CELLULAR
IMMUNOLOGY
145, 146-155 (1992)
Stress Stimuli-Induced
Lymphocyte
Activation
HARRY M. LANDER,*‘~” DANIEL M. LEVINE,*,? AND ABRAHAM NOVOGRODSKY*~~$.~ *The Rogosin Institute and the Departments qf tBiochemistry and $Medicine, Cornell University Medical College, 1300 York Avenue, Box 135, New York, New York 10021 Received March 18. 1992; accepted August 13, I992 Oxidants, heavy metals, and heat shock, collectively known as stressstimuli, induce the synthesis of a variety of proteins, termed stress proteins, and enhance glucose uptake. In this study, we have demonstrated that stressstimuli enhance protein tyrosine phosphorylation (PTyr-P), modulate protein tyrosine phosphatase (PTPase) activity, activate the src family protein tyrosine kinase (PTK), p56”‘, and enhance glucose uptake in human peripheral blood mononuclear cells. The heavy metal Hg2+ and heat shock stimulated PTPase activity at an optimal dose, whereas the oxidant phenylarsine oxide (PAO) was only marginally stimulatory. Treatment of lymphocytes with stress stimuli at a dose which activated PTPase did not produce discernable PTyr-P using Western blotting techniques. PTyr-P was only seen at doses of stressstimuli which were associated with an inhibition of PTPase activity. We could demonstrate a correlation between the dose of stress stimuli effective in increasing PTPase activity and ~56’~~ activation using heat shock and Hg2+ as stress stimuli. On the other hand, much lower concentrations of PA0 were effective in activating PTPase than those effective in eliciting ~56’~~ activation. We could not demonstrate a correlation between an effectivedose inducing PTyr-P and glucose uptake. Our data do not permit us to draw a simple correlation between enhancement of PTPase activity, activation of p561ck, induction of PTyr-P, and induction of the biological response. It is possible that both stimulation and inhibition of PTPase could regulate PTyr-P by either activating the src family PTKs or preventing dephosphorylation of target proteins which are involved in the biological response. Our data may also provide the biochemical basis for the previously reported mitogenic effectsof Hg*+ on lymphocytes. 0 1992 Academic Press, Inc.
INTRODUCTION We have previously shown that hemin, an oxidant, is mitogenic for human lymphocytes (1, 2). In studying the mechanism of its stimulatory effect on lymphocytes, we have found (submitted for publication) that hemin induced an insulinomimetic effect-namely an enhancement of glucose transport and an increase in protein tyrosine phosphorylation (PTyr-P). These effects were associated with stimulation of protein tyrosine phosphatase (PTPase) activity and activation of the SK family protein tyrosine kinase (PTK), ~56’“~. We postulated that the latter phenomena were related because of the finding that SK kinases are negatively regulated by tyrosine phosphorylation of ’ To whom correspondence should be addressed. * Current address: The Rogoff Institute, Beilinson Medical Center, Petah-Tikva, 49100, Israel. 146 0008-8749192 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reprcductmn in any form reserved
STRESS
STIMULI
ACTIVATE
~56”’
147
the carboxy terminus (3, 4). Protein tyrosine phosphorylation was implicated to play a central role in mediating signal transduction in lymphocytes (5- 10). Oxidants, heavy metals, and heat shock have all been shown to induce the synthesis of heat shock proteins (11, 12). We also found that hemin induced hsp 70 mRNA expression in human lymphocytes. These stresses also have other inductive effects in various cell types such as enhancement of glucose uptake and gene expression including c-jun, c-fos, ubiquitin, and multidrug resistance genes (13- 15). In addition, heat shock and Hz02 was shown to induce PTyr-P in cultured cells (16, 17). In this study, we investigated the response of human peripheral blood mononuclear cells (PBM) to various stress stimuli and, in addition, investigated whether these agents mimic hemin in activation of the PTyr-P signal transduction pathway. MATERIALS Cell Isolation
AND METHODS
and Culture
Human PBM were isolated from healthy volunteers by Ficoll-Hypaque density gradient centrifugation. The murine lymphoma BW5147 (CD45+) and mutant BW5 147(T200-a)5.1 (CD45-) cell lines were a kind gift of Dr. Robert Hyman (The Salk Institute, San Diego, CA). The CD45- cells expressed less than 1% of CD45 antigen in the membrane or cytoplasm (18, 19). These cells were maintained in DMEM with high glucose (JRH Bioscience, Lenexa, KS) containing 10% heat-inactivated horse serum. The human erythroleukemia K562 was maintained in RPM1 1640 containing 10% heat-inactivated fetal calf serum. Cell viability was greater than 95% after stress stimuli treatments as assessed by trypan blue exclusion. All reagents were purchased from Sigma Chemicals (St. Louis, MO). Preparation
of Particulate
Fraction
PBM or murine lymphoma cells were washed in PBS and resuspended to approximately 40 X lo6 cells/ml in 25 mM Hepes, 5 mA4 Na lactate, 1 ,mg/ml BSA, and aliquoted for treatment. After appropriate incubation time, cells were washed twice with wash buffer (25 mA4 Hepes, pH 7.4,5 mM KCl, 150 mM NaCl) and resuspended with 100 ~1 of sonication buffer (25 nGt4 Tris, pH 7.5, 1 mM MgC12, 1 mM PMSF, 100 pg/ml aprotinin, 10 pg/ml leupeptin). They were then sonicated in a bath sonicator (Laboratory Supplies, Hicksville, NY) for 20 set and put on ice for 40 sec. This was repeated 10 times or until inspection of aliquots under a microscope showed complete disruption. The tubes were spun at 1200 t-pm for 5 min to pellet debris and the supernatants spun for 1 hr at 100,OOOg at 4°C. The supernatants were discarded and the pellets resuspended in 100 ~1 of sonication buffer by vortexing. This particulate fraction was put on ice and assayed for protein by the method of Lowry as modified by Markwell et al. (20). Protein Tyrosine Phosphatase Assay
Particulate fractions were assayed exactly as described by Streuli et al. (2 1) using the 32P-Raytide substrate. Raytide (Oncogene Science, Manhasset, NY), a gastrin analog, was tyrosine phosphorylated in the presence of [T-~~P]ATP with the v-AH protein (Oncogene Science). 32P-Tyr-Raytide was recovered by TCA precipitation with extensive washing to remove unincorporated label.
148
LANDER, LEVINE, AND NOVOGRODSKY
Five microliters of sample (0.1-0.5 mg/ml in sonication buffer) was mixed with 5 ~1 32P-Tyr-Raytide (l-5 X lo4 cpm) in 50 ~1 buffer (25 mM Hepes, pH 7.3, 50 mM EDTA, 100 mM DTT). Sample concentration was varied such that no more than 10% of the substrate was destroyed by the enzyme during the 10 min incubation at 37°C. To terminate, 750 ~1 of acidic charcoal was added, tubes were centrifuged in a microfuge for 5 min, and 400 ~1 supernatant was counted. The assay was linear at 10 min. Data are expressed as pmol PO; released per milligram protein per minute and data points represent the average values from three experiments in which samples were assayed in duplicate. Determination of Protein Tyrosine Phosphorylation
Cells were prepared for analysis via Western blotting in the following manner: 20 X lo6 cells/ml in 1 ml of RPM1 1640 containing 5% FCS were treated under the appropriate conditions. After washing twice with PBS, cells were pelleted in a microfuge for 1 min. The medium was decanted, the pellets were frozen in liquid nitrogen, and 500 ~1 extraction buffer (1 mM Tris, pH 7.4, 1% SDS, 1 mM NaV03, 0.1 mM NazMo04, 1 mM PMSF) was added. The pellets were immediately boiled for 5 min and transferred to glass test tubes and sonicated in a bath sonicator until DNA was sheared (3-4 min). Protein levels were determined and 5X Laemmli buffer (22) (containing 20 mM DTT) was added, boiled for 3 min, and 200 pg protein/lane was loaded onto 3-27% SDS polyacrylamide gels with a 3% stack. Electrophoresis was carried out at 100 V/gel until the dye front nearly ran off. The gel was soaked in transfer buffer (0.05 M glycine, 0.05 M Tris, pH 8.8) for 15 min and electrotransferred onto 0.22pm nitrocellulose paper for 22 hr at 35 V at 4°C. After transfer, the nitrocellulose was soaked in PBS containing 2 mg/ml BSA for 1 hr at 22°C washed twice in wash buffer (PBS, BSA, 0.05% Tween 20) and soaked in wash buffer containing anti-phosphotyrosine antibody (1: 1000) (PY20 clone, ICN Biomedicals, Costa Mesa, CA). As a control, we blotted in the presence of 20 m/t4 0-phosphotyrosine and preimmune serum and found that no PTyr-P was evident. After washing five times with wash buffer, it was soaked in wash buffer containing 0.2 &i/ml ‘251-protein G (Amersham, Arlington Heights, IL) for 2 hr, again washed five times, and exposed to XOMAT (Kodak, Rochester, NY) film for 16 hr at -70°C with intensifying screens. 14C-protein standards (Amersham) were included on all gels to estimate molecular masses. Assay of p561ckActivity
Activity of ~56’“~ was assessed as follows. PBM (20 X lo6 cells) were treated as indicated. Cells were pelleted, washed three times with PBS, and lysed in 250 ~1 antiCD4 buffer ( 1% NP-40, 150 mJ4 NaCl, 1 miM NaMo3, 0.4 mM NaVo4, 5 mM MgC12, 1 mM PMSF, 10 pg/ml leupeptin) containing 200 U DNase. After 30 min at 4°C 5 pg anti-CD4 (OKT4, Ortho Diagnostics), which coprecipitates ~56’~~ (23), was added to each sample and after another hour at 4°C 25 ~1 of a 1: 1 suspension of Sepharoselinked protein G was added. After 1 hr, samples were washed three times with antiCD4 buffer and 30 ~1 containing 25 mMHepes, 0.1% NP-40, and 10 &i [Y-~~P]ATP was added. After 5 min at 25°C 30 ~1 SDS sample buffer was added and samples were boiled. Samples were electrophoresed and transferred as described above.
STRESS STIMULI PA0 (PM): 0.2
ACTIVATE
2
20
~56”’
149
200
kD 200 97 69 46 -
14 -
FIG.I. Effect of phenylarsine oxide on protein tyrosine phosphorylation. Human PBM were treated with the indicated concentration of phenylarsine oxide for 1 hr followed by analysis of protein tyrosine phosphorylation via Western blotting as described under Materials and Methods. 2-Deoxy-D-glucose Uptake
Fresh PBM were resuspended to 10 X lo6 cells/ml in PBS containing 1 mg/ml BSA and 5 mM Na lactate. Cells (0.5 ml per data point) were treated appropriately and after incubation at 37°C were put on a shaker and 10 ~1 of 5 mA4 2-DOG containing 5 &I [3H]-2-DOG (Amersham) was added. After 5 min, 200 ~1 was removed and layered on top of dibutyl phthalate:mineral oil (4: 1) and spun in a microfuge for 30 sec. The tip (with the cell pellet) was cut and placed in 1 ml of 1% Triton X- 100 and 0.1 N NaOH, vortexed, and counted with 15 ml Ultrafluor (National Diagnostics, Manville, NJ) in a liquid scintillation counter. The assay was linear at 5 min. RESULTS StressStimuli Induce PTyr-P
We investigated the effects of stress stimuli on tyrosine phosphorylation in human lymphocytes. We examined the effects of the oxidant PAO, heat shock, and the heavy
HgCI2(pM):
I 0
0.2
(PM) 2
20
200
1 2mM
200 97694630-
14-
FIG.2. Effect of Hg*+ on protein tyrosine phosphorylation. Human PBM were treated with the indicated concentration of HgCl, for 1 hr followed by analysis of protein tyrosine phosphorylation via Western blotting as described under Materials and Methods.
150
LANDER,
LEVINE,
AND
NOVOGRODSKY
metal HgZf on PTyr-P. We found that treatment of PBMC with 200 PM PA0 for 1 hr (Fig. l), 200 pA4 and 2 mM Hg2+ for 1 hr (Fig. 2) or heat shock at 50 and 56°C for 30 min (Fig. 3) induced PTyr-P as detected by using Western blotting with an anti-phosphotyrosine antibody. In addition, heat shock induced PTyr-P in an erythroleukemia cell line K562 (Fig. 3). Stress Stimuli
Modulate
PTPase Activity
Previous studies indicated that the oxidants H202 and phenylarsine oxide (PAO) induced PTyr-P and was associated with inhibition of PTPase activity. Our data with hemin found an association between hemin-induced PTyr-P and an increase in PTPase activity. Following these results, we investigated whether the stress stimuli PAO, Hg*+, and heat shock could modulate PTPase activity. We found that the stress stimuli at low intensity stimulated PTPase activity to different degrees. Heat shock at 40°C for 30 min slightly increased PTPase activity (Fig. 4A). Hg2+ maximally activated PTPase at 0.2 PM after 1 hr treatment and PA0 had a marginal stimulatory effect at 0.2 PM (Fig. 4B). Stress stimuli at high intensity invariably resulted in marked inhibition of PTPase activity. The PTPase assay involved measuring the release of 32P from a tyrosine phosphorylated peptide substrate (2 1). Stimulation of PTPase activity could also be demonstrated in a murine lymphoma cell line, BW5 147, treated with PA0 or Hg*’ (Figs. 4C and 4D). Mutants of the lymphoma cell line lacking CD45 failed to be stimulated by these agents. PTPase activity of the parental cells ranged from 0.0 16 1 + 0.00 15 to 0.0 155 f 0.0011 pmol PO;/mg protein/min and the mutants 0.0114 -t 0.0016 to 0.0096 -+ 0.0009 pmol PO:/mg protein/min. * Temp. (“C): 22 45 50 56 22 45 50 56
97694630-
PBM FIG. 3. Effect of heat shock on protein tyrosine phosphorylation. cells were heat shocked for 30 min at the indicated temperatures phosphorylation via Western blotting as described under Materials
K562 Human PBM or erythroleukemia followed by analysis of protein and Methods.
K562 tyrosine
STRESS STIMULI
ACTIVATE
151
~56’~~
u
0 0
i
20
30
40
50
Temperature
('C)
60
PA0 HgCI,
I
0
0.01
0.1
1
Stress 200
10
1001000
Stimulus
D
(JJM)
0
d
l
w45+ CD45-
150
/::
100
6 'y-
\oLb
50 I 1 I I
0 0.001
I
I
I
I
0.01
0.1
1
10
PA0
I
0 0
I 0.01
(PM)
I 0.1
a I 1
WI,
I 10
1 100
(IJM)
FIG. 4. Effect of stress stimuli on PTPase activity in human lymphocytes. Human PBM were incubated for 30 min with heat (A) or for 1 hr with phenylarsine oxide (PAO) or H&l2 (B), followed by measurement of protein tyrosine phosphatase activity as described under Materials and Methods. Parental murine lymphoma CD45+ and mutant CD45- cells were treated with phenylarsine oxide (C) or Hg’+ (D) at the indicated concentrations for 1 hr and then were assayed for protein tyrosine phosphatase activity as described under Materials and Methods.
Stress Stimuli
Induce Glucose Uptake in Human PBMC
Tyrosine phosphorylation induced by insulin and oxidants is associated with an enhancement of glucose transport. We therefore investigated whether the stress stimuli, which we found to alter tyrosine phosphorylation in human lymphocytes, could also enhance glucose uptake. We found that each of the stress stimuli had a concentration range which significantly stimulated glucose uptake (Figs. 5 and 6) beyond which glucose transport was inhibited. Stress Stimuli Activate ~56”~ Kinase Activity Protein tyrosine kinases of the STCfamily were implicated to play a role in signal transduction in lymphocytes. These kinases are negatively regulated by tyrosine phos-
152
LANDER,
LEVINE, AND NOVOGRODSKY
300 0
0
PA0
I 0
10-2
I 10-l Stress
I 100
I 10’
Stimulus
I
I
102
103
(j~b.4)
FIG. 5. Effect of stress stimuli on glucose uptake. Human PBM were treated for 1 hr with PA0 or HgCl* at the indicated concentrations and then analyzed for the rate of glucose uptake as described under Materials and Methods. Values for control cells were 7.2 k 0.8 pmol [3H]-2-deoxy-D-glucose/106 cells/min and set equal to 100%.
phorylation at the carboxy terminus. In view of our findings that stress stimuli enhanced PTPase activity, we investigated whether p561ck, a SK kinase family member, was activated upon treatment of the cell with stress stimuli. Utilizing the immune complex
10
20
40 Temperature
50
60
70
80
(‘C)
FIG. 6. Effect of heat shock on glucose uptake. Human PBM were treated for 30 min at the indicated temperature and then were analyzed for the rate of glucose uptake as described under Materials and Methods.
STRESS STIMULI
ACTIVATE
~56’~”
153
kinase assay, we found that ~56’~~ which was coimmunoprecipitated with anti-CD4 from cells treated with PA0 (0.2 and 200 pA4), HgCl* (0.2 and 200 pw or heat (40°C 30 min) had increased kinase activity as evidenced by an increase in autophosphorylation (Fig. 7). This assay is based on the ability of activated and immunoprecipitated ~56’~~ to autophosphorylate in the presence of [T-~~P]ATP. DISCUSSION PTyr-P is intimately involved in regulating cellular functions such as growth, differentiation, and signal transduction (24, 25). We have examined the effect of various stress stimuli on PTyr-P in human lymphocytes. PTyr-P is an early event in T cell activation (26, 27). Cellular targets for PTyr-P include the T-cell antigen receptor { chain (28), phospholipase Cyl (29) and other proteins (30, 31). Oxidants such as H202 and PA0 were previously shown to induce PTyr-P (17, 32) and this effect was associated with inhibition of PTPases. We have previously found that another oxidant, hemin, stimulated PTyr-P in human lymphocytes, but surprisingly this effect was associated with stimulation of PTPase activity. We have suggested that activation of a src family PTK, which is negatively regulated by tyrosine phosphorylation of the carboxy terminus, mediated the enhanced PTyr-P induced by hemin. Oxidants, heavy metals, and heat shock, collectively known as stress stimuli, have been shown to induce the synthesis of a variety of proteins termed stress proteins. In this study, we have demonstrated that the stress stimuli share with hemin its ability to enhance PTyr-P, modulate PTPase activity, and activate ~56’~~. Each stress stimulus tested stimulated PTPase activity at an optimal dose, beyond which activity was inhibited. Treatment of PBM with stress stimuli at a dose which activated PTPase did not produce discernable PTyr-P using Western blotting techniques. PTyr-P was only seen at doses of stress stimuli which were associated with an inhibition of PTPase activity. We could demonstrate a correlation between the dose of stress stimuli effective in increasing PTPase activity and ~56’~~ activation using heat shock and Hg2+ as stress stimuli. On the other hand, much lower concentrations of PA0 were effective in activating PTPase than those effective in eliciting ~56’~~ activation. We could not demonstrate a correlation between an effective dose inducing PTyr-P and glucose uptake (Table 1). Our data do not permit us to draw a simple correlation between enhancement of PTPase activity, activation of p56 Ick, induction of PTyr-P, and induction of the biological response. It is possible that both stimulation and inhibition of PTPase could regulate PTyr-P by either activating the src family PTKs or by preventing dephosphorylation of target proteins which are involved in the biological response (Fig. 8).
PA0
HgCI,
H.S.
(@+A):0 ’ 0.2 200”0.2 200’ 4o” 56 kd a CD4 FIG. 7. Effect of stress stimuli on ~56~‘~ kinase activity. Human PBM were treated with the indicated concentration of phenylarsine oxide (PAO) or HgC12 for I hr or heat for 30 min prior to analysis of immune complex kinase activity as described under Materials and Methods.
154
LANDER,
LEVINE, AND NOVOGRODSKY TABLE I
Doses of Stress Stimuli Inducing Cellular Events Glucose
PTPase (t)
PTPase (4)
uptake
stimulus
PTyr-P (t)
Heat shock PA0 Hg*+
50 “C 200/.1M 200 pM
40°C 0.2 pM 0.2 pM
45°C 200/.tM 200 FM
45°C 0.2 PM 200 FM
Stress
~56”’
(t)
(t,
40°C 200pM 0.2, 200 pM
Phosphorylation of these target proteins might have been induced at low doses of stress stimuli but not detected by the method employed. In this study we have only examined one of the SYCPTKs, p56 Ick. It should be noted that the biological response, such as glucose uptake, could be mediated by other src proteins such as fyn or yes. It is important to note that several of the assays were performed at cell densities >lO’ cells/ml. Cell densities such as this can stress cells and it is possible that we examined cells that were already stressed. This is unlikely for two reasons: (1) although the cells were initially at high density, they still responded to the stress stimuli and (2) cells at high density but otherwise untreated had biochemical parameters similar to those of resting cells at low density. Tyrosine phosphorylation was implicated to mediate insulin-induced glucose uptake in many cell types (33). Resting human lymphocytes do not bear the insulin receptor (34). Our data indicate that modulation of PTPase and/or PTyr-P may be involved in enhancing glucose uptake in cells lacking the insulin receptor. Heavy metals have been previously shown to elicit a mitogenic signal to lymphocytes (35, 36). Our results indicating that the heavy metal Hg2+ induced PTyr-P may provide insight into this phenomenon. The generalization of the mechanism proposed above to other cell types and its role in other oxidation-mediated cellular events such as tumor promotion remain to be elucidated.
71 t p
t
PTPase
!
PTPase
4
(CD45?)
OR
+ Protein
~
Y
)
Protein-Tyr-P
f Biological
Response
FIG. 8. Summarizing scheme shows possible mechanism for stress stimuli-induced cellular responses.
STRESS
STIMULI
ACTIVATE
~56’~~
155
ACKNOWLEDGMENT This work
was supported
in part by a grant from
the HRC
Foundation.
REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Stenzel, K. H., Rubin, A. L., and Novogrodsky, A., J. Immunol. 127, 2469, 1981. Novogrodsky, A., Suthanthiran, M. K., and Stenzel. K. H., J. Immunol. 143, 3981, 1989. Amrein, K. E., and Sefton, B. M., Proc. Natl. Acad. Sci. USA 85, 4247, 1988. Marth, J. D., Cooper, J. A., King, C. S., Ziegler, S. F., Tinker, D. A., Overell, R. W., Krebs, E. G., and Perlmutter, R. M., Mol. Cell Biol. 8, 540, 1988. Cooke, M. P., Abraham, K. M., Forbush, K. A., and Perlmutter. R. M., &l/65, 281, 1991. Rudd, C. E., Immunol. Today 11, 400, 1990. Veillette. A., Bookman, M. A.. Horak, E. M., Samelson, L. E., and Bolen, J. B.. Nature338,257, 1989. Kiausner, R. D., and Samelson, L. E., Cell 64, 875, 199 I. Glaichenhaus, N., Shastri, N., Littman, D. R., and Turner, J. M., Cell 64, 5 I 1, 199 I. Horak, I. D., Cress. R. E., Lucas, P. J.. Horak, E. M., Waldman, T. A., and Bolen, J. B., Proc. N&l.
Acad. Sci. USA 88, 199, 199 I. I I. Freeman, M. L., Scidmore, N. C., Malcolm, A. W., and Meredith, M. J., Biochem. Pharmacol. 36, 21, 1987. 12. Lindquist, S., Annul. Rev. Biochem. 55, I 15 I, 1986. 13. Bukh, A., Martinez-Valdez, H., Freedman, S. J., Freedman, M. H., and Cohen, A., J. Immunol. 144, 4835, 1990. 14. Muller-Taubenberger, A., Hagmann, J., Noegel, A., and Gerish, G., J. Cell. Sci. 90, 5 I. 1988. 15. Chin, K-V., Tanaka, S., Darlington, G., Pastan, I., and Gottesman, M. M., J. Biol. Chem. 265, 22 I, 1990. 16. Maher, P. A., and Pasquale, E. B., J. Cell Biol. 108, 2029, 1989. 17. Heffetz, D., Bushkin, I., Dror, R., and Zick, Y.. J. Biol. Chem. 265, 2896, 1990. 18. Hyman, R., and Trowbridge, I., Immunogentics 12, 5 11, 1981. 19. Ostergaard, H. L.. Shackelford, D. A., Hurley, T. R., Johnson, P., Hyman. R., Sefton, B. M., and Trowbridge. 1. S.. Proc. Natl. Acad. Sci. USA 86, 8959. 1989. 20. Markwell, M. A. K., Haas, S. M., Bieber. L. L., and Tolbert, N. E., Anul~~f. Biochem. 87, 206, 1978. 21. Streuli, M., Kreuger, N. X., Thai, T., Tang, M., and Saito. H., EMBO J. 9, 2399, 1990. 22. Laemmli, U. K., Nuture 227, 680, 1970. 23. Horak, I. D., Popovic, M., Horak. E. M., Lucas. P. J., Gress, R. E., June, C. H., and Bolen, J. B., Nature 348,557, 1990. 24. Ulhich, A., and Schlessinger, J.. Gel/ 61, 203, 1990. 25. Fischer, E. H., Charbonneau, H., and Tonks, N. K., Science 253, 401. 1991. 26. Wedner, H. J., and Bass, G., J. Immunol. 136, 4226, 1986. 27. Samelson, L. E., O’Shea, J. J.. Luong, H., Ross, P., Urdahl. K. B., Klausner, R. D., and Bluestone, J.. J. Immunol. 139, 2708, 1987. 28. June, C. H., Fletcher, M. C., Ledbetter, J. A., and Samelson, L. E., J. Immunol. 144, 1591, 1990. 29. Nishibe. S., Wahl, M. I., Hernandez-Sotomayor, S. M. J., Tonks, N. K.. Rhee, S. G., and Carpenter, G., Science 250, 1253, 1990. 30. Cooper, J. A., Bowen-Pope, D. F., Raines, E., Ross, R.. and Hunter, T., Ce/l31, 263, 1982. 31. Stanley, J. P., Gorczynski, R., Huang. C. K., Love, J.. and Mills, G. B., J. Immztnol. 145, 2189. 1990. 32. Garcia-Morales, P., Minami, Y., Luong, E.. Klausner, R. D., and Samelson, L. E.. Proc. N&l. ilcad. Sci. USA 87, 9255, 1990. 33. Rosen, 0. M., Science 237, 1452, 1987. 34. Gozes, Y., Caruso, J., and Strom, T. B., Diabetes 30, 314, 198 1. 35. Warner, G. L., and Lawrence, D. A., Cell. Immunol. 101, 425, 1986. 36. Reardon, C. L., and Lucas, D. O., Immunobiology 175, 455, 1987.
IMMUNOLOGY
145, 146-155 (1992)
Stress Stimuli-Induced
Lymphocyte
Activation
HARRY M. LANDER,*‘~” DANIEL M. LEVINE,*,? AND ABRAHAM NOVOGRODSKY*~~$.~ *The Rogosin Institute and the Departments qf tBiochemistry and $Medicine, Cornell University Medical College, 1300 York Avenue, Box 135, New York, New York 10021 Received March 18. 1992; accepted August 13, I992 Oxidants, heavy metals, and heat shock, collectively known as stressstimuli, induce the synthesis of a variety of proteins, termed stress proteins, and enhance glucose uptake. In this study, we have demonstrated that stressstimuli enhance protein tyrosine phosphorylation (PTyr-P), modulate protein tyrosine phosphatase (PTPase) activity, activate the src family protein tyrosine kinase (PTK), p56”‘, and enhance glucose uptake in human peripheral blood mononuclear cells. The heavy metal Hg2+ and heat shock stimulated PTPase activity at an optimal dose, whereas the oxidant phenylarsine oxide (PAO) was only marginally stimulatory. Treatment of lymphocytes with stress stimuli at a dose which activated PTPase did not produce discernable PTyr-P using Western blotting techniques. PTyr-P was only seen at doses of stressstimuli which were associated with an inhibition of PTPase activity. We could demonstrate a correlation between the dose of stress stimuli effective in increasing PTPase activity and ~56’~~ activation using heat shock and Hg2+ as stress stimuli. On the other hand, much lower concentrations of PA0 were effective in activating PTPase than those effective in eliciting ~56’~~ activation. We could not demonstrate a correlation between an effectivedose inducing PTyr-P and glucose uptake. Our data do not permit us to draw a simple correlation between enhancement of PTPase activity, activation of p561ck, induction of PTyr-P, and induction of the biological response. It is possible that both stimulation and inhibition of PTPase could regulate PTyr-P by either activating the src family PTKs or preventing dephosphorylation of target proteins which are involved in the biological response. Our data may also provide the biochemical basis for the previously reported mitogenic effectsof Hg*+ on lymphocytes. 0 1992 Academic Press, Inc.
INTRODUCTION We have previously shown that hemin, an oxidant, is mitogenic for human lymphocytes (1, 2). In studying the mechanism of its stimulatory effect on lymphocytes, we have found (submitted for publication) that hemin induced an insulinomimetic effect-namely an enhancement of glucose transport and an increase in protein tyrosine phosphorylation (PTyr-P). These effects were associated with stimulation of protein tyrosine phosphatase (PTPase) activity and activation of the SK family protein tyrosine kinase (PTK), ~56’“~. We postulated that the latter phenomena were related because of the finding that SK kinases are negatively regulated by tyrosine phosphorylation of ’ To whom correspondence should be addressed. * Current address: The Rogoff Institute, Beilinson Medical Center, Petah-Tikva, 49100, Israel. 146 0008-8749192 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reprcductmn in any form reserved
STRESS
STIMULI
ACTIVATE
~56”’
147
the carboxy terminus (3, 4). Protein tyrosine phosphorylation was implicated to play a central role in mediating signal transduction in lymphocytes (5- 10). Oxidants, heavy metals, and heat shock have all been shown to induce the synthesis of heat shock proteins (11, 12). We also found that hemin induced hsp 70 mRNA expression in human lymphocytes. These stresses also have other inductive effects in various cell types such as enhancement of glucose uptake and gene expression including c-jun, c-fos, ubiquitin, and multidrug resistance genes (13- 15). In addition, heat shock and Hz02 was shown to induce PTyr-P in cultured cells (16, 17). In this study, we investigated the response of human peripheral blood mononuclear cells (PBM) to various stress stimuli and, in addition, investigated whether these agents mimic hemin in activation of the PTyr-P signal transduction pathway. MATERIALS Cell Isolation
AND METHODS
and Culture
Human PBM were isolated from healthy volunteers by Ficoll-Hypaque density gradient centrifugation. The murine lymphoma BW5147 (CD45+) and mutant BW5 147(T200-a)5.1 (CD45-) cell lines were a kind gift of Dr. Robert Hyman (The Salk Institute, San Diego, CA). The CD45- cells expressed less than 1% of CD45 antigen in the membrane or cytoplasm (18, 19). These cells were maintained in DMEM with high glucose (JRH Bioscience, Lenexa, KS) containing 10% heat-inactivated horse serum. The human erythroleukemia K562 was maintained in RPM1 1640 containing 10% heat-inactivated fetal calf serum. Cell viability was greater than 95% after stress stimuli treatments as assessed by trypan blue exclusion. All reagents were purchased from Sigma Chemicals (St. Louis, MO). Preparation
of Particulate
Fraction
PBM or murine lymphoma cells were washed in PBS and resuspended to approximately 40 X lo6 cells/ml in 25 mM Hepes, 5 mA4 Na lactate, 1 ,mg/ml BSA, and aliquoted for treatment. After appropriate incubation time, cells were washed twice with wash buffer (25 mA4 Hepes, pH 7.4,5 mM KCl, 150 mM NaCl) and resuspended with 100 ~1 of sonication buffer (25 nGt4 Tris, pH 7.5, 1 mM MgC12, 1 mM PMSF, 100 pg/ml aprotinin, 10 pg/ml leupeptin). They were then sonicated in a bath sonicator (Laboratory Supplies, Hicksville, NY) for 20 set and put on ice for 40 sec. This was repeated 10 times or until inspection of aliquots under a microscope showed complete disruption. The tubes were spun at 1200 t-pm for 5 min to pellet debris and the supernatants spun for 1 hr at 100,OOOg at 4°C. The supernatants were discarded and the pellets resuspended in 100 ~1 of sonication buffer by vortexing. This particulate fraction was put on ice and assayed for protein by the method of Lowry as modified by Markwell et al. (20). Protein Tyrosine Phosphatase Assay
Particulate fractions were assayed exactly as described by Streuli et al. (2 1) using the 32P-Raytide substrate. Raytide (Oncogene Science, Manhasset, NY), a gastrin analog, was tyrosine phosphorylated in the presence of [T-~~P]ATP with the v-AH protein (Oncogene Science). 32P-Tyr-Raytide was recovered by TCA precipitation with extensive washing to remove unincorporated label.
148
LANDER, LEVINE, AND NOVOGRODSKY
Five microliters of sample (0.1-0.5 mg/ml in sonication buffer) was mixed with 5 ~1 32P-Tyr-Raytide (l-5 X lo4 cpm) in 50 ~1 buffer (25 mM Hepes, pH 7.3, 50 mM EDTA, 100 mM DTT). Sample concentration was varied such that no more than 10% of the substrate was destroyed by the enzyme during the 10 min incubation at 37°C. To terminate, 750 ~1 of acidic charcoal was added, tubes were centrifuged in a microfuge for 5 min, and 400 ~1 supernatant was counted. The assay was linear at 10 min. Data are expressed as pmol PO; released per milligram protein per minute and data points represent the average values from three experiments in which samples were assayed in duplicate. Determination of Protein Tyrosine Phosphorylation
Cells were prepared for analysis via Western blotting in the following manner: 20 X lo6 cells/ml in 1 ml of RPM1 1640 containing 5% FCS were treated under the appropriate conditions. After washing twice with PBS, cells were pelleted in a microfuge for 1 min. The medium was decanted, the pellets were frozen in liquid nitrogen, and 500 ~1 extraction buffer (1 mM Tris, pH 7.4, 1% SDS, 1 mM NaV03, 0.1 mM NazMo04, 1 mM PMSF) was added. The pellets were immediately boiled for 5 min and transferred to glass test tubes and sonicated in a bath sonicator until DNA was sheared (3-4 min). Protein levels were determined and 5X Laemmli buffer (22) (containing 20 mM DTT) was added, boiled for 3 min, and 200 pg protein/lane was loaded onto 3-27% SDS polyacrylamide gels with a 3% stack. Electrophoresis was carried out at 100 V/gel until the dye front nearly ran off. The gel was soaked in transfer buffer (0.05 M glycine, 0.05 M Tris, pH 8.8) for 15 min and electrotransferred onto 0.22pm nitrocellulose paper for 22 hr at 35 V at 4°C. After transfer, the nitrocellulose was soaked in PBS containing 2 mg/ml BSA for 1 hr at 22°C washed twice in wash buffer (PBS, BSA, 0.05% Tween 20) and soaked in wash buffer containing anti-phosphotyrosine antibody (1: 1000) (PY20 clone, ICN Biomedicals, Costa Mesa, CA). As a control, we blotted in the presence of 20 m/t4 0-phosphotyrosine and preimmune serum and found that no PTyr-P was evident. After washing five times with wash buffer, it was soaked in wash buffer containing 0.2 &i/ml ‘251-protein G (Amersham, Arlington Heights, IL) for 2 hr, again washed five times, and exposed to XOMAT (Kodak, Rochester, NY) film for 16 hr at -70°C with intensifying screens. 14C-protein standards (Amersham) were included on all gels to estimate molecular masses. Assay of p561ckActivity
Activity of ~56’“~ was assessed as follows. PBM (20 X lo6 cells) were treated as indicated. Cells were pelleted, washed three times with PBS, and lysed in 250 ~1 antiCD4 buffer ( 1% NP-40, 150 mJ4 NaCl, 1 miM NaMo3, 0.4 mM NaVo4, 5 mM MgC12, 1 mM PMSF, 10 pg/ml leupeptin) containing 200 U DNase. After 30 min at 4°C 5 pg anti-CD4 (OKT4, Ortho Diagnostics), which coprecipitates ~56’~~ (23), was added to each sample and after another hour at 4°C 25 ~1 of a 1: 1 suspension of Sepharoselinked protein G was added. After 1 hr, samples were washed three times with antiCD4 buffer and 30 ~1 containing 25 mMHepes, 0.1% NP-40, and 10 &i [Y-~~P]ATP was added. After 5 min at 25°C 30 ~1 SDS sample buffer was added and samples were boiled. Samples were electrophoresed and transferred as described above.
STRESS STIMULI PA0 (PM): 0.2
ACTIVATE
2
20
~56”’
149
200
kD 200 97 69 46 -
14 -
FIG.I. Effect of phenylarsine oxide on protein tyrosine phosphorylation. Human PBM were treated with the indicated concentration of phenylarsine oxide for 1 hr followed by analysis of protein tyrosine phosphorylation via Western blotting as described under Materials and Methods. 2-Deoxy-D-glucose Uptake
Fresh PBM were resuspended to 10 X lo6 cells/ml in PBS containing 1 mg/ml BSA and 5 mM Na lactate. Cells (0.5 ml per data point) were treated appropriately and after incubation at 37°C were put on a shaker and 10 ~1 of 5 mA4 2-DOG containing 5 &I [3H]-2-DOG (Amersham) was added. After 5 min, 200 ~1 was removed and layered on top of dibutyl phthalate:mineral oil (4: 1) and spun in a microfuge for 30 sec. The tip (with the cell pellet) was cut and placed in 1 ml of 1% Triton X- 100 and 0.1 N NaOH, vortexed, and counted with 15 ml Ultrafluor (National Diagnostics, Manville, NJ) in a liquid scintillation counter. The assay was linear at 5 min. RESULTS StressStimuli Induce PTyr-P
We investigated the effects of stress stimuli on tyrosine phosphorylation in human lymphocytes. We examined the effects of the oxidant PAO, heat shock, and the heavy
HgCI2(pM):
I 0
0.2
(PM) 2
20
200
1 2mM
200 97694630-
14-
FIG.2. Effect of Hg*+ on protein tyrosine phosphorylation. Human PBM were treated with the indicated concentration of HgCl, for 1 hr followed by analysis of protein tyrosine phosphorylation via Western blotting as described under Materials and Methods.
150
LANDER,
LEVINE,
AND
NOVOGRODSKY
metal HgZf on PTyr-P. We found that treatment of PBMC with 200 PM PA0 for 1 hr (Fig. l), 200 pA4 and 2 mM Hg2+ for 1 hr (Fig. 2) or heat shock at 50 and 56°C for 30 min (Fig. 3) induced PTyr-P as detected by using Western blotting with an anti-phosphotyrosine antibody. In addition, heat shock induced PTyr-P in an erythroleukemia cell line K562 (Fig. 3). Stress Stimuli
Modulate
PTPase Activity
Previous studies indicated that the oxidants H202 and phenylarsine oxide (PAO) induced PTyr-P and was associated with inhibition of PTPase activity. Our data with hemin found an association between hemin-induced PTyr-P and an increase in PTPase activity. Following these results, we investigated whether the stress stimuli PAO, Hg*+, and heat shock could modulate PTPase activity. We found that the stress stimuli at low intensity stimulated PTPase activity to different degrees. Heat shock at 40°C for 30 min slightly increased PTPase activity (Fig. 4A). Hg2+ maximally activated PTPase at 0.2 PM after 1 hr treatment and PA0 had a marginal stimulatory effect at 0.2 PM (Fig. 4B). Stress stimuli at high intensity invariably resulted in marked inhibition of PTPase activity. The PTPase assay involved measuring the release of 32P from a tyrosine phosphorylated peptide substrate (2 1). Stimulation of PTPase activity could also be demonstrated in a murine lymphoma cell line, BW5 147, treated with PA0 or Hg*’ (Figs. 4C and 4D). Mutants of the lymphoma cell line lacking CD45 failed to be stimulated by these agents. PTPase activity of the parental cells ranged from 0.0 16 1 + 0.00 15 to 0.0 155 f 0.0011 pmol PO;/mg protein/min and the mutants 0.0114 -t 0.0016 to 0.0096 -+ 0.0009 pmol PO:/mg protein/min. * Temp. (“C): 22 45 50 56 22 45 50 56
97694630-
PBM FIG. 3. Effect of heat shock on protein tyrosine phosphorylation. cells were heat shocked for 30 min at the indicated temperatures phosphorylation via Western blotting as described under Materials
K562 Human PBM or erythroleukemia followed by analysis of protein and Methods.
K562 tyrosine
STRESS STIMULI
ACTIVATE
151
~56’~~
u
0 0
i
20
30
40
50
Temperature
('C)
60
PA0 HgCI,
I
0
0.01
0.1
1
Stress 200
10
1001000
Stimulus
D
(JJM)
0
d
l
w45+ CD45-
150
/::
100
6 'y-
\oLb
50 I 1 I I
0 0.001
I
I
I
I
0.01
0.1
1
10
PA0
I
0 0
I 0.01
(PM)
I 0.1
a I 1
WI,
I 10
1 100
(IJM)
FIG. 4. Effect of stress stimuli on PTPase activity in human lymphocytes. Human PBM were incubated for 30 min with heat (A) or for 1 hr with phenylarsine oxide (PAO) or H&l2 (B), followed by measurement of protein tyrosine phosphatase activity as described under Materials and Methods. Parental murine lymphoma CD45+ and mutant CD45- cells were treated with phenylarsine oxide (C) or Hg’+ (D) at the indicated concentrations for 1 hr and then were assayed for protein tyrosine phosphatase activity as described under Materials and Methods.
Stress Stimuli
Induce Glucose Uptake in Human PBMC
Tyrosine phosphorylation induced by insulin and oxidants is associated with an enhancement of glucose transport. We therefore investigated whether the stress stimuli, which we found to alter tyrosine phosphorylation in human lymphocytes, could also enhance glucose uptake. We found that each of the stress stimuli had a concentration range which significantly stimulated glucose uptake (Figs. 5 and 6) beyond which glucose transport was inhibited. Stress Stimuli Activate ~56”~ Kinase Activity Protein tyrosine kinases of the STCfamily were implicated to play a role in signal transduction in lymphocytes. These kinases are negatively regulated by tyrosine phos-
152
LANDER,
LEVINE, AND NOVOGRODSKY
300 0
0
PA0
I 0
10-2
I 10-l Stress
I 100
I 10’
Stimulus
I
I
102
103
(j~b.4)
FIG. 5. Effect of stress stimuli on glucose uptake. Human PBM were treated for 1 hr with PA0 or HgCl* at the indicated concentrations and then analyzed for the rate of glucose uptake as described under Materials and Methods. Values for control cells were 7.2 k 0.8 pmol [3H]-2-deoxy-D-glucose/106 cells/min and set equal to 100%.
phorylation at the carboxy terminus. In view of our findings that stress stimuli enhanced PTPase activity, we investigated whether p561ck, a SK kinase family member, was activated upon treatment of the cell with stress stimuli. Utilizing the immune complex
10
20
40 Temperature
50
60
70
80
(‘C)
FIG. 6. Effect of heat shock on glucose uptake. Human PBM were treated for 30 min at the indicated temperature and then were analyzed for the rate of glucose uptake as described under Materials and Methods.
STRESS STIMULI
ACTIVATE
~56’~”
153
kinase assay, we found that ~56’~~ which was coimmunoprecipitated with anti-CD4 from cells treated with PA0 (0.2 and 200 pA4), HgCl* (0.2 and 200 pw or heat (40°C 30 min) had increased kinase activity as evidenced by an increase in autophosphorylation (Fig. 7). This assay is based on the ability of activated and immunoprecipitated ~56’~~ to autophosphorylate in the presence of [T-~~P]ATP. DISCUSSION PTyr-P is intimately involved in regulating cellular functions such as growth, differentiation, and signal transduction (24, 25). We have examined the effect of various stress stimuli on PTyr-P in human lymphocytes. PTyr-P is an early event in T cell activation (26, 27). Cellular targets for PTyr-P include the T-cell antigen receptor { chain (28), phospholipase Cyl (29) and other proteins (30, 31). Oxidants such as H202 and PA0 were previously shown to induce PTyr-P (17, 32) and this effect was associated with inhibition of PTPases. We have previously found that another oxidant, hemin, stimulated PTyr-P in human lymphocytes, but surprisingly this effect was associated with stimulation of PTPase activity. We have suggested that activation of a src family PTK, which is negatively regulated by tyrosine phosphorylation of the carboxy terminus, mediated the enhanced PTyr-P induced by hemin. Oxidants, heavy metals, and heat shock, collectively known as stress stimuli, have been shown to induce the synthesis of a variety of proteins termed stress proteins. In this study, we have demonstrated that the stress stimuli share with hemin its ability to enhance PTyr-P, modulate PTPase activity, and activate ~56’~~. Each stress stimulus tested stimulated PTPase activity at an optimal dose, beyond which activity was inhibited. Treatment of PBM with stress stimuli at a dose which activated PTPase did not produce discernable PTyr-P using Western blotting techniques. PTyr-P was only seen at doses of stress stimuli which were associated with an inhibition of PTPase activity. We could demonstrate a correlation between the dose of stress stimuli effective in increasing PTPase activity and ~56’~~ activation using heat shock and Hg2+ as stress stimuli. On the other hand, much lower concentrations of PA0 were effective in activating PTPase than those effective in eliciting ~56’~~ activation. We could not demonstrate a correlation between an effective dose inducing PTyr-P and glucose uptake (Table 1). Our data do not permit us to draw a simple correlation between enhancement of PTPase activity, activation of p56 Ick, induction of PTyr-P, and induction of the biological response. It is possible that both stimulation and inhibition of PTPase could regulate PTyr-P by either activating the src family PTKs or by preventing dephosphorylation of target proteins which are involved in the biological response (Fig. 8).
PA0
HgCI,
H.S.
(@+A):0 ’ 0.2 200”0.2 200’ 4o” 56 kd a CD4 FIG. 7. Effect of stress stimuli on ~56~‘~ kinase activity. Human PBM were treated with the indicated concentration of phenylarsine oxide (PAO) or HgC12 for I hr or heat for 30 min prior to analysis of immune complex kinase activity as described under Materials and Methods.
154
LANDER,
LEVINE, AND NOVOGRODSKY TABLE I
Doses of Stress Stimuli Inducing Cellular Events Glucose
PTPase (t)
PTPase (4)
uptake
stimulus
PTyr-P (t)
Heat shock PA0 Hg*+
50 “C 200/.1M 200 pM
40°C 0.2 pM 0.2 pM
45°C 200/.tM 200 FM
45°C 0.2 PM 200 FM
Stress
~56”’
(t)
(t,
40°C 200pM 0.2, 200 pM
Phosphorylation of these target proteins might have been induced at low doses of stress stimuli but not detected by the method employed. In this study we have only examined one of the SYCPTKs, p56 Ick. It should be noted that the biological response, such as glucose uptake, could be mediated by other src proteins such as fyn or yes. It is important to note that several of the assays were performed at cell densities >lO’ cells/ml. Cell densities such as this can stress cells and it is possible that we examined cells that were already stressed. This is unlikely for two reasons: (1) although the cells were initially at high density, they still responded to the stress stimuli and (2) cells at high density but otherwise untreated had biochemical parameters similar to those of resting cells at low density. Tyrosine phosphorylation was implicated to mediate insulin-induced glucose uptake in many cell types (33). Resting human lymphocytes do not bear the insulin receptor (34). Our data indicate that modulation of PTPase and/or PTyr-P may be involved in enhancing glucose uptake in cells lacking the insulin receptor. Heavy metals have been previously shown to elicit a mitogenic signal to lymphocytes (35, 36). Our results indicating that the heavy metal Hg2+ induced PTyr-P may provide insight into this phenomenon. The generalization of the mechanism proposed above to other cell types and its role in other oxidation-mediated cellular events such as tumor promotion remain to be elucidated.
71 t p
t
PTPase
!
PTPase
4
(CD45?)
OR
+ Protein
~
Y
)
Protein-Tyr-P
f Biological
Response
FIG. 8. Summarizing scheme shows possible mechanism for stress stimuli-induced cellular responses.
STRESS
STIMULI
ACTIVATE
~56’~~
155
ACKNOWLEDGMENT This work
was supported
in part by a grant from
the HRC
Foundation.
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