Characterization of mice deficient in the Src family nonreceptor tyrosine kinase Frk/rak.

Abstract

Frk/rak belongs to a novel family of Src kinases with epithelial tissue-specific expression. Although developmental expression patterns and functional overexpression in vitro have associated these kinases with growth suppression and differentiation, their physiological functions remain largely unknown. We therefore generated mice carrying a null mutation in iyk, the mouse homolog of Frk/rak. We report here that frk/rak(-/-) mice are viable, show similar growth rates to wild-type animals, and are fertile. Furthermore, a 2-year study of health and survival did not identify differences in the incidence and spectrum of spontaneous tumors or provide evidence of hyperplasias in frk/rak(-/-) epithelial tissues. Histological analysis of organs failed to reveal any morphological changes in epithelial tissues that normally express high levels of Frk/rak. Ultrastructural analysis of intestinal enterocytes did not identify defects in brush border morphology or structural polarization, demonstrating that Frk/rak is dispensable for intestinal cytodifferentiation. Additionally, frk/rak-null mice do not display altered sensitivity to intestinal damage induced by ionizing radiation. cDNA microarray analysis revealed an increase in c-src expression and identified subtle changes in the expression of genes regulated by thyroid hormones. Significant decreases in the circulating levels of T3 but not T4 hormone are consistent with this observation and reminiscent of euthyroid sick syndrome, a stress-associated clinical condition.

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MOLECULAR AND CELLULAR BIOLOGY, July 2002, p. 5235–5247
0270-7306/02/$04.00?0 DOI: 10.1128/MCB.22.14.5235–5247.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 22, No. 14
Characterization of Mice Deficient in the Src Family Nonreceptor
Tyrosine Kinase Frk/rak
Subhashini Chandrasekharan,1,2† Ting Hu Qiu,2Nawal Alkharouf,2Kelly Brantley,2
James B. Mitchell,3and Edison T. Liu2*
Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
275991; Section of Cell Signaling and Oncogenesis, Division of Clinical Sciences, National Cancer Institute, National
Institutes of Health, Gaithersburg, Maryland 208772; and Radiation Biology Branch, National Cancer Institute,
National Institutes of Health, Bethesda, Maryland 208923
Received 12 November 2001/Returned for modification 14 January 2002/Accepted 13 March 2002
Frk/rak belongs to a novel family of Src kinases with epithelial tissue-specific expression. Although devel-
opmental expression patterns and functional overexpression in vitro have associated these kinases with growth
suppression and differentiation, their physiological functions remain largely unknown. We therefore generated
mice carrying a null mutation in iyk, the mouse homolog of Frk/rak. We report here that frk/rak?/?mice are
viable, show similar growth rates to wild-type animals, and are fertile. Furthermore, a 2-year study of health
and survival did not identify differences in the incidence and spectrum of spontaneous tumors or provide
evidence of hyperplasias in frk/rak?/?epithelial tissues. Histological analysis of organs failed to reveal any
morphological changes in epithelial tissues that normally express high levels of Frk/rak. Ultrastructural
analysis of intestinal enterocytes did not identify defects in brush border morphology or structural polariza-
tion, demonstrating that Frk/rak is dispensable for intestinal cytodifferentiation. Additionally, frk/rak-null
mice do not display altered sensitivity to intestinal damage induced by ionizing radiation. cDNA microarray
analysis revealed an increase in c-src expression and identified subtle changes in the expression of genes
regulated by thyroid hormones. Significant decreases in the circulating levels of T3 but not T4 hormone are
consistent with this observation and reminiscent of euthyroid sick syndrome, a stress-associated clinical
condition.
Protein tyrosine kinases (PTKs) are a large and diverse
multigene family evolved to perform functions that regulate a
range of cellular processes, including cell growth, differentia-
tion, death, motility, adhesion, and cell-to-cell communication
in multicellular organisms. PTKs can be broadly classified into
two groups, receptor tyrosine kinases (RTKs) and nonreceptor
tyrosine kinases (NRTKs), based on their intracellular loca-
tions. RTKs, examples of which include EGFR and PDGFR,
have transmembrane domains, which allow them to be integral
parts of the plasma membrane and extracellular domains,
which are exposed to the external milieu. The extracellular
domains bind specific ligands (or growth factors), resulting in
activation of the intracellular catalytic kinase domain. The
second group of kinases, the NRTKS, possess only the intra-
cellular kinase domain and are usually targeted to the cyto-
plasmic face of the plasma membrane by N-terminal posttrans-
lational modifications. The Src family kinases (SFKs) are a
group of related NRTKs, the prototype of which, c-src, was
identified as the normal cellular counterpart of the transform-
ing protein v-src from the oncogenic Rous sarcoma virus (45).
c-src encodes a 60-kDa phosphoprotein with kinase activity
that is expressed ubiquitously (25). Based on amino acid se-
quence homology and structural similarity, 7 additional mem-
bers of this family, Fyn, Yes, Fgr, Hck, Lck, Blk, and Lyn have
been isolated. The prototypical SFKs are composed of six
structurally and functionally distinct domains called Src homol-
ogy (SH) domains, which are characterized by specific se-
quence motifs and regulatory residues (8). The modular struc-
ture of the SFKs allows them to interact with a diverse group
of proteins, creating highly complex signal transduction net-
works. SFKs are activated by a variety of extracellular signals
and regulate an equally extensive set of cellular functions.
SFKs have been demonstrated to be key downstream elements
in signaling pathways emerging from RTKs, integrins, cad-
herins, G protein-coupled receptors, GPI-linked receptors,
voltage- (Ca2?and K?) and ligand-gated channels, cytokine
receptors, and immune recognition receptors (1, 49). Cellular
functions regulated by SFKs include mitosis, cell spreading,
adhesion, motility, cell death, survival, and differentiation (49).
Further division of the SFKs into groups A and B has been
suggested based on their tissue expression profiles. Group A
includes Src, Fyn, Hck, and Yes, which are expressed ubiqui-
tously, although with various levels in different cell types. In
contrast, SFK group B, consisting of Hck, Fgr, Lck, Blk, and
Lyn, is expressed primarily in hematopoietic cells (42).
Less is known about the function of a third group of SFKs,
the Frk/rak family of kinases. To date, two genes, frk/rak and
brk, which share 60% amino acid homology with one another,
have been assigned to this family. These kinases are expressed
primarily in epithelial tissues and show the highest homology
of ?50% at the amino acid level to Fyn. The first member of
* Corresponding author. Present address: Genome Institute of Sin-
gapore, 1 Capricorn 05-01, Science Park II, Singapore 117528, Singa-
pore. Phone: 65-68275212. Fax: 65-68275202. E-mail: gisliue@nus.edu
.sg.
† Present address: Department of Cystic Fibrosis and Pulmonary
Research, University of North Carolina at Chapel Hill, Chapel Hill,
NC 27599-7248.
5235

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this family, Rak, was identified in human breast tumors and the
mammary epithelial cell line 600PE in a screen for novel ty-
rosine kinases expressed in breast cancer. The complete cDNA
was isolated from the BT-20 cDNA library, and analysis of the
primary sequence revealed ?51% identity to Fyn (9). The
identical kinase was independently isolated from a human hep-
atoma cell line (24) and named Frk, an acronym for fyn-related
kinase. Chromosomal localization studies reveal that fyn and
frk/rak are in fact linked genes located within 1.3 centimorgans
of each other on chromosome 6q (11; unpublished data). The
mouse homolog iyk, an acronym for intestinal tyrosine kinase,
was identified as a novel PTK in a screen for SFKs expressed
in resting mature mouse mammary glands. The complete
cDNA was isolated from 31E-mouse epithelial cells encoding a
57-kDa protein with 89% identity at the amino acid level to
Frk/rak (50). This gene was also independently isolated from
the mouse insulin-producing cell line ?-TC1 and therefore
named bsk, i.e., beta-cell src-like kinase (37). gtk, an acronym
for gut tyrosine kinase, is the rat homolog of frk/rak and iyk and
was identified as a novel src-like kinase gene expressed in rat
intestinal epithelial cells. The primary structure of Gtk has a
49% homology to fyn and 88% identity to Frk/rak at the amino
acid level (16). In the interest of simplicity, both the human
gene and its mouse and rat homologs will be referred to as
frk/rak in this report. A related gene, brk, breast tumor kinase,
was identified as a novel src-like kinase expressed in metastatic
human breast cancer (32, 33). brk encodes a 52-kDa protein
with ?60% homology to Frk/rak and ?40% homology to Fyn
and Src. The mouse homologue of this kinase, sik, was isolated
from a mouse duodenum cDNA library and encodes a 52-kDa
protein with 80% identity to Brk (53).
Similar to all members of the SFKs, Frk/rak kinases possess
the classic SH domain structure and conserved autoregulatory
tyrosine residues in their catalytic domains. However, they
differ significantly in certain structural features, the most strik-
ing of which is the presence of a putative bipartite nuclear
localization signal (KRXXXXXFFXXRRR motif) in the SH2
domain. This consensus nuclear localization signal motif is
present both in the mouse and rat homologs but not retained
in Brk (and its mouse homologue Sik). Consistent with the
suggestion that the subcellular localization of these kinases
may differ from that of other SFKs, the critical glycine residue
in the consensus myristoylation motif MGXXXS/T, necessary
for the conjugation of myristate and plasma membrane target-
ing is absent in human Frk/rak (19). The conserved serine
residue (S) is replaced with a glutamine (Q) in Frk/rak at
position 6. Furthermore, the consensus motif required for
palmitylation (CXXC or CXC) is only partially retained.
Taken together, these findings suggest that Frk/rak is unlikely
to be targeted to the plasma membrane. Recently, it has been
demonstrated that Frk/rak protein is in fact localized to the
juxtanuclear region in a number of epithelial cell lines, colo-
calizing with markers specific to the Golgi (58,000 molecular
weight [58K]) and ?-tubulin centrosomal compartments (C. D.
Carter, L. D. Miller, and E. T. Liu, unpublished data). The
consensus myristoylation sequences are however retained in
the mouse and rat homologs (Iyk and Gtk), suggesting poten-
tial membrane targeting. In support of this, Gtk is found to be
myristoylated in vitro and high kinase activity is detected in the
membrane fraction of intestinal epithelial cells, suggesting lo-
calization to the plasma membrane (16, 17). Additional distin-
guishing structural features of Frk/rak are, a 7-amino-acid in-
sert in the SH3 domain (also present in the mouse and rat
homologs, Iyk and Gtk) and a conserved DLAARN motif in
subdomain VI of the kinase domain found in both members of
the family.
The expression profile of Frk/rak and its homologs shows
considerable similarity. The expression of frk/rak is higher in
epithelial tissues and cell lines, with the highest expression
detected in the kidney and liver (9, 11; C. D. Carter and E. T.
Liu, unpublished data). In the mouse, the expression of frk/rak
mRNA was detected primarily in epithelial organs, with high-
est expression in the intestine, particularly the jejunum and
ileum (37, 50). During embryonic development of the rat,
expression of Frk/rak in the gut started to increase on day 11
and peaked between days 15 and 18, concomitant with the
conversion of the multilayered undifferentiated epithelium to a
polarized monolayer (16). Similarly, the highest expression of
brk and its homolog sik are detected in different regions of the
gastrointestinal tract, i.e., the stomach, duodenum, and colon
(26, 35)
To date, attempts to assign a function to Frk/rak have relied
mainly on the study of intracellular localization and the impact
of functional overexpression of the gene on growth and differ-
entiation in various cell lines. Surprisingly, and in contrast to
prototypical SFKs, overexpression of Frk/rak in a number of
cell lines of epithelial and mesenchymal origin resulted in a
potent growth arrest (10, 11; R. Craven and L. D. Miller,
unpublished observations). Inducible expression of Frk/rak in
HeLa cells results in growth suppression, potentially due to an
arrest in the G1phase of the cell cycle (Carter et al, unpub-
lished data). A putative mechanism is suggested by the obser-
vation that Frk/rak was found to associate with hyperphospho-
rylated and hypophosphorylated forms of retinoblastoma
during the G1and S phases of the cell cycle in vitro (11). The
expression of Frk/rak protein is highest in the G1and S phases
of the cell cycle and dramatically reduced during mitosis (10,
11). Frk/rak is expressed at the centrosomes during all stages in
the cell cycle except mitosis, and expression reappears during
the late telophase, suggesting a potential role for Frk/rak in
centrosome structure and/or function, particularly in the con-
text of regulation of cell division (C. D. Carter and E. T. Liu,
unpublished data). Overexpression of constitutively activated
forms of mouse Frk/rak results in suppression of growth of
NIH 3T3 fibroblasts and RINm5F pancreatic cells. Cell cycle
analysis studies indicate that activated Frk/rak suppresses
growth by inducing a G1arrest, possibly by preventing entry
into the S phase of the cell cycle (3, 38). Interestingly, an
increase in the levels of the cdk inhibitor p27 is associated with
this growth arrest. Increases in p27 levels have been associated
with the differentiation of various cell types and is potentially
required for maintenance of the differentiated state (13, 51). A
recent study demonstrated increased nerve growth factor-in-
dependent neurite growth in PC-12 cells stably expressing wild-
type mouse Frk/rak, a process reminiscent of neuronal differ-
entiation (2). The expression of Brk mRNA and protein
increases during confluence-induced differentiation of CaCo-2
intestinal epithelial cells (26). The murine homolog, Sik, is
rapidly activated upon Ca2?-induced differentiation of mouse
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Page 3

primary keratinocytes, and functional overexpression of wild-
type Sik in EMK cells increased the expression of fillagrin, a
marker of keratinocyte differentiation (54). Interestingly,
genes in invertebrate organisms with the highest similarity to
this family of Frk/rak-related kinases are srk1, which mediates
regeneration in fresh water sponges (39), stk1 in Hydra (7), and
dsrc41, a src-like kinase that regulates ectodermal differentia-
tion in flies (47). Human frk/rak is localized to 6q21-23 (11), a
region that undergoes loss of heterozygosity in 30% of breast
cancers and ovarian carcinomas (23, 43). This region has also
been defined as a senescence locus by using microcell-medi-
ated chromosome transfer experiments in breast cancer cell
lines (35). These observations, in conjunction with in vitro
growth-suppressive functions, suggest that frk/rak is potentially
a novel tumor suppressor gene for epithelial cancers regulating
growth and/or morphological differentiation of epithelial cells.
In order to investigate these possibilities, we decided to
elucidate the physiological function of Frk/rak using gene dis-
ruption. In this study we present the characterization of mice
bearing a targeted mutation in the mouse frk/rak gene. We
demonstrate that the frk/rak-null mice are viable and do not
show any histological abnormalities in epithelial tissues or de-
velop any pathological and/or metabolic disorders associated
with the failure of epithelial organs. Analysis of the tumor
spectrum in animals observed for over 2 years did not reveal
increased incidence of spontaneous tumors in epithelial or-
gans. No abnormalities were detected in the cellular morphol-
ogy or the polarization of intestinal epithelial cells. We also
assessed the survival of frk/rak-null mice following radiation-
induced intestinal damage and found that frk/rak-null mice do
not have an altered sensitivity to ionizing radiation. Finally,
using large-scale cDNA microarray expression analysis, we
have identified sets of differentially regulated genes, which led
to the discovery of a subtle physiological defect in frk/rak-null
mice resembling euthyroid sick syndrome (ESS).
MATERIALS AND METHODS
Generation of frk/rak-null mice. Genomic clones for frk/rak were isolated from
a 129 Sv/Ev mouse genomic DNA library, kindly provided by David C. Lee
(University of North Carolina—Chapel Hill, Chapel Hill). Libraries were
screened with a 1.1-kb murine frk/rak cDNA probe that encodes the entire open
reading frame, except the kinase domain, to avoid cross-reaction with other
SFKs. A 13.5-kb genomic clone containing exons 1a and b, which encode the 5?
untranslated region and the N-terminal and SH3 domains, respectively, was used
to generate the two flanking arms. The 4.5- and 3.8-kb arms were cloned into the
targeting vector pJNS2, kindly provided by Beverly H. Kolller (University of
North Carolina—Chapel Hill), on either side of the neomycin phosphotransfer-
ase cassette (see Fig. 2A). The linearized targeting vector was electroporated
into E14TG2a embryonic stem (ES) cells, and then they were placed under
neomycin and ganciclovir selection to obtain 64 resistant clones (34). ES clones
were analyzed by Southern blotting with a [32P]dCTP labeled 1.8-kb genomic
fragment (see Fig. 2A) and a neomycin cDNA probe to confirm correct targeting.
Two of the three targeted ES clones identified were injected into 3.5-day-old
C57BL/6J blastocysts and implanted in C57BL/6J pseudopregnant mothers to
generate chimeras (34). Chimeras were bred with C57BL/6J males and females
(Charles River Laboratories) for germ line transmission of the targeted allele.
Transmitting chimeras (males) were also bred to 129 Sv/Ev females (Taconic,
Germantown, N.Y.) to establish heterozygotes in the 129 inbred genetic back-
ground. Heterozygotes obtained in the 129-BL/6 mixed genetic and 129 inbred
backgrounds were intercrossed to generate ?/?, ?/?, ?/? mice. All the analysis
described in this report was done with cohorts of mice maintained on the
129-BL/6 mixed genetic background
Genotyping of mice. DNA was isolated from 1-cm-long tail biopsies of weaned
mice by using the high-salt precipitation method for Southern blotting (31) or the
QIAmp tail DNA isolation kit (Qiagen, Inc.) for PCR screening. For Southern
blotting, genomic DNA was digested with EcoRV and electrophoresed on 0.8%
agarose gels. DNA was transferred to nylon Hybond membranes (Amersham
Ltd.) by capillary transfer and cross-linked to the membranes by UV cross-
linking in a Stratalinker (Stratagene, La Jolla, Calif.). Membranes were probed
with a [32P]dCTP random primer-labeled 1.8-kb Kpn-EcoRV genomic fragment
(5? end probe) (see Fig 2A). The primer pairs used for PCR-based genotyping
are sense primer iyk 1 (5?-CACCATGGGCAGCGTCTGTGTGAGA-3?) and
antisense primer iyk 12 (5?-CGCCACGTAATTGGAAGGAATGTAG-3?),
which generate a 343-bp band corresponding to the wild-type allele. The primer
pair consisting of sense primer pNeo1 (5?-TGCTCGACGACGTTGTCACT-3?)
and antisense primer pNeo3 (5?-TCATCCTGATCGACAAGACC-3?) was used
to generate a 196-bp band corresponding to the targeted allele. All PCRs for
genotyping were performed with the Taq PCR master mix kit (Qiagen Ltd.).
RNA isolation and Northern analysis. The organs of interest were isolated and
snap frozen in liquid nitrogen. Total RNA was isolated from frozen organs
homogenized in Trizol reagent (GIBCO-BRL Ltd.) according to the manufac-
turer’s protocol. Twenty micrograms of total RNA was electrophoresed on 1.2%
formaldehyde–agarose gels and then transferred to Hybond nylon membranes
(Amersham Ltd.) by capillary transfer. Blots were probed with [32P]dCTP-la-
beled full-length murine frk/rak, fyn, and src cDNA probes and PCR-amplified
murine c-yes and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA
probes. Membranes were hybridized in Express Hyb (Clontech) at 68°C over-
night, washed in 2? SSC (1? SSC is 0.15 M NaCl plus 0.015 M sodium citrate)
and 0.1% sodium dodecyl sulfate (SDS) at room temperature, and washed in
0.1? SSC and 0.1% SDS at 50°C. Membranes were then exposed to XO-
MAT-AR (Kodak) autoradiography film. Autoradiographs were scanned, and
quantitation of the signal was performed with National Institutes of Health
(NIH) IMAGE, version 6.2, software to obtain normalized ratios of expression
for SFK genes. The average value of change in SFK gene expression for three
frk/rak-null mice compared to age-matched wild-type controls was calculated.
Western blotting analysis. Total protein from tissues of interest was obtained
by homogenization with a tissue homogenizer (Fisher Scientific Ltd.) in lysis
buffer (20 mM HEPES, 0.15 M NaCl, 2 mM EDTA, 2 mM EGTA, 1% Triton
X-100, 500 ?M sodium orthovanadate, 50 ?M sodium molybdate, 10 ?g of
aprotinin/ml, 10 mM sodium fluoride, 10 ?g of leupeptin/ml). Lysates were
electrophoresed on SDS–10% polyacrylamide gel electrophoresis (PAGE) gels,
transferred to polyvinylidene difluoride membranes (Millipore), and probed with
polyclonal rabbit antibodies directed against the SH2 and SH3 domains of
murine Frk/rak (gift of Michael Welsh, Uppsala University, Uppsala, Sweden).
Rabbit polyclonal antibodies directed against beta-casein were used at a 1:500
dilution (gift of Nancy Hynes, Friedrich Meischer Institute, Basel, Switzerland).
Sheep anti-rabbit secondary antibodies (Amersham Ltd.) were used at a 1:5,000
dilution and membranes were developed using the enhanced chemiluminescence
(ECL) reagent (Amersham Ltd.). Membranes were then exposed to Hyperfilm
(Amersham Ltd.) for the detection of the signal.
Histological analysis and electron microscopy. All organs were fixed in 10%
normal-buffered formalin overnight; embedded in paraffin and sectioned at 4-mi-
cron thickness (American Histolabs, Gaithersburg, Md.). Sections were stained
with hematoxylin and eosin. Histological analysis of all tissues was performed by
Miriam Anver, National Cancer Institute (NCI), Frederick Cancer Research and
Development Center, Frederick, Md. Tissue preparation for electron micro-
scopic ultrastructural studies was performed as described previously (14). Pieces
of freshly isolated mouse jejunum were initially fixed in 4% paraformaldehyde
and 2% glutaraldehyde in phosphate-buffered saline (pH 7.4). The tissues were
postfixed, dehydrated, and embedded in epoxy resin. The cured embedded
blocks were sectioned at approximately 50 to 60 nm with the Ultracut microtome
(Leica, Bannockburn, Ill.). Thin sections were mounted on a naked copper mesh
grid and stained with uranyl acetate and lead citrate solution to enhance the
contrast. Sections were examined and photographed with an H7000 electron
microscope (Hitachi, Tokyo, Japan) operated at 75 kV. All transmission electron
microscopy (TEM) and scanning electron microscopy (SEM) analyses were per-
formed by Kunio Nagashima, NCI, Frederick Cancer Research and Develop-
ment Center, NIH.
Irradiation of animals for intestinal injury. Cohorts of 9.5- to 11-week-old
males and females comprising all three genotypes were obtained by heterozygous
intercrosses and housed under normal conditions. Animals were placed in Lucite
boxes (five at a time) and exposed to a single dose of 9, 10, 11, or 12 Gy of
whole-body gamma-irradiation delivered from a cesium-137 irradiator at a rate
of 1 Gy/min. Animals were observed for 10 days and monitored daily for health
and mortality. The number of surviving animals of each genotype was recorded
on day 10 postirradiation to calculate the LD50/10survival percentages. LD50/10
is defined as the dose that results in 50% mortality of animals in 10 days.
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Radiation injury and survival experiments were performed in triplicate for the
10- and 11-Gy doses and in duplicate for the 9-Gy dose with 6 to 12 animal
cohorts per genotype. The day 10 percent survival per dose represents the
average value with standard error for all experiments.
Statistical analysis. All statistical analysis for the radiation survival experi-
ments was kindly performed by Seth M. Steinberg, Biostatistics and Data Man-
agement Section, Center for Cancer Research, NCI. The comparisons of the
fractions of wild-type, heterozygous, and mutant frk/rak animals, which did not
survive past 10 days, were done with the chi-square test or Fisher’s exact test as
appropriate. All P values indicated are two tailed. The Kaplan-Meir method was
used for the calculation of the median survival of mice.
cDNA microarray expression analysis. The organs of interest were isolated
from 3- to 4-month-old frk/rak?/?and frk/rak?/?littermate males maintained
under normal conditions and frozen immediately in liquid nitrogen. Frozen
tissues were homogenized in Trizol reagent (GIBCO-BRL Ltd), and total
RNA was isolated. In order to control for variability between animals and
increase reproducibility, comparisons were performed for four pairs of frk/
rak?/?and frk/rak?/?littermates and in triplicate for each pair (including a
reciprocal experiment). Three micrograms of total RNA was used for RNA
amplification performed with the modified method of Phillips and Eberwine
(41). Briefly, first-strand cDNA synthesis was performed with Superscript II
reverse transcriptase (GIBCO-BRL) and a T7-oligo(dT) primer and second-
strand cDNA was synthesized with DNA polymerase I. In vitro transcription
to amplify RNA was performed with the T7 Megascript kit (Ambion Ltd.) by
following the manufacturer’s instructions. Ten micrograms of amplified RNA
from each sample (frk/rak?/?or frk/rak?/?) was reverse transcribed, incor-
porating either Cy3 or Cy5 dUTP (NEN) nucleotides to generate fluores-
cently labeled probes. The 2,700 cDNA element mouse expression microar-
rays used in the experiments described here were printed at the NCI array
facility, Advanced Technology Center, NCI, NIH. The Cy3- and Cy5-labeled
probes (corresponding to frk/rak?/?and frk/rak?/?samples) were mixed
together, denatured at 100°C, and applied to the chips in hybridization buffer
(25% formamide, 5? SSC, and 0.1% SDS). Microarrays were hybridized
overnight at 42°C, washed sequentially in 2? SSC, 0.1% SDS, 1? SSC, 0.1%
SDS, 0.2? SSC, and 0.5? SSC, air dried, and scanned at wavelengths corre-
sponding to the two fluorochromes with a Genepix 400A microarray scanner
(Axon Instruments, Union City, Calif.). The intensities of the Cy3 and Cy5
signals for every feature on the array were recorded, and normalized signal
ratios (after background subtraction) were obtained for each element with
the GenePix Pro 3.0 microarray analysis software (Axon Instruments). The
complete data set for each array was deposited into the NCI microarray
database, maintained by the Center for Information Technology, NCI, NIH.
All genes that displayed a concordant change in expression of 1.2-fold (base-
line changes) or more and, conversely, of 0.8-fold and below (calculated as
the normalized ratio of expression in frk/rak?/?to frk/rak?/?) in at least two
of the three pairwise comparisons per animal pair were selected to generate
a primary list of potential Frk/rak-responsive candidates. The primary data-
base of all potential candidates was analyzed to identify genes that consis-
tently displayed a change in expression of 1.5-fold or more in at least three
out of four frk/rak?/?animals. All higher level analysis, such as comparison
of expression patterns of genes in multiple arrays or over multiple tissues and
organs, was performed with web-based multiarray analytical tools available
and custom bioinformatics tools developed in the laboratory.
Measurement of thyroid hormone levels. Two- and 3-month-old frk/rak?/?
and frk/rak?/?littermates were starved overnight, and whole blood was collected
by cardiac puncture. Blood was allowed to clot at room temperature for at least
1 h, and serum was collected after centrifugation at 10,000 ? g for 10 min at 4°C.
The total circulating levels of the thyroid hormones T3, T4, and thyrotropin
(TSH) (free and bound fractions) were quantitated by enzyme-linked immuno-
assay (AniLytics, Inc., Gaithersburg, Md.). To assess statistical significance, a
Wilcoxon signed rank test was performed on the sibling-paired difference be-
tween the log values of total circulating levels of T3 (and T4) in frk/rak?/?and
matched frk/rak-null littermates. Statistical analysis was kindly performed by
Dominic T. Moore at the Biostatistics Shared Resources Group, Lineberger
Comprehensive Cancer Center, University of North Carolina—Chapel Hill.
RESULTS
Analysis of Frk/rak expression in mouse tissues. Previous
studies indicate that frk/rak expression is restricted to tissues of
epithelial origin. To extend these studies and identify tissues
that are likely to be affected by loss of frk/rak, we examined
Frk/rak protein expression in a panel of adult tissues. The
highest levels of Frk/rak protein were detected in the gastro-
intestinal tract and the pancreas. Frk/rak could also be easily
detected, albeit at lower levels, in the kidney, ovary, lung, and
liver. Levels of Frk/rak were extremely low in the whole brain,
virgin breast, and spleen, and no Frk/rak could be detected in
the heart (Fig. 1A). While Frk/rak is barely detectable in the
prepubescent and resting virgin mammary gland, Frk/rak pro-
tein levels increase during pregnancy (Fig. 1B and C), with
expression peaking at day 16 of pregnancy. Maximal Frk/rak
expression is detected after 24 h of involution, and levels return
to those detected in the virgin and resting gland by day 5 of
involution.
Analysis of frk/rak-null mice. We designed a targeting con-
struct that, upon homologous recombination with the wild-type
frk/rak allele, results in replacement of exons 1a and b with the
neomycin gene. As exon 1b encodes the 5? untranslated region,
the translation initiation codon, N-terminal sequences, and the
complete SH4 and SH3 domains of Frk/rak, we expected that
this recombination event would generate a null frk/rak allele
(Fig. 2A). Mouse lines were derived from two ES cell clones
carrying the frk/rak-null allele. No differences were detected in
the phenotypes of these two lines. Heterozygous males and
females exhibited no obvious abnormalities. frk/rak?/?pups
were obtained at the expected Mendelian ratio in litters ob-
tained from heterozygous intercrosses of mice both on the
mixed and 129 genetic backgrounds (Table 1). Northern anal-
ysis of total RNA derived from the kidney, small intestine, and
colon of frk/rak?/?mice failed to detect any of the three
transcripts encoding Frk/rak (Fig. 2C). Similarly, Western
analysis revealed the absence of Frk/rak protein in tissues
derived from frk/rak?/?mice (Fig. 2D). Loss of frk/rak did not
affect neonatal development, as frk/rak?/?pups could not be
distinguished from their wild-type littermates by differences in
size or appearance. No obvious changes in cage exploration,
social behavior, or response to stimuli were observed in frk/
rak?/?mice. frk/rak?/?adult males and females were fertile
and yielded litters of sizes comparable to those generated by
wild-type mice after mating to C57BL/6J animals. Additionally
frk/rak?/?females were able to bear and sustain litters as
efficiently as their wild-type and heterozygous littermates.
Comparisons of frk/rak-null and control animals did not reveal
any obvious changes in size or growth from weaning to 12
months of age. Age-matched 2-, 5-, 8-, and 12-month-old frk/
rak?/?and frk/rak?/?littermates from the F2generation were
euthanized, and a complete (greater than 20 organs) his-
topathological analysis was performed. No differences were
observed between the three different groups in the number or
types of lesions present in various organs (data not shown). A
nearly 200-animal cohort (F2generation) consisting of ?/?,
?/?, and ?/? animals was monitored for health and mortality
up to 28 months of age. No significant increase in the incidence
of any pathological lesions, including spontaneous tumors, was
detected in frk/rak?/?animals (data not shown).
Analysis of the ultrastructure of the intestinal epithelium in
frk/rak-null mice. The rat homolog of Frk/rak has been re-
ported to localize to the apical membranes (brush borders) of
intestinal enterocytes. Expression levels and subcellular local-
ization of Frk/rak correlated with cytodifferentiation of the
intestinal epithelium. Furthermore, Frk/rak has been re-
5238 CHANDRASEKHARAN ET AL.MOL. CELL. BIOL.

Page 5

ported to colocalize with c-met in the brush borders of rat
intestinal enterocytes. Frk/rak activity increased in response
to hepatocyte growth factor (HGF) stimulation in vitro (17),
suggesting that Frk/rak is downstream of the HGF receptor
and could potentially participate in epithelial morphogenic
programs mediated by HGF signaling. We therefore initi-
ated a detailed investigation of the morphology of the in-
testinal mucosa. Histological analysis of sections of the
small intestine derived at different ages from age-matched
frk/rak?/?and frk/rak?/?littermates did not reveal any dif-
ference in the morphology of the epithelium. To determine
if the ultrastructure of intestinal enterocytes and their apical
membranes was altered in frk/rak-null mice, SEM and TEM
were performed on sections of the jejunum. The apico-basal
polarity of the enterocytes was found to be structurally in-
tact, as ascertained by proper intracellular positioning of
organelles like the nucleus and Golgi apparatus (Fig. 3A).
No significant differences were observed in the length, di-
ameter, arrangement, and packing of the microvilli in the
brush border (Fig. 3B). Frk/rak protein (endogenous and
ectopically expressed) has been localized to the Golgi and
centrosomal compartments in a number of cell lines. TEM
analysis also failed to reveal any abnormalities in either the
intracellular localization or the ultrastructure of centrioles
in frk/rak?/?enterocytes (Fig. 3C and D).
FIG. 1. Expression of Frk/rak in adult murine tissues. Total protein was isolated from a panel of organs derived from 2-month-old male and
female C57BL/6J mice (Charles River Laboratories). Two hundred micrograms of total protein was electrophoresed on SDS–10% PAGE gels.
(A) Western blots were probed with anti-Frk antibodies used at a 1:500 dilution (Michael Welsh, Uppsala University) and then with donkey
anti-rabbit horseradish peroxidase-conjugated secondary antibody (Amersham) used at a 1:5,000 dilution. Blots were developed with ECL reagent
(Amersham). Mouse Frk/rak is detected as a 57-kDa band. Lane 1, 50 ?g of NmuLi cell lysate control; lane 2, brain; lane 3, breast; lane 4, colon;
lane 5, heart; lane 6, kidney; lane 7, liver; lane 8, lung; lane 9, ovary; lane 10, pancreas; lane 11, skeletal muscle; lane 12, small intestine; lane 13,
spleen; lane 14, stomach; lane 15, testis. (B) Expression of Frk/rak protein increases during normal murine mammary gland differentiation. Murine
mammary glands were isolated at different time points during the developmental cycle of healthy C57BL/6J female mice. Tissues were homog-
enized, 200 ?g of total protein was electrophoresed on SDS–10% PAGE gels, and transferred to Immobilon-P (Millipore) membranes. Blots were
incubated with primary Bsk-specific polyclonal antibodies used at a 1:500 dilution (Michael Welsh, Uppsala University) and with donkey anti-rabbit
horseradish peroxidase-conjugated secondary antibody (Amersham) used at a 1:5,000 dilution. Blots were developed with ECL reagent (Amer-
sham). Top panel lanes: 1, 4-week-old virgin; 2, 6-week-old virgin; 3, 10-week-old virgin; 4, day 6 of pregnancy; 5, day 9 of pregnancy; 6, day 14
of pregnancy; 7, day 16 of pregnancy; 8, day 3 of lactation; 9, day 8 of lactation; 10, day 1 of involution; 11, day 5 of involution; 12, day 10 of
involution; 13, 50-?g NmuLi cell lysate (control for Frk/rak). For the results shown in the bottom panel, lysates were probed with rabbit polyclonal
antibodies directed against beta-casein used at a 1:1,000 dilution (Nancy Hynes, Friedrich Meischer Institute) Lanes 1 to 12, same as for top panel;
lane 13, 100-?g lysate of HC11 cells induced to differentiate for 10 days with prolactin (control for beta-casein antibody).
TABLE 1. Genotypic analysis of offspring from heterozygous
intercrosses
Genetic background Gender group
No. (%) of genotype:
?/??/??/?
129-BL/6 Male
Female
Combined
37 (26.8)
37 (25.7)
74 (26.2)
71 (51.4)
66 (45.8)
137 (48.6)
30 (21.7)
41 (28.5)
71 (25.2)
129ola ? 129SvEvMale
Female
Combined
12 (26.1)
9 (26.5)
21 (26.3)
22 (47.8)
16 (47.1)
38 (47.5)
12 (26.1)
9 (26.5)
21 (26.3)
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FIG. 2. Scheme for generation of a targeted disruption in frk/rak. (A) A targeting construct was generated such that homologous recombination
would result in the replacement of exons 1a and b with the neomycin phosphotransferase selection marker cassette. This design results in the
removal of the first methionine and the translation initiation site of the protein, indicated ATG. The replacement of exons 1a and b with the
neomycin cassette also results in the introduction of a novel EcoRV site as shown (bold E). Sites for restriction enzymes are shown as follows: E,
EcoRV; S, SalI; K, KpnI; N, NheI. The 5? probe, a 1.8-kb KpnI-EcoRV fragment, thus detects a 5.8-kb fragment from the mutant allele compared
to a 7.2-kb fragment detected from the wild-type allele (untargeted) in EcoRV-digested genomic DNA. (B) Representative Southern blot of mice
generated by heterozygous crosses. Tail DNA was digested with EcoRV, and Southern blots were probed with the 5? probe. Wild-type,
heterozygous, and homozygous mutant mice are identified based on the presence of the 7.2-kb (wild type) and 5.8-kb (mutant) EcoRV bands. The
disruption introduced in frk/rak produces a null mutation. (C) Northern blot analysis was performed on total RNA derived from the small intestine,
colon, and kidney of 4-month-old age-matched littermates. Twenty micrograms of total RNA was electrophoresed on 1.2% formaldehyde–agarose
gels. Northern blots were probed with [32P]dCTP-labeled frk/rak complete cDNA. (D) Western blot analysis was performed on total protein
isolated from organs derived from age-matched littermates. Two hundred micrograms of the total cellular protein isolated was analyzed with
polyclonal murine anti-Frk antibodies. Lanes 1 to 6, frk/rak?/?mice; lanes 7 to 12, frk/rak?/?mice; lanes 1 and 7, liver; lanes 2 and 8, lung; lanes
3 and 9, kidney; lanes 4 and 10, stomach; lanes 5 and 11, colon; lanes 6 and 12, spleen; lane 14, NmuLi cell lysate (control for murine anti-Frk
antibody).
5240 CHANDRASEKHARAN ET AL.MOL. CELL. BIOL.

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Histological analysis of other organs, such as the kidney and
lung, which contain polarized epithelia and brush borders, also
did not reveal any morphological defects (data not shown). In
addition, despite high levels of expression in the differentiating
mammary gland, female frk/rak?/?mice successfully nursed
litters, and normal branching and alveolar differentiation was
detected in their mammary glands (data not shown).
Survival of frk/rak-null mice after radiation-induced intes-
tinal injury. Intestinal epithelial cells are extremely sensitive to
ionizing radiation, and the loss of epithelial integrity primarily
contributes to the death of animals within 10 days of exposure
to 10- to 12-Gy doses of whole-body radiation. A functional
role for Frk/rak in epithelial cell death, damage repair, and/or
proliferation and differentiation might alter the radiation sen-
sitivity and survival of frk/rak?/?mice following acute injury.
To address this, age-matched (9.5 to 11 week old) cohorts of
mice consisting of frk/rak?/?, frk/rak?/?, and frk/rak?/?mice
were exposed to a single dose of 9, 10, 11, or 12 Gy of whole-
body ionizing radiation, and the percent survival of each ge-
notype was determined on day 10 postirradiation. Exposure to
12 Gy caused complete mortality of all genotypes before day 10
(data not shown). At 11 Gy, approximately 50% survival was
observed in the control groups (?/? and ?/? mice) The
LD50/10survival of frk/rak?/?mice did not differ significantly
from that of the frk/rak?/?(59.5% ? 13.4% versus 52.2% ?
27.7%, P ? 0.68) and frk/rak?/?(59.5% ? 13.4% versus 53%
? 18.3%, P ? 0.84) animals (Table 2). The survival of all three
groups increased after exposure to 10 and 9 Gy of radiation,
and no significant differences were observed in the day 10
percentage survival of frk/rak?/?mice compared to the control
groups at both of these doses (Table 2). The median survival of
frk/rak-null mice over 2 weeks following exposure to 10 Gy also
did not differ significantly from that of the control groups
(Table 3). Furthermore, qualitative histological analysis of ap-
optosis and mitosis performed on sections of the jejunum iso-
lated from frk/rak?/?mice 1, 3, 7, and 10 days after exposure
to 10 Gy revealed no significant differences in the morphology
of the regenerating intestinal epithelium compared to that of
the controls (data not shown).
Expression analysis of SFK genes in frk/rak-null mice. The
failure of Frk/rak disruption to result in defects in growth and
morphology of intestinal epithelial cells could be the conse-
quence of compensatory changes in the expression of SFK
genes or other cellular pathways. To examine this possibility,
we compared the global gene expression profiles in various
organs of frk/rak?/?and frk/rak?/?mice by using cDNA mi-
croarrays. While the majority of changes were in the range of
1.2-fold, alterations in the expression (even as low as 1.5-fold)
of certain genes were consistently detected in multiple tissues
of frk/rak-null mice (data not shown). The mouse cDNA arrays
used in the analysis included SFKs (src, fyn, yes) and members
of the Frk/rak family (sik). Interestingly, a 2.2- and 2.1-fold
increase in c-src expression was detected in the intestine and
kidney, respectively, of frk/rak?/?mice (Fig 4A). Additionally,
a modest (1.38-fold) increase in yes expression was detected in
the colon of frk/rak-deficient mice. To verify these findings,
Northern blot analysis of the expression of SFKs, src, fyn, and
yes was performed with total RNA isolated from the colon,
kidney, and jejunum of frk/rak?/?and frk/rak?/?mice. While
expression of c-src increased by 1.9-fold in the intestine and
1.4-fold in the colon (Fig. 4 B), no changes in the expression of
yes and fyn were detected in either organ (data not shown).
Investigation of the thyroid hormone status of frk/rak-null
mice cDNA expression analysis provides clues to biochemical
phenotypes. An analysis of all genes identified with a differen-
tial expression of 1.2-fold or more in frk/rak?/?animals sur-
prisingly revealed a subset of genes in common to those iden-
tified by microarray expression analysis of rat pituitary cells
treated with T3 hormone in vitro (43). A qualitative compar-
ison was therefore performed between all genes differentially
expressed in frk/rak?/?mice and in a panel of organs isolated
from mice injected with T3 hormone (L. D. Miller, unpub-
lished data). Consistent with our earlier observation, genes
identified in frk/rak-null mice were also represented in this set
of T3-responsive candidates, which included genes and gene
families regulated at the transcriptional level by thyroid hor-
mones and those associated with T3 signaling. This finding
presented the intriguing possibility that thyroid hormone levels
or signaling may be altered in frk/rak-null mice. Circulating
levels of T3, T4, and TSH were therefore measured for 10 pairs
of age-matched frk/rak?/?and frk/rak?/?littermates. Total
circulating levels of T3 were decreased in 80% (8 out of 10
pairs, P ? 0.019) of frk/rak?/?animals compared to their
frk/rak?/?littermates (Fig. 5A). However, no significant
changes were detected in the circulating levels of T4 (P ? 0.84)
(Fig. 5B) and TSH (data not shown).
DISCUSSION
This study clearly demonstrates that frk/rak is not essential
for embryonic development, as viable frk/rak-null mice are
obtained at the predicted Mendelian frequency in different
genetic backgrounds. Furthermore, histopathological analysis
of over 20 organs derived from age-matched frk/rak-null and
wild-type littermates, failed to identify any deficits in the de-
velopment of epithelial tissues that normally express high lev-
els of this kinase. Also, no overt defects in the physiological
functions of all major organs were identified, as illustrated by
the good health and normal growth of frk/rak?/?animals.
Human frk/rak localizes to chromosome 6q21-23, and the
TABLE 2. Comparison of survival of frk/rak-null mice after
exposure to single doses of ionizing radiationa
Dose
(Gy)
% Survival ? SEM on day 10 (n) of genotype:
Statistical
significance
?/??/??/?
1152.2 ? 27.2 (13)53 ? 18.4 (31) 59.5 ? 13.4 (23)0.80b
0.84c
0.68d
0.75b
0.69c
0.37d
N/Ab,e
0.24c
0.23d
1082.3 ? 16.6 (25) 78.7 ? 11.2 (37) 69.9 ? 21.2 (31)
9100 (21)100 (25) 87.5 ? 2.2 (25)
aData is cumulative from two separate trials (9 and 11 Gy) and three separate
trials (10 Gy). Cohorts consist of 9.5- to 11-week-old age-matched littermates
generated by heterozygous intercrosses with nearly 50% male and females.
bP value for ?/? versus ?/? mice is given.
cP value for ?/? versus ?/? mice is given.
dP value for ?/? versus ?/? mice is given.
eN/A, not applicable.
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mouse gene iyk localizes to the syntenic region on proximal
mouse chromosome 10. The 6q21-23 region has been identi-
fied as a tumor suppressor and/or senescence locus (35, 43).
Taken in conjunction with the finding that overexpression of
Frk/rak causes a potent growth arrest in vitro both in human
and in murine systems, it was hypothesized that the loss of
frk/rak would lead to epithelial hyperplasias or neoplasias.
However, to date (animals observed for greater than 2 years),
no spontaneous primary tumors have been observed in the
colon, small intestine, kidney, pancreas, and mammary gland
FIG. 3. Morphological analysis of intestinal epithelium. frk/rak?/?enterocytes have normal structural polarization. TEM analysis was per-
formed on ultrathin sections of jejunum derived from age-matched frk/rak?/?and frk/rak?/?littermates. (A) As revealed by the low-magnification
analysis shown above, frk/rak?/?enterocytes from the villus maintain normal apico-basal polarity, as defined by the intracellular positioning of
organelles like the nucleus and Golgi apparatus. (B) TEM analysis of the brush borders of frk/rak?/?mice. Sections of the jejunum isolated from
3- to 4-month-old frk/rak?/?and frk/rak?/?littermates were fixed appropriately for TEM. Ultrathin sections were cut and analyzed at high
resolution to determine the structural integrity, organization, and dimensions of the microvilli that constitute the brush border. TEM analysis
reveals normal ultrastructure and dimensions of microvilli in frk/rak?/?enterocytes compared to that of their wild-type littermates. For frk/rak?/?
mice, the average length is 1.6 ?m and the average width is 0.1 ?m. For Frk/rak?/?mice, the average length is 1.6 ?m and the average width is
0.1 ?m. (C) Subcellular positioning of centrioles in frk/rak?/?enterocytes. Ultrathin sections of jejunum obtained from age-matched frk/rak?/?and
frk/rak?/?littermates were analyzed by TEM to determine the position and morphology of centrioles. A representative section from an frk/rak?/?
enterocyte demonstrating characteristic apical localization of centrioles (closer to the plasma membrane) is shown. (D) Centriole structure in
frk/rak?/?mice enterocytes. Ultrathin sections were analyzed by TEM to verify the ultrastructure of the centrioles. The panel shows normal
centriolar structure in frk/rak?/?mice compared to that of frk/rak?/?mice, as seen by the arrangement of microtubule triplets.
5242 CHANDRASEKHARAN ET AL.MOL. CELL. BIOL.

Page 9

of frk/rak-null mice. Our studies therefore fail to support a
tumor suppressor role for frk/rak in the development of epi-
thelial cancers.
Expression studies performed in the developing rat intestine
demonstrate that frk/rak is detected at low levels in the cyto-
plasm of the undifferentiated and rapidly dividing intestinal
epithelium. Expression levels of the protein increase concom-
itant with the onset of polarization and formation of early
microvilli in the intestinal epithelium. In the adult rat, Frk/rak
is detected abundantly in the brush borders of the intestines
and at lower levels in the brush borders of polarized epithelial
cells in the kidney and lung (16). It was hence speculated that
Frk/rak has a functional role in epithelial cytodifferentiation.
Histological analysis of adult intestinal tissue derived from
frk/rak-null mice revealed normal morphological differentia-
tion of the epithelium. The ultrastructure of frk/rak?/?entero-
cytes is normal, and the appropriate subcellular localization of
organelles indicates that the apical-basal polarity of cells is
intact. The normal dimensions and packing of microvilli re-
vealed by TEM and SEM experiments further indicate that
frk/rak is not essential for determining or maintaining the
structural integrity of the brush border.
Frk/rak protein has been localized to the Golgi apparatus
and centrosomes (Carter et al., unpublished data). The cen-
trosomal localization of Frk/rak is microtubule independent
and cell cycle regulated, suggesting that it is a resident centro-
somal protein with a potential role in cell cycle regulation.
Centrioles of murine intestinal enterocytes are positioned api-
cally, within 1 to 3 ?m of the brush border, and the spatial
uncoupling of the centrosome from the Golgi apparatus is
integral to enterocyte differentiation (21, 22). Using TEM, we
have demonstrated that centrioles are apically localized in in-
testinal frk/rak?/?enterocytes and that their ultrastructure is
also normal. Our studies thus demonstrate that Frk/rak is not
an essential structural component of centrosomes. However,
we cannot yet exclude the possibility that minor defects in
some aspect of centriolar function may exist in frk/rak?/?cells.
To test whether Frk/rak had a role in intestinal regeneration,
as suggested by its differential expression along the length of
the crypt-villus axis, we exposed frk/rak-null mice to a range of
doses of whole-body radiation that cause acute intestinal dam-
age. Radiation-induced injury and readaptation have been ex-
tensively characterized (15, 28a). Doses of 9 Gy and above
result in lethality within 10 days of exposure due to severe
gastrointestinal toxicity. Response to intestinal injury involves
an immediate wave of apoptosis to overcome DNA damage
TABLE 3. Comparison of median survival of frk/rak-null mice over
14 days after exposure to 10 Gy of radiationa
Dose (Gy)
Median survival in days for
genotype:
Statistical significance
?/??/??/?
1010.710.710.9
?0.50b
?0.50c
?0.50d
0.99e
aData is cumulative from two separate trials. Cohorts consist of 9.5- to 11-
week-old age-matched littermates generated by heterozygous intercrosses.
bP value for ?/? versus ?/? mice is given.
cP value for ?/? versus ?/? mice is given.
dP value for ?/? versus ?/? mice is given.
eP value for ?/? and ?/? versus ?/? mice is given.
FIG. 3—Continued.
VOL. 22, 2002CHARACTERIZATION OF frk/rak?/?MICE 5243

Page 10

followed by proliferation and differentiation of the enterocytes
to reconstitute the epithelium. A defect in any of these cellular
processes would result in altered sensitivity to ionizing radia-
tion, consequently altering the survival of the animals. For
example, Ku80?/?mice display extreme hypersensitivity to low
doses of ionizing radiation, exhibiting complete mortality
within 2 weeks of exposure compared to their wild-type coun-
terparts (36). Similarly, PARP?/?mice and mice bearing the
scid mutation are exquisitely sensitive to gamma-irradiation
radiation-induced damage (4, 52). Our study clearly demon-
strates that frk/rak?/?mice do not show significant differences
in percent or median survival compared to the control groups,
particularly at the LD50/10dosage of 11Gy. We therefore infer
that the biological processes responsible for DNA damage
repair (apoptosis and cell cycle checkpoints) and regeneration
of the intestinal epithelium are not impaired in these animals.
It is, however, possible that the frk/rak-null mutation, in com-
bination with deficiencies in other SFK and Frk genes, which
are highly expressed in the colon and intestine, would result in
altered radiation sensitivity. Also, loss of frk/rak may elicit a
different response in other models of intestinal injury, such as
5-fluorouracil- or indomethacin-mediated gut toxicity.
The advent of cDNA expression arrays has facilitated
broader phenotypical analysis of mouse models with targeted
gene disruptions. We performed comparative gene expression
profiling of frk/rak-null and wild-type mice in order to identify
molecular phenotypes, which could provide a first approxima-
tion of cellular or physiological states, detect potential com-
pensatory mechanisms, and/or provide clues to phenotypes
which may become apparent after specific stresses. While the
absolute values of severalfold changes in gene expression de-
tected were not dramatic (the majority of genes with a 1.2- to
2.0-fold change), the consistency in the pattern of change
across multiple pairwise comparisons strongly suggests that
some candidates may indeed represent real responses to frk
disruption. Interestingly, the number of differentially expressed
genes identified in each tissue also correlated with the level of
endogenous frk/rak expression, with the greatest number of
outliers detected in the intestine and the least number dect-
ected in the heart (data not shown).
A possible explanation for the lack of an overt defect in
frk/rak-deficient mice is that its functions may be redundant or
overlap with those of other members of the Frk/rak and Src
families. This does not come completely as a surprise, espe-
cially since SFKs have been demonstrated to have redundant
functions both in vitro and in vivo. Over the last decade mouse
models have been generated with targeted disruptions in all 8
prototypical SFKs. Although in vitro experimentation has as-
sociated these versatile proteins with a multitude of biological
processes (1, 49), analysis of mice deficient in SFKs reveals
FIG. 4. Expression of c-src in organs of frk/rak-null mice. (A) The expression of c-src was compared in the colon, intestine, and kidney of three
pairs of age-matched frk/rak?/?and frk/rak?/?littermates. Twenty micrograms of total RNA for each sample was electrophoresed on 1.2%
formaldehyde–agarose gels. Northern blots were probed with a full-length mouse c-src cDNA probe (provided by Philip Soriano) and with murine
GAPDH cDNA (bottom panels) for normalization. (B) Comparison of changes in SFK gene expression by microarray and Northern blot analysis.
The normalized ratios of change in gene expression for c-src in the intestine, colon, and kidney of frk/rak-null animals is shown as detected by
Northern blotting and microarray analysis separately. Similar analyses were performed for c-fyn and c-yes with Northern blotting and microarray
analysis. A ratio of 1.0 indicates equivalent gene expression in wild-type and null animals. ?, increase (n-fold) in gene expression in frk/rak?/?mice
(GAPDH-normalized ratios averaged over three pairs); #, increase (n-fold) in gene expression in frk/rak?/?mice (background-normalized ratios
averaged for three out of four pairs analyzed).
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Page 11

lesions in restricted cell types and defects in specific cellular
processes, as exemplified by the src- and fyn-null mice (12, 28,
44). A high level of functional redundancy between SFK genes
is supported by evidence of compensatory changes in expres-
sion, activity, and subcellular localization of other SFKs in
mice bearing single mutations, possibly accounting for the min-
imal phenotypes observed (28, 48). The fact that mice with
deficiencies in multiple SFK genes often develop more-severe
phenotypes or novel and complex phenotypes not observed in
the single mutants is also consistent with this. For instance,
expression and activity of hck is elevated in src?/?osteoclasts
and src?/?hck?/?mice develop a more-severe osteopetrosis
phenotype (27). Another example pertinent to our study is that
fyn?/?mice have minor phenotypes, and yes?/?mice have no
overt defects. The fyn?/?yes?/?mice, however, exhibit in-
creased perinatal lethality, and viable double-null animals de-
velop severe glomerulonephritis (46).
Interestingly, the intestine and colon, which are physiologi-
cally similar, express the highest endogenous levels of frk/rak as
well as high levels of sik, c-src and c-yes. An approximately
2.0-fold increase in the expression of c-src detected in frk/
rak?/?intestines by cDNA microarray and Northern blot anal-
ysis suggests a compensatory role for c-src in the intestine.
Elevated expression of c-src mRNA (1.4-fold) is also detected
in frk/rak?/?colon tissue, further supporting this hypothesis.
Although Src and frk/rak may have distinct subcellular local-
izations, c-src could potentially compensate for frk/rak by par-
ticipating in the same or a complementary signaling pathway
through interactions with common downstream elements.
While no changes in the expression of sik were detected in our
analysis, it remains possible that changes in Sik levels or activ-
ity may compensate for loss of frk/rak function.
The observation that some of the differentially expressed
genes identified by microarray analysis also appear as potential
T3 hormone-responsive genes presented the intriguing possi-
bility that the thyroid hormone status of frk/rak-deficient mice
may be altered. These included novel candidates and others
reported to be transcriptionally regulated by thyroid hormones
such as IGFII, ferritin, and peptidyl-alpha-amidating-monoox-
ygenase (PAM) (30). Elevated expression of PAM RNA has
been demonstrated in hypothyroid and euthyroid rats (40). An
increase in PAM gene expression was detected in the intestine,
colon, and kidney of frk/rak-null mice, suggestive of a hypothy-
roid state (data not shown). Similarly, decreased expression of
ferritin detected in frk/rak?/?intestines was consistent with a
defect in T3 hormone-dependent transcriptional regulation
(18; data not shown). Quantitative measurements of circulat-
ing levels of thyroid hormones indeed revealed a statistically
significant (P ? 0.019) decrease in T3 levels of frk/rak?/?
animals but no significant changes in the circulating levels of
T4 or TSH compared to their age-matched wild-type litter-
mates. The circulating thyroid hormone profile observed in
frk/rak-deficient mice is reminiscent of that described for clin-
ical syndromes such as the ESS or the low-T3 syndrome. These
conditions are characterized by lower circulating T3 levels and
minor increases or no changes in the levels of T4 and TSH.
ESS is usually associated with high systemic stress resulting
from a variety of conditions, including systemic shock, hepati-
tis, renal disease, and aging (29). ESS-type syndromes are
thought to be the result of impaired extrathyroidal peripheral
FIG. 5. frk/rak-null mice have decreased circulating levels of T3 hor-
mone. A scatter plot analysis of total circulating levels of T3 (A) and T4
(B) hormones in serum obtained from age-matched frk/rak?/?and frk/
rak?/?littermates derived by heterozygous intercrosses. Levels of T3 and
T4 hormone in serum were assayed by AniLytics, Inc., as described in
Materials and Methods. Comparisons were performed between both
male and female littermates of each genotype. n (total) ? 10; n (12-week-
old mice) ? 4 (F); n (8-week-old mice) ? 6 (E). Connector lines indicate
the actual pairing of frk/rak?/?and frk/rak?/?littermates.
VOL. 22, 2002CHARACTERIZATION OF frk/rak?/?MICE5245

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metabolism, primarily involving the 5?-deiodinases, which con-
vert T4 into metabolically active T3 (20). It is also not clear at
present whether ESS-type syndromes represent protective or
adaptive physiological mechanisms or are pathological. While
stress- or shock-induced animal models for the low-T3 syn-
drome and ESS have been characterized (5, 20), evidence for
genetically determined components associated with ESS is lim-
ited (6). Inherited defects in enzymes involved in hormone
metabolism (such as the 5?-deiodinases) or T3 and T4 trans-
porter and binding proteins are suggested as possibilities. The
discovery of a thyroid hormone phenotype in frk/rak-deficient
animals is unexpected because expression studies and in vitro
characterization to date do not support such a function. Al-
though the mechanisms for Frk/rak involvement in thyroid
hormone homeostasis or the compensation of the thyroid hor-
mone axis remain unclear, the alteration of thyroid hormone-
responsive gene expression suggests a pharmacodynamic effect
on the end organs. It is therefore conceivable that subtle ge-
netic defects (such as deficiency of Frk/rak) combined with the
appropriate environmental factors could manifest themselves
physiologically in ESS-type syndromes.
The results presented here provide the first report on the
role of Frk/rak family kinases in development and organ func-
tion in vivo. The minimal effect of frk/rak disruption on epi-
thelial tissues indicates that Frk/rak does not have an essential
function in epithelial cell homeostasis, specifically in negative
growth regulation and differentiation, as suggested by in vitro
experimentation. However, our results do suggest that com-
pensatory events such as the up-regulation of c-src may miti-
gate the effects of Frk/rak loss. The discovery of changes in the
thyroid hormone profile of frk/rak-null mice with symptoms
resembling ESS, while surprising, has opened a novel avenue
for the investigation of its function. Finally, the generation of
mouse models bearing combinations of mutations in other
SFKs (src, fyn, and yes) and Frk family kinases (sik) coupled
with the use of comparative gene expression profiling will help
elucidate the physiological functions of Frk/rak family kinases.
ACKNOWLEDGMENTS
We thank Michael Welsh for generously providing the murine Frk/
rak antibodies. We thank Anastasia Sowers for technical assistance in
the intestinal radiation injury studies. We also thank Anne Latour for
generation of targeted ES clones. We are also extremely grateful to
Beverly H. Koller for extensive discussion and critical reading of the
manuscript.
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