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INTRODUCTION |
The pim family of proto-oncogenes encodes three
serine-threonine kinases, Pim-1, -2, and -3 (1-3). Little is known
about the functional role of the Pim kinases in vivo,
although a number of observations have pointed to a potential role for
Pim-1 in signal transduction. Pim-1 has been shown to phosphorylate the transcriptional co-activator p100 and activate the transcription factor
c-Myb downstream of Ras (4). Moreover, Pim-1 has been implicated in
glycoprotein 130-mediated induction of cell proliferation and
protection from apoptosis downstream of signal transducer and activator
of transcription 3 (5). In addition, pim-1-deficient mast cells exhibit decreased responsiveness to interleukin-3 in vitro (6). Finally, the Pim kinases have recently been shown to
associate with and stabilize the suppressor of cytokine signaling protein, SOCS-11 (7). SOCS-1
has been shown to be a potent inhibitor of JAK kinase activity and to
play an important role in regulating the responsiveness of cells to
cytokine stimulation (8-11). These observations suggest that the Pim
kinases may play an important role in cytokine signaling.
Expression of the Pim kinases in normal cells is highly regulated, and
several mechanisms by which Pim-1 expression is controlled have been
characterized. Pim-1 mRNA levels are regulated by
transcriptional attenuation of pim-1 (12) as well as by
induction of transcription of pim-1 upon mitogenic
stimulation of cells (13). Pim-1 expression is rapidly induced by a
number of cytokines including interferon-
, interleukin-2, and
interleukin-3 in hematopoietic cells, and by synaptic stimulation in
neurons (3, 13, 14). Pim-2 transcription, like that of Pim-1, is
cytokine-inducible, whereas Pim-3 and Pim-1 transcription are induced
by synaptic activity (3, 15). Levels of pim-1 mRNA are
also regulated post-transcriptionally by modulation of mRNA
stability (13, 16). Expression of the Pim kinases is therefore highly
regulated by multiple mechanisms.
Dysregulation of Pim expression has been implicated in the pathogenesis
of several different forms of leukemia and lymphoma. Pim-1
was originally identified as a common proviral insertion site of
Moloney murine leukemia virus in T cell lymphomas in mice (1), and
viral activation of pim-2 (2) and transgenic overexpression of pim-1 and pim-2 (15, 17) also result in T cell
lymphoma development. The Pim kinases have been implicated in the
development of human hematopoietic malignancies as well. High levels of
Pim-1 protein have been seen in acute myeloid, lymphoid, and erythroid leukemia (18-20).
The observation that transgenic mice overexpressing wild-type Pim-1 or
Pim-2 develop tumors demonstrates that Pim-mediated transformation can
occur as a consequence of dysregulated Pim expression (15, 17). Diverse
mechanisms have been identified in spontaneous tumors that lead to
increased Pim-1 expression. Enhanced Pim-1 mRNA stability has been
observed in numerous transformed cell lines (21) and the half-life of
the Pim-1 protein has been shown to be prolonged in several tumor cell
lines (22, 23). Therefore disruption of the regulation of Pim-1
transcript levels and Pim-1 protein stability have been shown to be
associated with Pim-mediated transformation.
Protein phosphatase 2A (PP2A) is a highly conserved serine-threonine
phosphatase expressed in all eukaryotic cells that is involved in a
multitude of cellular functions including transcription and
translation, cell cycle progression, and cytokine signaling (reviewed
in Ref. 24). In the present study, PP2A is demonstrated to be a binding
partner and regulator of the protein stability of the Pim kinases. PP2A
associates with Pim-1 and Pim-3 in vivo and dephosphorylates
Pim-3 in vitro. Furthermore, overexpression of PP2A results
in a decrease in the expression levels of Pim-1 and Pim-3 protein, and
inhibition of PP2A activity by okadaic acid results in stabilization of
the Pim-1 protein. Finally, PP2A inhibits the effects of the Pim
kinases on the SOCS-1 protein. These data demonstrate a novel mechanism
for the regulation of Pim protein expression and function and suggest a
role for PP2A in Pim-mediated transformation.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Reagents--
The 293T human embryonic kidney
cell line is a gift from Dr. Chris Schindler, Columbia University, New
York, and was maintained in Iscove's modification of DMEM containing
10% fetal calf serum unless otherwise noted. BALB/c mice were
purchased from Charles River, Wilmington, MA, and splenocytes and
thymocytes isolated from the mice were maintained in RPMI 1640 medium
supplemented with 10% fetal calf serum, 5 µM
-mercaptoethanol, 1 mM sodium pyruvate, 2 mM
L-glutamine, 10 mM HEPES, and nonessential
amino acids. Cycloheximide, okadaic acid, PMA, and ionomycin were
purchased from Sigma. Recombinant PP2A was purchased from Upstate
Biotechnology, Lake Placid, NY.
Plasmids--
The SOCS-1, Pim-1, and Pim-2 expression vectors
have previously been described (7). The human Pim-3 expression vector
was a gift from Dr. Vicki Cohan of Pfizer Inc., Groton, CT. Kinase inactive Pim-3 was generated by site-directed mutagenesis of lysine 69 of human Pim-3 to methionine using a PCR-based approach. Murine PP2A
was subcloned into the mammalian expression vector pcDNA3.1 His-C,
purchased from Invitrogen, San Diego, CA.
Antibodies--
The anti-Pim-1 and anti-Pim-3 antibodies were a
gift from Dr. Vicki Cohan, Pfizer Inc., Groton, CT. The anti-His
antibody was purchased from Santa Cruz Biotechnology, Santa Cruz, CA.
The anti-HA antibody was purchased from Roche Molecular Biochemicals. The anti-PP2A antibody used in immunoprecipitation was purchased from
Upstate Biotechnology, Lake Placid, NY. The anti-PP2A antibody used in
Western blotting was purchased from Transduction Laboratories, Lexington, KY. The anti-
-actin and anti-Flag antibodies were purchased from Sigma.
Whole Cell Extracts, Transfections, and
Immunoprecipitation--
Calcium-phosphate transient transfections of
293T cells were performed as previously described (7). For
cycloheximide time course experiments, cycloheximide was used at a
concentration of 10 µg/ml. For okadaic acid treatments of cells,
okadaic acid was used at a concentration of 25 nM. For LLnL
treatments of cells, LLnL was used at a concentration of 10 µM. Whole cell extracts were prepared and
immunoprecipitations were performed also as previously described
(7).
In Vitro Kinase and in Vitro Phosphatase Assays--
In
vitro kinase assays were performed as previously described (6,
22). In vitro kinase assays that were subjected to in
vitro phosphatase reactions were performed as above except that,
after washing twice in buffer containing 20 mM PIPES and 14 mM
-mercaptoethanol, the immunoprecipitates were washed
one time in phosphatase buffer containing 50 mM HEPES, pH
7.5, 0.5% bovine serum albumin, and 1 mM dithiothreitol,
and then resuspended in 40 µl of phosphatase buffer (25). Recombinant
PP2A was then added, with or without 5 µM okadaic acid,
and reactions were incubated at room temperature for 45 min with
agitation. Reactions were terminated by washing twice in 50 mM HEPES, pH 8.0.
Pulse-Chase--
293T cells grown in DMEM supplemented with 10%
fetal calf serum (cDMEM) were transfected by calcium-phosphate
transfection in 6-cm dishes. After 24 h, cells were washed once
with phosphate-buffered saline and cultured in cDMEM for 24 h.
Cells were then washed twice with phosphate-buffered saline and
cultured in methionine-free DMEM supplemented with 10% dialyzed fetal
calf serum for 1 h. 0.5 mCi per plate of 35S-labeled
methionine was then added to the transfections for 30 min. Cells were
then washed twice with phosphate-buffered saline and cultured in cDMEM
for the indicated amounts of time, after which cells were harvested and
lysed. Whole cell extracts were immunoprecipitated with anti-Pim-3
antibody, immunoprecipitates were run on SDS-PAGE, and the gels were
dried and subjected to autoradiography.
Northern Blotting--
Total cellular RNA was isolated using the
RNeasy MiniKit purchased from Qiagen, Valencia, CA, and Northern blots
were prepared using standard techniques. Pim-1 was detected with a
full-length cDNA probe radiolabeled by the NEBlot Kit purchased
from New England Biolabs, Beverly, MA.
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RESULTS |
Pim Protein Kinases Associate with the Serine-threonine Phosphatase
PP2A in Vivo--
The three Pim kinases and the catalytic subunit of
PP2A have previously been identified as proteins that can bind the
regulator of cytokine signaling, SOCS-1 (7). To determine whether PP2A and the Pim kinases can directly associate, His-tagged PP2A and His-tagged Pim-1 were expressed alone or together in 293T cells, and
lysates were subjected to immunoprecipitation with anti-PP2A antibody.
His-tagged Pim-1 was detected in PP2A immunoprecipitates in extracts of
cells expressing both Pim-1 and PP2A, but not in extracts of cells
expressing Pim-1 alone (Fig.
1A). Similar results were
observed when Pim-3 and PP2A were co-expressed in 293T cells (data not
shown). SOCS-2 was used as a negative control for the immunoprecipitations. In extracts of cells co-expressing His-tagged SOCS-2 and His-tagged PP2A, SOCS-2 did not co-immunoprecipitate with
PP2A (Fig. 1A). Thus, in an overexpression system, the Pim protein kinases and PP2A are capable of interacting in
vivo.

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Fig. 1.
PP2A associates with the Pim kinases in
vivo. A, 293T cells were transfected with 15 µg of His-tagged PP2A and 15 µg of His-tagged Pim-1 expression
constructs as indicated, and cell lysates were immunoprecipitated with
antibody to PP2A, then immunoblotted with His antibody. His-PP2A
co-immunoprecipitated with His-Pim-1 in lysates coexpressing His-PP2A
and His-Pim-1 (lane 3) but not in lysates expressing
His-PP2A or His-Pim-1 alone (lanes 1 and 2).
Antibody to PP2A did not immunoprecipitate His-SOCS-2, either in the
presence or absence of coexpressed His-PP2A (lanes 4 and
5). B, splenocytes (S) and thymocytes
(T) from wild-type BALB/c mice were cultured with or without
50 ng/ml PMA and 500 ng/ml ionomycin for 4 h, and protein
complexes were immunoprecipitated with a polyclonal antibody to PP2A
followed by immunoblotting with polyclonal Pim-1 (upper
panel) and monoclonal PP2A (lower panel)
antibodies. Pim-1 expression in whole cell extracts (WCE)
was dramatically induced by PMA and ionomycin (lanes 1-4),
and was detected in immunoprecipitates of lysates from cells cultured
with, but not cells cultured without, PMA and ionomycin (lanes
5-8).
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To confirm that endogenous Pim kinases and PP2A can associate, the
ability of Pim-1 to co-immunoprecipitate with PP2A in lysates from
primary mouse cells was determined. High levels of Pim-1 are induced by
culturing of either mouse spleen or thymus with PMA and ionomycin (26).
Therefore, extracts from primary splenocytes and thymocytes,
unstimulated or stimulated with 50 ng/ml PMA and 500 ng/ml ionomycin,
were immunoprecipitated with antibody to PP2A, and the presence of
co-immunoprecipitated Pim-1 protein was detected by Western blotting.
Pim-1 was detected in PP2A immunoprecipitates of extracts from
stimulated, but not unstimulated cells (Fig. 1B). Thus,
endogenous Pim-1 protein is capable of interacting with PP2A in
vivo.
Pim Kinases Are Direct Substrates of PP2A Phosphatase
Activity--
The Pim kinases are known to autophosphorylate in
vitro (2, 3, 27), and when immunoprecipitates of wild-type and
kinase-inactive Pim-3 were incubated in in vitro kinase
reactions, a band corresponding to phosphorylated Pim-3 was detected in
wild-type but not kinase-inactive Pim-3 immunoprecipitates (Fig.
2, lanes 1 and 3).
To determine whether PP2A can directly dephosphorylate the
autophosphorylation sites on the Pim kinases, Pim-3 immunoprecipitates
were subjected to in vitro kinase assays followed by
in vitro phosphatase assays. When wild-type Pim-3
immunoprecipitates were subjected to in vitro kinase-in vitro phosphatase reactions, recombinant PP2A was
able to dephosphorylate the autophosphorylation sites on Pim-3 (Fig. 2,
lanes 4 and 5). This effect of PP2A was blocked
by addition of okadaic acid, an inhibitor of PP2A catalytic activity,
to the phosphatase reactions (Fig. 2, lanes 7 and
8). These data show that the Pim kinases are in
vitro substrates of PP2A phosphatase activity and suggest that
PP2A may regulate the phosphorylation state of the Pim kinases in
vivo.

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Fig. 2.
Recombinant PP2A dephosphorylates Pim-3
in vitro. 293T cells were transfected with 15 µg of wild-type or kinase-inactive Pim-3, and lysates were
immunoprecipitated with Pim-3 antibody. Wild-type Pim-3
immunoprecipitates were incubated with 10 µCi of
[ -32P]ATP in in vitro kinase reactions, and
then incubated with 0, 0.5, and 2 units of recombinant PP2A
(rPP2A) in in vitro phosphatase reactions.
Autophosphorylated Pim-3 was detected by autoradiography (upper
panel), and immunoprecipitates were immunoblotted with Pim-3
antibody (lower panel). Wild-type but not kinase-inactive
Pim-3 underwent autophosphorylation (lanes 1 and
3), and incubation of wild-type Pim-3 immunoprecipitates
with rPP2A decreased the intensity of labeling of Pim-3 (compare
lane 3 to lanes 4 and 5).
Co-incubation of wild-type Pim-3 immunoprecipitates with rPP2A and
okadaic acid inhibited the dephosphorylation of Pim-3 by rPP2A
(lanes 7 and 8).
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Overexpression of PP2A Results in the Decreased Expression of Pim
Kinases and Co-expressed SOCS-1--
As the activity of the Pim
kinases is controlled by regulation of protein expression, the effect
of overexpression of PP2A on Pim protein levels was examined. 293T
cells were co-transfected with Pim-1 and increasing amounts of PP2A in
the presence of SOCS-1, and Pim-1 levels were detected by Western
blotting. A lacZ expression vector was also
co-transfected to control for transfection efficiency, protein loading,
and the nonspecific effects of overexpressing PP2A. With increasing
expression of PP2A, the levels of expression of Pim-1 decreased (Fig.
3A), and similar results were
seen for Pim-3 (Fig. 3B). These data suggest that
overexpression of PP2A decreases expression of the Pim kinases and
suggest that PP2A may regulate pim kinase expression
in vivo.

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Fig. 3.
Overexpression of PP2A decreases the
steady-state levels of the Pim proteins, and SOCS-1, in 293T
cells. 3 µg of His-tagged lacZ and 3 µl of
Flag-tagged SOCS-1 were transfected into 293T cells alone or together
with 10 µg of HA-tagged Pim-1 and 0, 0.1, 0.5 or 1.0 µg of
His-tagged PP2A (A) or 3 µg of untagged Pim-3 and 0, 0.1, 0.5, 1.0, 2.5, or 5.0 µg of His-tagged PP2A (B), and
lysates were immunoblotted with HA (upper panel,
A) and Pim-3 (upper panel, B) antibodies,
His (second and fourth panels, A
and B), and Flag (third panels, A
and B). Co-expression of SOCS-1 and Pim-1 (A),
and of SOCS-1 and Pim-3 (B), resulted in increased
expression of SOCS-1 and the appearance of slower mobility forms of
SOCS-1 (A and B, lane 2).
Co-expression of SOCS-1, Pim-1, and increasing amounts of PP2A
(A), and of SOCS-1, Pim-3, and increasing amounts of PP2A
(B), resulted in a decrease in the steady-state levels of
the Pim kinases, and in a decrease in the steady-state levels and
appearance of the slower mobility forms of SOCS-1 (A,
lanes 3-5; B, lanes 3-6). LacZ
served as a control for transfection efficiency, protein loading, and
nonspecific effects of overexpression of Pim kinases and PP2A.
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Previous work has demonstrated that the Pim kinases are capable of
phosphorylating and stabilizing the SOCS-1 protein (7). The observation
that overexpression of PP2A decreases the steady-state levels of
expression of the Pim kinases therefore implies that PP2A can modulate
the function of the Pim kinases. Co-expression of SOCS-1 and Pim-1
resulted in an increase in the levels of expression of SOCS-1 and in
the appearance of multiple hyperphosphorylated forms of SOCS-1 of
slower mobility (Fig. 3A, lanes 1 and
2). Upon co-expression of PP2A, however, the levels of
SOCS-1, and the presence of the slower mobility forms of SOCS-1,
decreased with increasing PP2A expression (Fig. 3A,
lanes 3-5). Similar results were obtained when SOCS-1, Pim-3, and
increasing amounts of PP2A were co-expressed in 293T cells (Fig.
3B). Therefore overexpression of PP2A in 293T cells
modulates the increase in the steady-state levels of SOCS-1 and the
appearance of hyperphosphorylated forms of SOCS-1 induced by
co-expression of SOCS-1 with the Pim kinases.
To confirm that the effect of overexpression of PP2A on
SOCS-1 mobility is not a result of direct dephosphorylation of SOCS-1 by PP2A, the ability of PP2A to dephosphorylate SOCS-1 was determined. SOCS-1 was co-expressed with Pim-3, immunoprecipitated, and incubated in in vitro kinase assays, and SOCS-1 phosphorylated by
Pim-3 was then used as a substrate in in vitro phosphatase
assays with recombinant PP2A. Incubation of the labeled SOCS-1
immunoprecipitates with recombinant PP2A did not result in the
dephosphorylation of SOCS-1 (data not shown). Thus SOCS-1 is not a
direct substrate of PP2A, and the effects of overexpression of PP2A on
SOCS-1 mobility are therefore likely an indirect effect of the
decreased levels of Pim protein expressed upon overexpression of
PP2A.
Inhibition of Proteasome Function Results in Stabilization of the
Pim Kinases--
SOCS-1 has been shown to be involved in the
recruitment of signaling molecules to the proteasome (28-31).
Interestingly, the decrease in the expression levels of the Pim
proteins upon overexpression of PP2A requires the presence of SOCS-1.
When 293T cells were transfected with Pim-3 and increasing amounts of
PP2A in the absence of overexpressed SOCS-1, no decrease in the
steady-state levels of Pim-3 were observed (data not shown). The
observation that down-regulation of expression of the Pim kinases by
PP2A requires the expression of SOCS-1 raises the possibility that the
Pim kinases undergo proteasomal degradation and that they are targeted
for degradation by SOCS-1. To determine whether the Pim kinases are degraded by the proteasomal pathway, 293T cells were transfected with
Pim-3, the cells were treated with the proteasomal inhibitor LLnL, and
pulse-chase assays were performed to measure the effect of LLnL on the
stability of the Pim-3 protein. LLnL treatment resulted in enhancement
of stability of the Pim-3 protein (Fig. 4). These data, combined with the
observation that altering cellular levels of PP2A affects the levels of
expression of the Pim protein kinases, suggest that the Pim kinases are
targeted for proteasomal degradation in a SOCS-1-dependent
mechanism.

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Fig. 4.
Inhibition of the proteasome by LLnL
stabilizes the Pim-3 protein. 293T cells were transfected in 6-cm
plates with 0.5 µg of Pim-3, and cells were starved of methionine,
then cultured in methionine-free medium supplemented with
35S-labeled methionine, and then washed and cultured in
complete media in the absence (lanes 1-3) or presence
(lanes 4 and 5) of LLnL for the indicated times.
Lysates were immunoprecipitated with anti-Pim-3 antibody, and labeled
Pim-3 was detected by autoradiography (upper panel).
Steady-state levels of Pim-3 protein were detected by immunoblotting
with anti-Pim-3 antibody (lower panel).
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Inhibition of PP2A Activity in Primary Cells Results in an Increase
in the Stability of Endogenous Pim-1 Protein--
To determine whether
PP2A activity can regulate the stability of endogenous Pim proteins,
the effect of inhibition of PP2A on the half-life of Pim-1 in primary
cells was determined. Primary mouse splenocytes and thymocytes,
untreated or pretreated with 25 nM okadaic acid were
stimulated with PMA and ionomycin and subsequently treated with 100 µg/ml cycloheximide for various times, and the levels of Pim-1
protein expression were determined by Western blotting (Fig.
5). The concentration of okadaic acid used in these experiments has previously been shown to potently inhibit
PP2A but not the other major known serine-threonine phosphatases PP-1,
PP2B/calcineurin, and PP2C (32). Stimulation of splenocytes with PMA
and ionomycin resulted in induction of expression of two, 44 and 35 kDa, isoforms of Pim-1, and both isoforms displayed half-lives of less
than 5 min (Fig. 5A, lanes 1 and
3-8). Upon pretreatment of the splenocytes with okadaic
acid, however, the half-lives of both isoforms of Pim-1 increased. In
cells pretreated with okadaic acid, the levels of the 44-kDa isoform of
Pim-1 did not begin to decrease until after 30 min of cycloheximide
treatment, and the protein was still clearly detectable at 60 min (Fig.
5A, lanes 12-14). Furthermore, in untreated
cells, the levels of the 35-kDa isoform of Pim-1 were barely detectable
after 30 min of cycloheximide treatment, whereas in cells pretreated
with okadaic acid the levels of the 35-kDa isoform were still
detectable after 45 min of cycloheximide treatment (Fig.
5A, lanes 6 and 13). Okadaic acid
alone, however, did not induce expression of Pim-1 in the splenocytes
(Fig. 5A, lane 2).

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Fig. 5.
Inhibition of PP2A activity enhances the
stability of Pim-1 in primary cells. Primary mouse splenocytes
(A) and thymocytes (B) were untreated, treated
with okadaic acid (25 nM), PMA (50 ng/ml), and ionomycin
(500 ng/ml), or PMA, ionomycin, and okadaic acid, and then treated with
cycloheximide (100 µg/ml) for the indicated times. Cell lysates were
then immunoblotted with Pim-1 antibody (A and
B, upper panels). Pim-1 levels were barely
detectable in untreated cells (A and B,
lane 1) and okadaic acid treatment alone did not increase the
expression of Pim-1 (A and B, lane 2).
PMA and ionomycin dramatically increased Pim-1 levels (A and
B, lane 3), and Pim-1 was rapidly degraded in the
presence of the protein synthesis inhibitor cycloheximide (A
and B, lanes 4-8). Pretreatment of PMA and
ionomycin-cultured cells with okadaic acid resulted in sustained
expression of Pim-1 upon cycloheximide treatment (A and
B, compare lanes 4-8 to 10-14).
-Actin (A and B, lower panels)
served as a control for protein loading and for nonspecific effects of
okadaic acid.
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As PMA and ionomycin also induce Pim-1 expression in the thymus, the
effect of okadaic acid on Pim-1 protein stability was confirmed in
thymocytes. In thymocytes, okadaic acid treatment had a similar effect
on the stability of the 35-kDa isoform of Pim-1 (Fig. 5B).
In cells not pretreated with okadaic acid, the levels of the 35-kDa
isoform of Pim-1 were significantly decreased after 15 min of
cycloheximide treatment (Fig. 5B, lane 4). In cells pretreated with okadaic acid, however, the levels of the 35-kDa
isoform of Pim-1 did not begin to decrease until after 60 min of
cycloheximide treatment (Fig. 5B, lane 10). The
44-kDa isoform of Pim-1 has a longer half-life in thymocytes than in splenocytes (Fig. 5B, lanes 3-6), and the levels
of the 44-kDa isoform of Pim-1 did not begin to decrease until after 60 min of treatment of the cells with cycloheximide (Fig.
5B, lane 6). Okadaic acid treatment did not
significantly affect the half-life of the 44-kDa isoform of Pim-1
expressed in thymocytes (Fig. 5B, lanes 7-10).
Therefore inhibition of PP2A activity by okadaic acid preferentially
enhances the protein stability of the most labile isoforms of Pim-1
expressed in splenocytes and thymocytes.
Inhibition of PP2A Activity Does Not Alter the Steady-state Levels
of pim-1 mRNA--
To confirm that the effects of okadaic acid on
the levels of expression of Pim-1 are not the result of increased
steady-state levels of pim-1 mRNA, the effect of okadaic
acid on the levels of pim-1 mRNA was determined.
Splenocytes untreated or pretreated with 25 nM okadaic acid
were stimulated with PMA and ionomycin and subsequently treated with
cycloheximide for various times, RNA was isolated from the cells, and
the level of pim-1 mRNA expressed under the different
conditions was determined by Northern blotting (Fig.
6). Low levels of pim-1
mRNA were expressed in untreated cells, and these levels were not
increased by treatment with okadaic acid alone (Fig. 6, lanes
1 and 2). PMA and ionomycin, however, induced the
expression of pim-1 mRNA, and the levels of
pim-1 mRNA induced by PMA and ionomycin, and by PMA,
ionomycin, and okadaic acid, were equivalent (Fig. 6, compare
lanes 3 and 7). Inhibition of PP2A results in an
increase in the stability of the Pim-1 protein, but no change in the
steady-state levels of pim-1 mRNA, in primary cells.

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Fig. 6.
Inhibition of PP2A activity has no effect on
the steady-state levels of Pim-1 mRNA in primary cells.
Primary mouse splenocytes were untreated, treated with okadaic acid (25 nM), PMA (50 ng/ml), and ionomycin (500 ng/ml), or PMA,
ionomycin, and okadaic acid, and then treated with cycloheximide (100 µg/ml) for the indicated times. Total cellular RNA was then isolated
and Northern blotted with Pim-1 probe (upper panel).
Pim-1 mRNA levels were barely detectable in untreated
cells (lane 1), and okadaic acid treatment alone did not
increase the levels of pim-1 mRNA (lane 2).
Stimulation with PMA and ionomycin increased pim-1
transcript levels equivalently in cells untreated (lane 3)
or pretreated with okadaic acid (lane 7). Equal amounts of
RNA were loaded as judged from the ethidium bromide-stained ribosomal
bands (lower panel).
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DISCUSSION |
In this study, the role PP2A plays in the regulation of the Pim
kinases was examined. The Pim kinases were found to associate with PP2A
in vivo, and were found to be substrates of PP2A in vitro. Moreover, PP2A was found to regulate the expression of the
Pim proteins. Overexpression of PP2A in 293T cells resulted in a
decrease in the expression levels of the Pim-1 and Pim-3 proteins, and
inhibition of PP2A activity in primary mouse splenocytes and thymocytes
by okadaic acid resulted in the stabilization of endogenous Pim-1
protein. Finally, overexpression of PP2A resulted in the inhibition of
Pim kinase function in 293T cells. The lower mobility forms of SOCS-1
induced by co-expression of SOCS-1 and the Pim kinases disappeared upon
co-expression of PP2A. Thus, regulation of Pim protein stability by
PP2A represents an additional mechanism by which Pim kinase expression
and function are regulated.
Dephosphorylation of the Pim kinases by PP2A may directly affect the
stability of the Pim kinases. Phosphorylation has been shown to
regulate the stability of many cellular proteins by modulating the
targeting of the proteins to degradation. Whereas phosphorylation has
most often been identified as an event that enhances protein degradation, several proteins, including the antiapoptotic protein Bcl-2, have recently been found to be protected from degradation by
phosphorylation (33). Dephosphorylation of the Pim kinases by PP2A may
therefore target the Pim proteins to proteasomal degradation. To
determine whether the phosphorylation status of the Pim kinases has a
direct effect on their stability, the half-lives of wild-type and
kinase-inactive Pim-3 protein, which has very weak autophosphorylation activity in vitro (Fig. 2, lane 1), were
examined. In cycloheximide time course experiments, wild-type and
kinase-inactive Pim-3 protein had similar half-lives (data not shown).
This implies that phosphorylation of the Pim kinases and, by extension,
dephosphorylation of the Pim kinases by PP2A, does not have a direct
effect on the stability of the Pim kinases. However, the Pim kinases
could be stabilized in vivo by phosphorylation at the same
or other sites than those that undergo autophosphorylation, and these
phosphorylation events could affect the stability of endogenous Pim kinases.
Alternatively, the regulation of the stability of the Pim kinases by
PP2A may not be a direct consequence of Pim dephosphorylation by PP2A
but may instead be an indirect result of PP2A regulating the
phosphorylation state and/or function of other cellular proteins that
regulate Pim protein stability. One gene that may be involved in the
PP2A-mediated targeting of the Pim kinases for degradation is SOCS-1.
As well as being a JAK kinase inhibitor, SOCS-1 has also been
identified as a potential tumor suppressor gene. SOCS-1 targets
TEL-JAK2, an oncogenic form of JAK (29-31) to degradation, thereby
inhibiting TEL-JAK2-mediated transformation. This targeting is thought
to involve the association of the SOCS box with elongin B and elongin
C, homologues of components of the ubiquitination pathway (34, 35).
Binding of SOCS-1 to elongin B and elongin C is thought to recruit
SOCS-1-binding proteins to proteasomal degradation. The observations
that SOCS-1 binds the Pim kinases (7) and that SOCS-1 is required for
the PP2A-mediated decrease in steady-state levels of Pim-1 and Pim-3 in
293T cells, suggest that SOCS-1 acts as an adapter between the Pim
kinases and the ubiquitination pathway.
Inhibition of the proteasome by LLnL enhances the stability of the Pim
kinases in 293T cells (Fig. 4). Furthermore, the proteasome has been
found to be phosphorylated, and phosphorylation of subunits of the
proteasome is thought to be involved in the regulation of the assembly
of proteasome complexes and the function of the proteasome (36-38).
PP2A may therefore regulate phosphorylation of the proteasome, or of
proteasome-associated factors, thereby modulating the activity of the
complex. As the stability of Pim-1 is known to be enhanced in several
Pim-1-expressing tumor lines (22, 23), this raises the possibility that
dysregulation of PP2A contributes to pim-mediated transformation.
The observation that inhibition of PP2A activity by okadaic acid
results in the stabilization of endogenous Pim-1 protein in primary
cells suggests that PP2A plays a role in vivo in the regulation of Pim protein stability. Okadaic acid has been shown to
preferentially inhibit PP2A at the low concentration used in these
experiments, and at this concentration, okadaic acid does not inhibit
the other major serine-threonine phosphatases PP1, PP2B/calcineurin,
and PP2C (32). This does not rule out, however, that other less well
characterized serine-threonine phosphatases, some of which share
greater sequence similarities with PP2A than do PP1, PP2B, and PP2C,
may also be inhibited under these conditions. The stabilization of
Pim-1 in primary cells by okadaic acid treatment may therefore result
from the inhibition of other cellular phosphatases than just PP2A.
However, the observation that increasing cellular PP2A activity by
overexpression of PP2A decreases the levels of expression of Pim-1 and
Pim-3 in 293T cells, whereas decreased cellular PP2A activity, as a
result of okadaic acid treatment, stabilizes the Pim-1 protein in
primary cells, strongly implicates PP2A in the modulation of the
stability and expression of the Pim kinases.
In conclusion, regulation of Pim protein stability is an important site
of regulation of Pim kinase function, and disruption of that regulation
may be an important mechanism by which the Pim kinases lead to
transformation. Mutations in the various genes involved in the
PP2A-mediated regulation of Pim protein stability may potentially
contribute to Pim-induced tumor formation.