Molecular mechanisms underlying
SHP-1
gene expression
Hing Wo Tsui
1
, Kathleen Hasselblatt
2
, Alberto Martin
3
, Samuel Chi-ho Mok
2
and Florence Wing Ling Tsui
1,4
1
Division of Cellular & Molecular Biology, Toronto Western Research Institute, University Health Network, Toronto, Ontario,
Canada;
2
Laboratory of Gynecologic Oncology, Department of Obstetric Gynecology and Reproductive Biology, Brigham and
Women’s Hospital, Dana-Farber Harvard Center, Boston, Massachusetts, USA;
3
Department of Cell Biology, Albert Einstein College
of Medicine, Bronx, New York, USA;
4
Department of Immunology, University of Toronto, Toronto, Ontario, Canada
SHP-1, a protein-tyrosine phosphatase with two src-
homology 2 domains, is expressed predominantly in
hematopoietic and epithelial cells and has been implicated in
numerous signaling pathways as a negative regulator. Two
promoters direct the expression of human and murine
SHP-1, and two types of transcripts (I) and (II) SHP-1,are
initiated from each of these promoters. The cDNA

phosphorylation. Among the known protein tyrosine
phosphatases, SHP-1 and SHP-2 are distinguished by the
presence of two tandem src-homology 2 domains. Src-
homology 2 domains interact with phospho-tyrosine resi-
dues in many growth factor receptors and thus play an
important role in directing the effects of tyrosine phos-
phorylation [1]. We [2] and others [3] showed that motheaten
mice have mutations in the SHP-1 gene. These mutant mice
thus provide insight into the role of SHP-1. Motheaten mice
die prematurely and have characteristics of both immuno-
deficiency and autoimmunity [4]. From analyses of moth-
eaten mice and other work in cell lines, SHP-1 functions
predominantly as a negative regulator in hematopoietic
signaling pathways [5].
SHP-1 is expressed predominantly in hematopoietic and
epithelial cells [6]. It has recently been shown that localiza-
tion of SHP-1 differs between hematopoietic and epithelial
cells (i.e. cytoplasmic in hematopoitic cells vs. nuclear in
epithelial cells) [7]. Two promoters direct the expression of
human [8] and murine SHP-1 [9], and two types of
transcripts are initiated from the promoters. Transcripts
that contain the 5¢-most exon [termed (I)SHP-1] encode
SHP-1 with the initial amino acid sequences being MLSRG
as compared to the MVR sequence encoded by transcripts
that contain the 3¢ exon 1 [termed (II)SHP-1]. As there are
minor to no enzymatic differences between (I) and (II)
isoforms [9], we favor the view that different forms have
arisen because of a need to regulate SHP-1 transcription
using distinct promoters. Very little is known regarding the
functionality of the two promoters and their usage in

P-labeled DNA
fragment containing sequences encoding the phosphatase
domain of SHP-1 and autoradiographed. The intensity of
bandswasmeasuredbydensitometryonanimager(Bio-
Rad Fluor-S
TM
multi-imager).
Electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared from cell lines [10]. Protein
concentrations of the nuclear extracts were determined
using the Coomassie protein assay reagent (Pierce). Five
lg of nuclear extracts were mixed with herring DNA
(BMC) and labeled oligonucleotide with and without
competitor DNA (500-fold excess) in a buffer containing
25 m
M
Tris/HCl pH 7.5, 50 m
M
KCl, 0.6 m
M
dithiothre-
itol, 1 m
M
EDTA, 0.5 m
M
spermidine, 12% glycerol for
20 min at room temperature. For supershift experiments,
the nuclear extracts were incubated with 2 lgofthe
antibody for 30 min at room temperature before adding
the labeled oligonucleotide. The reaction was subjected to

construct A or B and re-circularizing the constructs, one
copy of the 12-bp repeat was removed from each of the two
constructs to form constructs A)12 bp and B)12 bp.
Analysis of promoter function
SKOV3 cells were cotransfected with the (I)SHP-1 pro-
moter–luciferase constructs and pSV
2
bGal (pCH110) by
lipofection using Fugene 6 (Roche Molecular Biochem).
Forty-eight hours after transfection, fractions of each cell
extract were used for the b-galactosidase (bGal) [11] and
luciferase [12] assays. The conditions used for the luciferase
assay were within the linear range of the assay for the
promoters tested in this study. Each construct was tested in
three different transfection experiments, with triplicates for
each experiment.
RESULTS
Differential expression of SHP-1 isoform transcripts
in human vs. murine cell lines
In mouse as well as in human, SHP-1 proteins are detected
in both hematopoietic and epithelial cells. As the two SHP-1
protein isoforms only differ in the first few amino acids, it is
difficult to distinguish the two protein isoforms. Thus, it is
unclear whether the SHP-1 proteins are translated from the
(I)SHP-1 or (II)SHP-1 transcripts or both. To assess
whether both SHP-1 promoters are transcriptionally active
in hematopoietic and epithelial cells, we used RT-PCR to
specifically amplify either the (I)SHP-1 or (II)SHP-1
transcripts. Expression of (I)SHP-1 and (II)SHP-1 tran-
scripts were assessed using the primers (I)SHP-1-90-5¢ and

mouse cell lines and the relative abundance of these
isoforms are summarized in Table 1. In most human
(4/6) and mouse (6/8) hematopoietic cell lines, both SHP-1
isoform transcripts were detected. However, some cell lines
expressed only one of the two isoforms (Table 1). Of the cell
lines that expressed both isoforms, the ratio of (II)SHP-1
to (I)SHP-1 transcripts ranged from 0.3 : 1 to 63 : 1
(human) and 28 : 1 to 110 : 1 (mouse). Similarly, in mouse
epithelial cell lines (3/4), both isoform transcripts were
present, although the ratio of (II)SHP-1 to (I)SHP-1,
which ranged from 1.3 : 1 to 2 : 1 is much lower than that
found in hematopoietic cells. However, in human epithelial
cell lines (5/6), only (I)SHP-1 transcripts were detected.
As all the cell lines used for this study are transformed, we
asked whether the SHP-1 promoter usage is similar in
untransformed hematopoietic cells. For human, we used
3058 H. W. Tsui et al. (Eur. J. Biochem. 269) Ó FEBS 2002
tonsillar T cells grown in the presence of phytohemagglu-
tinin (TON-phytohemagglutinin) and for mouse, we used
splenic T cells as well as thymus. In all three cases,
both (II)SHP-1 and (I)SHP-1 transcripts were detected,
with the former isoform being the predominant species
(Table 1).
(I)SHP-1
transcripts were up-regulated by 4b-phorbol
12-myristate 13-acetate (PMA) in HL60 and SKOV3 cells
As human epithelial cells expressed only (I)SHP-1 tran-
scripts, these cells (such as SKOV3, an ovarian cancer cell
line) are ideal for the study of the distal promoter function of
SHP-1. We first wished to identify agent(s) that can

NFjB. In the distal promoter of human SHP-1,thereisa
putative NFjBsiteat)314 (GGGATTTTCC). We first
asked whether NFjB proteins can bind to this putative
NFjB consensus sequence. We carried out EMSAs using
SKOV3 nuclear extracts and a double-stranded oligonucle-
otide containing this consensus sequence as a probe. We
detected two specific DNA–protein complexes (Fig. 3, lane
2), both of which can be super-shifted using anti-NFjBIg
(p50) and anti-NFjB Ig (p65) (Fig. 3, lanes 3 and 4). If
(I)SHP-1 transcription is increased because PMA activated
NFjB, we would expect to find more NFjB binding to this
NFjB site located in the distal SHP-1 promoter. We thus
carried out EMSA using equal amounts of untreated and
PMA-treated SKOV3 nuclear extracts. As expected, we
found that nuclear extracts from PMA treated SKOV3 cells
had a 4–5-fold higher NFjB activity than those from
untreated cells (Fig. 3, compare lane 8 with lane 6). These
data suggest that the up-regulation of (I)SHP-1 transcrip-
tion by PMA is mediated via the NFjB site in the distal
promoter of SHP-1.
Fig. 1. Relative abundance of (I)SHP-1 and (II)SHP-1 transcripts in human vs. mouse cell lines. Raji is a Burkitt’s Lymphoma cell line (i.e.
hematopoietic); HeLa and HT1080 are human epithelial cancer cell lines. BW5147 is a mouse T-cell line and L cell are a mouse epithelial cell line.
RNA from the cell lines were reverse transcribed, and serial dilutions (shown below each lane) of the RT mixture were used in PCR for (I)SHP-1
with primer pair (I)SHP-1-90-5¢ and SHP-1-1859-3¢,or(II)SHP-1 with primer pair (II)SHP-1-74-5¢ and SHP-1-1859-3¢. The RT-PCR products
were separated by electrophoresis, transferred to nitrocellulose and probed with
32
P-labeled sequences of the phosphatase domain for SHP-1.
Arrows denote the SHP-1 transcripts which are translatable into proteins [9,13]. Densitometry was performed on this species of SHP-1 transcripts.
Bottom panels: Schematics showing the generation of (I)SHP-1 vs. (II)SHP-1 transcripts from the SHP-1 gene.
Ó FEBS 2002 Regulation of SHP-1 expression (Eur. J. Biochem. 269) 3059

IC21 977 24 41 : 1
70Z/3 1320 27 49 : 1
J558L 140 0
A20 520 0
Hematopoietic cells
Splenic T cells 82 1.2 68 : 1
Thymus 629 11 60 : 1
Fig. 2. Up-regulation of SHP -1 expression in
PMA treated HL60 (A) and SKOV3 (B) cells.
Relative abundance of (I)SHP-1 and
(II)SHP-1 transcripts in untreated or PMA
treated cells was estimated by quantitative
RT-PCR (as in Fig. 1).
3060 H. W. Tsui et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Functional deletional analyses of the distal promoter
of human SHP-1 in epithelial cells
To characterize further the distal promoter, we needed to
obtain a genomic segment containing the distal promoter.
From a human SHP-1-containing cosmid clone
(LL12NCOIN 143H6), we isolated the 5¢ flanking region
upstream of the first exon of (I)SHP-1. We sequenced the
region 986 bp upstream of the transcription initiation site,
and the sequence was identical to the published one [8]. To
test the functionality of the human SHP-1 distal promoter,
we generated three deletion constructs (A, B and C) which
were adjoined to a luciferase reporter gene (Fig. 4). These
constructs contained different amounts of 5¢ flanking DNA
and lacked the (I)SHP-1 AUG. They were individually
transfected into SKOV3 cells, and lysates were assayed for
luciferase activities. A bGal construct was cotransfected

activator(s) binds to this 12-bp repeat. The reasons for a
much larger effect on construct B will be considered in the
Discussion.
Fig. 3. EMSA and supershift analyses. The NFjBsite(TGTTAGG
GATTTCCTTA) from (I)SHP-1 promoter was used as a probe.
Lanes: 1 and 5, no nuclear extracts present in the reaction mix; lanes 2
and6,twospecificcomplexes(AandB)formedwhenthereactionmix
contains both nuclear extracts and labeled probe. The lowest shifted
band is nonspecific, as it cannot be competed out with excess unlabeled
oligonucleotide in the reaction mix (lane 7); Both complexes A and B
were supershifted when either anti-NFjB Ig, p50 or p65 (Rel A) were
included in the reaction mix; lane 8, more complexes A and B
were formed when nuclear extracts from PMA treated SKOV3 cells
were used.
Fig. 4. Schematic of the (I)SHP-1 deletion
constructs and luciferase activities of these
constructs in SKOV3 cells. Constructs A, B
and C contain various lengths of (I)SHP-1
promoter region. Construct D is promoterless
and was used as a negative control. Construct
E is a luciferase construct driven by the
SV40 promoter and enhancer; it served as a
positive control. Both copies of the 12-bp
repeat are present in constructs A and B, while
only one copy of the repeat is present in either
construct A)12 bp or B)12 bp. K, KpnI;
S, SstI.
Ó FEBS 2002 Regulation of SHP-1 expression (Eur. J. Biochem. 269) 3061
USF1 and USF2 bind to the 12-bp repeat in the
(I)SHP-1

extracts from both SKOV3 and MDA453 cells (Fig. 5, lanes
2 and 7). As Myc hetero-dimerizes with Max, the inability of
anti-Max Ig to supershift the complex would imply that
Myc, like Max, does not bind to the E-box sequences in the
12-bp repeat.
USFs are also known E-box binding proteins [17]. We
therefore asked whether the protein complexes formed,
contain USF1 and/or USF2 using the 12-bp repeat
oligonucleotide and nuclear extracts from SKOV3 and
MDA453 cells. As shown in Fig. 5 (lanes 3, 4, 8 and 9), one
of the protein complexes was supershifted using either anti-
USF1 Ig or anti-USF2 Ig. Therefore, both USF1 and USF2
proteins form a stable complexes with the 12 bp repeat.
We have not identified the proteins involved in the
formation of complexes X and Y.
DISCUSSION
Differential usage of
SHP1
promoters in mouse vs.
human epithelial cell lines
A previous report [8] showed that a few human hemato-
poietic cell lines expressed only (II)SHP-1 transcripts.
Contrary to their finding that HL60 cells expressed only
(II)SHP-1 transcripts, we found that HL60 cells not only
express (I)SHP-1 transcript, but also can be stimulated by
PMA to express up to 48-fold more (I)SHP-1 mRNA. In
addition, we found that most human (5/7) and mouse (8/10)
hematopoietic cells, expressed both SHP-1 transcript
isoforms, albeit with (II)SHP-1 transcripts being the
predominant species. In mouse hematopoietic cell lines

cleotide (TTGAGCTCCA GGGAGAG
CTCCAGGGA; lane 5) was included in the
reaction mix for EMSA.
3062 H. W. Tsui et al. (Eur. J. Biochem. 269) Ó FEBS 2002
localized in the nuclei [7]. As we showed that only (I)SHP-1
transcripts were expressed in human epithelial cells, it
appears that SHP-1 proteins derived from human
(I)SHP-1 transcript are localized in the nuclei and thus
might have different signaling substrates compared to that
of the cytoplasmic (II)SHP-1 proteins. In support of this
notion, tyrosine-phosphorylated stat-5b and SHP-1 com-
plex has been detected in the nuclei of growth hormone
stimulated liver cells in culture [18].
Activators of the distal promoter of human
SHP-1
Our deletional analyses of the distal promoter of SHP-1 (in
an ovarian cancer cell line, SKOV3) showed less promoter
activity with sequential deletion of the 5¢ flanking region.
This suggests that the distal promoter of SHP-1 is regulated
by multiple activators. Indeed, we found two motifs within
the distal promoter that were important for promoter
activity. One such motif was an E-box containing a 12-bp
repeat. Deletion of one copy of the repeat resulted in
significantly lower promoter activity (Fig. 4). The additional
region I (420 bp) in construct A presumably contains
redundant regulatory elements, thus masking the contribu-
tion of the 12-bp repeats in the comparison of construct A
vs. construct A)12 bp activities. It appears that the two
tandem E-boxes separated by 6 bp are crucial for presum-
ably high affinity binding of the activator(s) involved.

might contribute to the regulation of SHP-1 expression is a
NFjB site located 105 bp upstream of the E-box containing
12-bp repeat. EMSA and supershift experiments show that
NFjB p50 and p65 bind this NFjB consensus sequence
(GGGATTTTCC). It was previously shown that PMA
treatment of HL60 cells increased SHP-1 transcription [15].
We found that (I)SHP-1 transcripts were upregulated by
PMA in HL60 and SKOV3 cells. Furthermore, PMA-
treated SKOV3 nuclear extracts showed more NFjB
binding activity (fourfold to fivefold; Fig. 3) than those
from untreated cells. Thus, it is likely that PMA activates
NFjB proteins which in turn leads to higher expression of
(I)SHP-1 transcripts. Confirmation of this result can be
achieved by deleting the NFjB site in the promoter
construct and assessing whether this will render transfected
cells unresponsive to PMA.
Our analyses of the deletion constructs transfected into
SKOV3 cells indicated that deletion of the region I (the 5¢
420 bp sequences, Fig. 4) from the promoter construct
(construct B) resulted in a 54% reduction of luciferase
activity. Interestingly, no consensus sequences for known
nuclear factors are found in region I. This result indicates
that there might be novel nuclear factors (activators) which
contribute to (I)SHP-1 promoter activity.
Contrary to our results (i.e. progressively less promoter
activity with sequential deletion of the 5¢ flanking region), a
recent deletional study of the same distal promoter in
MCF7 cells (a breast cancer cell line), showed a dramatic
drop of promoter activity to  15% using a deletion
construct with 5¢ flanking sequences up to 60 bp upstream

protein-tyrosine phosphatase (Hcph) gene. Cell 73, 1445–1454.
4. Shultz, L.D., Coman, D.R., Bailey, C.L., Beamer, W.G. &
Sidman, C.L. (1984) ÔViable motheatenÕ, a new allele at the moth-
eaten locus. Am. J. Pathol. 116, 179–192.
5. Neel, B.G. (1997) Role of phosphatases in lymphocyte activation.
Curr. Opin. Immunol. 9, 405–420.
Ó FEBS 2002 Regulation of SHP-1 expression (Eur. J. Biochem. 269) 3063
6. Plutzky, J., Neel, B.G. & Rosenberg, R.D. (1992) Isolation of a src
homology 2-containing tyrosine phosphatase. Proc. Natl Acad.
Sci. USA 89, 123–1127.
7. Cragg, G. & Kellie, S. (2001) A functional nuclear localization
sequence in the C-terminal domain of SHP-1. J. Biol. Chem. 276,
23719–23725.
8. Banville, D., Stocco, R. & Shen, S.H. (1995) Human protein
tyrosine phosphatase 1C (PTPN6) gene structure: alternate pro-
moter usage and exon skipping generate multiple transcripts.
Genomics 27, 165–173.
9. Martin, A., Tsui, H.W., Shulman, M.J., Isenman, D. & Tsui,
F.W.L. (1999) Murine SHP-1 splice variants with altered src
homology 2 (SH2) domains: implications for the SH2-mediated
intramolecular regulation of SHP-1. J. Biol. Chem. 274,
21725–21734.
10. Dignam, J.D., Lebovitz, T.M. & Roeder, R.G. (1983) Accurate
transcription initiation by RNA polymerase II in a soluble extract
from isolated mammalian nuclei. Nucleic Acids Res. 11,
1475–1489.
11. Rosenthal, N. (1987) Identification of regulatory elements
of cloned genes with functional assays. In Methods in Enzy-
mology Vol. 152 (Berger, S.L. & Kimmel, A.R., eds)
pp. 704–720.

20. Xu, Y., Banville, D., Zhao, H F., Zhao, X. & Shen, S H. (2001)
Transcriptional activity of the SHP-1 gene in MCF7 cells is dif-
ferentially regulated by binding of NF-Y factor to two distinct
CCAAT-elements. Gene 269, 141–153.
21. Yip, S.S., Crew, A.J., Gee, J.M.W., Hui, R., Blamey, R.W.,
Robertson, J.F.R., Nicholson, R.I., Sutherland, R.L. & Daly, R.J.
(2000) Up-regulation of the protein tyrosine phosphtase SHP-1 in
human breast cancer and correlation with grb2 expression. Int. J.
Cancer. 88, 363–368.
22. Mok, S.C., Kwok, T.T., Berkowitz, R.S., Barrett, A.J. & Tsui,
F.W.L. (1995) Overexpression of the protein tyrosine phospha-
tase, nonreceptor type 6 (PTPN6), in human epithelial ovarian
cancer. Gynecol. Oncol. 57, 299–303.
3064 H. W. Tsui et al. (Eur. J. Biochem. 269) Ó FEBS 2002