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Institut National de la Santé et de la Recherche Médicale U440, Paris, France
| Abstract |
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| Introduction |
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In this respect, the function of CD2 is still a debated question. The
hypothesis that CD2 and CD3 share a common activation pathway was
supported by the observation that both CD2 and CD3 molecules apparently
triggered the same cascade of some of the earliest biochemical
events (8, 9). In particular, phosphorylation on tyrosine occurred
mostly on the same set of proteins after stimulation of both CD3 and
CD2 receptors (10), including phospholipase C
-1 (9, 11), in which
phosphorylation correlates with the activation of phosphoinositide
turnover (12, 13).
Alternatively, we and others have proposed that CD2 induces distinct signal transduction events that would, at least in part, differ from those initiated by the TCR/MHC complex engagement (14, 15, 16, 17, 18, 19). In support of this postulate, we showed previously (20) that the stimulation of a CD4+ T lymphocyte clone via CD2 was associated with a specific phosphorylation pattern of stathmin (21), differing from that induced in response to PMA, an activator of protein kinase C (PKC).4
Stathmin (22), also referred to as p19 (23), prosolin (24), p18 (25), pp20 (26), and Op18 (27), was identified in several cellular systems as a ubiquitous, conserved cytosolic phosphoprotein in which expression and phosphorylation are highly regulated in relation to cell proliferation and differentiation (for review see 21 . Therefore, it appeared to be an intracellular proteic relay integrating diverse intracellular signaling pathways (21). It was recently proposed that it might function by controlling the mitotic microtubule dynamics (28, 29, 30, 31, 32).
Stathmin is highly expressed in activated T-PBL as well as in transformed T lymphocytes, including Jurkat cells (24, 25). In intact cells, stathmin is a target for both cell cycle and cell surface receptor-regulated phosphorylation events that are initiated by the activation of diverse protein kinase systems such as the cAMP-dependent kinase (PKA), PKC, Ca2+-dependent kinases, cyclin-dependent kinases (cdks), mitogen-activated protein (MAP) kinases, and tyrosine kinases (reviewed in 21 (33, 34, 35, 36, 37, 38, 39). Site mapping studies performed in intact cells and in vitro revealed four phosphorylation sites, namely Ser16, Ser25, Ser38, and Ser63. PKA catalyzes the phosphorylation of stathmin on Ser63 and Ser16, Ser63 being the major target of this kinase (36, 40); MAP kinase and cdc2 kinase both induce phosphorylation on Ser25 and Ser38, respectively, but with an opposite site preference (Ser25 for MAP kinase, and Ser38 for cdc2) (36, 39). Moreover, phosphorylation of only these four sites and their specific combinations account for all the phosphoforms of stathmin identified so far in vivo in diverse biologic systems (36, 41). Thus, analysis of the phosphorylation site patterns of stathmin might give clues for the identification of intracellular pathways involved in various cell regulatory processes. Site-mapping studies of stathmin revealed that CD3 stimulation of Jurkat T cells resulted in an apparently PKC-independent activation of both the MAP kinase and the Ca2+/calmodulin-dependent kinase IV (Gr) (37, 38).
In the present study, to address specific protein kinase systems involved in the CD2 pathway of T cell activation, we analyzed the site-specific phosphorylation of stathmin in response to the stimulation of the CD2 receptor in the Jurkat T cell line. Our data show that stimulation of CD2 activated multiple signal transduction pathways, resulting in phosphorylation of distinct sites of stathmin, the combination of which only partially overlaps the CD3- and CD28-induced patterns. Indeed, in intact cells, prolonged CD2 stimulation induced a major phosphorylation of Ser16 without phosphorylation of Ser63, indicating the involvement of a kinase distinct from PKA. Furthermore, site-mapping studies show that purified Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) was able to phosphorylate Ser16 of recombinant stathmin in vitro. Studies performed in intact cells with CD2 and A23187 indicate that CaM kinase II might be responsible for the CD2-induced phosphorylation of stathmin on Ser16 in vivo.
| Materials and Methods |
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Forskolin, PMA, calmodulin, aprotinin, pepstatin, leupeptin,
DTT, temed, trizma base, and ammonium persulfate were from Sigma
Chemical Co. (St. Louis, MO). TPCK (tosylamido-2-phenylethyl
chloromethyl ketone)-treated trypsin was from Worthington
(Freehold, NJ); A23187 from Boehringer (Mannheim, Germany); okadaic
acid (OA) and thermolysin from Calbiochem (La Jolla, CA);
[
-32P]ATP from Amersham (Amersham, U.K.); TLC sheets
and cellulose plates from Kodak (New Haven, RI); acrylamide and
bisacrylamide (Acrylogel 2.6) from Merck (Darmstadt, Germany); SDS from
Serva (Heidelberg, Germany); ampholines from Pharmacia LKB (Uppsala,
Sweden); FCS from Life Technologies (Grand Island, NY); and
phosphate-free Eagles MEM from Flow Laboratories (Irvine, U.K.).
Methods
Monoclonal Abs.
Ag-specific recognition can be artificially triggered after binding of
the appropriate anti-CD3 or anti-TCR mAbs (2). The mAb UCHT1
(IgG1), specific for CD3
chain, kindly provided by Dr. P.
C. L. Beverley (Imperial Research Cancer Fund, London, U.K.) (42),
was used for stimulation experiments at a 1:500 dilution of an ascitic
fluid. Several distinct natural ligands have been described for the CD2
molecule (43), and artificial activation via CD2 requires the
appropriate combination of two mAbs directed against distinct epitopes
of the CD2 molecule. Anti-CD2 mAbs X11 (IgG1) and D66 (IgG2b) were
previously described: mAb X11 recognizes the T11.1 epitope of the CD2
molecule, whereas mAb D66, specific for a cryptic epitope on resting T
cells, is unmasked after X11 mAb binding (44). Thus, for
inducing mitogenic stimulation, these mAbs must be used in combination,
and the pair X11 + D66 will therefore be designated "anti-CD2
mAbs" throughout the text. For all stimulation experiments, both mAbs
were thus added together at saturating concentrations (50 µg/ml).
YTH655.5, a mAb (kindly provided by Drs. J. P. Revillard, Institut
National de la Santé et de la Recherche Médicale U80, Lyon,
France and H. Waldmann, University of Oxford, Oxford, U.K.)
directed against another cryptic epitope of the CD2 molecule expressed
only on activated T cells (45), was also tested in combination with the
X11 mAb. Anti-CD28 mAb (IOT28; Immunotech, Marseille, France) was used
at 20 µg/ml.
Cells. The leukemic CD2+CD3+ Jurkat T cell line, clone E-6.1, kindly given by Dr. A. Alcover (Institut Pasteur, Paris, France), was maintained in RPMI 1640 medium (Flow Laboratories) supplemented with 10% FCS, penicillin (50 U/ml), streptomycin (50 µg/ml), L-glutamine (2 mM), and sodium pyruvate (1 mM). To stimulate them when they are exponentially growing, cells were always diluted at a density of 0.5 x 106 cells/ml, 16 h before stimulation.
Protein preparations. Recombinant stathmin was expressed in Escherichia coli in its unphosphorylated form and purified as described (40). Briefly, the NcoI-BamHI fragment of the cDNA containing the entire sequence coding for human stathmin (46) was cloned into the expression vector pEt8c, and transfected into E. coli BL21 (DE3). Upon induction with IPTG (isopropyl-thio-ß-D-galactoside), stathmin was purified to homogeneity by a two-step procedure involving chromatography on DEAE-Sepharose CL-6B and gel-filtration on Superose 12 (Pharmacia LKB). CaM kinase II, purified from rat brain, was a generous gift from Dr. J. A. Girault (Institut National de la Santé et de la Recherche Médicale U114, Paris, France).
Phosphorylation of stathmin in vitro by CaM kinase II.
Recombinant stathmin (1 µg) was incubated at 30°C with 1 U CaM
kinase II (1 U is the amount of enzyme that catalyzes the
phosphorylation in 1 min of 1 pmol of synapsin I), and 370 kBq of
[
-32P]ATP (8.11 GBq/mmol), in 100 µl of
phosphorylation buffer: 50 mM Tris-HCl, pH 7.6, 10 mM
MgCl2, 0.4 mM EGTA, 10 mM CaCl2, and calmodulin
(0.1 mg/ml). The reactions were initiated by adding 20 µl of 250-µM
radioactive ATP (sp. act., 3 MBq/mmol), after preincubation of the
other components for 1 min at 30°C. After 5 min at 30°C, the
reactions were stopped by adding 50 µl of 3x STOP solution (Tris-HCl
(200 mM), pH 6.8; 7% SDS; 33% glycerol; 3% ß-mercaptoethanol), and
the samples were boiled and further submitted to two-dimensional PAGE
as described below. All of the relevant [32P]-labeled
stathmin spots were excised from the gels and pooled. After Cerenkov
counting, the gel pieces were rehydrated and the samples were prepared
for [32P]phosphopeptide mapping analysis as further
described below.
Radioactive labeling and pharmacologic treatments. 32PO43- labeling was performed by preincubating 5 x 106 cells in 250 µl of phosphate-free medium completed with 5.55 MBq 32PO43- (DuPont-New England Nuclear Research Products, Nemours, France), for 4 h. Test agents were added directly to the radioactive medium for the last 30 min, except for OA, which was added at the very beginning of the radioactive incubation. The labeling was stopped as described previously (47), preparing the samples for two-dimensional electrophoresis. The same amount of TCA-precipitable radioactivity was used for each sample within a given experiment, allowing direct comparison of autoradiograms.
Polyacrylamide gel electrophoresis. Two-dimensional PAGE was performed as described previously (47). The isoelectric focusing gels contained 2% total ampholines, pH 46, 57, and 310, in the proportion 2:2:1. The second dimension was run on 13% acrylamide gels. The fixed gels were dried and exposed for autoradiography with Kodak XAR-5 film. Quantification of stathmin phosphorylation was obtained by analysis of the gel and direct counting of the radioactivity in each relevant [32P]-labeled spot with an Instant Imager apparatus (Packard Instrument, Meriden, CT).
Phosphopeptide mapping. Two-dimensional thin layer phosphopeptide mapping of stathmin was conducted as described (48) with modifications (36): proteolysis was performed with trypsin at 75 µg/ml overnight followed with thermolysin at a concentration of 100 µg/ml, and the radioactive material was spotted in the middle, 4 cm from the bottom of the TLC sheet. Autoradiography was performed using Kodak XAR-5 film at -70°C with Kodak Quanta III intensifying screens. Instant Imager analysis of radioactive spots was used for quantification of phosphopeptides.
CaM kinase II assay.
CaM kinase II activity was assayed in cell lysates using CaM kinase II
assay kits (Upstate Biotechnology, Lake Placid, NY) and using
autocamtide 2 (KKALRRQETVDAL) as a peptide substrate with relative
selectivity for CaM kinase II. Exponentially growing Jurkat T cells,
stimulated or not, were lysed in assay dilution buffer (20 mM MOPS, pH
7.2, 25 mM ß-glycerol phosphate, 1 mM sodium orthovanadate, 1 mM DTT,
1 mM CaCl2). Cell lysates were centrifuged 15 min at 12,500
rpm at 4°C. The concentration of protein was determined in the
resulting supernatant by the Bradford assay (Bio-Rad, Hercules,
CA). The reaction mixture contained, following the
manufacturers instructions, 10 µl of the sample extract, 10 µl of
substrate mixture, 10 µl of a mixture containing inhibitors of other
serine/threonine kinases such as PKA and PKC, and 10 µl of
Mg2+/ATP mixture containing [
-32P]ATP. The
mixture was incubated at 30°C for 10 min, and the phosphorylated
substrate was separated from the residual [
-32P]ATP
using p81 phosphocellulose paper. The papers were washed with five
rinses of 0.75% phosphoric acid, then washed in acetone for 2
min, and the bound radioactivity was quantified with a scintillation
counter. Blanks to correct for nonspecific binding of
[
-32P]ATP and its breakdown products to the
phosphocellulose paper and controls for phosphorylation of endogenous
proteins in the sample were performed, and CaM kinase II activity was
expressed as pmol/min/mg protein.
| Results |
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We have previously shown that PKC-dependent and -independent pathways are responsible for the stathmin phosphorylation pattern observed in the P28D CD4+ T cell clone stimulated via the alternative pathway of T lymphocyte activation, which is triggered via the CD2 molecule. Indeed, when these T cells were treated with the phorbol ester PMA, the resulting pattern of the various phosphorylated forms of stathmin only partially mimicked the pattern observed after treatment with anti-CD2 mAbs (20). Moreover, we have previously identified specific phosphorylation sites of stathmin as hallmarks of specific kinase activities in vitro as well as in intact cells (36, 39).
To further characterize intracellular signaling pathways associated
with the stimulation via CD2 and to distinguish them from those related
to CD3 or CD28 triggering, Jurkat T cells were used in the present
study to identify stathmin phosphorylated sites in response to
treatment with either anti-CD2, anti-CD3, or anti-CD28
mAbs. Exponentially growing Jurkat cells, which express high levels of
stathmin (24), were prelabeled with [32P]orthophosphate
to achieve isotopic equilibrium and then submitted to treatments with
the various agonists. Following two-dimensional PAGE separation, the
radioactive phosphoproteins were revealed by autoradiography (see Fig. 2
), and the radioactivity incorporated in relevant spots was quantified
directly on the gels (see Materials and Methods). Figure 1
shows that stimulation of
[32P]-labeled Jurkat cells via CD2, CD3, or CD28 resulted
in a similar increase in phosphorylation of stathmin. The D66 mAb by
itself did not stimulate stathmin phosphorylation, although it was
shown to trigger a tyrosine kinase independently of the CD3-dependent
signaling pathway (18). However, this mAb has no mitogenic effect by
itself, i.e., without the cooperative action of the X11 mAb. Treatments
with pharmacologic agents known to partially stimulate T cell
activation, such as PMA, the Ca2+ ionophore A23187, or OA,
also increased the total amount of radioactive phosphate incorporated
in stathmin. The increased incorporation of [32P] into
stathmin in response to the various stimuli corresponded to major
changes in the actual phosphorylation state of stathmin rather than
only to an increased phosphate turnover, as it was previously shown in
Jurkat cells by direct protein detection of the unphosphorylated and
phosphorylated forms of stathmin on silver-stained two-dimensional gels
(41).
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These results illustrate that the various forms of stathmin might be used as hallmarks of the activation as well as of the interaction of distinct intracellular signaling pathways involved in the various stimulating modes of T cell activation. They further document the fact that CD2 and CD3 or CD28 activate intracellular pathways that are at least partially nonoverlapping.
Site-specific phosphorylation of stathmin in response to CD2 stimulation
We further investigated the CD2-dependent alternative pathway of T
cell activation and the related intracellular kinase pathways on the
basis of the kinase specificity of the four distinct phosphorylation
sites of stathmin accounting for all of the forms encountered in
numerous biologic systems and in response to diverse regulatory signals
(36). Experiments were performed to identify the specific sites of
stathmin involved and to quantify their phosphorylation in response to
CD2 stimulation. In control and in CD2-stimulated Jurkat cells, the
various phosphoforms of stathmin were resolved by two-dimensional PAGE,
and the corresponding spots were excised and analyzed by
two-dimensional phosphopeptide mapping (see Materials and
Methods). The excised spots from each gel were either pooled (Fig. 3
) or each spot was processed
individually (Fig. 4
), according to a
procedure yielding characteristic migration patterns for each of the
four previously identified phosphorylation sites of the protein (36).
The sites of stathmin phosphorylated after CD2 activation of T cells
(Fig. 3
) were the same four sites (Ser16,
Ser25, Ser38, and Ser63) for which
phosphorylation was described in other cellular systems. As compared
with the control, CD2-stimulation induced a strong phosphorylation of
Ser16, Ser25, and Ser38, and only a
very slight one of Ser63. This comprehensive method of
analysis of stathmin phosphoforms was coupled with the quantitative
evaluation of the level of phosphate in each serine phosphorylated in
each phosphoform (Fig. 4
). In control cells, stathmin was mostly
phosphorylated on single sites yielding spot P1 on two-dimensional
gels, or on two sites yielding spot P2 and low levels of spot
161 (see also Fig. 2
B). In control cells, the
kinase showing the highest basal activity was the kinase responsible
for the phosphorylation of Ser38 (Figs. 3
and 4
). CD2
treatment resulted in the incorporation of [32P] into
spots P1 and P2, as well as into both forms of each slowly migrating
set 16 and 17 resulting from the concurrent phosphorylation on
Ser16 and Ser25 (Figs. 2
and 4
). Accordingly,
Ser16 and Ser25 appeared overall severalfold
more phosphorylated than in control cells (Figs. 3
and 4
).
Phosphorylation on Ser38 was also stimulated after CD2
triggering, yielding some P2 but mostly 171 and
172 spots, whereas phosphorylation on Ser63 was
undetectable in control and minimal in CD2-treated cells.
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Altogether, since increased phosphorylation of Ser16 and Ser25 appeared predominant as compared with that of Ser38 in CD2-treated cells, it can be concluded from our results that the preferential kinase activities stimulated by CD2 treatment of Jurkat T cells displayed a preference for phosphorylation of stathmin on Ser16 and Ser25.
Differential regulation of phosphorylation of serines 16, 25, and 38 of stathmin in CD2-, CD3-, or CD28-stimulated Jurkat T cells
To characterize the protein kinase(s) responsible for stathmin
phosphorylation specifically in response to CD2 treatment, we compared
the phosphorylation of stathmin after stimulation of proliferating
Jurkat T cells via CD2, CD3, or CD28. Actually, it has been proposed
that Ser16, Ser25, Ser38, and
Ser63 of stathmin may be respective physiologic substrates
for either the CaM kinase Gr (38), MAP kinase (37, 39), members of the
cdc2 kinase family (36, 49), or PKA (34, 35, 36). The results obtained with
the treatments with various mAbs were compared with those corresponding
to stimulation of the cells with PMA or A23187, which are both known to
bypass the membrane transductional step and which have been related to
various kinase pathways regulating stathmin phosphorylation (24, 33, 34, 37, 50). Thus, all of the labeled phosphoforms of stathmin
generated in response to each treatment were processed together for
mapping studies, and the corresponding phosphopeptide patterns were
directly quantified. Stimulation of Jurkat T cells via CD2 and CD3 had
only partially overlapping effects on stathmin phosphorylation (Fig. 5
): whereas the relative phosphorylation
of Ser16 was much more enhanced after CD2-stimulation,
Ser38 was relatively much more phosphorylated in response
to CD3-treatment; induced phosphorylation of Ser25 was of
similar magnitude in CD2- and CD3-stimulated T cells. After CD28
triggering, the phosphorylation increase of Ser16 was
intermediate between that obtained after CD2 and CD3 treatments, and
phosphorylation of Ser25 was weaker than in both of the
latter treatments; induced phosphorylation on Ser38 was
similar to that following CD2 triggering. PMA strongly stimulated the
phosphorylation of Ser25 and, to a lesser extent,
Ser16, but poorly stimulated that of Ser38.
Finally, in A23187-treated cells, Ser38 was the most
phosphorylated residue, and the induced phosphorylation of
Ser25 was in about the same range as in CD2-stimulated
cells, whereas that of Ser16 was weaker in the condition
tested (30-min stimulation; see below).
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Therefore, our results suggest that specific combinations of relative phosphorylation levels of the same four specific serine sites of stathmin, resulting in the integration of different kinase/phosphatase activities, might correlate with different functional states of T lymphocytes.
Characterization of the "serine 16-kinase" activity
Our results (Fig. 5
) indicated that phosphorylation of
Ser16 corresponds to a pathway activated particularly in
Jurkat T cells in response to CD2 as compared with CD3 treatment.
Phosphorylation of Ser16 of stathmin has been previously
correlated with Ca2+-regulated kinase pathways (26, 33, 38). Indeed A23187 treatment of Jurkat cells resulted in
phosphorylation of Ser16 (Fig. 5
). The amino acid sequence
context of Ser16 fits the minimal consensus substrate
sequence of CaM kinases II and IV (CaM kinase Gr), Arg-X-X-Ser/Thr.
Both kinases have been proposed as being involved in T lymphocyte
activation (54, 55, 56). Recently, stathmin was proposed as an early
cytosolic target for CaM kinase IV activated following CD3 stimulation
of Jurkat T cells (38). Yet CaM kinase IV activity was shown to be
transient, declining after 1 min post-TCR-CD3 engagement (54) and,
indeed, Ser16 was not significantly phosphorylated in
Jurkat cells after 30 min of CD3 treatment (Fig. 5
). Therefore, we
investigated whether CaM kinase II might be responsible for the
Ser16-kinase activity stimulated by prolonged CD2 treatment
of Jurkat T cells. We compared the phosphorylation of Ser16
of stathmin at two times after stimulation of Jurkat cells with either
anti-CD2 mAbs or A23187. Our results, shown in Figure 6
, clearly demonstrate that both
treatments correlate with significant Ser16
phosphorylation, which remained significantly above control levels for
up to 30 min.
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| Discussion |
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Redundancy between CD3-dependent and costimulatory pathways: activation of MAP kinase and cdc2 kinase families
In the present study, Jurkat T cells were examined in exponential growth phase. Therefore, the basal levels of phosphorylation of Ser25 and Ser38 in unstimulated cells (control) are in agreement with previous data on the enhanced phosphorylation of stathmin during both the S phase and the mitotic phase of the cell cycle, which have been attributed to the activity of members of the cdc2 kinase family (61). All of the stathmin phosphorylation modifications observed in response to CD3 stimulation may be explained by the increased phosphorylation of Ser25 and Ser38. The p21ras/MAP kinase enzymatic cascade (62) has already been correlated with the CD3-induced phosphorylation of Ser25 of stathmin (37). Similarly, the phosphorylation of Ser25 of stathmin subsequent to CD2 as well as CD28 stimulation of T cells could be due to the stimulation of a MAP kinase activity downstream of the activation of p21ras (63, 64). The observed phosphorylation increase of Ser25 of stathmin induced by PMA and OA treatments is in agreement with the previously described activation of MAP kinase by these two pharmacologic agents in T cells (65, 66).
Although it has been shown that Ca2+-sensitive signals do not regulate MAP kinase activity in Jurkat T cells (62), phosphorylation of Ser25 of stathmin in response to A23187 treatment of Jurkat T cells could be due to stimulation of one of the MAP kinase-like parallel pathways (67). Alternatively, it might be speculated that, like the phosphorylation of Ser38, the phosphorylation of Ser25 in response to A23187 treatment results, at least partially, from stimulated cdc2 or cdk2 kinase activity, as these enzymes are known to phosphorylate Ser25 of stathmin, even if with an approximately fivefold lower efficacy than on Ser38 (36, 39).
Stimulation of CaM kinase II is specific to costimulatory pathways
T cell responses to the CD3 vs CD2 activation pathway, depending on the in vitro experimental systems examined, were most often found to be identical in terms of early responses (e.g., calcium mobilization (68), CD3 phosphorylation (69), tyrosine phosphorylation (9, 10, 11)), whereas they were found to be rather distinct when focusing on later responses to costimulatory second signals (e.g., cytokine responsiveness (70), pp19 dephosphorylation (17, 71), p67 phosphorylation (19)). We therefore chose to examine potential differences between CD3- and costimulation-induced signals at treatment times that revealed phosphorylation events distant from those occurring during very early responses.
Our previous studies showed that CD2 stimulation generated preferentially the less acidic spots, 161 and 171, of the 21- and 23-kDa phosphoisomers of stathmin in the normal P28D CD4+ T cell clone (20). Here, we show that a strong increase of stathmin phosphorylation on Ser16 occurred in response to a 30-min treatment of proliferating Jurkat cells with anti-CD2 mAbs, and to a lesser extent with anti-CD28 mAb, but not with anti-CD3 mAb. Indeed, in CD2-stimulated Jurkat T cells, the same major spots, 161 and 171, most likely resulted from the stimulated phosphorylation on Ser16 of the P1 phosphoform basally phosphorylated on Ser25 in control cells and of the P2 phosphoform basally phosphorylated on Ser25 and Ser38 in control cells, respectively.
We therefore also investigated the kinase activity responsible for the phosphorylation of Ser16 of stathmin in response to costimulatory signals. PKA was not a good candidate, as we have previously shown that Ser16 of stathmin was not a good in vitro substrate for PKA although it is within a consensus site for this enzyme in the stathmin sequence (36). CaM kinase IV was recently proposed as the kinase responsible for the early transient phosphorylation of Ser16 of stathmin in response to stimulation of the CD3 Ag (37, 38). We found that Ser16 is also an efficient substrate for CaM kinase II in vitro, in agreement also with the fact that CaM kinase II and CaM kinase IV have been shown to share several substrates in vitro (72). Moreover, CaM kinase II activity was enhanced in vivo following CD2 triggering of Jurkat cells. These observations suggest that CaM kinase II is likely to contribute to the phosphorylation of stathmin, in particular in response to the activation of costimulatory pathways.
The proposed involvement of CaM kinase IV (37, 38) is in agreement with previous data showing a peak at 1 min, followed by a rapid decline in CaM kinase IV autophosphorylation and both autonomous (Ca2+-independent) and Ca2+/CaM-dependent kinase activities, despite the persistence of elevated [Ca2+]i for at least 10 min following TCR-CD3 signaling (54). In line with these results is the lack of phosphorylation of Ser16 after 30 min of treatment with anti-CD3 mAb. Therefore, the fact that phosphorylation of Ser16 was high at 10 min and maintained for over 30 min after treatments with anti-CD2 mAbs, anti-CD28 mAb, or A23187, as opposed to stimulation via CD3, made us favor the hypothesis of the involvement of CaM kinase II, another potential target for Ca2+-signaling in lymphocytes (73), in the phosphorylation of Ser16 of stathmin in response to CD2 and CD28 treatment. Moreover, in the present report, the observed phosphorylation of Ser16 in unstimulated Jurkat cells might also not be attributed to minimal autologous or Ca2+/CaM-dependent catalytic activity of CaM kinase IV, as it has been shown that CaM kinase IV molecules isolated from unstimulated Jurkat cells exhibited negligible autonomous or Ca2+/CaM-dependent catalytic activity (54). Finally, phosphorylation of Ser16 after treatment of unstimulated Jurkat cells with OA also favors the hypothesis of CaM kinase II involvement. Indeed, previous studies have demonstrated that CaM kinase II activity was up-regulated by autophosphorylation and inhibited by the Mg2+-independent phosphatases 1 and 2A, whereas CaM kinase IV autophosphorylation and subsequent inhibitory effects on enzyme activity were strictly Mg2+ dependent, which raised the possibility that phosphatases 1 and 2A do not act on this enzyme (54, 57). Thus, OA, an inhibitor of phosphatases 1 and 2A, cannot stimulate phosphorylation processes via the stimulation of basal catalytic activity of CaM kinase IV by preserving autophosphorylation of the enzyme.
Enhanced and prolonged CaM kinase II activation at later times of cell stimulation has been associated with increased cytosolic availability of calmodulin due to its release from calmodulin-binding proteins after their PKC-dependent phosphorylation or after treatment with high doses of Ca2+ ionophore (74). This process could thus account for the phosphorylation of stathmin Ser16 in response to both PMA and A23187 in Jurkat cells. Moreover, this mode of regulation of CaM kinase II activation could also account for the contrasting results on Ser16 phosphorylation after CD3 triggering and via CD2 or CD28 stimulation. Indeed, arachidonic acid metabolites have been shown to be responsible for sustained activation of some PKC isotype(s) (75). We have previously reported that CD2 and not CD3 stimulation of the P28D T cell clone generated lipid messenger molecules due to a phospholipase A2 activation (14, 15). Interestingly, CD28 has recently also been shown to transduce the activation signal through phospholipase A2 and 5-lipoxygenase activation (76). Therefore, in CD2- and CD28-stimulated cells, prolonged CaM kinase II activity might be due, indirectly, to sustained activation of certain PKC isotype(s) by arachidonic acid metabolites. In contrast, the transient PKC activation observed after CD3 stimulation (20) could not positively regulate CaM kinase II activity.
The CD2-dependent pathway has been shown, according to the activation state of peripheral blood T cells, to direct them either toward proliferation or toward apoptosis (56, 77). In Jurkat T cells, CaM kinase II has been shown to induce an IL-2 transcriptional block, independently of the Ca2+/calmodulin-responsive phosphatase, calcineurin (55). Accordingly, cyclosporin A, a calcineurin inhibitor, had no effect on early (15 min) phosphorylation of stathmin in OKT3-induced activation of freshly isolated PBL (78). Phosphorylation of stathmin has been associated with both activation and down-regulation of cellular proliferation. When proliferating leukemic cells were induced to undergo terminal differentiation in culture, they stopped proliferating, and their stathmin underwent rapid phosphorylation (79). Comparatively, when naive PBL were stimulated with OKT3, phosphorylation preceded DNA synthesis and cell proliferation, whereas in proliferating cells challenged with phorbol esters, they stopped proliferating and the level of phosphorylation of stathmin increased (24, 78). Similarly, stathmin expression appeared after the first mitotic peak following hepatectomy in the rat, when cells that reenter the cell cycle need to be slowed down to prevent overgrowth of the regenerating liver (80). The increased expression of stathmin was regulated also in relation to the limitation of cell overgrowth at the stage preceding the differentiation of C2 myoblasts in myotubes, depending on cell-cell interactions most likely mediated by cell adhesion molecules such as cadherins (81). We have therefore proposed that stathmin could be expressed to play a general role in the control of cell activation, proliferation, and differentiation, through integrated phosphorylation on its various phosphorylation sites (21, 81).
In summary, it might be speculated that CaM kinase II phosphorylation of stathmin in T cells stimulated by CD2 triggering could be associated, according to the cells resting/proliferating state, either with an activating or a retroinhibiting process of T cell proliferation. The latter, associated with a stimulated CaM kinase II activity, could direct the cells either toward decreased proliferation or toward activation-induced cell death, according to whether the cells were in a nonmalignant or malignant state.
| Acknowledgments |
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| Footnotes |
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2 Current address: INSERM U99 and Laboratoire dImmunologie Biologique, Hôpital Henri Mondor, 51, avenue du Maréchal de Lattre de Tassigny, 94010 Créteil cèdex, France. ![]()
3 Address correspondence and reprint requests to Dr. Andre Sobel, INSERM U440, 17 rue du Fer à Moulin, 75005 Paris, France. E-mail address: ![]()
4 Abbreviations used in this paper: PKC, protein kinase C; PKA, cAMP-dependent protein kinase A; MAP kinase, mitogen-activated protein kinase; CaM kinase II, Ca2+/calmodulin-dependent kinase II; OA, okadaic acid; cdk, cyclin-dependent kinase. ![]()
Received for publication November 26, 1997. Accepted for publication March 25, 1998.
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