fragment was isolated by agarose gel electrophoresis, digested with BamHI and EcoRI, ligated into the vector pBS (Stratagene) that was digested with the same enzymes, and transformed into JM109. 144 colonies were prepared for double-stranded sequencing of their plasmid DNA. Redundant clones were identified and eliminated by single-base sequencing all PCR inserts by the dideoxy chain termination method using Sequenase (U. S. Biochemical Corp.) and ddTTP. The non-redundant PCR inserts were sequenced in their entirety and compared to Genbank using the BLAST program of the NCBI (National Institutes of Health). One clone was novel and was pursued. The pcDNA3-based C57MG cDNA library was screened using the PCR fragment as a probe and hybridization buffer (500 mM Na(2)HPO(4), 7% SDS, 1 mM EDTA, 0.2% BSA) at 65 °C. A clone containing a 3.6-kb cDNA insert was isolated. This cDNA was sequenced by creating an ExoIII deletion series(16) , and gaps in the sequence were filled using oligonucleotide primers. The human homolog of this cDNA was isolated from the pcDNAI-based human ovary cDNA library using a 1.2-kb BamHI fragment of the mouse cDNA as a probe and hybridization buffer at 55 °C. The human cDNA was sequenced similarly using the chain termination method.

Northern Blot Analysis

Either total or poly(A) RNA from mouse organs, tissues, mammary tumors, or cell lines was resuspended in 1 times gel running buffer (20 mM MOPS, pH 7.0, 8 mM sodium acetate, and 1 mM EDTA, pH 8.0) containing 2% formaldehyde, 35% deionized formamide, and 1 µg/ml ethidium bromide and was electrophoresed on a 1% agarose, 2% formaldehyde gel in 1 times gel running buffer. Gels were blotted onto Hybond N (Amersham Corp.) in 10 times SSC and UV cross-linked with a Stratalinker (Stratagene). For the experiment depicted in Fig. 4A, 10 µg of each total RNA sample was denatured, fractionated by electrophoresis in a 1.3% agarose, 2.2 M formaldehyde slab gel, and capillary blotted onto a nylon filter for hybridization. All hybridizations were performed at 65 °C in hybridization buffer, and blots were washed with hybridization wash buffer (40 mM Na(2)HPO(4), 1 mM EDTA, 0.1% SDS). Probes were prepared with a Rediprime random priming kit (Amersham). Quantitations were performed with a Molecular Dynamics PhosphorImager. Blots were stripped in 70% formamide, 1 times SSC at 65 °C for 60 min with one change.


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.



SKY Epitope Tag and Kinase Deletion Constructions

The hemagglutinin (HA) epitope (YPYDVPDYA) was inserted in-frame via PCR at the extreme 3` terminus of the open reading frame in the human SKY cDNA, using a 3`-oligonucleotide primer complementary to the human SKY cDNA that also encoded the HA epitope and an XbaI restriction site. The 5`-PCR primer ended at base pair 2592 of the human SKY cDNA. The resulting 150-bp PCR fragment was digested with XbaI and Bpu1102I, an internal unique site. This fragment then was used to reconstruct the human SKY cDNA in the pcDNA3 vector. The resulting construct was sequenced to ensure that no mutations were introduced. Since this HA-tagged construct removed the entire 3`-untranslated region of the SKY cDNA, an analogous untagged version of SKY was made by truncating the SKY cDNA at the unique AflII restriction site at bp 3054. An in-frame internal deletion in the conserved kinase domain was created in both the HA-tagged and untagged versions of SKY by removing a BspEI restriction fragment from these constructs and religating. This internal deletion removes amino acids 590-754, inclusive of the SKY open reading frame.

Alteration of Human SKY 5`-End

To reduce the GC content of the 5`-end of the human SKY cDNA, the following complementary oligonucleotides were synthesized (Oligos Etc. Inc.): 5`-AATTCCGCCGATGGCATTAAGAAGAAGTATGGGAAGACCAGGATTACCACCATTACCATTACCACCACCACCGC-3` and 5`-GGTGGTGGTGGTAATGGTAATGGTGGTAATCCTGGTCTTCCCATACTTCTTCTTAATGCCATCGGCGG-3`. When annealed, the duplex creates EcoRI and SacII restriction site overhangs (underlined). This duplex was then ligated into the pcDNA3-based human SKY HA-tagged and untagged constructs at the unique SacII site at bp 70.

Immunoprecipitations and Immunoblots

Transfected QT6 cells were lysed 36-48 h post-transfection in TENT buffer (50 mM Tris, pH 8.0, 2 mM EDTA, 150 mM NaCl, and 1% Triton X-100) or RIPA buffer (50 mM Hepes, pH 7.4, 1% deoxycholic acid, 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 1 mM EDTA). Immunoprecipitations with the anti-HA monoclonal antibody (12CA5E; Babco) were performed by adding 2 µl of 12CA5E ascites fluid to TENT or RIPA lysates and incubating for 1 h at 4 °C. Protein A-Sepharose (Sigma) was added subsequently, and the incubation was allowed to proceed for an additional 30 min. Immunocomplexes were washed with TENT or RIPA buffer, resuspended, and boiled in SDS-electrophoresis sample buffer containing beta-mercaptoethanol. Samples were electrophoresed on a 7.5% SDS-PAGE gel and transferred to nitrocellulose (Schleicher and Schuell) by electroblotting. Blots were blocked in 2% BSA in Tris-buffered saline (TBS) (20 mM Tris, pH 8.0, 150 mM NaCl). All washes were performed with TBS and 0.2% Tween 20 (TBST). 12CA5E was diluted 1:2000 in 1% BSA in TBST, with probing at room temperature for 1 h or overnight at 4 °C. Goat anti-mouse IgG conjugated to peroxidase (Boehringer Mannheim) diluted 1:20,000 in TBST was used as the secondary antibody. Incubations were performed at room temperature for 1 h, and blots were developed by enhanced chemiluminescence (ECL) (Amersham) and exposed to autoradiography film (Hyperfilm, Amersham). For anti-phosphotyrosine analysis, 50-100 µg of whole cell lysate was electrophoresed on a 7.5% SDS-PAGE gel and immunoblotted. Blots were blocked in 2% BSA in TBST. The anti-phosphotyrosine antibody 4G10 was diluted 1:4 in 2% BSA in TBST, with probing and detection identical to the conditions described above for 12CA5E, except that the secondary antibody was diluted in 2% BSA in TBST.

In Vitro Kinase Assay

QT6 lysates were prepared in RIPA buffer and immunoprecipitated with 12CA5E as described above. Immunocomplexes were washed three times with RIPA buffer and two times with in vitro kinase buffer (20 mM Tris, pH 7.4, 10 mM MnCl(2), and 10 µM ATP). For autophosphorylation assays, 10 µCi of [-P]ATP was added, and the reaction was allowed to proceed for 5 min at room temperature. For exogenous substrate reactions, 10 µg of poly(Glu,Tyr) was included. Samples were quenched with electrophoresis sample buffer, boiled, and electrophoresed on a 7.5% (Fig. 9) or 10% (Fig. 6) SDS-PAGE gel. Gels were fixed, dried, and exposed to autoradiography film (Kodak X-AR or Amersham Hyperfilm).


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 DeltaKinase (lane4) SKY expression construct alone or in combination with 10 µg of the untagged versions of the DeltaKinase (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. DeltaK, 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 DeltaKinase (SKYDeltaK-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; DeltaK, 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; DeltaK, deletion of kinase subdomains V to IX.



Retrovirus-mediated Gene Transfer into Fibroblasts

An EcoRI to XbaI (blunted) fragment of murine Sky-HA and SkyDeltaK-HA was ligated into the HindIII (blunted) site of the pSRMSVTKNEO vector(17) . An EcoRI-HindIII fragment from pMV7-SrcA was inserted into the pSRMSVTKNEO vector between its EcoRI and HindIII sites. These constructs were cotransfected into 293T cells with pSV-psi-E-MLV, a construct which expresses the murine leukemia virus ecotropic envelope protein(17) . Virus was collected over 3 days and used to infect RatB1a fibroblasts for 2 h in the presence of 4 µg/ml polybrene. 2 days later, cells were trypsinized and counted. 500 and 5000 cells were assayed for colony formation in soft agar (see below), 250,000 cells were passaged to observe morphology, and 10^6 cells (in 100 µl) were injected into both of the hind flanks of two nude mice to assay for tumor formation. The remaining cells were analyzed for Sky protein expression by lysing in RIPA and precipitating with concanavalin A followed by anti-HA immunoblot analysis. For concanavalin A precipitation, RIPA lysis buffer was supplemented with 2 µg/ml MnCl(2), and the cleared lysates were incubated with 50 µl of concanavalin A-Sepharose for 1 h at 4 °C. Soft agar assays were performed in 6-well dishes. Cells (in 1.25 ml) were mixed 1:1 with 0.7% low melting point agarose (Seaplaque, FMC BioProducts) in complete DME H-21 media and layered over a cushion (2.5 ml) of solidified 0.7% agarose in media. After solidification, the agarose/cell layer was covered with an additional 2.5 ml of 0.7% agarose in media. Cells were incubated at 37 °C for 12 days before photographs were taken.

Tunicamycin Treatment

QT6 cells were transfected as described above. Approximately 48 h post-transfection, cells were pretreated with various concentrations of tunicamycin (Sigma) for 30 min at 37 °C in DME media lacking cysteine. The media was then replaced with DME lacking cysteine, supplemented with 2% dialyzed FBS and 0.4 mCi/ml [S]cysteine (Amersham) and antibiotics. Tunicamycin again was added at various concentrations, and the cells were incubated for 4 h at 37 °C. Cells were harvested and immunoprecipitated as described above, except that the cell lysates were preincubated with protein A-Sepharose beads for 30 min at 4 °C prior to the addition of the 12CA5E antibody and fresh beads.


RESULTS

Identification and Isolation of Murine sky and Human SKY cDNAs

We used PCR to identify receptor tyrosine kinase genes expressed in the mouse mammary epithelial cell line C57MG(9) . To bias our search against members of the src gene family, the upstream degenerate oligonucleotide primer encoded the amino acids HRDLAAR from the conserved region of the kinase domain of tyrosine kinases(2, 18) . The downstream primer encoded the kinase domain residues DVMS(F/Y)G(V/I). A cDNA library constructed from C57MG poly(A) RNA was used as the DNA substrate for the amplification reaction. Sequence analysis of the PCR subclones revealed identities to known receptor tyrosine kinase (platelet-derived growth factor receptor, fibroblast growth factor receptor, epithelial growth factor receptor, insulin-like growth factor receptor, and insulin receptor), to putative receptor kinases encoded by the murine ryk gene and the mouse homolog of the chicken klg gene, as well as to the cytoplasmic tyrosine kinases Csk and Fer. One of the 144 PCR products analyzed appeared to be a fragment of a novel gene and was studied further. Using the PCR product insert as a probe, a 3607-base pair cDNA clone was isolated from the C57MG cDNA library and sequenced. This murine clone was used to probe a cDNA library constructed from human ovary poly(A) RNA, and a 3702-bp human cDNA was isolated and sequenced. During the course of this work, the sequences for both the murine and human cDNAs were published under the names sky(11) and rse(12) . Since sky was the initial designation reported, that convention will be used in this paper.

The sky Gene Is Expressed during Postnatal Mammary Development

As an initial effort to determine the role, if any, of sky in mammary gland development, we examined the expression of this gene in mammary glands isolated from virgin females as well as from females at various stages of pregnancy, lactation, and mammary gland involution (Fig. 1). Northern blot analysis of poly(A) RNA isolated from these glands revealed low levels of sky RNA in mammary glands from virgin females (lane1). sky RNA levels increased slightly during pregnancy (lanes2-4), decreased during lactation (lanes5-8), and achieved highest levels during mammary gland involution (lanes 9-11). To assess the integrity of the RNA and to control for variations in the amounts of RNA loaded, the blot was hybridized with a probe for the housekeeping gene GAPDH. However, since expression of GAPDH is known to decrease during lactation(19) , the blot also was hybridized with a probe for mRNA encoding the secreted milk protein beta-casein. RNA harvested from C57MG cells was included to verify that sky is expressed in the cell-type from which its cDNA was cloned (lane12). The levels of sky, beta-casein, and GAPDH RNA in C57MG cells most closely resemble those observed in mammary glands isolated during the latter stages of involution (compare lane12 to lanes10 and 11), consistent with the fact that this cell line was derived from the glands of a retired breeder C57Bl/6 female mouse(9) .

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).



The Murine sky mRNA Is Abundant in Mouse Mammary Tumors

In view of the fact that expression of sky may be regulated during postnatal mammary gland development (Fig. 1), we asked whether sky RNA levels vary during mammary tumorigenesis. As a model for mammary tumorigenesis, we chose transgenic and bitransgenic mice expressing the wnt-1 and fgf-3 proto-oncogenes in their mammary glands. Mice expressing the wnt-1 transgene have a high risk of developing mammary adenocarcinomas(8) , while fgf-3 transgenic mice develop mammary tumors infrequently(20, 21) . Mice expressing both transgenes develop mammary tumors more frequently and earlier in life than mice carrying either transgene individually(22) . We harvested RNA from mammary tumors isolated from these transgenic and bitransgenic animals. We also purified RNA from wnt-1 transgenic mammary glands exhibiting preneoplastic hyperplasia (all 10 mammary glands of wnt-1 transgenic mice exhibit a marked alveolar and ductal hyperplasia, while only a few glands develop tumors (8) ). The identification of mammary tissue as hyperplastic or tumorous was verified by histological analysis (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).

Human SKY RNA Levels Are Elevated in a Tumorigenic Human Breast Cell Line

Since our results showed that murine sky transcripts are abundant in mammary tumors but not in preneoplastic glands we wished to examine whether the human homolog exhibits an analogous expression pattern at different stages of neoplasia. Normal HMEC (23) derived from reduction mammoplasty tissue have been manipulated to give rise to a series of cell lines that exhibit a progression toward a tumorigenic phenotype (reviewed in (15) ), providing a useful cell culture model for human breast tumors. To generate these lines, an epithelial cell strain with a finite lifespan (termed 184) was treated with the chemical mutagen benzo[a]pyrene to create an immortal but nontumorigenic cell line (184A1; (24) ). When the 184A1 cells were passaged in media lacking specific growth factors, a pseudotriploid nontumorigenic cell line was isolated (184A1N4). Finally, the oncogenes v-Ha-ras and SV40 large T antigen were introduced, resulting in a cell line (184A1N4-TH) that is tumorigenic in nude mice(25) . When we tested RNA isolated from the various HMEC lines by Northern blot (Fig. 4A), human SKY transcripts were detected only in the tumorigenic line 184A1N4-TH (lane4) and not in any of the precursor lines (lanes1-3).

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.

The SKY Gene Product Is an Active Tyrosine Kinase

Although the predicted amino acid sequence of the murine and human SKY gene products strongly suggests that this protein is a tyrosine kinase, biochemical data were required to verify this prediction. We transiently transfected an eukaryotic expression construct of the SKY cDNA into the quail fibrosarcoma cell line QT6. This cell line was chosen due to its high transfection efficiency, which allows for relatively high levels of protein production. Transfections also were performed in COS cells and yielded results identical to those in QT6 cells (data not shown). Although only data derived from experiments with human SKY are presented in this section, identical results were obtained with the murine homolog of SKY.

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 DeltaKinase, that should be enzymatically inactive. The DeltaKinase 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 DeltaKinase 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 DeltaKinase 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 DeltaKinase 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 DeltaKinase protein was unable to phosphorylate poly(Glu,Tyr) (lane6), confirming that it is enzymatically nonfunctional. Both the full-length and DeltaKinase 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.

Overexpression of Murine sky in Fibroblasts Leads to Transformation

Since the Sky tyrosine kinase is constitutively active in transfected QT6 cells, we wished to determine whether overexpression of this kinase in fibroblasts would result in transformation, as has been observed for other receptor tyrosine kinases (reviewed in (5) ). Murine Sky-HA and SkyDeltaK-HA were introduced into RatB1a fibroblasts by retrovirus-mediated gene transfer. Activated c-src (SrcA) was introduced separately as a control. RatB1a cells overexpressing Sky-HA were refractile and adopted a rounded cellular morphology (Fig. 7A, panel2), similar to cells expressing SrcA (panel4). Cells expressing SkyDeltaK-HA (panel3) maintained a flat, cuboidal morphology, identical to that of uninfected control cells (panel1).


Figure 7: Sky overexpression can transform fibroblasts. RatB1a fibroblasts were infected with recombinant retroviruses expressing murine Sky-HA, SkyDeltaK-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 SkyDeltaK-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. DeltaK, 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 SkyDeltaK-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 SkyDeltaK-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.

The SKY Gene Product Is Glycosylated

The conceptual translation of the murine and human SKY cDNAs predicts that the SKY protein possesses a signal sequence and could be glycosylated at up to seven asparagine residues in its putative ectodomain, suggesting that SKY is a cell surface protein. To test for N-linked glycosylation of SKY, we metabolically labeled transfected QT6 cells in the presence of increasing concentrations of tunicamycin, a drug known to block the formation of protein N-glycosidic linkages. In these experiments, we transfected the HA-tagged full-length (SKY-HA) and deleted kinase domain (SKYDeltaK-HA) versions of SKY. In the absence of tunicamycin, the SKY-HA protein migrated at approximately 110 kDa (Fig. 8, lane1). When tunicamycin was added during the radiolabeling, the SKY-HA protein migrated at approximately 95 kDa (lanes2-4). We observed a similar shift in migration pattern, from approximately 90 to 75 kDa, when the DeltaKinase-HA form was radiolabeled in the presence of tunicamycin (lanes5-8). In addition, both the full-length and DeltaKinase forms of SKY were precipitable with concanavalin A-Sepharose (data not shown). Taken together, these data suggest strongly that the SKY protein normally is glycosylated as it traverses the secretory pathway to the cell surface.


Figure 8: SKY is a glycoprotein. HA-tagged wild-type (SKY-HA) and DeltaKinase (SKYDeltaK-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 DeltaKinase SKY proteins after tunicamycin treatment. DeltaK, deletion of kinase subdomains V to IX.



The SKY Tyrosine Kinase Functions as a Dimer

It is known that many receptor kinases function as dimers, especially in the presence of their ligands(6) . To address whether SKY could function as a dimer, we performed in vitro kinase assays with immunoprecipitates from lysates of QT6 cells cotransfected with the HA-tagged SKY and the untagged DeltaKinase (SKY-HA + SKYDeltaK) and the converse combination (SKY + SKYDeltaK-HA). As controls, SKY-HA and SKYDeltaK-HA were transfected alone. When immunocomplexes from cells cotransfected with both the full-length and DeltaKinase version of SKY were incubated with [-P]ATP, both forms of SKY became radiolabeled (Fig. 9, lanes2 and 3). As expected, the autophosphorylated SKY protein migrates at 110,000 daltons (lane1), and no radiolabeled band was observed from the immunocomplex with the DeltaKinase-HA form alone (lane4), although immunoblot analysis confirmed that the protein was synthesized (data not shown).

These data lead to several conclusions. First, the presence of both species within the same immunocomplex strongly suggested that the full-length and DeltaKinase 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 DeltaKinase subunit was HA-tagged and the full-length subunit was not (lane3). Under these conditions, only heterodimers and inactive DeltaKinase 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 DeltaKinase SKY proteins during immunoprecipitation since phosphorylation of both species was not observed when lysates from cells transfected separately with SKY-HA and SKYDeltaK-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.


DISCUSSION

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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant CA-39832. 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.

§
A Damon Runyan-Walter Winchell Cancer Research Fund Fellow.

Contributed equally to this work.

**
Supported by a Centennial Fellowship from the Medical Research Council of Canada.

§§
Received support from National Institutes of Health Grants CA-54247 and CA-24844 as well as from the Office of Energy Research, Office of Health and Environmental Research, U. S. Dept. of Energy under Contract DE-AC0376SF00098.

¶¶
Formerly an American Cancer Society Research Professor. To whom correspondence should be addressed: NCI, National Institutes of Health, Bldg. 49, Rm. 4A56, 9000 Rockville Pike, Bethesda, MD 20892. Tel.: 301-496-0429; Fax: 301-496-0332.

(^1)
The abbreviations used are: PTKs, protein tyrosine kinases; PCR, polymerase chain reaction; HA, hemagglutinin; FBS, fetal bovine serum; MEF, mouse embryo fibroblasts; HMEC, human mammary epithelial cells; DME media, Dulbecco's minimal essential media; bp, base pair(s); kb, kilobase(s); MOPS, 4-morpholinepropanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.


ACKNOWLEDGEMENTS

We thank Lucy Godley, Linda Yuschenkoff, and Helen Kwan for assistance in dissecting mouse organs and mammary tumors; Ken Kaplan for primary, immortalized, and oncogene-transformed mouse embryo fibroblast cells; Martin Scott and David Baltimore for 293T cells; Daniel Afar and Owen Witte for retroviral vectors; Caroline Alexander and John Ashkenas for the beta-casein probe; Don Ganem for support; Don Macrae for computer advice; Steve Chuck for helpful comments on the manuscript; and the surviving members of the Varmus lab at UCSF for many useful discussions.

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.


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