Cellular Signalling 26 (2014) 2721–2729

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Effects of protein tyrosine phosphatase-PEST are reversed by Akt in T cells Yutaka Arimura a,b,⁎, Kazuhiko Shimizu c, Madoka Koyanagi a, Junji Yagi b a b c

Host Defense for Animals, Nippon Veterinary and Life Science University, 1-7-1 Kyonan, Musashino, Tokyo, 180-8602, Japan Microbiology and Immunology, Tokyo Women's Medical University School of Medicine, 8-1 Kawada, Shinjuku, Tokyo, 162-8666, Japan Anatomy and Developmental Biology, Tokyo Women's Medical University School of Medicine, 8-1 Kawada, Shinjuku, Tokyo, 162-8666, Japan

a r t i c l e

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Article history: Received 29 July 2014 Accepted 17 August 2014 Available online 22 August 2014 Keywords: Protein Tyrosine Phosphatase (PTP) PTP-PEST Cell death Growth factor signaling Akt

a b s t r a c t T cell activation is regulated by a balance between phosphorylation and dephosphorylation that is under the control of kinases and phosphatases. Here, we examined the role of a non-receptor-type protein tyrosine phosphatase, PTP-PEST, using retrovirus-mediated gene transduction into murine T cells. Based on observations of vector markers (GFP or Thy1.1), exogenous PTP-PEST-positive CD4+ T cells appeared within 2 days after gene transduction; the percentage of PTP-PEST-positive cells tended to decrease during a resting period in the presence of IL-2 over the next 2 days. These vector markers also showed much lower expression intensities, compared with control cells, suggesting a correlation between the percent reduction and the low marker expression intensity. A catalytically inactive PTP-PEST mutant also showed the same tendency, and stepwise deletion mutants gradually lost their ability to induce the above phenomenon. On the other hand, these PTP-PEST-transduced cells did not have an apoptotic phenotype. No difference in the total cell numbers was found in the wells of a culture plate containing VEC- and PTP-PEST-transduced T cells. Moreover, serine/threonine kinase Akt, but not the anti-apoptotic molecules Bcl-2 and Bcl-XL, reversed the phenotype induced by PTP-PEST. We discuss the novel mechanism by which Akt interferes with PTP-PEST. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Tyrosine phosphorylation is a classic post-translational protein modification that is crucial for various fundamental cellular functions [1–3], especially responses to external stimulation and intercellular communication. Tyrosine phosphorylation is kept in balance by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). The human genome encodes 107 PTPs [4]. Among them, more than 60 PTPs are estimated to be expressed in immune cells [5]. PTP-PEST (Ptpn12) belongs to the non-receptor PTP subfamily, specifically a group of three PTPs also containing LYP/PEP and BDP1/PTP-HSCF (human/mouse orthologs, respectively). PTP-PEST has an N′-terminal phosphatase domain and several proline-rich sequences at the C′-terminal region that contribute to substrate specificity through the protein–protein interaction [6]. PTP-PEST has been mainly analyzed in fibroblasts and, to a lesser extent, in lymphocytes. PTP-PEST is known to associate either directly or indirectly with Cas [6,7], Paxillin [8–10],

Abbreviations: Ab, antibody; PTP, Protein Tyrosine Phosphatase. ⁎ Corresponding author at: Host Defense for Animals, Nippon Veterinary and Life Science University, 1-7-1 Kyonan, Musashino, Tokyo, 180-8602, Japan. Tel.: +81 422 31 4151; fax: +81 422 33 2094. E-mail address: [email protected] (Y. Arimura).

http://dx.doi.org/10.1016/j.cellsig.2014.08.014 0898-6568/© 2014 Elsevier Inc. All rights reserved.

Hic-5 [11], FAK [12], Pyk2/CAKβ [13], Grb2 [14], Shc [15,16], Csk [17], Filamin-A [18], PSTPIP1/2 [19–23], and WASP [24]. Thus, PTP-PEST seems to regulate cytoskeletal reorganization, cell adhesion, cell polarity and migration. In lymphocytes, similar to LYP/PEP and BDP1/PTP-HSCF [25,26], PTP-PEST and Csk cooperatively and negatively regulate the activity of Src family PTKs, such as Lck [17,27]. In human transformed T cells, we also reported that PTP-PEST negatively regulates naïve T cell function by dephosphorylating Lck at Y394 [28]. Conventional PTP-PEST-deficient mice are embryonic lethal [29,30]. Thus, Davidson et al. prepared T cell-specific conditional knockout mice, and reported that PTP-PEST-deficient T cells demonstrated a defect in a secondary T cell response [31]. Given that Pyk2 was hyperphosphorylated in PEST-deficient T cells, Pyk2 seems to be a substrate and to contribute to T cell anergy [31–33]. In response to antigens, T cells become activated through signals via TCR and CD28 that are essential for IL-2 secretion and massive expansion; once the antigen has been cleared, the T cells start to decline in vivo, mainly because of IL-2 exhaustion. Thus, T cell survival in the periphery is maintained by common γ chain (γc)-utilizing cytokines and other survival/growth factors. Survival cytokines elicits multiple signals. JAK-STAT, PI3K-Akt, MAPK, or adaptors like Shc deliver positive signals from receptors for cytokines [34]. Then, these signals subsequently upregulate or maintain anti-apoptotic molecules of the Bcl-2

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family, such as Bcl-2, Bcl-xL, and Mcl-1 [35,36]. On the other hand, IL-2 exhaustion leads to the induction of a pro-apoptotic molecule Bim; in turn, Bim triggers downstream apoptotic processes [37–39]. Among PTPs, TCPTP dephosphorylates JAK1/3 [40], while PTP1B targets JAK2 and TYK2 [41]. SHP-1 negatively regulates STAT6 activation through IL-4 and IL-13 [42]. SHP2 affects the STAT5 phosphorylation level [43]. PTPεC also inhibits JAK-STAT signals via IL-6 and IL-10 [44,45]. PTPPEST is reportedly cleaved by caspase-3 during apoptosis [46,47]; however, the direct involvement of PTP-PEST in the regulation of cytokine signals or T cell apoptosis has not been previously reported. In this report, to explore the physiological function of PTP-PEST, we utilized a milder gene transduction method, i.e., retrovirus-mediated gene transfer, than electroporation into mouse T cells. Upon overexpression, PTP-PEST-transduced T cells appeared within 2 days posttransduction, but during another 2 days of culture in the presence of IL-2, these T cells decreased to about half their previous level. Here, we discuss whether the enforced expression of PTP-PEST induces T cell death by suppressing growth/survival signals or other mechanisms that result in the reduction of the expression level of an internal marker of the retroviral vector that was used. 2. Materials and methods 2.1. Abs and reagents For the flow cytometric analysis, PE- or FITC-conjugated anti-mouse Thy1.1 mAb (CD90.1: HIS51), FITC-anti-mouse CD4 (RM4-5), FITC-antimouse CD11a (M17/4), PE-anti-mouse CD69 (H1.2F3), and anti-mouse CD16/32 (Fc block: 93) were purchased from eBioscience (San Diego, CA). FITC-anti-mouse CD44 (IM7), FITC-anti-mouse CD69 (H1.2F3), and PE-Cy5-anti-mouse CD4 (RM4-5) were purchased from BD Biosciences (San Diego, CA). Anti-class II I-Ab/d (28-16-8S) and anti-CD8 (83.12.5) have been described previously [48]. Anti-mouse CD3/CD28 Abs-coated beads were purchased from Dynal Biotech (Oslo, Norway). Anti-mouse CD3ε (145-2C11) was purchased from eBioscience, and anti-mouse CD28 (37.51) was purchased from BD Biosciences. Goat anti-mouse IgG was purchased from Jackson ImmunoResearch (West Grove, PA), and goat anti-hamster IgG Ab was purchased from Cappel/ MP Biomedicals (Santa Ana, CA). PE-conjugated anti-phospho-Y402Pyk2 Ab was obtained from BD Biosciences, and PE-anti-phosphoY694-STAT5 Ab (L68-1256.272) was obtained from eBioscience. The pan-caspase inhibitor Boc-Asp-fmk (BAF) was purchased from Calbiochem/Merck Milliporre (Billerica, MA), and the caspase-3 inhibitor z-DEVD-fmk (DEVD) was purchased from MBL International (Woburn, MA). Carboxyfluorescein diacetate succinimidyl ester (CFSE) was purchased from Molecular Probes (Leiden, The Netherlands). 7Aminoactinomycin D (7-AAD) and Stem count FluoSphere beads were obtained from Beckman Coulter (Brea, CA). 2.2. Mice and cells C57BL/6 and BALB/c mice were purchased from Japan SLC (Hamamatsu, Japan) and were maintained in our animal facility. The animal experiments in the present study were approved by the ethical review committee of Tokyo Women's Medical University. Spleen CD4+ and CD8+ T cells were purified using anti-class II Ab plus guinea pig C′ (52) or using MACS mouse T cell isolation kits according to the manufacturer's instructions (Miltenyi Biotec, Auburn, CA). WEHI-231 cells and BW5147 cells, which represented B cell lymphoma and thymoma cell lines, respectively, were maintained in RPMI1640 medium with 10% FCS, and were also used for retrovirus-mediated gene transfer.

constructs [23] with the restriction enzymes Bam HI and Xho I and then inserting them into pMXs retrovirus vector with eGFP or Thy1.1 markers [49,50]. Other PTP-PEST constructs, such as N′- and C′-terminal halves (1–303, 304–775 a.a., respectively) with an HA tag, and the stepwise C′-terminal truncated mutants delta-Pro 2 (dP2, 1–350 a.a.), -Pro 3 (dP3, 1–507 a.a.), -NPLH (dNPLH, 1–590 a.a.), and -Pro 4 (dP4, 1–670 a.a.) were also prepared by inserting PCR-amplified fragments into the pMX vector. Mouse Bcl-2, Bcl-XL, and p27Kip1 were prepared. The nucleotide sequences for these constructs were verified by DNA sequencing. The Akt constructs (wild type and constitutively active E40K) were described previously [51]. 2.4. Retrovirus-mediated gene transfer To introduce the PTP-PEST construct into mouse primary CD4+ T cells, retrovirus-mediated gene transfer was conducted as described previously [51]. Initially, the plasmid expression vectors were transfected into packaging cells for retrovirus, Plat-E [52] (a gift from Dr. T. Kitamura, The University of Tokyo, Japan), using the calcium phosphate method or using the Lipofectamine 2000 reagent, according to the manufacturer's instructions (Invitrogen/Life Technologies, Carlsbad, CA). The culture supernatant containing recombinant retrovirus was collected at 1 or 2 days after transfection. To achieve infection, 2 × 106/mL of CD4+ T cells that had been preactivated with anti-CD3/28 Abs-coated beads or plate-bound anti-CD3ε (1 μg/mL)/anti-hamster IgG (10 μg/mL) for 1 day were incubated with virus-containing supernatant from Plat-E supplemented with polybrene (10 μg/mL; Sigma-Aldrich, St. Louis, MO) and human rIL-2 (10 U/mL), then centrifuged for 1 h at 2000 rpm and incubated for 4–6 h at 37 °C. An equal volume of fresh DMEM with 10% FCS was added. The following day, the cells were optionally re-infected as described above. One day later, the infected cells were expanded/ rested in the presence of rIL-2 (100 U/mL) for an additional 2–3 days and were then subjected to the following experiments. 2.5. Intracellular staining of phospho-Pyk2 Mouse CD4+ T cells transduced with control retrovirus vector or PTP-PEST expression vector were first fixed with 1% paraformaldehyde for more than 1 h at 4 °C to prevent the leakage of GFP protein, then stained with anti-phospho-Y402 Pyk2 Ab using the intracellular cytokine staining kit (BD Biosciences) according to the manufacturer's instructions. 2.6. Cell division assay with CFSE staining Mouse T cells were stained with CFSE (5 μM) for 5 min at 37 °C, and cold complete RPMI1640 medium was then added to stop the staining reaction; the cells were then washed 3 times to remove excess CFSE. The stained T cells were subjected to stimulation, as described above, to monitor cell division. Then, the samples were measured using the Epics XL flow cytometer (Beckman Coulter, Miami, FL) and were analyzed using WinMDI 2.8 free software (The Scripps Institute, La Jolla, CA). 2.7. Statistical analysis Data were analyzed using the Mann–Whitney U-test. Values of P b 0.05 were considered significant. 3. Results

2.3. Plasmid constructs

3.1. PTP-PEST-introduced T cells disappear during IL-2 incubation

Fragments of wild type and an enzymatically-inactive mutant (C231S) of mouse PTP-PEST were obtained by digesting corresponding

To elucidate the role of PTP-PEST in the immune system, a mouse PTP-PEST gene or a control empty vector was introduced into mouse

Y. Arimura et al. / Cellular Signalling 26 (2014) 2721–2729

spleen CD4+ T cells once (day 1) or twice (days 1 and 2) using a bicistronic retrovirus vector with Thy1.1 as an internal marker, following anti-CD3 stimulation (Fig. 1A). Then, the activated T cells were rested for 2–4 days in culture medium with IL-2. In Fig. 1C, the percentage of PTP-PEST-introduced T cells rose to 36.0% on day 3 (2 days after introduction); however, it markedly decreased to 14.6% on day 5 (day 2 of resting in IL-2), corresponding to a 60% loss of the introduced cells. Nevertheless, this percentage never decreased to zero. In contrast, cells introduced with the control vector (VEC) were largely stable (from 45.6% to 42.4%). In addition, the Thy1.1 expression intensity in the PTPPEST-introduced cells was markedly lower (47.0–51.8), compared with that for the control vector (89.4–132.5). The transfection efficiencies into Plat-E (virus packaging cells) were comparable between the control and PTP-PEST (57.2% vs. 51.4%)(Fig. 1B). These results raised several possibilities as follows: i) the overexpression of PTP-PEST may induce the cell death of the introduced cells, ii) PTP-PEST may internalize or degrade cell surface marker Thy1 molecules, or iii) PTP-PEST may suppress the basal long terminal repeat (LTR) promoter activity of the retrovirus vector.

these results suggest that PTP-PEST probably does not internalize or degrade the Thy1.1 molecule. We also tried to use a phosphatase-dead mutant (C231S) of PTP-PEST and found that it also showed a similar decrease in the introduced cells, indicating that enzymatic activity is dispensable for this phenomenon (Fig. 2D). On the other hand, upon infection with PTP-PEST retrovirus, transformed T cells (BW5147) and B cells (WEHI-231) also showed similar expression patterns, such as a lower infection percentage and a lower fluorescence intensity compared with those for a control empty virus (Fig. 2E). BW5147 appeared to be more sensitive to this phenomenon than WEHI-231. These results suggest that PTP-PEST behaves in a similar manner in these transformed cells as well. 3.3. PTP-PEST-introduced cells do not show an apoptotic phenotype We next examined the possibility that the overexpression of PTPPEST induces the cell death of its introduced cells. Using 7-AAD, which binds DNA and detects apoptotic cells, we tried to see whether PTPPEST-introduced CD4+ T cells contained more 7-AAD-positive cells than the control cells but failed to find any clear difference (Fig. 3A). Furthermore, caspase inhibitors, such as DEVD and BAF, did not block the decrease in %Thy1.1+ cells in PTP-PEST-introduced CD4+ T cells (Fig. 3B). Furthermore, during the period when the percentage was decreasing (day 1 of IL-2 incubation), we compared the phosphorylation level of Pyk2, which is a substrate for PTP-PEST in T cell-conditional knockout mice, and found no alteration (Fig. 3C). Thus, these data indicate that the enforced expression of PTP-PEST was not accompanied by an apoptotic phenotype, supporting the notion that PTP-PEST does not induce the cell death of the introduced T cells.

3.2. Phenotypes of PTP-PEST-introduced cells Next, we tested the second possibility mentioned above: whether PTP-PEST may internalize or degrade cell surface Thy1. Thy1.1 is an internal marker of the retrovirus vector, while Thy1.2 is endogenously expressed on T cells from C57BL/6 mice. The Thy1.2 expression level was the same between the control empty vector and the PTP-PESTintroduced cells (Fig. 2A), indicating that PTP-PEST does not affect Thy1 expression itself. We also examined other phenotypes of the PTP-PEST introduced cells, such as the expression levels of T cell activation markers (such as CD44, CD69, and CD11a) and the forward scatter (FSC), which indicates the cell size. On day 5, the expression level of CD69 was higher in PTP-PEST-introduced cells than in control cells, while differences in CD44, CD11a, and FSC were negligible (Fig. 2B). On day 3, all the markers exhibited the same expression levels between the control and the PTP-PEST-introduced cells (not shown). Moreover, when we used a retrovirus vector with the GFP marker, the same decrease was observed (from 19.5% to 5.0%) (Fig. 2C). Taken together,

A

Day 0 1 2 3 CD3

4 5 6

3.4. No difference in total cell number in wells of VEC- and PTP-PEST-introduced T cells Given that the possibility of cell death induced by PTP-PEST was unlikely, we decided to simply count the total cell numbers in wells containing control vector or PTP-PEST-introduced T cells and then to compare the numbers. The percentage of Thy1.1+ cells in the wells of PTP-PEST-introduced CD4+ T cells following one or two retrovirus

C

CD4

Day 1 infection

IL-2

VEC

infection

Day 3

Day 1+2 infect.

PTP-PEST

45.6%

Day 5

Day 1 infect.

B

2723

42.4%

(89.4)

36.0% (51.8)

Plat-E VEC 57.2% (99.4)

PTP-PEST 51.4% (106.6)

(132.5)

14.6% (47.0)

Fig. 1. PTP-PEST-introduced T cells disappear during IL-2 incubation. (A) Time course for retrovirus-mediated gene introduction into T cells. Using a retrovirus vector with Thy1.1 as an internal marker, a mouse PTP-PEST gene or control empty vector (VEC) was introduced into mouse spleen CD4+ T cells once (day 1) or twice (day 1 and day 2) following anti-CD3 stimulation (bold line). Then, the activated T cells were rested in the presence of IL-2 for 2–3 days (thin line). The arrow indicates the time(s) of infection. (B and C) Transfection efficiency into virus packaging cells Plat-E (B) and CD4+ T cells (C) is shown based on the internal marker Thy1.1 expression. The numerals in parentheses indicate the mean fluorescence intensity. The infection efficiency into CD4+ T cells was estimated on day 3 (anti-CD3) and day 5 (day 2 of IL-2 incubation). Data are the representative results of at least ten independent experiments.

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A

B Day 5

VEC PEST

VEC

124

CD11a

Thy1.2

D

PEST (CS)

Day 3

21.0% (27.2)

12.0% (26.8)

Day 5

8.6% (23.4)

6.0% (24.6)

E

VEC (GFP)

PEST (GFP)

75.2% (61.6)

14.6% (25.4)

(-)

(-)

5.0% (19.1)

FSC

GFP

(-)

WEHI-231

19.5% (26.6)

PEST (WT)

Thy1.1

Day 3

PEST (GFP)

Day 6

C

CD69

BW5147

129

PEST

CD44

VEC PEST

81.2% (411.8)

45.7% (85.9)

GFP

Fig. 2. Phenotypes of PTP-PEST-introduced cells. (A and B) Surface expression levels of Thy1.2, CD44, CD69, and CD11a and cell size (FSC) in VEC- and PTP-PEST-introduced CD4+ T cells on day 5 (day 2 of IL-2 incubation). Thy.1.1+ cells were gated for comparison purposes. The numerals indicate the mean fluorescence intensity of Thy1.1. The red and black lines in the histogram show the VEC and PTP-PEST-introduced cells, respectively. (C) Instead of Thy1.1 as an internal marker, a GFP-containing vector was also used to monitor the gene-introduced T cells. The percentages of GFP+ cells are shown. The numerals in parentheses indicate the mean fluorescence intensity of GFP. (D) The wild-type (WT) and an enzymatically -inactive mutant (CS: C231S) of PTP-PEST were introduced into T cells, and the number of positive cells was monitored based on Thy1.1 expression on days 3 and 6. The percentages of Thy1.1+ cells are shown. The numerals in parentheses indicate the mean fluorescence intensity of Thy1.1. (E) PTP-PEST or a control vector with a GFP marker was introduced into a transformed T cell line (BW5147) and a B cell line (WEHI-231), and the percentages of GFP+ cells and the fluorescence intensity were then monitored.

infections decreased consistently (one infection, from 34% to 11%; two infections, from 57% to 20%) (Fig. 4A). In contrast, the total cell numbers in wells containing control vector or PTP-PEST-introduced T cells were comparable. The cell number was much larger than the expected numerical value based on the decrease in Thy1.1+ cells (Fig. 4B), suggesting that PTP-PEST-introduced T cells did not die. In addition, the cell cycle inhibitor p27 Kip1 was also introduced and its effect was similarly estimated. The percentage of GFP+ cells in Kip1 also decreased from day 4 to 6 (Fig. 4C, left panel). The cell number of wells containing Kip1-introduced cells clearly decreased (Fig. 4C, right panel), in contrast to the case of PTP-PEST. That indicates cell cycle arrest in the Kip1introduced cells. Furthermore, control and PTP-PEST-introduced CD4+ T cells were examined for cell division on day 3 (Fig. 5A) or 4 (Fig. 5B) post-stimulation and after an additional 7 h of incubation in IL-2 (Fig. 5C) using CFSE staining, enabling cell division to be monitored. The control cells show 2–4 cell divisions, and the PTP-PEST-introduced T cells also showed a similar cell division rate, although the Thy1.1 intensity was very low. Thus, these results indicate that PTP-PESTintroduced cells do not have a tendency to undergo cell cycle retardation or arrest.

constructs, such as N′- and C′-terminal halves with an HA tag (N′ (HA) and C′ (HA)), delta Pro 2, 3, 4 motifs (dP2, 3, 4), and a delta NPLH motif (Fig. 6A). Following infection, the GFP intensities and changes in the percentages of GFP+ cells of these constructs from day 3 to 5 or from day 4 to 6 were measured. The changes in GFP+ cells were then normalized against that of the control empty vector pIG(−) because of their variation. The most notable observation was that the GFP intensity and the change in GFP+ cells showed a very similar pattern, in which the full length or longer constructs largely resulted in a lower GFP intensity and a much greater reduction in GFP+ cells (Fig. 6A and B). The C′ (HA) construct completely lost the ability to induce the above-mentioned phenomena and behaved like a control vector. On the other hand, the N′ (HA) construct had an intermediate activity, although it was the shortest construct. These results indicate that longer constructs are more capable of inducing these two phenomena (a lower GFP intensity and a much greater reduction in GFP+ cells) and that the substrates responsible for these phenomena might either be the same or might overlap. 3.6. Akt, but not Bcl-2 and Bcl-XL, reverses the suppression of Thy1.1 intensity by PTP-PEST

3.5. Effect of stepwise deletion mutants of PTP-PEST To identify the responsible site of PTP-PEST required for the above phenomena, we prepared stepwise deletion mutants of retrovirus

In relation to the above-mentioned observation in which the PTPPEST-introduced T cells did not seem to die, we attempted to examine whether anti-apoptotic molecules could block the phenomenon

Y. Arimura et al. / Cellular Signalling 26 (2014) 2721–2729

A

VEC

C

PEST 1.0%

7.1%

1.5%

(-)

0.8%

2725

3.2

82.9

4.2

92.5

VEC (GFP+)

7-AAD

2.9% 10.6%

3.9%

2.3%

PEST (GFP+) Thy1.1 (-)

Thy1.1

Day 3

B

(-) Day 5

p-Pyk2

12.1% (33.5)

BAF (10 µM)

6.9% (29.5)

7.1% (28.4)

DEVD (2 µM) 5.9% (26.7)

(-) Fig. 3. PTP-PEST-introduced cells do not show an apoptotic phenotype. (A) The VEC- and PTP-PEST-introduced CD4+ T cells on day 3 were stained with PE-anti-Thy1.1 and 7-AAD, which can be used to detect apoptotic cells. (B) PTP-PEST-introduced CD4+ T cells were rested/incubated for 3 days in IL-2, and caspase inhibitors such as DEVD (2 μM) and BAF (10 μM) were used to monitor the alteration in %Thy1.1+ cells in PTP-PEST-introduced CD4+ T cells. The numerals in parentheses indicate the mean fluorescence intensity of Thy1.1. (C) The tyrosine phosphorylation level of Pyk2 was examined using intracellular staining without (red line) or with anti-phospho-Y402-Pyk2 Ab (black line) in GFP+ CD4+ T cells after 1 day of incubation in IL-2.

induced by PTP-PEST. The CD4+ T cells were co-introduced with the anti-apoptotic factors Bcl-2 or Bcl-XL (both GFP+) in combination with PTP-PEST or a control empty vector pIT(−) (both Thy1.1+). Compared with a control combination (pIT(−) plus Bcl-2) (Fig. 7A, left panel), both Thy1.1 intensity and percentage of the PTP-PEST-introduced T cells were much lower, irrespective of the presence of Bcl-2 or Bcl-XL, indicating that these Bcl-2 family members did not have any effect on this phenomenon (Fig. 7A and B). In contrast, in co-introduced cells with PTP-PEST and a constitutively active form of the serine/threonine kinase Akt E40K (right upper corner of quadrant in Fig. 7A, right panel), the Thy1.1 intensity was markedly higher than that in the PTP-PESTalone-introduced cells (left upper corner). These results indicate that Akt reverses the effect of PTP-PEST, suggesting that PTP-PEST does not induce cell death but somehow suppresses Thy1.1 or GFP expression from the retrovirus vector. 4. Discussion In this report, exogenous PTP-PEST-positive (GFP+ or Thy1.1+) T cells appeared 2 days post-retroviral gene transfer, usually reached a maximal expression level on days 2–3, and then decreased during the

next 2 days of a resting period in the presence of IL-2, at which time the TCR signal was absent. The easiest interpretation explaining this observation is that the cell death was caused by PTP-PEST. The observations also suggest the possibility that PTP-PEST suppresses particular signal pathway(s) from IL-2 or other growth/survival factors included in the culture medium. Indeed, another cytosolic PTP, SHP-2, has been reported to have a negative impact on hematopoietic cell survival through the dephosphorylation of STAT5, which is a downstream molecule of IL-2 as well, under the condition of growth factor deprivation [43]. PTP-PEST has also been reported to be cleaved by caspase-3 under apoptosis-inducing conditions [46]. To confirm the possibility of cell death, we tried multiple staining experiments, such as 7-AAD (Fig. 3), Annexin V, and propidium iodide (not shown), to show an apoptotic phenotype, but could not obtain clear evidence of a promoted tendency for cell death in the PEST-positive cells. In Fig. 4, we counted the absolute cell number in each well containing the control vector or PTP-PEST-introduced T cells using flow cytometry with FluoSphere beads. Unexpectedly, we found no significant difference in the cell number. In contrast, a reduced cell number of Kip1-introduced cells was observed, indicating that this method was sufficient to detect cell death or cell cycle arrest. Taken collectively,

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Day 1 infection pIT (-) pIT-PEST

Day 2 infection pIG(-)

90

40

60

20

30

0

0 day-3

C

day-5

day-4

day-6

24.0% (248)

27.2% (493)

32.7% (319)

17.2% (260)

B Cell no. (x105)

80

pIT (-) pIT-PEST n.s.

60

240

n.s.

%GFP+

40

10

0

20 0

0 day-4

day-5

expected

*

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40 day-3

*

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80

0

pIG(-) Kip1

80

30

120

20

Cell no. (x105)

100

200 160

40

Kip1

Day 4

%Thy1.1+

60

Day 1+2 infection

Day 6

A

day-6

day-4

day-2 day-4 day-6

day-6

Fig. 4. No difference in total cell number in wells of VEC- and PTP-PEST-introduced T cells. (A) The percentage of control empty vector pIT(−)- and PTP-PEST-introduced CD4+ T cells following one (day 1) or two (day 1 and 2) retrovirus infection(s) were estimated by staining with anti-Thy1.1 on days 3 and 5 (for cells infected once) or days 4 and 6 (for cells infected twice). The gene introduction was performed in triplicate. (B) The total cell numbers in wells of pIT(−)- and PTP-PEST-introduced T cells were determined with flow cytometry using FluoSphere beads. The closed and open bars indicate the control empty vector pIT(−)- and the PTP-PEST-introduced CD4+ T cells, respectively. The dashed lines indicate the expected cell number based on the decrease in the percentage of Thy1.1 + cells in PTP-PEST-introduced cells (38.2 × 105 cells and 116.0 × 105 cells for left and right panels, respectively). (C) The GFP-containing control empty vector pIG(−) or the Kip1 expression vector was introduced on day 2, and the percentage of GFP+ cells and the total cell numbers were estimated on days 4 and 6, as above. The closed and open bars indicate the pIG(−)- and Kip1-introduced CD4+ T cells, respectively. The dashed line indicates the expected cell number (51.2 × 105 cells). *, P b 0.05; n.s., not significant.

A

C

Day 1 infection PEST

Day 1+2 infection 18.6 35.3 31.3 14.8

Thy1.1

VEC Day 4

50

Day 3 (D1-inf)

Day 3 (D1+2-inf) 50

40

40

30

30

20

20

10

10

0

R6

R5

R4

R3

R2

0

Day 3 + 7h (D1-inf)

PEST 18.9 32.6 32.8 15.7

B

7.0 39.2 38.2 15.6

Thy1.1

9.5 41.8 34.7 14.0

Day 3

(-)

VEC

50

40

40

30

30

20

20

10

10 0 R6 R5 R4 R3 R2

R7 R6 R5 R4 R3 R2

Day 4 (D1+2-inf)

50

0

VEC PEST

VEC PEST

R7 R6 R5 R4 R3 R2

Cell division (CFSE) Cell division (CFSE) Fig. 5. No difference in cell division of VEC- and PTP-PEST-introduced T cells. VEC or PTP-PEST was introduced once (A) or twice (B) into CD4+ T cells stained with CFSE prior to transfection, and the cell division rates were estimated based on dilution of the CFSE intensity. The numerals indicate the percentage of each cell division. (C) The percentages in each cell division of CFSE staining in Fig. 5A and B were summarized as bar graphs. Squares (i.e., R2 to R7) indicate gate regions that approximately correspond to 2–7 cell divisions. The closed and open bars indicate the control VEC- and PTP-PEST-introduced cells, respectively.

Y. Arimura et al. / Cellular Signalling 26 (2014) 2721–2729

these results strongly suggest that the reduction in PTP-PESTtransduced cells was not caused by cell death, but by some other mechanism(s). Other pieces of evidence that do not support cell death are the negligible effects of Bcl-2 and Bcl-XL. In thymocytes, Bcl-2 and Bcl-XL are repressors of a wide variety of cell death, such as γ-irradiation, glucocorticoids, and anti-CD3 treatment [35,37]. When simultaneously transfected with PTP-PEST into peripheral T cells, neither of these repressors was able to affect the phenomenon induced by the enforced expression of PTP-PEST (Fig. 7). Caspase inhibitors also failed to block the reduction of PTP-PEST-positive cells (Fig. 3). Recently, a new type of cell death, named programmed necrosis or necroptosis, has been reported; this type of cell death occurs in the presence of the pan-caspase inhibitor z-VAD-fmk [53]. However, since this new type of cell death can be blocked by Bcl-2 family members [54], the reduction of PTP-PESTpositive cells is probably not caused by cell death, including programmed necrosis. Thus, the reduction of PTP-PEST-positive cells was likely caused by other mechanisms, such as the transcriptional suppression of LTR, translational suppression, or the destabilization of proteins. Surprisingly, Akt reversed the low Thy1.1 intensity induced by PTPPEST (Fig. 7). How was Akt able to do this? Akt is known to play various roles in cells, exerting not only an anti-apoptotic function [55], but also amplifying cytokine production in T cells [51]. However, this reversing effect was not mediated through cytokines, which might have been secreted by T cells, because the Thy1.1 intensity was still low in PTP-PESTalone-introduced cells, even in the same well of a cell culture plate containing PTP-PEST/Akt doubly introduced cells with a higher intensity (Fig. 7). Thus, cytokines that might have been increasingly produced by Akt did not affect singly transfected T cells in a paracrine fashion. Rather, Akt directly modulated a particular signal leading to the upregulation of the marker Thy1.1. Upon TCR triggering, the transmembrane protein tyrosine phosphatase CD45 is known to dephosphorylate tyrosine 505

A

P1 P2

PEST

P3

NPLH

P4 CTH

2727

of Lck, releasing the C-terminal portion; in turn, Lck becomes activated, phosphorylates the TCRζ chain thereby recruiting ZAP-70, and leads to the sequential generation of multiple downstream signaling pathways. In this process, the TCR signal may suppress the inhibitory effect of PTP-PEST, since the percentage of extrinsic PTP-PEST-positive cells was maintained at a relatively high level during TCR stimulation. When the T cells were transferred to the culture medium in IL-2 for resting, the percentage started to decline. Which signal contributes to this observation remains obscure. Akt, which is activated upon TCR stimulation, might play a role. In this report, the C231S enzymatically inactive mutant of PTPPEST still had the same effect as the wild type (Fig. 2D), indicating that the enzyme activity was dispensable for this phenomenon. Since C to S and D to A mutants are often used for substrate trapping, the C231S mutant might still retain its capacity to bind to, but not to dephosphorylate, substrates. For instance, the pseudophosphatase MK-STYX, which does not possess phosphatase activity, can still interact with G3BP, which is a regulator of Ras signaling, and continues to be capable of exerting a physiological function [56]. On the other hand, PKA and PKC phosphorylate Ser39 of PTP-PEST, and PTP-PEST loses its binding affinity for substrates resulting in a decrease in enzymatic activity [57]. The amino acid sequence around Ser39 of PTP-PEST fits the RxxS/T motif for Akt substrates. To the best of our knowledge, whether Akt indeed phosphorylates this site and affects the function of PTP-PEST has not been previously investigated. However, Akt might interact with PTP-PEST, affecting the phosphatase activity. Taken together, these considerations suggest that the C231S mutant, but not the Ser39-phosphorylated form (potentially induced by Akt), might be able to induce a reduction in the GFP or Thy1.1 intensity and its percentage by binding and sequestrating substrate molecules from the specific signal transduction pathway that is involved, if the substrate is an important molecule delivering

B

PTP

dP2

PTP

dP3

PTP

dNPLH

PTP

dP4

PTP

MFI of GFP

800

PTP

day 4 day 6 600

400

Changes in %GFP+ normalized to pIG(-)

C

From Day 4 to Day 6

From Day 3 to Day 5 1.2

1.2

1

1

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

0

0

Fig. 6. Effect of stepwise deletion mutants of PTP-PEST. (A) Schema of full-length PTP-PEST and its stepwise deletion mutants, such as N′- and C′-terminal halves with HA tag (N′ (HA) and C′ (HA)), delta Pro 2, 3, 4 motifs (dP2, 3, 4), and a delta NPLH motif. PTP, phosphatase domain; CTH, C′-terminal homology region; Slash, HA tag. (B and C) Following infection, the GFP intensities and the changes in the percentages of GFP+ cells for these constructs from days 3 to 5 or from days 4 to 6 were measured. (C) The percentage of GFP+ cells on day 5 was divided by that on day 3 (ratio, changes in the percentage of GFP+ cells). Similarly, the value on day 6 was divided by that on day 4. These ratios were normalized against that of the control empty vector pIG(−).

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Y. Arimura et al. / Cellular Signalling 26 (2014) 2721–2729

pIT(-)+Bcl2

pIT(-)

33.9% 4.1% (139) (172)

PEST+Bcl2 8.1% (82.3)

1.3% (95.1)

PTP-PEST

A

PEST+BclXL 2.5% (46.2)

Bcl-2 200 180 160

MFI (Thy1.1)

PEST+Akt (E40K) 3.8% (73.9)

13.9% (165)

Day 4

Bcl-2

B

4.7% (55.0)

Bcl-XL (-) Bcl2

160

350

(-) BclXL

140 120

140 120

100

100

80

80

60

Akt (E40K)

300

(-) E40K

250 200 150

60

100

40

40

50

20

20 0

0

VEC PEST

0

VEC PEST

VEC PEST

Fig. 7. Akt, but not Bcl-2 and Bcl-XL, reverses the suppression of Thy1.1 intensity by PTP-PEST. (A) The CD4+ T cells were simultaneously co-introduced with Bcl-2 or Bcl-XL (both GFP+) in combination with a control empty vector pIT (−) or PTP-PEST (both Thy1.1+); 4 days after the initial T cell stimulation, the cells were stained with PE-conjugated anti-Thy1.1. The numerals in parentheses indicate the mean fluorescence intensity of Thy1.1. (B) The Thy1.1 intensity of the dot plots in Fig. 6A is summarized as bar graphs. The closed and open bars indicate the Thy1.1 intensities in the control vector or PTP-PEST alone and those in combination with Bcl-2, Bcl-XL or Akt E40K-introduced cells, respectively.

a positive signal for this phenomenon. Further experiments in this area are required. In T cell-specific conditional PTP-PEST-deficient mice, Pyk2 was hyperphosphorylated, indicating that Pyk2 was a bona fide substrate for PTP-PEST in T cells [31]. Reportedly, Pyk2 is also a positive regulator of IL-2 [58]. In our studies, the tyrosine-phosphorylation level of Pyk2 was not different between the VEC- and the PTP-PESTtransduced T cells, based on the results of an intracellular staining method (Fig. 3C). We do not know whether the reason for this discrepancy can be ascribed to differences in the experimental methods that were used in the studies involving the gene-deficient mice and the gene-overexpressing cells, in which the amounts and species of associated molecules might have differed. If PTP-PEST modulates signals from cytokines or growth factors, what pathway is affected by the enforced expression of PTP-PEST? Among non-receptor PTPs, TCPTP negatively regulates signals from IL-2 and IFNs [40], and SHP-1 also suppresses signals from IL-4 and IL-13 [42]. PTPεC selectively inhibits IL-6 and IL-10 [45]. Thus, PTP-PEST might also regulate downstream molecules from cytokines and growth factors. A substrate-trapping method for PTP-PEST might be worth testing in our experimental setting. On the other hand, which region of PTP-PEST is responsible for the current phenomenon? The C′-terminal half of PTP-PEST harbors several unique motifs for protein interactions, such as four proline-rich sequences (Pro 1–4), an NPxH sequence, and a C′-terminal homology region (CTH). To date, PTP-PEST is known to associate directly or indirectly with Cas (via Pro 1); Paxillin, FAK, and Pyk2 (via Pro 2); Shc (via NPxH); Csk (via Pro 4); and PSTPIP1/2, c-Abl, and WASP (via CTH). As shown in Fig. 6, stepwise deletion mutants gradually lost their effect on T cells as PTP-PEST was truncated more severely. The effect of the deletion of P4 and NPLH was relatively smaller, while that of the deletion of P2 and P3 was relatively larger. The C′-terminal half construct completely abrogated the effect. Thus, the full-length PTP-PEST

had the strongest binding affinity, while the truncated constructs gradually lost their ability to capture the substrate. Nevertheless, the boundary and the specific region that are important and required to capture the substrate remain uncertain. A similar type of binding manner has been observed between Grb2 and PTP-PEST, in which multiple binding sites contribute simultaneously in a synergistic fashion [14]. Since Grb2 is a well-known positive regulator of growth factors, such as EGF, Grb2 is likely to be the primary candidate responsible for the presently described phenomenon. To understand the whole picture, further extensive and discrete experiments are needed. 5. Conclusion We explored a role for PTP-PEST in T cells, and found that PTPPEST-transduced T cells appeared to decrease during their resting period. Enzymatic activity of PTP-PEST was dispensable for the phenomenon. PTP-PEST with stepwise deletion gradually failed to induce the above finding. In addition, these PTP-PEST-transduced T cells did not have an apoptotic phenotype. Consistent with that, Akt, but not anti-apoptotic molecules Bcl-2 and Bcl-XL, reversed the effect induced by PTP-PEST. These results suggest that cell death reportedly caused by PTPs should be reevaluated carefully. Competing financial interests The authors declare no competing financial interests. Acknowledgments This study was supported by grants from our university and the Ministry of Education, Culture, Sports, Science and Technology (#23570173). We are grateful to Dr. J. F. Côté for the PTP-PEST expression vectors (pcDNA3.1). We thank Dr. T. Kitamura for the pMXs

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