Phosphorylation-Dependent Regulation of PSF
by GSK3 Controls CD45 Alternative Splicing
Florian Heyd1and Kristen W. Lynch1,*
1Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, 422 Curie Boulevard, Philadelphia, PA
Signal-induced alternative splicing of the CD45 gene
in human T cells is essential for proper immune func-
tion. Skipping of the CD45 variable exons is
controlled, in large part, by the recruitment of PSF
to the pre-mRNA substrate upon T cell activation;
however, the signaling cascade leading to exon
exclusion has remained elusive. Here we demon-
strate that in resting T cells PSF is directly phosphor-
ylated by GSK3, thus promoting interaction of PSF
with TRAP150, which prevents PSF from binding
CD45 pre-mRNA. Upon T cell activation, reduced
releasing PSF from TRAP150 and allowing it to bind
CD45 splicing regulatory elements and repress
exon inclusion. Our data place two players, GSK3
and TRAP150, in the complex network that regulates
CD45 alternative splicing and demonstrate a para-
digm for signal transduction from the cell surface to
the RNA processing machinery through the multi-
functional protein PSF.
Signal-induced alternative splicing is a primary, but poorly
understood, mechanism for regulating protein isoform expres-
sion in response to changing cellular environments (Lynch,
2007; Shin and Manley, 2004). In humans, examples of signal-
induced splicing regulation are known to play essential roles in
diverse cellular responses including neuronal depolarization,
insulin signaling, and T cell activation (An and Grabowski,
2007; Chalfant et al., 1995; Lee et al., 2007; Lynch, 2007).
However, surprisingly few cases have been studied in detail
(Shin and Manley, 2004; Lynch, 2007). Therefore, despite the
functional importance of linking extracellular stimuli to pre-
mRNA processing, the mechanisms governing this regulation
are mostly unknown.
A well-documented example of signal-induced splicing regu-
lation is the transmembrane tyrosine phosphatase CD45, which
encodes at least five isoforms as a result of tightly controlled
alternative splicing (Figure 1A; Hermiston et al., 2002). CD45 is
expressed on all nucleated hematopoietic cells and has regula-
tory functions in a variety of signal transduction pathways,
including cytokine-, interferon-, and antigen receptor-mediated
signaling (Hermiston et al., 2002). In T cells, where CD45 plays
an essential role in signal transmission from the T cell receptor
ping of three exons, thereby increasing expression of the small-
est CD45 isoform, CD45R0 (Figure 1A; Hermiston et al., 2002).
This differential CD45 isoform expression in naive versus
activated and memory T cells has long been used as the defining
marker of these T cell states. At a functional level, the activation-
induced exon exclusion in CD45 has been suggested to play an
important role in the homeostasis of the immune system as the
resulting CD45R0 protein forms catalytically inactive dimers
that attenuate T cell signaling (Hermiston et al., 2002; Xu and
Weiss, 2002). Consistently, a silent point mutation in CD45
exon 4 that disrupts the essential splicing regulatory element
ESS1 (exonic splicing silencer 1; Figure 1A) results in aberrant
exon inclusion, loss of CD45R0 isoform expression, and
increased susceptibility to several autoimmune diseases in
humans (Jacobsen et al., 2002; Lynch, 2004).
In recent work, we and others have demonstrated that PSF
and hnRNP L-like (hnRNP LL) bind to the ESS1 regulatory
element in stimulated cells and mediate the increased skipping
of the CD45 variable exons observed upon T cell activation
(Melton et al., 2007; Oberdoerffer et al., 2008; Topp et al.,
2008; Wu et al., 2008; Motta-Mena et al., 2010). Both PSF and
hnRNP LL are RNA-binding proteins that have been shown to
regulate the alternative splicing of many genes in addition to
CD45 (Hung et al., 2008; Oberdoerffer et al., 2008; Shav-Tal
and Zipori, 2002). Moreover, PSF is a highly abundant nuclear
protein that has functions in a range of RNA biogenesis
processes from basic splicing catalysis to transcription to
nuclear export (Shav-Tal and Zipori, 2002). However, how the
various activities of PSF are regulated in cells is not yet clear.
In terms of CD45 alternative splicing, the stimulation-specific
activity of PSF and hnRNP LL is due to the fact that these
latory sequence in activated versus resting T cells (Melton et al.,
2007; Motta-Mena et al., 2010; Topp et al., 2008). In the case of
hnRNP LL, this activation-induced binding is readily attributed to
an increase in protein expression in activated T cells (Topp et al.,
2008). By contrast, nuclear PSF expression remains unchanged
upon activation (Melton et al., 2007), suggesting that its binding
to the CD45 pre-mRNA is regulated at a posttranslational level.
However, the underlying signaling cascade regulating PSF
function has thus far not been characterized.
126 Molecular Cell 40, 126–137, October 8, 2010 ª2010 Elsevier Inc.
Glycogen synthase kinase 3 (GSK3) was initially described as
an enzyme regulating glucose metabolism but has since
attracted much interested due to its involvement in many other
cellular processes (Cohen and Frame, 2001). For example,
GSK3 has been linked to the innate immune system (Martin
et al., 2005) and is involved in regulating neuronal cell fate
and development of tauopathies (Plattner et al., 2006). In the
acquired immune system, specifically T cells, GSK3 activity is
decreased upon antigen stimulation through phosphorylation
on serine 9 (Ohteki et al., 2000; Welsh et al., 1996). The
reduced GSK3 activity has been shown to be involved in medi-
ating the CD28 costimulatory signal and is thus required for an
optimal T cell response (Diehn et al., 2002). In our present
work, we have identified an additional role of GSK3, the modu-
lation of alternative splicing. We show that in resting T cells,
GSK3 directly phosphorylates the splicing regulatory protein
PSF. In this phosphorylated form, PSF is sequestered in
a complex with TRAP150, precluding it from binding the
ESS1 sequence in CD45 alternatively spliced exons. Upon
T cell stimulation, reduced GSK3 activity leads to reduced
PSF phosphorylation, thereby releasing PSF from TRAP150
and allowing it to participate in activation-induced CD45 exon
skipping. Thus, we have now identified the complete signaling
cascade linking T cell receptor engagement with PSF-mediated
exclusion of alternatively spliced CD45 exons and have impli-
cated GSK3 and TRAP150 as two critical regulators of this
PSF T687 Is Differentially Phosphorylated in Resting
versus Activated T Cells
Previously we have demonstrated that PSF is uniquely recruited
to the ESS1 regulatory element of CD45 variable exons in
response to cell stimulation, even though this protein is present
at equal concentration in the nuclei of resting and activated cells
(Melton et al., 2007; Motta-Mena et al., 2010). Importantly, PSF
purified from activated cells has silencing activity on the CD45
variable exons in in vitro splicing assays, while PSF purified
from resting cells does not (Melton et al., 2007). Moreover, using
the MS2 system to tether PSF to a model exon, we find that
forced recruitment of PSF results in exon exclusion under both
resting and activated conditions (data not shown), suggesting
that the activity of PSF is primarily regulated at the level of
RNA binding. Taken together, these data led us to test the
hypothesis that PSF itself is differentially modified in resting
versus activated T cells in a manner that controls its ability to
bind to CD45 exons and cause repression.
Figure 1. Phosphorylation of PSF on T687 Is Decreased upon T Cell Stimulation
(A) Schematic of CD45 showing expressed isoforms, ESS1 silencer sequence, and core motif common to ESS1 and other variable exons through which PSF
(B) Endogenous PSF immunoprecipitated from nuclear extract from either untreated or PMA-stimulated JSL1 cells (72 hr), or IP with unrelated control antibody,
western blotted for total PSF or with antiphosphothreonine (P-Thr) or antiphosphotyrosine (P-Tyr) antibody. Size of PSF in anti-P blots is as indicated.
(C) JSL1 cells stably expressing indicated Flag-tagged PSF variants were lysed, subjected to anti-Flag immunoprecipitation, and blotted for P-Thr or Flag.
Nonspecific band is labeled with an asterisk.
TRAP150 Binds to PSF to Limit Splicing Activity
Molecular Cell 40, 126–137, October 8, 2010 ª2010 Elsevier Inc. 127
We first compared possible posttranslational modifications of
PSF precipitated from resting versus activated JSL1 cells that
we and others have demonstrated to be a faithful model for
antigen-induced alternative splicing of CD45 when stimulated
with the phorbol ester PMA (Lynch and Weiss, 2000;
Oberdoerffer et al., 2008). Using an antibody specific for phos-
phothreonine, we observed a substantial decrease in threonine
phosphorylation under activated conditions (Figure 1B and see
below). In contrast, global tyrosine phosphorylation of PSF is
weak and does not change in the presence of PMA
(Figure 1B). A database search (http://www.phosphosite.org/)
revealed only one site of predicted threonine phosphorylation,
namely T687. Consistent with this prediction, either deletion of
antiphosphothreonine reactive band (Figure 1C). This result is
further supported by a previous study which mapped threonine
phosphorylation in PSF to the C-terminal third of the protein
(Shav-Tal et al., 2001). Therefore, while we cannot entirely rule
out the presence of additional phosphorylated threonine
residues which may escape the detection with the antibody,
we conclude that T687 is the primary site of threonine phosphor-
ylation in PSF.
PSF Specifically Associates with TRAP150/BTF in
a Phosphorylation-Dependent Manner
Strikingly, the immunoprecipitations of PSF derivatives also
revealed a protein of around 135 kD that coprecipitated with
the wild-type PSF and the T687D mutant but was largely absent
in the precipitate of the T687A mutant when detected by P-Thr
antibody or silver stain (Figure 2A and data not shown). Although
a few additional PSF-associated proteins were also observed to
react with the P-Thr antibody, only the 135 kD species differs in
association between the T687A and T687D mutant (see
Figure S1A available online). As the T687A mutant mimics the
dephosphorylated state of PSF that is observed in activated
cells, we reasoned that differential association with the 135 kD
protein could explain differential RNA-binding capability in
resting and activated cells. We therefore subjected the 135 kD
band to mass spectroscopy analysis and identified specific
peptidesfor twonuclear proteins,
(Figure S1B), which comigrate in SDS-PAGE and share
extensive sequence homology (Figure 2B). Using specific
antibodies, we confirmed that both TRAP150 and BTF
coprecipitate with PSF fromnuclear extract and that thisinterac-
tion is reduced in the T687A mutant whencompared to either the
phosphomimic T687D or wild-type PSF (Figure 2C). Consistent
Figure 2. Phosphorylation of PSF on T687 Regulates Association with TRAP150/BTF
(A) Anti-P-Thr blot of Flag-PSF immunoprecipitates as in Figure 1C, looking at coprecipitated proteins of around 150 kD.
(B) Schematic of TRAP150 and BTF showing location of RS (yellow) and homologous (orange) domains.
(C) Immunoprecipitates of Flag-PSF variants, similar to Figure 1C, blotted with antibodies specific to TRAP150 or BTF.
(D) Immunoprecipitates of Flag-PSF from resting (?PMA) versus stimulated (+PMA, 72 hr) JSL1 cells, blotted with antibodies specific to the indicated proteins.
Samples in lanes 6 and 7 were treated with 20 U RNase T1 and 20 mg RNase A for 15 min at 37?C prior to IP. Lanes 1 and 2 correspond to 30% input. See also
TRAP150 Binds to PSF to Limit Splicing Activity
128 Molecular Cell 40, 126–137, October 8, 2010 ª2010 Elsevier Inc.
with reduced phosphorylation of T687 upon T cell stimulation,
the interaction of PSF with TRAP150 and BTF is also significantly
stronger in resting (?PMA) versus activated (+PMA) JSL1 cells
(Figure 2D). Importantly, we observe no significant change in
the threonine phosphorylation status of TRAP150 itself upon
PMA treatment (Figure S1C). Furthermore, the interaction
between PSF and TRAP150/BTF in resting cells is independent
of RNA, as addition of RNase A and T1 did not decrease the
observed coprecipitation (Figure 2D, lane 4 versus 7). In fact,
we observe a reproducible increase in the association of
TRAP150 and BTF with PSF following RNase treatment, sug-
gesting that loss of RNA association makes PSF more acces-
sible to bind TRAP150/BTF. Finally, we note that the interaction
of TRAP150/BTF with PSF is unlikely to be directly mediated by
T687, as deletion of the C-terminal 40 amino acids of PSF,
encompassing T687, is also permissive for interaction with
TRAP150 and BTF (Figure 2A, Figure S1D). We hypothesize,
therefore, that phosphorylation of T687 drives association with
TRAP150 and/or BTF in an allosteric manner as discussed
The function of both TRAP150 and BTF remains poorly char-
acterized. TRAP150 was initially cloned as a component of the
transcription mediator complex (Fondell et al., 1996). However,
consistent with subsequent studies suggesting that TRAP150
is not a primary mediator subunit (Conaway et al., 2005), we
do not observe an interaction of PSF with TRAP220, a core
component of the mediator complex (Figure 2D). More recent
studies have implicated TRAP150 and BTF in RNA processing
(Merz et al., 2007), either as components of a complex with
SNIP1 involved in mRNA stability (Bracken et al., 2008) or in
a loosely associated complex of splicing regulatory factors
including nucleolin and hnRNP A1 (Li et al., 2003). We do detect
SNIP1, nucleolin, and hnRNP A1 in precipitates with PSF.
However, unlike TRAP150 and BTF, these proteins associate
with PSF equally in resting and stimulated cells, and the addition
of RNase completely abolishes this interaction (Figure 2D).
Together these data demonstrate a specific and phosphoryla-
tion-dependent interaction of TRAP150/BTF with PSF and
suggest that this TRAP150-BTF-PSF complex is distinct from
other assemblies in which TRAP150 or BTF have been charac-
terized thus far.
Interaction of TRAP150 with PSF Inhibits CD45 Exon
Toinvestigate thefunctionalroleof thePSF-TRAP150-BTF inter-
action with respect to CD45 alternative splicing, we next
knocked down TRAP150 and BTF using morpholino oligos.
Remarkably, knockdown of TRAP150 results in a dose-depen-
dent decrease in CD45 variable exon inclusion in resting cells,
which at the highest level of TRAP150 depletion (Figure 3A) is
similartothatobservedupon Tcell activation(+PMA).This effect
of TRAP150 depletion on CD45 alternative splicing is dependent
on the presence of the ESS1 regulatory element through which
PSF functions as inclusion of CD45 variable exon 4 from a stan-
dard minigene construct is decreased upon depletion of
TRAP150, while splicing of an analogous construct lacking the
ESS1 regulatory sequence is unaffected by TRAP150 knock
down (Figure S2A). Notably, however, this change in splicing
occurs without any alterations in the expression of the ESS1
regulatory proteins, PSF, hnRNP L, or hnRNP LL (Figure S2B).
To initially investigate if the ESS1-dependent splicing change
we observe upon TRAP150 depletion is a result of altered
TRAP150-PSF interaction, we also tested the effect of disrupting
this interaction by mutating PSF. We predict that the T687A
mutant of PSF, which interacts more weakly with TRAP150
(Figures 2A and 2C), should promote CD45 exon repression in
a manner similar to that observed upon knockdown of
TRAP150. Strikingly, we observe a significant decrease in the
inclusion of wild-type CD45 exon 4 upon overexpression of
PSF-T687A in resting cells (Figure 3B). In contrast, expression
of WT PSF had no effect on exon 4 splicing, and neither protein
mimic PSF-T687D had no effect on exon 4 splicing in either
resting or stimulated cells (Figure 3B and data not shown),
consistent with the notion that the T687 phosphorylated pool of
PSF is itself nonfunctional for CD45 exon repression and does
not block the accumulation and activity of the hypophosphory-
lated form of endogenous PSF upon stimulation (see below). A
functional role for the TRAP150-PSF interaction is further sup-
ported by the finding that the majority of wild-type, endogenous
PSF is associated with TRAP150 in resting cells, as indicated
by the efficiency of coimmunoprecipitation (Figure 3C). In
contrast, the bulk of PSF is not precipitated by TRAP150
following stimulation (Figure 3C). Taken together, these data
confirm a significant and regulated interaction between endoge-
nous TRAP150 and PSF, and are consistent with this interaction
playing a critical role in regulating PSF function in CD45 splicing.
not substantially change CD45 alternative splicing either on its
own (Figure S2C) or in combination with TRAP150 depletion
(data not shown). As BTF and TRAP150 have been shown to
associate (Bracken et al., 2008), we reasoned that TRAP150
could be the functionally relevant PSF binding partner, with
BTF only recruited to PSF indirectly via TRAP150. Consistent
with this prediction, we find that upon knockdown of TRAP150,
BTF no longer coprecipitates with PSF, although total protein
levels remain unchanged (Figures S2B and S2D). In contrast,
the association of TRAP150 with PSF does not require BTF, as
knockdown of BTF does not weaken TRAP150 coprecipitation
with PSF (Figure S2D).Moreover,we observe a direct interaction
in vitro between purified recombinant TRAP150 and PSF (data
not shown). Therefore we conclude that TRAP150 is the primary
functionally relevant PSF-associated protein and have focused
our subsequent studies on characterizing the regulation and
consequence of this TRAP150-PSF interaction.
TRAP150 Directly Inhibits Binding of PSF to the ESS1
Theaboveresults,together withourpreviousdemonstration that
T cell activation induces PSF’s association with the ESS1 regu-
latory sequence in CD45 (Melton et al., 2007; Motta-Mena et al.,
2010), suggest a mechanism in which the interaction between
TRAP150 and PSF limits exon skipping by blocking the interac-
tion of PSF with ESS1. We therefore assayed the association of
PSF with the ESS1 regulatory element in an RNA pull-down
TRAP150 Binds to PSF to Limit Splicing Activity
Molecular Cell 40, 126–137, October 8, 2010 ª2010 Elsevier Inc. 129
an increase in the association of PSF with ESS1 RNA that is
highly similar to that observed upon T cell activation (Melton
et al., 2007; Motta-Mena et al., 2010). Moreover, PSF associated
with ESS1 RNA does not react efficiently with the P-Thr antibody
unless TRAP150 is depleted (Figure 4A, P-Thr), suggesting that
the phosphorylated population of PSF, bound by TRAP150, is
hindered from binding to ESS1. In further support of this model,
we are unable to detect any TRAP150 itself, or BTF, among the
ESS1-associated proteins (Melton et al., 2007; and data not
We next sought to determine whether TRAP150 directly regu-
lates the binding of PSF to the CD45 ESS1 RNA using purified
proteins (Figure S3A). Purified PSF binds specifically to radiola-
beled ESS1 RNA in a UV crosslinking assay, as shown by
competition with nonspecific versus ESS1 RNA (Figure S3B).
Importantly, PSF purified from resting or stimulated cells binds
equally well to ESS1 RNA, demonstrating that the RNA-binding
capability of PSF itself is not altered upon stimulation
(Figure 4B). However, the addition of purified TRAP150 specifi-
cally inhibits the RNA-binding capability of PSF purified from
resting cells (Figure 4B, top), with little impact on the RNA
binding of PSF purified fromstimulated cells (Figure 4B, bottom).
We further observe that nuclear extract differentially inhibits
RNA bindingof PSFfrom
(Figure 4B). This result provides an explanation for our previous
observation that PSF purified from resting cells cannot repress
CD45 exon usage when added to nuclear extract in an in vitro
splicing assay (Melton et al., 2007), as the nuclear extract would
nuclear extracts from resting or activated T cells are equally
effective in inhibiting RNA binding of PSF, demonstrating that
stimulation alters the inherent susceptibility of PSF to binding
extract (Figure 4B and Figure S3C). Moreover, the inhibitory
against TRAP150, but not by a variety of control antibodies (Fig-
ure 4C and data not shown). Taken together, our data from inter-
action, knockdown, and RNA-binding studies demonstrate that
directly blocks PSF from binding ESS1 in resting T cells, thereby
limiting the repression of CD45 exons prior to T cell activation.
Figure 3. The Majority of Cellular PSF Is Bound to TRAP150 to Inhibit CD45 Exon Skipping in Resting Cells
(A) Representative RT-PCR and quantitation (n = 3) of CD45 isoform expression in JSL1 cells under resting (?) or stimulated (+PMA, 72 hr) conditions or after
treatment of resting cells with control or anti-TRAP150 morpholino oligomers. Error bars correspond to standard deviation. Western blot confirmation of
TRAP150 knockdown shown in lower right.
(B) Quantitation (n = 4) of splicing of CD45 exon 4-derived minigenes in cells transfected with the WT, T687A, or T687D version of Flag-PSF (see western). Error
bars correspond to standard deviation, p value for difference between WT and TA PSF is shown. Minigene E4-WT contains the complete native CD45 exon
4 sequence, while in E4-DESS the ESS1 motif has been replaced by an unrelated sequence of similar length (Melton, et al., 2007).
(C) Analysis of PSF in pellet (P) versus supernatant (S) following precipitation with antibody to TRAP150 or control antibody (IgG). See also Figure S2.
TRAP150 Binds to PSF to Limit Splicing Activity
130 Molecular Cell 40, 126–137, October 8, 2010 ª2010 Elsevier Inc.
GSK3 Directly Phosphorylates PSF and Increases
PSF-TRAP150 Interaction and CD45 Variable Exon
In order to link the TRAP150-dependent regulation of PSF to
T cell signaling, we next sought to uncover how T cell stimulation
allows for reduced phosphorylation of PSF. The Ser/Thr kinase
GSK3 has several of the hallmarks expected of a signal-respon-
sive regulator connecting T cell activation to PSF phosphoryla-
leads to increased phosphorylation of the autoinhibitory S9,
thereby reducing GSK3 activity (Diehn et al., 2002; Figure S4A);
and T687 in PSF is surrounded by several prolines, which is
a preferred substrate context for GSK3 (Hooper et al., 2008).
We therefore purified a HA-tagged version of the constitutively
active GSK3beta S9A mutant from HEK293 cells for use in an
in vitro kinase assay with PSF. In this assay, GSK3-S9A does
(Figure 5A) and JSL1-expressed (Figure S4B) recombinant
PSF. Importantly, in vitro phosphorylation of bacterially ex-
pressedrecombinantPSF byGSK3-S9A indicates thata priming
phosphate is not required for this activity, as is sometimes
observed for GSK3 substrates. We further show that threonine
phosphorylation of PSF by GSK3 primarily occurs on residue
T687, as this phosphorylation is detected by the P-Thr antibody
and is markedly decreased in the T687A mutant of PSF
To examine the functional effects of GSK3, we next produced
JSL1 cell lines stably overexpressing the activated HA-GSK3-
S9A mutant. Consistent with the in vitro kinase results, we
observed increased phosphorylation of endogenous PSF in
Figure 4. TRAP150 Directly Inhibits RNA Binding of
(A) RNA affinity purification using biotinylated 60 nt ESS1
sequence and nuclear extracts of JSL1 cells treated with
control or anti-TRAP150 morpholino oligomers, blotted
for endogenous PSF, P-Thr, or hnRNP L as loading
(B) PSF was purified from resting or stimulated (PMA,
72 hr) JSL1 cells stably expressing Flag-PSF. UV cross-
linking of recombinant PSF purified from resting (R) or
stimulated (S) JSL1 cells to ESS1 RNA alone (?) or in the
presence of anti-Flag eluate from control lysate that lacks
Flag-TRAP150 (mock), purified TRAP150, or nuclear
(C) Addition of anti-TRAP150 or control antibody, to UV
crosslinking experiment as in (B). Lane 9 is the same as
8 except for the addition of more NE, demonstrating that
the increase in PSF binding is a consequence of the anti-
body titrating an inhibiting factor in NE (i.e., TRAP150).
See also Figure S3.
these cell lines (Figure 5C). Remarkably, this
increase in PSF phosphorylation correlates
with an increased interaction between PSF
and TRAP150 and a dramatic decrease in
CD45 variable exon skipping (Figures 5C and
5D). As for the TRAP150 knockdown, the effect
of GSK3-S9A expression on CD45 splicing is dependent on the
presence of the ESS1 element, as determined by minigene
experiments (data not shown and see below). Since expression
levels of the other known regulators of CD45 alternative splicing
were not affected by GSK3-S9A expression (Figure S4C), we
conclude that the change in CD45 splicing induced by GSK3-
S9A is due to the increased interaction of PSF with TRAP150
sequestering PSF away from the CD45 pre-mRNA.
Reduced GSK3 Activity Leads to CD45 Exon Exclusion
in JSL1 Cells and in Primary Human T Cells
To confirm that GSK3 is in fact an endogenous regulator of PSF,
we took advantage of the widely used GSK3 inhibitor SB216763.
Consistent with the studies above, treatment of JSL1 cells with
SB216763 results in a decrease in PSF threonine phosphoryla-
tion, with no change in the expression of PSF itself or any other
known CD45 regulatory protein (Figure 6A, Figure S5A). More-
over, SB216763 induces a robust increase in CD45 exon
skipping in a manner that mimics the effect of PMA stimulation
(Figure 6B). Such exon repression in response to SB216763 is
dependent on the presence of the ESS1 regulatory element, as
substitution of the ESS1 in a CD45 exon 4 minigene greatly
Figure S5B). Two other independent methods to reduce GSK3
activity in resting T cells also resulted in increased CD45 variable
pholino oligos (Figure 6D) or treatment of cells with LiCl, another
GSK3 inhibitor (data not shown). Therefore, regardless of
method, reduction of GSK3 activity in resting cells closely
mimics the effect of PMA stimulation on PSF phosphorylation
and CD45 exon skipping, suggesting that GSK3 inhibition is
TRAP150 Binds to PSF to Limit Splicing Activity
Molecular Cell 40, 126–137, October 8, 2010 ª2010 Elsevier Inc. 131
the primary mode by which PMA stimulation causes PSF-medi-
ated changes in CD45 splicing. Consistently, treatment of cells
simultaneously with SB216763 and PMA does not substantially
increase the magnitude of exon skipping over PMA alone
(Figure S5C), suggesting that SB216763 and PMA act on the
same pathway in order to achieve CD45 exon exclusion.
Finally, in order to confirm the physiological relevance of our
data, we examined whether GSK3 regulates CD45 alternative
splicing in primary cells. To this end, we cultured purified
CD4+ or CD8+ primary human T cells in the presence of
SB216763 or solvent control and analyzed CD45 splicing after
24 or 64 hr. Consistent with our observations in JSL1 cells,
SB216763 induces repression of CD45 variable exons in both
CD4+ and CD8+ T cells at both time points (Figure 6E). There-
fore, we conclude that our data from JSL1 T cells indeed
accurately reflect the in vivo pathway from T cell activation to
CD45 alternative splicing.
Accumulation of Unphosphorylated PSF upon T Cell
Activation Requires De Novo Protein Synthesis
The kinetics of the effect of SB216763 on CD45 splicing is
remarkably similar to that observed upon PMA stimulation of
JSL1 cells or in response to activation of primary T cells
(Figure 6B and Lynch and Weiss, 2000). However, we note that
this time course is markedly slower than the kinetics with which
most signaling events are thought to occur. We previously have
demonstrated that the delayed response of CD45 splicing to
PMA treatment is due, at least in part, to a requirement for de
novo protein synthesis (Lynch and Weiss, 2000). Given that the
GSK3 inhibitor displays similar kinetics, we conclude that the
rate-limiting step in this pathway must be downstream of
(Diehn et al., 2002; Sengupta et al., 2007).
One model to explain the delay in alternative splicing following
GSK3 inhibition would be a highly stable Phospho-T687 form of
Figure 5. GSK3 Directly Phosphorylates PSF, Thereby Regulating CD45 Alternative Splicing
(A) SDS-PAGE of in vitro kinase assay using the constitutively active GSK3beta S9A mutant purified by HA-tag from HEK293 cells (or control mock purification)
incubated with recombinant PSF in the presence of32P-gamma-ATP. Radiolabeled protein corresponding to the size of PSF is indicated.
(B) In vitro kinase assay as in (A) with PSF (WT) and PSF T687A (TA) purified from transiently transfected HEK293 cells. Reactions were performed inthe presence
of cold ATP, and phosphorylation was detected by antiphosphothreonine western blot.
(C) JSL1 cells stably expressing HA-GSK3beta S9A blotted for P-Thr and TRAP150 in precipitates of PSF.
(D) Representative RT-PCR and quantitation (n = 3) of CD45 isoform expression in two representative stable GSK3beta S9A cell lines versus control, all under
resting conditions. Error barscorrespondtostandard deviation.Expressionof theHA-taggedGSK3beta S9Aconfirmedby western blotwithhnRNP U asloading
control (lower panel). See also Figure S4.
TRAP150 Binds to PSF to Limit Splicing Activity
132 Molecular Cell 40, 126–137, October 8, 2010 ª2010 Elsevier Inc.
PSF, perhaps aided by being associated with TRAP150. In this
model, the kinetics of changes in CD45 splicing would be limited
bythehalf-lifeof PSF,as turnoverof PSF in the absence of GSK3
activity would lead to a slow but steady accumulation of PSF un-
phosphorylated at T687 that is not able to be sequestered by
TRAP150. Consistent with this hypothesis, PSF itself and
TRAP150 are both highly stable, with a half-life of over 24 hr (Fig-
ure S6A; A.A. Melton, F.H., and K.W.L., unpublished data), and
a strong loss of PSF phosphorylation is not observed until after
24 hr poststimulation (Figure S6B). Moreover, although the pres-
ence of TRAP150 does not change the overall half-life of PSF
(Figure S6A), it does stabilize the threonine phosphorylation of
PSF as preincubation with recombinant TRAP150 inhibits the
Figure 6. Inhibition of Endogenous GSK3 Causes a Stimulation-like Change in CD45 Splicing
(A) Western blot of endogenous PSF precipitated from JSL1 cells treated with 10 mM SB216763 (SB) or DMSO vehicle control.
(B) Quantitation (n = 3) of CD45 isoform expression in cells treated with SB216763 (10 mM) or PMA (20 ng/ml) for times indicated.
(C) Quantitation (n = 3) of splicing of minigenes (described in Figure 3) in cells treated with SB216763 or DMSO control.
(D) Quantitation (n = 3) of endogenous CD45 isoform expression in cells treated with a morpholino oligo against GSK3. Western blot confirmation of knockdown
relative to U1A loading control shown at top.
(E) Quantitation (n = 3) of endogenous CD45 isoform expression in primary CD4+ or CD8+ T cells purified from human blood and cultured in the absence (DMSO)
or presence of SB216763 for the times indicated. For all panels, error bars correspond to standard deviation. See also Figure S5.
TRAP150 Binds to PSF to Limit Splicing Activity
Molecular Cell 40, 126–137, October 8, 2010 ª2010 Elsevier Inc. 133
A further prediction of the above model is that the relative
amount of phosphorylated PSF would remain high in the
absence of protein synthesis following stimulation. Indeed,
wefindthatPMAtreatmentfails toinduce achangeinPSF phos-
(Figure 7B, Figure S6B), consistent with the block in activation-
induced alternative splicing of CD45 conferred bycycloheximide
(Lynch and Weiss, 2000). We have further shown previously that
blocking PSF translation with a morpholino oligomer abrogates
the ability of PMA to induce CD45 exon skipping (Melton et al.,
2007). Thus, taken together, our data suggest that de novo
PSF synthesis, in the absence of GSK3 activity, is likely the
rate-limiting step by which the proper temporal control of
CD45 splicing is achieved.
In this study, we have identified the complete signaling pathway
that leads from T cell receptor engagement to RNA binding of
PSF and CD45 exon exclusion (Figure 7C). We show that
TRAP150, a protein of largely unknown function, and GSK3,
a versatile signaling mediator, are key components of this
pathway. Our data also provide a molecular explanation for the
built-in ‘‘time lag’’ that is a hallmark of CD45 signal-induced
splicing. This study thus reveals substantial insight into the
mechanism for signal-induced alternative splicing in T cells,
While numerous examples of signal-induced alternative
splicing have been described in a variety of cell types, the
signaling pathways that connect membrane-bound receptors
to the nuclear RNA processing machinery have been identified
in only very few cases (Lynch, 2007; Shin and Manley, 2004).
Some notable examples in T cells are the Sam68-mediated
splicing of CD44 and the TIA-1 regulated Fas alternative splicing
(Izquierdo and Valca ´rcel, 2007; Matter et al., 2002). In these
cases, direct phosphorylation of Sam68 or TIA-1 was shown to
occur rapidly upon activation of signaling pathways and to
directly increase the activity of these proteins bound to RNA.
Such a direct effect of phosphorylation on splicing activity is
markedly different from the mechanism we demonstrate here
in which phosphorylation of PSF indirectly regulates its accessi-
bility to the target RNA via protein-protein interactions. There-
fore, the GSK3-dependent regulation of a mutually exclusive
interaction between PSF and either TRAP150 or CD45 RNA is
a unique paradigm for connecting intracellular signaling path-
ways to RNA processing events.
In our model, the crucial regulatory point is the phosphoryla-
tion-dependent interaction of PSF with TRAP150, which
prevents PSF from binding to the ESS1 RNA. We were able to
map the functionally relevant phosphorylation in PSF to a single
threonine residue, T687, as a T687A point mutant shows
substantially reduced interaction with TRAP150 and bypasses
the regulatory pathway leading to CD45 exon exclusion in
resting cells. We do detect a residual interaction of the PSF
T687A mutant with TRAP150, suggesting that phosphorylation
strongly facilitates this interaction but is not an absolute require-
ment. Furthermore, the finding that deletion of the C-terminal 40
amino acids does not prevent interaction of PSF with TRAP150
argues against direct interaction of TRAP150 with the phos-
phorylated T687 residue. Rather, our data suggest that phos-
phorylation of T687 drives conformational changes regulating
the interaction with TRAP150. In such a model, a small
Figure 7. Stability of P-Thr Form of PSF and Model of Pathway from GSK3 Activity to CD45 Splicing
(A) Western blot of P-Thr form of PSF following in vitro incubation of purified PSF with CIP in the absence or presence of recombinant TRAP150.
(B) Western blot of PSF precipitated from cells treated with PMA (20 ng/ml), PMA + cycloheximide (CHX, 20 mM), or DMSO vehicle control (?) for 48 hr. Samples
were normalized for total PSF in each lane.
(C) Schematized model for signal-induced regulation of CD45 in resting and stimulated T cells as described in text. See also Figure S6.
TRAP150 Binds to PSF to Limit Splicing Activity
134 Molecular Cell 40, 126–137, October 8, 2010 ª2010 Elsevier Inc.
proportion of the T687A mutant or the nonphosphorylated WT
PSF could still be in the conformation permissive to TRAP150
interaction, thereby explaining a basal level of interaction in
the nonphosphorylated state. Future studies to characterize
the interface between TRAP150 and PSF are thus predicted
to uncover complex and important intra- and intermolecular
A further broad implication of the data presented here is in
characterizing the regulation of PSF T687 phosphorylation, and
defining PSF as a substrate of GSK3. In many cases GSK3-
mediated phosphorylation requires a priming phosphate located
four amino acids toward the C terminus from the target site
(Cohen and Frame, 2001). PSF does contain a tyrosine at posi-
tion 691, which could potentially serve as a priming phosphate
of GSK3-mediated T687 phosphorylation. However, as GSK3
phosphorylates PSF purified from bacteria, we conclude that
a priming phosphate is not strictly required, limiting the potential
functional relevance of Y691. Finally, as PSF is a mostly nuclear
protein and GSK3 resides mostly in the cytoplasm, the simplest
model for GSK3-mediated PSF phosphorylation would be that it
occurs immediately upon translation, prior to nuclear import,
although we cannot rule out other possible models for the
location of this activity.
Interestingly, reduced GSK3 activity has been previously
shown to regulate the CD28 costimulatory signal in activated
T cells (Diehn et al., 2002). These data, combined with our
work, suggest that a decrease in GSK3 activity is required early
on CD45 alternative splicing to increase the threshold for activa-
tion, thereby leading to T cell attenuation. Obviously it is impor-
tant for proper immune function to have the attenuation step
temporally delayed with respect to initial activation. In general,
direct phosphorylation or dephosphorylation of proteins occurs
rapidly upon receptor engagement and are thus not suited to
exert a delayed response. In our model the appropriate temporal
response is accomplished by a mostly stable phosphorylation of
PSF and its interaction with TRAP150, which is only released by
de novo synthesis of PSF in conditions with reduced GSK3
Our data regarding the requirement for de novo protein
synthesis in order to induce reduced PSF phosphorylation and
CD45 exon skipping are fully consistent with the model we
have proposed. We cannot formally rule out that cycloheximide
blocks synthesis of a phosphatase required to dephosphorylate
PSF under activated conditions. However, as we have demon-
strated that changing the activity of GSK3 alone is sufficient to
change the phosphorylation state of PSF in the absence of any
phosphatase inhibitors (Figure 5, 6), we favor a model in which
the predominant factor determining the phosphorylation state
of PSF is GSK3. This notion is further supported by our data
showing that experimental decrease of GSK3 activity in resting
conditions is sufficient to induce a CD45 splicing pattern resem-
bling that of activated cells. In addition, if PMA stimulation would
mainly act through increasing expression of a phosphatase,
treatment of cells simultaneously with PMA and SB216763
should have an additive effect. The fact that this is not the
case again suggests that the contribution of a phosphatase, if
any, is minor. Nevertheless, it is possible that there may be
alternative models that are consistent with our data, and further
studies are necessary to work out the complete details of the
molecular interactions between GSK3, PSF, and TRAP150.
number of genes that are alternatively spliced in response to
T cell activation, several of which are regulated in an ESS1-
dependent manner in concert with CD45 (Ip et al., 2007; Roth-
rock et al., 2003). Consistent with PSF binding specifically to
the ESS1 sequence motif, we have shown PSF to be involved
in the regulation of several of the ESS1-containing exons studied
thus far (Motta-Mena et al., 2010; and data not shown). PSF is
also known to have multiple functions in the nucleus, including
roles in transcription and nuclear retention of RNA (Shav-Tal
and Zipori, 2002). It therefore is likely that in addition to influ-
encing CD45 alternative splicing in T cells, the regulation of
PSF by GSK3 and TRAP150 influences a wide spectrum of
PSF-mediated gene expression events impinging on numerous
cellular functions. In light of the many roles GSK3 has been
shown to play in a variety of cell types, it will be interesting to
elucidate the contribution of PSF in mediating these functions.
Cell Culture and Reagents
Growth and stimulation of the JSL1 cell line was done as described previously
(Lynch and Weiss,2000). The GSK3 inhibitor SB216763 (Tocris, 10 mM), cyclo-
heximide (Sigma, 20 mM), or DMSO solvent controls were used for the times
indicated. Purified human primary CD4+ and CD8+ T cells were obtained
from the University of Pennsylvania Human Immunology Core (IRB protocol
#811028). Cells were cultured in RPMI with 10% FBS at a density of 5 3 105
cells/ml. HEK293 cells were cultured in DMEM with Pen/Strep and 5% FBS.
For knockdown experiments, 10 3 106JSL1 cells were transfected with
5–20 nmol of a morpholino oligo (Gene Tools) blocking the translation start
site of the respective target using electroporation. Twenty-four hours post-
transfection, fresh media was added and cells were grown for an additional
48 hr. Morpholino sequences were as follows: BTF, CTAGAATTGGAGCGA
CCCATTTCTT; TRAP150, GTTTTTGACATCTCTGAAGAGAGGA; GSK3beta,
GGCCGCCCTGACATGATCACTCTCT; and PSF, GTCGAGGCAAAAGCGAA
Stable cell lines were produced as described (Rothrock et al., 2003). For
minigene assays, cells were transfected as above with 1 mg of a CD45 exon
4 minigene with WT or mutated ESS1 sequence (Rothrock et al., 2003) and
10 mg expression construct where indicated. In the case of testing expression
of recombinant forms of PSF, a morpholino directed against the 50UTR of
PSF was also cotransfected to reduce endogenous PSF expression (Melton
et al., 2007).
For overexpression of proteins, HEK293 cells were transfected with
Lipofectamine 2000 using standard procedures. Cell lysates were prepared
48 hr posttransfection.
Immunoprecipitation and Protein Purification
Whole-cell lysates (WCE) were prepared in lysis buffer (25 mM Tris [pH 7.4],
150 mM NaCl, 1 mM CaCl2, 1% Triton X-100), and nuclear extracts (NE)
were prepared as described (Melton et al., 2007). For IPs from JSL1 cells,
100 mg NE was incubated with 5 mg PSF antibody (Sigma) or TRAP150 anti-
body (Abcam) for 1 hr and an additional 1 hr with protein G Sepharose
at 4?C under rotation. Beads were spun down, washed six times with 200 ml
13RIPA buffer, and eluted with sample buffer. To compare bound and
unbound protein fractions, the supernatant after the first spin was saved and
analyzed by western blot.
Flag-tagged proteins or WCE from transiently transfected 293 cells was
TRAP150 Binds to PSF to Limit Splicing Activity
Molecular Cell 40, 126–137, October 8, 2010 ª2010 Elsevier Inc. 135
immunoprecipitated as above using M2-Flag affinity gel. Elution of bound
tration of 500 mg/ml in GFB100 (20 mM Tris [pH 7.5], 100 mM KCl, 1 mM EDTA)
withgentle shaking. Forlarge-scale protein purification, Flag-PSF was purified
from JLS1 cells, or His-PSF from E. coli, using affinity chromatography as
described in Melton et al. (2007).
The following antibodies were used throughout the manuscript as noted:
PSF (Sigma P2860 for IP, abnova 269–362 for WB), phosphothreonine
(42H4, Cell Signaling), TRAP150 (ab71985, abcam), SNIP1 (ab19611, abcam),
TRAP220 (ab64965, abcam), GSK3beta (ab31826, abcam), GSK3alpha
(ab28833, abcam), Flag (2368, Cell Signaling), BTF (ab51758, abcam), hnRNP
L (4D11, abcam), hnRNP LL (ARP41102, Aviva Systems Biology), hnRNP U
(3G6, abcam), and phosphotyrosine (P-Tyr-100, Cell Signaling).
RNA was isolated from JSL1 cells using RNA-Bee (Tel-Test) according to the
prepared using T7 polymerase as in Rothrock et al. (2005); cold competitor
RNAs were chemically synthesized by Dharmacon.
32P-labeled ESS1 RNA for UV crosslinking was
Radioactive CD45 RT-PCR was performed as previously described (Lynch
and Weiss, 2000). Briefly, 1 mg of total RNA was reverse transcribed using
a primer binding the junction of CD45 exons 9 and 10 (E9/10). Low-cycle
PCR was then performed with a radiolabeled primer hybridizing to CD45
exon 3 and unlabeled E9/10 (sequences in Topp et al., 2008). PCR products
were separated on denaturing 5% PAGE and visualized using autoradiog-
raphy. Quantification was performed using a Phosphorimager (Typhoon
9200, GE Healthcare) and ImageQuant software.
RNA pull-down assays with biotinylated ESS1 RNA (Dharmacon) were per-
formed as described in Melton et al. (2007). UV crosslink assays were carried
out as described (Rothrock et al., 2005). Briefly, 1 3 105cpm uniformly32P-
labeled ESS1 RNA was incubated with purified proteins and/or nuclear
extracts or cold competitor RNAs. Reaction mixtures were crosslinked with
254 nm UV light for 20 min, RNase digested, and analyzed by 8% SDS-
PAGE. Purified proteins were mixed and preincubated at 30?C for 2 min prior
to the addition of hot RNA probe. Similarly, mixtures including cold RNA
competitor were incubated 2 min at 30?C before32P-labeled RNA was added.
In Vitro Kinase Assay
HEK293 cells expressing HA-tagged constitutively active GSK3beta mutant
(S9A) were lysed in lysis buffer (as above but 0.1% Triton X-100), and
500 mg protein were used for HA immunoprecipitation in lysis buffer. The
precipitate was washed three times in lysis buffer and three times in kinase
buffer (25 mM HEPES [pH 7.5], 25 mM MgCl2,1 mM DTT; 10 mM ATP; adopted
from Wu et al., 2009). The final pellet was resuspended in kinase buffer, mixed
with purified PSF variants as indicated, and incubated for 1 hr at 37?C in the
presence of 5 mM ATP or 25 uCi32P-gamma-ATP.
Supplemental Information includes six figures and can be found with this
article online at doi:10.1016/j.molcel.2010.09.013.
oftheLynchlabforcritical readingofthemanuscript. Thisworkwassupported
by U.S. National Institutes of Health grant R01 GM067719 (to K.W.L.). F.H. is
funded by a fellowship from the Deutsche Forschungsgemeinschaft.
Received: February 18, 2010
Revised: May 17, 2010
Accepted: July 16, 2010
Published: October 7, 2010
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