Extracellular domain splice variants of a transforming protein tyrosine phosphatase ? mutant differentially activate Src-kinase dependent focus formation

Article (PDF Available)inGenes to Cells 12(1):63-73 · February 2007with11 Reads
DOI: 10.1111/j.1365-2443.2006.01034.x · Source: PubMed
The extracellular domains of receptor-type protein-tyrosine phosphatases (PTPs) contain a diverse range of protein modules like fibronectin- or immunoglobulin-like structures. These are frequently expressed in a tissue- and development specific manner as splice variants. The extracellular domain of PTPalpha is rather short and heavily glycosylated. Two splice variants are known, which it differs by an exon encoding nine amino acids within the extracellular domain. We have analyzed the expression pattern of both variants and found that the smaller form is ubiquitously expressed while the larger form was found at an increased level only in brain, some skeletal muscle and differentiating cells like granule neurons, adipocytes and myotubes. The phosphatase activity of both forms was similar when tested in vitro using para-nitrophenylphosphate as a substrate and in a transient expression system with the substrates c-Fyn or c-Src. In a quantitative focus formation assay the capability of the larger form to activate Src-dependent focus formation in intact cells was increased more than twofold whereas the capability to dephosphorylate the insulin receptor in a BHK cell system was similar. We conclude that the two splice variants of PTPalpha are expressed differentially and regulate c-Src activity in different ways.
© 2006 The Authors
Genes to Cells (2007)
, 63–73
Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
DOI: 10.1111/j.1365-2443.2006.01034.x
Blackwell Publishing IncMalden, USAGTCGenes to Cells1356-9597© The Author. Journal compilation © by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
xxxOriginal ArticleSrc kinase activation by PTP
splice variantsK Kapp et al.
Extracellular domain splice variants of a transforming
protein tyrosine phosphatase αα
mutant differentially activate
Src-kinase dependent focus formation
Katja Kapp
, Jan Siemens
, Peter Weyrich
, Jörg B. Schulz
, Hans-Ulrich Häring
and Reiner Lammers
Medical Clinic IV, Otfried-Müller Str.10, 72076 Tübingen, Germany
Department of Neurology, Medical School, University of Tübingen, Hoppe-Seyler-Str. 3, 72076 Tübingen, Germany
The extracellular domains of receptor-type protein-tyrosine phosphatases (PTPs) contain a diverse
range of protein modules like fibronectin- or immunoglobulin-like structures. These are frequently
expressed in a tissue- and development specific manner as splice variants. The extracellular
domain of PTPαα
is rather short and heavily glycosylated. Two splice variants are known, which
it differs by an exon encoding nine amino acids within the extracellular domain. We have analyzed
the expression pattern of both variants and found that the smaller form is ubiquitously expressed
while the larger form was found at an increased level only in brain, some skeletal muscle and
differentiating cells like granule neurons, adipocytes and myotubes. The phosphatase activity of
both forms was similar when tested
in vitro
using para-nitrophenylphosphate as a substrate and in
a transient expression system with the substrates c-Fyn or c-Src. In a quantitative focus formation
assay the capability of the larger form to activate Src-dependent focus formation in intact cells
was increased more than twofold whereas the capability to dephosphorylate the insulin receptor
in a BHK cell system was similar. We conclude that the two splice variants of PTPαα
are expressed
differentially and regulate c-Src activity in different ways.
Protein tyrosine phosphorylation is a major mechanism
of cellular signal transduction and is regulated by protein
tyrosine kinases (PTKs) and protein tyrosine phosphatases
(PTPs) (Schlessinger 2000; Ostman & Bohmer 2001).
There are currently 113 vertebrate PTPs known (Andersen
et al.
2001), which are either cytoplasmic or membrane-
bound enzymes. Alternative splicing is a common feature
of receptor type PTPs including CD45 (Johnson
et al.
1989), PTP
et al.
1994), PTP
and LAR
et al.
1995), as well as PTP
et al.
1990), GLEPP1 (Aguiar
et al.
1999) and PTPRR
et al.
2004). For CD45, alternative splicing of
three exons close to the amino-terminus would result in
eight isoforms, of which several have been identified
et al.
2003). A tissue-specific regulation of
alternative splicing has been shown (Hathcock
et al.
1993) and, additionally, an impact of the varying extra-
cellular domains on protein function (Leitenberg
et al.
1996). More recently, it was reported that the alter-
natively spliced isoforms homodimerize differentially,
resulting in modified protein activity (Xu & Weiss 2002).
The extracellular domains of PTP
and PTP
not encode characteristic domains but are heavily
glycosylated. For PTP
, three splice variants have been
described. One variant contains an insert of 36 amino
acids in the phosphatase domain (Matthews
et al.
Further, there exist two isoforms due to an alternative
splicing of a 27 bp mini-exon localized in the juxtam-
embrane extracellular domain. The resulting isoforms
have an extracellular domain of 123 or 132 amino acids
in the mature protein (Kaplan
et al.
1990; Krueger
et al.
1990; Matthews
et al.
1990; Sap
et al.
1990) and here it
is referred to as small and large isoform or PTP
123 and
Communicated by
: Carl-Henrik Heldin
: E-mail: reiner.lammers@med.uni-tuebingen.de
Both authors contributed equally to this work.
Present address
: ZMBH (Center for Molecular Biology at the
University Heidelberg), University Heidelberg, Im Neuen-
heimer Feld 282, 69120 Heidelberg, Germany.
K Kapp
et al.
Genes to Cells (2007)
, 63–73
© 2006 The Authors
Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
132, respectively. The tissue-specific expression pattern
of the isoforms has not yet been evaluated in detail. Daum
et al.
(1994) found mainly the small form when inspect-
ing three different tissues or cell lines. Analyzing PTP
expression in the major insulin target tissues, Norris
et al.
(1997) detected a general, quite high expression of the
smaller isoform whereas the larger isoform was expressed
in fat but hardly in other tissues like liver, skeletal muscle
and endothelial. However, a possible functional difference
of the isoforms has not been looked into.
In the present study, we investigated the differential
expression of the isoforms and the difference in their acti-
vation potential towards Src kinases and the insulin receptor
that are known substrates of PTP
. While PTP
123 was
expressed in most tissues, expression of PTP
132 was
generally low but up-regulated in some tissues like brain
and skeletal muscle. Using focus formation as a cell-based
quantitative assay, the larger isoform was more efcient
to activate Src-dependent focus formation whereas inacti-
vation of the insulin receptor signal in a different functional
and quantitative assay was similar for both isoforms.
Tissue-specific expressions of the PTPαα
Expression analysis of PTP
through Northern blotting
using murine and human tissues revealed a ubiquitous
expression, with murine brain and kidney having the
highest expression levels (Matthews
et al.
1990; Sap
et al.
1990) whereas human adult brain showed little expression
et al.
1990). To verify the expression in human
tissues and to evaluate the contribution of the individual
splice variants to total expression, we performed
RT-PCR of several tissues and cell lines with primers,
which amplified a region covering the alternatively
spliced exon (Fig. 1A). Using this approach, we simulta-
neously detected both isoforms in a single reaction and
could directly compare their relative expression. As size
controls, PCRs using the cDNAs of human PTP
and PTP
132 were performed in parallel. We detected
the smaller splice variant PTP
123 as the predominant
variant in most sources used, which is similar to the findings
of Daum
et al.
(1994). Although mostly at a low level, the
amplification product for the larger splice variant, PTP
was found at a significantly higher level in human brain
and some, but not all, human skeletal muscle derived
cDNAs (SKM II and muscle biopsy; Fig. 1B).
With the determination of a higher expression of the
larger PTP
isoform in brain and muscle, we noted that
both tissues develop through differentiation of precursor
cells. Since Fang
et al.
(1996) have described a varying
expression of PTP
in the cerebellum, we wanted to
investigate a possible time course of isoform-specific
expression. To this end, we prepared cerebellum cDNA
from rats at different postnatal stages of development
for RT-PCR analysis. As expected, we found two PCR
fragments, showing that PTP
also occurs with two
isoforms in rat. During the first 14 days, the expression
of the smaller form remained constant whereas expres-
sion of the larger form increased to a similar level as the
smaller form (Fig. 1C). The time dependent variation of
expression in rat cerebellum suggested an increasingly
important role of this splice variant during rat develop-
ment. This would correlate with the migration and
differentiation of granule neurons. Therefore, we tested
this hypothesis using primary cultures of granule neurons
prepared from rats sacrificed at postnatal day 7. The
incubation of these cultures with cytosine-arabinoside
arrested the growth of non-neuronal cells, so that more
than 95% of the cultured cells belonged to the granule
neuron population. One week after preparation, the
cultures displayed a dense meshwork of dendritic con-
nections reflecting differentiation of the neurons. At
days 1 and 7 after preparation, the cells were lysed and
the expression pattern of the PTP
-isoforms analyzed.
Figure 1C demonstrates that the granule neuron popu-
lation expressed increasing amounts of the larger splice
variant of PTP
in a differentiation dependent manner.
Since a higher expression of the larger splice variant
was also revealed in skeletal muscle, we investigated the
expression pattern of PTP
in the murine
in vitro
entiating C2C12 myoblasts. In addition, 3T3L1 cells
were included which are a model cell line for the differ-
entiation to adipocytes. Again, a similar level of the smaller
phosphatase splice variant was detected in both cell lines
independent of the differentiation status, whereas PTP
expression increased during differentiation (Fig. 1D). In
summary, both splice variants occur in human, mouse
and rat, and the expression of the larger splice variant
132 can increase at specific stages in development
or differentiation of some tissues.
splice variants and their phosphatase activity
in vitro
and towards the insulin receptor
To determine a possible difference in the enzymatic
activity of the splice variants, we first used p-nitrophenyl
phosphate (pNPP) as a substrate. The splice variants of
were transiently over-expressed in human 293
cells and the lysates directly used for the dephosphorylation
assay. In addition, we included PTP
mutants of the
carboxyl-terminal tyrosine residue to phenylalanine in
Src kinase activation by PTP
splice variants
© 2006 The Authors
Genes to Cells (2007)
, 63–73
Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
this assay, since our previous experiments indicated an
important role in PTP
function (Lammers
et al.
As shown in Fig. 2, both splice variants and their mutants
of the carboxyl-terminal tyrosine showed similar activity,
with the observed minor differences not being statistically
Our previous work has shown that wild-type PTP
and the Y798F mutant have a similar activity towards the
insulin receptor (Lammers
et al.
1998). We next investigated
a possibly different activity of the two splice variants against
the insulin receptor in a functional cellular assay. BHK-
cells over-expressing the insulin receptor detach from the
cell culture dish upon treatment with insulin. However,
this effect can be abrogated by transfection of PTP
et al.
1995). Applying this system, we did not
detect a significant difference of the splice variants in the
ability to rescue the cells (data not shown).
splice variants differentially activate the Src
family kinases in intact cells
Next, we employed a known protein substrate of PTP
the Src kinase family member c-Fyn, to investigate the
phosphatase activity in intact cells. c-Fyn was transiently
Figure 1 Tissue-specific expression of PTPα splice variants. (A) Schematic representation of PTPα and its alternatively spliced exons.
The upper panel shows the exon–intron boundaries based on the published sequence (GenBank Acc. No. HSDJ534B8). Horizontal
arrows indicate the position of the primers used for RT-PCR amplification, resulting in fragments of 310 or 283 bp for human RNA.
The bottom panel shows the DNA and protein sequence of the alternatively spliced 27 bp miniexon (bold, vertical arrows) including its
surrounding sequence. SP, signal peptide; TM, transmembrane segment; D1 and D2, the phosphatase domains. (B) PCR-generate
fragments were separated on a 5% polyacrylamide gel and silver stained. Templates included vector controls (left lane), and cDNAs o
reverse transcribed RNAs of the indicated human cell lines and tissues (origin of cell lines: 293, kidney; TF-1, bone marrow, erythroblast;
Calu 6, lung carcinoma; MCF-7, mammary gland; LAN-1 and Kelly, neuroblastoma; U373 MG and U-87, glioblastoma). M, DNA size
marker (bp); SKM, skeletal muscle. (C) RNA from rat cerebellum was isolated at postnatal days 1, 4 and 14. Granule neurons were isolate
from rat cerebellum at postnatal day 7 and differentiated in vitro for 1 or 7 days. PCR fragments of 302 or 275 bps were separated as
described above. P, postnatal day. (D) RNA from 3T3-L1 and C2C12 cells was isolated from undifferentiated () or differentiated cells
(+). RNA was reverse transcribed and subjected to PCR with species-specific primers yielding fragments of 261 bp and 234 bp.
Electrophoretic separation was done as described above.
K Kapp et al.
Genes to Cells (2007) 12, 63–73
© 2006 The Authors
Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
over-expressed in 293 cells either alone or together with
one of the two splice variants of PTPα and cell lysates
were analyzed by immunoblotting. The top panels of
Fig. 3 show that similar amounts of the splice variants
of PTPα and c-Fyn were expressed. Investigating the
tyrosine phosphorylation of c-Fyn, we found that overall
phosphorylation was reduced upon co-expression of
either variant of PTPα. The use of phosphopeptide-
specific antibodies to c-Fyn revealed that Y531 at the
carboxyl-terminus was specifically dephosphorylated in
the presence of either of the two PTPα forms whereas
Y420 in the activation loop of c-Fyn remained phospho-
rylated under these conditions. Using c-Src as a substrate
in this system, similar data were obtained (not shown).
Thus, under these conditions both splice variants behaved
We have previously used focus formation as a cell-
based assay to determine the activity of different PTPα
mutants under physiological conditions. Infection with
retroviruses encoding PTPα132-Y798F but not the
wild-type phosphatase led to transformation of fibroblast
cells stably over-expressing a moderate level of c-Src,
indicating that the tyrosine mutant strongly activated
c-Src (Lammers et al. 2000). We have now employed this
assay to determine the activity of the splice variants
of PTPα. Previously, Lin et al. (1995) have observed a
significant anchorage-independent growth and tumori-
genicity when murine in contrast to chicken c-Src was
over-expressed in NIH3T3 cells. Therefore, we used
murine and chicken c-Src in parallel to rule out any
effect of PTPα based on the origin of c-Src. PTPα was
introduced into the cells by retroviral infection with sim-
ilar multiplicities of infection for the different isoforms
and mutants. Figure 4A shows that both wild-type iso-
forms, PTPα123 and PTPα132, did not lead to focus
formation, whereas PTPα123-Y789F and PTPα132-
Y798F activated c-Src (murine origin). Similar data
were obtained, when using fibroblasts over-expressing
a moderate amount of chicken c-Src (data not shown).
As controls, we either infected parental NIH3T3 or used
c-Src over-expressing cells, which were not infected
with PTPα encoding retroviruses. In both cases, we did
not observe any focus formation (data not shown). To
quantitatively analyze the effect of the splice variants, we
performed a series of five independent experiments that
are summarized in Fig. 4B,C. The activation potential
of the isoform PTPα132-Y798F exceeded the potential
of the isoform PTPα123-Y789F by 181% for murine
and 357% for chicken Src, with PTPα123-Y789F
representing 100%, respectively. To ensure that expression
of both variants was similar, NIH3T3 cells were infected
at similar m.o.i. with the corresponding retroviruses,
grown to confluence under selection and harvested. A
similar expression was detected by Western blotting for
Figure 2 In vitro phosphatase activity of PTPα isoforms. 293 cells
were transiently transfected with empty plasmid (control),
PTPα123, PTPα132, PTPα123-Y789F or PTPα132-Y798F.
Cells were lysed and PTP activity was assayed using pNPP as
substrate. The activity was corrected for PTPα expression. The
histogram represents an experiment done in quadruplicates an
shows the phosphatase activity as means ± SEM.
Figure 3 Both isoforms of PTPα dephosphorylate c-Fyn. 293
cells were transfected with 1 µg c-Fyn and 1 µg PTPα123 o
PTPα132 expression plasmid as indicated in the figure. Cells were
lysed, proteins were separated by SDS-PAGE and analyzed b
immunoblotting with the antibodies as indicated.
Src kinase activation by PTPα splice variants
© 2006 The Authors Genes to Cells (2007) 12, 63–73
Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
both splice variants (Fig. 5C, lower panel). We conclude
that the large PTPα isoform more efficiently leads to
Src-dependent focus formation, and that the activation
is stronger with c-Src derived from chicken.
As a further step, we investigated the activation of c-Src
at the molecular level. To this end, we isolated several
foci from the focus formation assays described above.
After amplification of the cells, lysates were analyzed by
immunoblotting using phosphopeptide-specific anti-
bodies. Figure 5A shows a representative set of the c-Src
over-expressing cell line and focus derived cell popula-
tions (from more than 20 foci analyzed). In control cells
expressing murine c-Src, which also serves as a loading
control, phosphorylation was found on Y527 but not on
Y416. In foci from chicken or murine c-Src expressing
cells infected with PTPα132-Y798F, the level of Y527
phosphorylation was reduced and the phosphorylation
of Y416 enhanced. The analysis of cells over-expressing
PTPα123-Y789F yielded a similar result, showing less
phosphorylation of Y527 and enhanced phosphorylation
of Y416. Densitometric scanning of the samples shown
(Fig. 5B) and analysis of additional foci confirmed that
despite of different expression levels of the two PTPα
splice variants in the individual foci derived cell lines, the
phosphorylation status of c-Src was similar. This was
reflected in a Src-kinase assay using parental NIH3T3
cells infected with retrovirus encoding either PTPα
isoform. The cells were grown as a pool under selection,
and after lysis endogenous Src was immunoprecipitated.
One half was assayed with enolase as a substrate, while the
other half was directly loaded on the gel to verify similar
amounts of Src protein in each immunoprecipitation
(Fig. 5C). In addition, the presence of PTPα in the lysate
was verified. We conclude that c-Src can be activated
by both splice variants; however, the large form has a
higher efficiency as represented by the higher number
of foci.
One reason for the different behavior could be a dif-
ference in the formation of dimers by the two isoforms.
To test this, we made use of PTPα extracellular domain
mutant P137C, which induces a permanent dimerization
due to the formation of stable disulfide bonds (number-
ing according to Jiang et al. (1999) which includes the
signal peptide). The authors showed that this mutant had
a decreased ability to activate c-Src in vitro kinase assays;
however, the effect was only shown for the smaller PTPα
isoform. To investigate a dimerization-dependent, differ-
ent focus formation potential of the splice variants, we
constructed P137C mutants for both splice variants
(PTPα123 P137C, Y789F and PTPα132 P137C, Y798F),
generated retroviruses and used them for focus forma-
tion assays as above. Interestingly, the number of foci
generated by the viruses encoding the additional P137C
mutant was similar to the number generated by the
PTPα123-Y789F or PTPα132-Y798F form (data not
shown). Since this result was unexpected, we performed
Figure 4 PTPα132-Y798F carries a higher c-Src activation
potential. (A) NIH3T3 cells over-expressing murine c-Src were
infected with retroviruses encoding the PTPα form indicated.
Cells were cultured for 21 days and foci were stained with crystal
violet. NIH3T3 cells over-expressing murine (B) or chicken c-Src
(C) were infected with retroviruses encoding the indicated PTPα
form; control cells were not infected. The histograms represent the
average of foci/10e6 retroviruses, given as means ± SEM from five
independent experiments. Statistics were done by analysis o
variance and Student’s t-test. The P values < 0.05 (*) and < 0.01
(**), as indicated in the figure, were considered to be significant
with respect to any other column. The number of foci from
control experiments in Fig. 4C as well as SEM from NIH3T3 cells
over-expressing chicken c-Src and infected with PTPα132 are too
small to be visualized.
K Kapp et al.
Genes to Cells (2007) 12, 63–73
© 2006 The Authors
Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
Figure 5 Analysis of c-Src activity. (A) Individual foci were picked, cells expanded and their lysates analyzed by immunoblotting. Dat
are representative for foci isolated from three independent focus formation assays. control, NIH3T3 cells over-expressing murine c-Src.
(B) The relative amount of phosphotyrosine on residues 416 and 527, as shown in A, was quantified. (C) NIH3T3 cells were infecte
with retrovirus encoding the tyrosine mutant of either splice variant, grown to confluence under selection and Src-kinase activit
determined using enolase as a substrate (upper panel). An aliquot of the immunoprecipitated Src is shown in a control blot (middle panel),
as is an aliquot of the cell lysate where PTPα is detected (lower panel). (D) The indicated isoforms of PTPα were transiently over-
expressed in 293 cells, cells lysed, proteins cross-linked and detected by Western blot analysis. DSS, disuccinimidyl suberate.
Src kinase activation by PTPα splice variants
© 2006 The Authors Genes to Cells (2007) 12, 63–73
Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
a transient expression of the PTPα mutants and analyzed
their potential to dimerize by cross-linking of the pro-
teins in the cell lysate. We did not detect a difference
between the splice variants, and dimerization was enhanced
by the P137C mutant (Fig. 5D). The slightly stronger
dimerization that appears for PTPα123-Y789F, P137C
is caused by a somewhat higher expression, as becomes
apparent on shorter exposures. Thus, phosphatase dimeri-
zation and the P137C mutants are not suitable to explain
the differential behavior of the splice variants during
focus formation.
In this study, we have investigated the expression of
extracellular splice variants of PTPα and their possible
physiological role. One isoform, PTPα123, was ubiqui-
tously and constitutively expressed, whereas a higher
expression of the second isoform with an additional
exon, PTPα132, was tissue-specific and dependent on
the differentiation status. The RT-PCR based expression
analysis points towards a possible role for the larger splice
variant in brain development. In support of this, in
chicken cerebellum the expression of PTPα is enhanced
during a comparable stage of development (Fang et al.
1996). However, alternative splicing products have not
been detected for chicken PTPα, but the described iso-
form is the orthologue of PTPα132. PTPα expression
was also described in zebra fish, especially in the nervous
system (van der Sar et al. 2001), and the importance of
PTPα for retinal development was shown (van der Sar
et al. 2002). More recently and using PTPα knock-out
mice, the role of PTPα in hippocampal neuronal migra-
tion and long-term potentiation was described (Petrone
et al. 2003) as well as a function in learning and other
forms of neuroplasticity (Skelton et al. 2003). It will be
interesting to see whether these functions are isoform
Alternative splicing during neuronal development was
also shown for PTP LAR. There are six alternatively
spliced isoforms that differ by small exons ranging from
12 bp (LASE-b), to 27 bp (LASE-c), 33 bp (LASE-a) and
75 bp (LASE-d). The alternatively spliced exons of the
LASE-c and LASE-d isoforms also occur in the extracel-
lular domain, and for LASE-c, the expression decreases
during CNS development but is up-regulated in NGF-
induced PC12 cell differentiation (Zhang & Longo
In addition to brain, PTPα132 is expressed in some
but not all skeletal muscle derived probes. In the C2C12
cell line an increase of PTPα132 correlated with the dif-
ferentiation from myoblasts to the myotube-like stage.
Using multiprobe RNase protection assays Norris et al.
(1997) detected the highest amounts of this isoform in
fat and not in skeletal muscle. A likely explanation for
this difference is that PTPα expression is different in
various types of skeletal muscle, as we have shown in
Fig. 1B. The general importance of PTPα expression for
differentiation of skeletal muscle has been demonstrated
by Lu et al. (2002). In parallel to the analysis of a cellular
model for skeletal muscle we investigated PTPα expres-
sion in a model for adipose tissue and also found an
increased expression of the larger phosphatase form
upon differentiation. A function of PTPα in adipose cells
was investigated by Cong and co-workers showing that
PTPα inhibits translocation of GLUT-4 upon insulin-
stimulation (Cong et al. 1999). In addition to these tissues,
we have recently presented data that both splice variants
of PTPα also occur in pancreatic β-cells and that PTPα
over-expression can down-regulate insulin secretion
(Kapp et al. 2003).
Functional analysis of the two splice forms in vitro
and in a cell based system with the insulin receptor as a
substrate revealed no major differences for the PTPα
isoforms. This confirms our previous observation that
down-regulation of the insulin signal is not affected by
mutation of the carboxyl-terminal tyrosine (Lammers
et al. 1998). Further, in agreement with den-Hertog et al.
(1994) mutation of the carboxyl-terminal tyrosine to
phenylalanine did not change the in vitro activity of the
As a second physiological substrate, we have focused
on Src family kinases. In vitro analyses showed that upon
transient over-expression the Src family kinases c-Fyn
and c-Src were dephosphorylated by both phosphatase
isoforms on the regulatory carboxyl-terminal tyrosine
residue. Under these conditions, expression of only the
Src kinases revealed that a fraction was constitutively
activated and autophosphorylated at Y420, likely because
of the limiting amount of endogenous Csk that is not
sufficient to phosphorylate the carboxyl-terminal c-Src
tyrosine thereby inactivating the kinase. Co-over-expressed
PTPα dephosphorylated the carboxyl-terminal tyrosine
residue of the inactive fraction, which should lead to
autophosphorylation and thus yield an increase in
phosphorylation of the activation loop tyrosine residue.
We did not detect such an increase; however, it has been
reported that PTPα can dephosphorylate both tyrosine
residues and thus itself limits the activation range of Src
like kinases (den-Hertog et al. 1993).
For further characterization of c-Src kinase activity,
focus formation as a functional, quantitative and cellular
assay was employed. By biochemical analysis of individ-
ual foci, we could show that the tyrosine mutants of both
K Kapp et al.
Genes to Cells (2007) 12, 63–73
© 2006 The Authors
Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
isoforms, PTPα123-Y789F and PTPα132-Y798F, were
able to activate c-Src (Fig. 5A). However, as demon-
strated by the increased number of foci, the mutant of
the larger isoform, PTPα132-Y798F, was more efficient
in activating c-Src. Whether this reflects a direct activa-
tion of the Src kinase that is not detected in an in vitro
assay or involves other mechanisms is currently unclear.
Although in the transient over-expression system the
wild-type phosphatases efficiently dephosphorylated
the Src-family kinases, only the mutants of the carboxyl-
terminal tyrosine were able to lead to cellular transfor-
mation in NIH3T3 cells, as shown before (Lammers et al.
2000). This is in contrast to the proposed phosphotyrosine
displacement mechanism of Zheng et al. (2000) where
the presence of a phosphorylated tyrosine residue at the
carboxyl-terminus of PTPα is essential for the dephos-
phorylation and activation of c-Src. As possible reasons
for this strikingly different result we cannot exclude cell
type-specific effects, like differences in the expression of
other c-Src activity regulating proteins like PKCδ (Brandt
et al. 2003), Sin (Yang et al. 2002) or Srcasm (Seykora et al.
2002). However, Yang et al. (2002) also demonstrated
c-Src activation by the mutant phosphatase, which
should not be possible according to the phosphotyrosine
displacement mechanism. In addition, these authors
found that neurite outgrowth and induction of transin
RNA in PC12 cells after EGF stimulation was stronger
in mutant than in wild-type PTPα over-expressing cells.
For the tyrosine phosphatase CD45, differential func-
tions of the isoforms also have been described and were
explained by a differential homodimerization of the
alternatively spliced isoforms (Xu & Weiss 2002). A
similar scenario could be possible for PTPα, since
dimerization has been shown to regulate its phosphatase
activity (Bilwes et al. 1996; Blanchetot et al. 2002).
We therefore employed the constitutively dimerizing
PTPαP137C mutant in the focus formation assay. How-
ever, we did not find a dimerization related significant
difference in foci generation between the splice variants.
This result indicates that a differential dimerization
potential does not explain the higher efficiency of the
larger phosphatase form to activate c-Src.
In conclusion, the splice variants of PTPα are
expressed in a tissue and differentiation dependent
manner and likely have distinct physiologic roles.
Experimental procedures
Plasmids and antibodies
Human PTPα132 and its mutant PTPα132-Y798F have been
described (Kaplan et al. 1990; Lammers et al. 1998). The isoform
PTPα123 was kindly provided by N.P.H. Møller (Bagsværd,
Denmark), the mutants of PTPα were generated by standard
cloning procedures. For transient expression the pRK5-vector
containing a CMV promoter was used. To generate retroviruses,
cDNAs were cloned into the vector pLXSN (Clontech).
The monoclonal antibody mab29 is directed against the
amino-terminal phosphatase domain of PTPα (kindly pro-
vided by N.P.H. Møller). To detect c-Fyn and c-Src, a rabbit
polyclonal serum directed against the carboxyl-terminal 15
amino acids was used. For the kinase assay, monoclonal antibody
327 (Calbiochem) was used. Phosphotyrosine was detected with
the antibody 4G10 (Upstate), the Phospho-Src (Tyr416) anti-
body (#2102, Cell Signaling) and the Phospho-Src (Tyr527)
antibody (#2105, Cell Signaling). Secondary antibodies were
horseradish peroxidase-coupled anti-rabbit or anti-mouse IgGs
(Sigma). Proteins were visualized with chemiluminescence
(ECL, Amersham Biosciences).
Cell lines and expression analysis
293 cells and BOSC23 cells were grown in Dulbecco’s modified
Eagle’s medium/F12 medium containing 10% fetal calf serum and
2 mm l-glutamine. NIH3T3 cells were grown in Dulbecco’s
modified Eagle’s medium with the same supplements and 1 g/L
glucose. GP + E cells, C2C12 and 3T3-L1 cells were grown in
Dulbecco’s modified Eagle’s medium with the same supplements
and 4.5 g/L glucose.
For differentiation of the mouse 3T3-L1 preadipocytes into
adipocytes, the cells were treated with insulin, dexamethasone,
and 3-isobutyl-1-methylxanthine for 5–7 days. Differentiation of
myoblast C2C12 cells to myotubes was initiated by reducing fetal
calf serum in the culture medium to 0.5%. For preparation of
primary cerebellar granule neurons, rats were sacrificed at postnatal
day 7 (P7) and neurons prepared by dissecting cerebella and
mechanically dissociating the cells in the presence of trypsin and
DNAse as described previously (Schulz et al. 1996). A 5 × 10
were plated on polylysine-coated 60-mm dishes and maintained
in Eagle’s basal medium containing 10% fetal calf serum, 2 mm
glutamine, and 20 µg/mL gentamycin. Twenty-four hours after
preparation, cytosin-arabinoside was added to the cultures at a final
concentration of 10 µm in order to arrest the growth of non-neuronal
cells. Cells were lysed for RNA isolation 1 or 8 days later.
Expression analysis
Total RNA was prepared from various cell lines by lysing the cells
before reaching confluence in 1 mL of guanidine thiocyanate/
phenol-based solution (PeqLab) per 60 mm dish. The RNA was
isolated according to the supplier’s instructions and dissolved in
DEPC-treated water. To assay the quality of the RNA, an aliquot
was analyzed by gel electrophoresis. For RT-PCR, 2 µg of total
RNA was denatured for 15 min at 65 °C and reverse-transcribed
in a total volume of 20 µL using the First Strand Synthesis kit
(Roche Diagnostics). Specific PCR reactions were carried out in
a total volume of 50 µL containing template (control: 10 ng of
each PTPα splice variant cloned into pRK5; cDNA libraries:
Src kinase activation by PTPα splice variants
© 2006 The Authors Genes to Cells (2007) 12, 63–73
Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
100 ng DNA; reverse-transcribed single-stranded cDNA as described
above: 5 µL; negative control: 0.5 µg total RNA), 2.5 µm primers
mouse reverse: 5-CGGAAAGAGTTGGAATGACTCC-3; rat
forward: 5-CTGATAACCAGTTCACGGATGC-3, rat reverse:
otides, 2.5 units Taq polymerase in PCR buffer (PeqLab) with 35
cycles. DNA fragments were separated on a 5% acrylamide gel and
silver stained according to standard protocols.
Lysis of cells and blotting
Transfections were performed using the method of Chen &
Okayama 1987 and analyzed as described (Lammers et al. 1993).
Cells derived from foci were lysed in Laemmli buffer, boiled
and proteins size-separated by SDS-polyacrylamide (PAGE)
gel electrophoresis, transferred to nitrocellulose filters and
analyzed by immunoblotting. Densitometric analysis of Western
blots was done using the Kodak digital science 1D Image Analysis
For cross-linking, an aliquot of the lysate was treated for 2 h at
4 °C with the cross-linking reagent disuccinimidyl suberate (DSS,
Pierce) that was dissolved in dimethyl sulfoxide and used at
0.5 mm. The reactions were terminated by adding Tris buffer,
pH 7.5, to a final concentration of 50 mm Tr is.
Phosphatase and kinase activity assays
Lysates from transiently transfected 293 cells were diluted with the
same volume of phosphate buffered saline. Twenty microliters
were incubated at 30 °C with pNPP-assay buffer (62.5 mm NaCl;
12.5 mm DTT; 60 mm K-acetate, pH 5.5) containing 8.67 mm
pNPP. Reactions were stopped after 20 min by the addition of
NaOH (100 µL, 0.4 M) and the OD
405 nm
was measured directly.
Experiments were done in quadruplicates. In parallel, aliquots of
the lysates were loaded on a SDS-PAGE gel, the gel was blotted
and PTPα amounts were quantified using the Kodak digital
science 1D Image Analysis Software. The phosphatase activity was
corrected for the amount of PTPα present in the lysates, and is
therefore referred to as relative phosphatase activity. The in vitro
kinase assay was performed as described by Yang et al. (2002).
Focus formation assay
NIH3T3 cells over-expressing a moderate amount of either
murine or chicken c-Src were used as described (Lammers et al.
2000). Briefly, 75 000 cells were seeded into a six-well dish and
16 h later infected for 6–7 h with equal amounts of retroviruses
(5 × 10
to 10
) in the presence of 6 µg/mL polybrene. Forty-eight
hours later, cells were trypsinized and seeded into a 10-cm dish in
Dulbecco’s modified Eagle’s medium (1 g/L glucose) containing
4% fetal calf serum. The medium was changed every other day for
3 weeks and the cells were stained with crystal violet (0.5% crystal
violet in 20% methanol).
Inactivation of the insulin signal in BHK cells
BHK cells over-expressing the insulin receptor were transfected
and treated as described (Moller et al. 1995). After 18 days, the cells
were stained with crystal violet and staining evaluated using the
TotalLab software from Nonlinear Dynamics.
Statistical analysis
Statistics were done by analysis of variance (ANOVA), followed
by Student’s t tests for unpaired groups. The statistical software
package JMP (SAS Institute) was used.
This work was supported by a grant from the DFG to R.L.
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Received: 11 October 2005
Accepted: 9 October 2006
    • "Although we cannot exclude the possibility that both antibodies displace an inhibitory molecule from the extracellular domain of CDCP1 or induce a conformational change that stabilizes CDCP1 complexes in lipid rafts, the lack of effect of a Fab fragment of RG7287 strongly suggests that the molecular basis of this agonistic behavior is antibodyinduced cross-linking of CDCP1. Recently, Cooper and Qian (2008) proposed that dimerization provides a robust activation mechanism for SFK-dependent transmembrane signaling by receptors that lack intrinsic catalytic activity, if the following conditions are met: i) the receptor has SFK phosphorylation sites, to which SFKs bind in a phosphorylation dependent manner ii) an SFK bound to one receptor can phosphorylate the second receptor or its associated SFK in a dimer, and iii) dephosphorylation occurs via an unregulated protein tyrosine phosphatase (PTP) (Kapp et al., 2007). Although presently nothing is known about the PTP involved, CDCP1 fulfills the first two conditions and we hypothesize that any bivalent anti-CDCP1 antibody that does not sterically interfere with receptor dimerization will induce CDCP1 activation. "
    [Show abstract] [Hide abstract] ABSTRACT: CUB-domain-containing-protein-1 (CDCP1) is an integral membrane protein whose expression is up-regulated in various cancer types. Although high CDCP1 expression has been correlated with poor prognosis in lung, breast, pancreas, and renal cancer, its functional role in tumor formation or progression is incompletely understood. So far it has remained unclear, whether CDCP1 is a useful target for antibody therapy of cancer and what could be a desired mode of action for a therapeutically useful antibody. To shed light on these questions, we have investigated the cellular effects of a therapeutic antibody candidate (RG7287). In focus formation assays, prolonged RG7287 treatment prevented the loss of contact inhibition caused by co-transformation of NIH3T3 cells with CDCP1 and Src. In a xenograft study, MCF7 cells stably overexpressing CDCP1 reached the predefined tumor volume faster than the parental MCF7 cells lacking endogenous CDCP1. This tumor growth advantage was abolished by RG7287 treatment. In vitro, RG7287 induced rapid tyrosine phosphorylation of CDCP1 by Src, which was accompanied by translocation of CDCP1 to a Triton X-100 insoluble fraction of the plasma membrane. Triggering these effects required bivalency of the antibody suggesting that it involves CDCP1 dimerization or clustering. However, this initial activation of CDCP1 was only transient and prolonged RG7287 treatment induced internalization and down-regulation of CDCP1 in different cancer cell lines. Antibody stimulated CDCP1 degradation required Src activity and was proteasome dependent. Also in three different xenograft models with endogenous CDCP1 expression RG7287 treatment resulted in significant tumor growth inhibition concomitant with substantially reduced CDCP1 levels as judged by immunohistochemistry and Western blotting. Thus, despite transiently activating CDCP1 signaling, the RG7287 antibody has a therapeutically useful mode of action.
    Article · Sep 2013
    • "the biological system (see Supplementary data): Y789F mutation reduces but does not eliminate RPTPa-Src binding (Vacaru and den Hertog, 2010a), and some activation of Src in vivo can be detected when RPTPa(Y789F) is retrovirally expressed at high levels (Kapp et al, 2007). Additionally, third-party proteins may act in lieu of pTyr789 in some situations. "
    [Show abstract] [Hide abstract] ABSTRACT: Receptor protein tyrosine phosphatase α (RPTPα)-mediated Src activation is required for survival of tested human colon and oestrogen receptor-negative breast cancer cell lines. To explore whether mutated RPTPα participates in human carcinogenesis, we sequenced RPTPα cDNAs from five types of human tumours and found splice mutants in ∼30% of colon, breast, and liver tumours. RPTPα245, a mutant expressed in all three tumour types, was studied further. Although it lacks any catalytic domain, RPTPα245 expression in the tumours correlated with Src tyrosine dephosphorylation, and its expression in rodent fibroblasts activated Src by a novel mechanism. This involved RPTPα245 binding to endogenous RPTPα (eRPTPα), which decreased eRPTPα-Grb2 binding and increased eRPTPα dephosphorylation of Src without increasing non-specific eRPTPα activity. RPTPα245-eRPTPα binding was blocked by Pro210 → Leu/Pro211 → Leu mutation, consistent with the involvement of the structural 'wedge' that contributes to eRPTPα homodimerization. RPTPα245-induced fibroblast transformation was blocked by either Src or eRPTPα RNAi, indicating that this required the dephosphorylation of Src by eRPTPα. The transformed cells were tumourigenic in nude mice, suggesting that RPTPα245-induced activation of Src in the human tumours may have contributed to carcinogenesis.
    Full-text · Article · Jul 2011
    • "In agreement with previous results with PTPa793(Y789F) (Zheng et al. 2000), they found that overexpressed Tyr789 fi Phe mutants alone did not transform normal NIH3T3 cells (Lammers et al. 2000; Kapp et al. 2007). However, they found that both PTPa793(Y789F) and PTPa802 (Y798F) did induce foci in NIH3T3 cells that had been primed by over-expression of Src (Kapp et al. 2007). This is not surprising, because the reduced ability of the Tyr789 fi Phe mutants to activate Src (Zheng et al. 2000) was compensated by the over-expression of Src itself. "
    [Show abstract] [Hide abstract] ABSTRACT: Two isoforms of the transmembrane protein tyrosine phosphatase PTPalpha, which differ by nine amino acids in their extracellular regions, are expressed in a tissue-specific manner. Over-expression of the shorter isoform transforms rodent cells, and it has previously been reasonable to assume that this was a direct consequence of its dephosphorylation and activation of Src. Transformation by the longer wild-type isoform has not previously been studied. We tested the activities of both isoforms in NIH3T3 cells and found that, while both dephosphorylated and activated Src similarly, only the shorter isoform induced focus formation or anchorage-independent growth. Differences in phosphorylation of PTPalpha at its known regulatory sites, Grb2 binding to PTPalpha, phosphorylation level of focal adhesion kinase by PTPalpha, or overall localization were excluded as possible explanations for the differences in transforming activities. The results suggest that transformation by PTPalpha involves at least one function other than, or in addition to, its activation of Src and that this depends on PTPalpha's extracellular domain. Previous studies have suggested that PTPalpha might be a useful target in breast and colon cancer therapy, and the results presented here suggest that it may be advantageous to develop isoform-specific therapeutic reagents.
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