- Email: [email protected]
Diminished activation of the MAP kinase pathway in CD3-stimulated T lymphocytes from old mice
Mechanisms of Ageing and Development 94 (1997) 71 – 83
Diminished activation of the MAP kinase pathway in CD3-stimulated T lymphocytes from old mice Gayle Gorgas a, Elizabeth R. Butch b, Kun-Liang Guan b,c, Richard A. Miller a,c,d,* a
Department of Pathology, Uni6ersity of Michigan School of Medicine, Ann Arbor, MI 48109, USA b Department of Biological Chemistry, Uni6ersity of Michigan School of Medicine, Ann Arbor, MI 48109, USA c Institute of Gerontology, Ann Arbor, MI 48109, USA d Ann Arbor DVA Medical Center, Ann Arbor, MI 48109, USA
Received 2 November 1996; received in revised form 14 December 1996; accepted 14 December 1996
Abstract Stimulation of the ERK family of protein kinases (‘extracellular signal regulated kinases’, also known as MAP kinases) plays an important role in the activation of many cell types, including T lymphocytes. ERKs are activated when they are phosphorylated by an upstream activator, the dual-specific protein kinase MEK. To see if aging leads to an impairment of MEK activation in mouse T cells, we used a mobility shift assay in which activation of MEK leads to phosphorylation and altered mobility of ERK-2 kinase. Similarly, we monitored mobility of pp90rsk, a known ERK substrate, as an indication of ERK function. We found an age-related decline in the ability of mouse T cells to activate both MEK and ERK function in response to stimulation by antibodies to the CD3 chain of the T cell receptor. Aging did not alter the kinetics of enzyme activation, but did diminish (by about 2-fold) the maximal level of substrate converted into the slower migrating form. Naive and memory CD4 T cells from young mice were equally able to convert ERK2 to its slower migrating form, suggesting that the decline in MEK function is not likely to be attributable to the shift, with age, from naive to memory T cell predominance. Our data suggest that age-dependent declines in gene activation, including genes for key cytokines like IL-2, may be due to * Corresponding author. Present address: University of Michigan, MSRB-3 Room 6301, Box 0642, 1150 W. Medical Center Drive, Ann Arbor, MI 48109-0642, USA; Tel.: +1 313 9362122; fax: + 1 313 9369220; e-mail: [email protected] 0047-6374/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 0 4 7 - 6 3 7 4 ( 9 6 ) 0 1 8 5 7 - X
72
G. Gorgas et al. / Mechanisms of Ageing and De6elopment 94 (1997) 71–83
declines in the upstream signals that lead to activation of the MEK/ERK protein kinase cascade. © 1997 Elsevier Science Ireland Ltd. Keywords: ERK; Immune senescence; MAP kinase; MEK; T lymphocytes; Signal transduction
1. Introduction T lymphocytes from old mice, when compared to cells from young mice, show abnormalities in the activation process within the first few min of exposure to mitogenic lectins or antibodies. These defects include lower levels of free calcium ion concentration [1 – 3] and trans-membrane calcium influx [4], declines in tyrosinespecific protein kinase function [5–8], and lower levels of phosphorylation of a wide range of proteins regulated by calcium and protein kinase C levels [9]. These changes early in the activation cascade seem likely to contribute to the age-related decline in expression of the growth-promoting cytokine interleukin 2 (IL-2) [10,11] and its receptor [12,13]. Indeed, enrichment of those T cells in old mice that are able to generate calcium signals also strongly enriches for cells that can produce and respond to IL-2 [14]. The accumulation in old age of memory T cells, and the parallel decline in naive T cells [15,16] can account for many of these changes, in that memory cells tend to show smaller changes in calcium signal generation [2,17] than naive cells, and are also hyporesponsive in tests for mitogen induction of tyrosine-specific protein phosphorylation [7]. Some age-related changes in protein kinase activity, however, are demonstrable in both naive and memory T cell subsets [9]. Stimulation of the T-cell receptor (TCR) initiates a signal transduction cascade that results in the release of numerous cytokines [18], including IL-2. The TCR complex has no intrinsic protein tyrosine kinase (PTK) activity, but induction of PTK activity as a result of TCR stimulation initiates two principal signal transduction pathways, involving activation of at least three PTKs, Fyn, Lck and ZAP-70 [18 – 21]. Both of these pathways begin with PTK dependent induction of phospholipase C (PLC) activity[18]. The hydrolysis of phosphatidylinositol 4,5-bisphosphate by PLC results in the production of inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). This PLC hydrolysis forms a branch point for the two divergent signaling pathways, the first of which depends on increased intracellular calcium (Ca + + ) resulting from IP3 production and leading to an increase in Ca + + / calmodulin dependent serine phosphatase (calcineurin). The calcineurin in turn activates the transcription factor NF-AT thus leading to increased IL-2 gene expression [18]. The DAG produced by PLC is an activator of protein kinase C (PKC) which initiates the second signal transduction pathway. Increased PKC activity results in activation of Ras which in turn activates Raf-1, a protein kinase that can in turn directly activate MEK (‘MAPK or ERK kinase’) [18,22]. MEK
G. Gorgas et al. / Mechanisms of Ageing and De6elopment 94 (1997) 71–83
73
activation results in phosphorylation and increased function of a family of extracellular signal-regulated kinases (ERKs), also known as mitogen activated protein kinases (MAPKs). Activation of the Ras/MEK pathway, in synergy with the Ca + + signal, leads ultimately to increased cytokine production [22]. Members of the ERK family are important components of various signaling pathways in many cell types. They can phosphorylate numerous substrates and may be a key component in linking growth factor receptor activation to serine/ threonine protein phosphorylation [23–25]. ERK activation can be induced by a variety of mitogenic stimuli, including phorbol esters, cytokines, and T-cell antigens, as well as by tyrosine-kinase coupled receptors to growth factors including insulin, epidermal growth factor, fibroblast growth factor and platelet-derived growth factor [25 – 28]. Stimulation of ERK in various non-lymphoid cell types leads to a multitude of cellular responses including phosphorylation of microtubule-associated proteins involved in microtubule rearrangement [29,30] and phosphorylation resulting in subsequent activation of transcription factors TCF and STAT [31 – 33]. In lymphoid cells, activation of ERK results in stimulation of T-cells to produce IL-2 and other cytokines [34–36]. ERK is itself activated by another protein kinase, MEK. MEK is a dual specific kinase which can phosphorylate ERK2 on both the threonine 183 and tyrosine 185 [37]. Two mammalian isoforms of MEK (MEK1 and MEK2), identified by molecular cloning techniques, can phosphorylate and activate ERK [38 – 44]. ERK is the only known substrate for MEK with all other proteins tested thus far being phosphorylated at least 1000-fold less efficiently by MEK [45]. Thus assays based upon the phosphorylation of ERK provide a useful gauge of MEK function. One of the known physiological targets of activated ERK is a serine/threonine specific protein kinase, pp90rsk, that can phosphorylate the 40S ribosomal protein S6[46 – 48]. Previous studies revealed that a Xenopus homolog of the pp90rsk family, which had been inactivated by a protein phosphatase, could be partially reactivated by insulin stimulated ERK2 [48]. Thus, phosphorylation of RSK can be used as a physiological indicator of ERK activity in cells. Since induction of the MEK–ERK system plays a critical role in T cell activation, we undertook a series of experiments to see if aging led to alterations in the ability of cross-linked anti-CD3 antibody to induce MEK and ERK activity in murine splenic CD4 + T lymphocytes. We monitored MEK function using a mobility shift assay, in which altered mobility of ERK2 in lysates of activated T cells provides an index of MEK function. Similarly, ERK activity was monitored by altered mobility of one of its substrates, the ribosomal S6 protein kinase pp90rsk. We report here that aging leads to a decline, of approximately 2-fold, in both MEK and ERK induction by anti-CD3 signals. We also show that both naive and memory T cells from young mice are equally susceptible to induction of MEK function, and thus conclude that the age-related decline in MEK function is unlikely to result simply from an accumulation of memory T cells in old age.
74
G. Gorgas et al. / Mechanisms of Ageing and De6elopment 94 (1997) 71–83
2. Materials and methods
2.1. Mice Specific pathogen-free CB6F1 male mice were purchased from the National Institute of Aging colonies maintained by the Charles River Laboratories (Kingston, NY). After shipment, they were housed under specific-pathogen free conditions for at least one week before use. The mice received rodent chow and water ad libitum. The terms ‘young’, ‘middle-aged’, and ‘old’ refer to mice aged 3 – 6 months, 12 – 14 months, and 18–22 months, respectively. Mice with splenomegaly or visible tumors were not used.
2.2. Cell preparations Spleens were removed aseptically and rubbed between frosted glass slides in Hanks balanced salt solution containing 0.2% BSA (BSS–BSA) to obtain a cell suspension. Debris and clumps of tissue were eliminated by passage of the suspension through nylon mesh. Erythrocytes were then removed by centrifugation over a Lympholyte-M cushion (Cedar lane Laboratories, Hornby, Ontario, Canada). T cell enrichment was accomplished by panning the cell suspension on petri dishes coated with anti-mouse IgG to remove B cells. This procedure regularly yields at least 90% pure T cells. CD4 + memory and CD4 + naive T cells were prepared by negative selection. To prepare CD4 + memory (i.e. CD45RBlo) cells, T cells were incubated with a mixture of anti-CD8 ascites (clone 53-6.7) at 1:200 and 5 mg/ml anti-CD45RB (clone 16A; Pharmingen, San Diego) for 40 min at 4°C. After removal of unbound antibody, cells were incubated for 40 min at 4°C with magnetic beads coated with anti-rat Ig at a bead:cell ratio of 50:1. A magnetic field was then applied at least 3 times to remove all cells bound to beads and free beads. CD4 naive cells (CD44lo) were prepared in the same way, substituting 5 mg/ml anti-CD44 (clone IM7; Pharmingen) for the anti-CD45RB antibody. The resulting preparations were respectively 85% CD4 + /CD44hi and 95% CD4 + /CD45RBhi as judged by FACS analysis. These cells are referred to as CD4 + ‘naive’ and CD4 + ‘memory’ T cells, respectively.
2.3. Cell stimulation Cells (3 ×106) were resuspended in 1 ml of RPMI-1640 supplemented with 10% FCS. Partially purified anti-CD3 ascites was added at a concentration of 6.6 mg/ml and incubated on ice for 30 min. The unbound antibody was removed and the anti-CD3 was crosslinked by the addition of goat anti-hamster Ig at a concentration of 5 mg/ml at 37°C for 0, 1, 2, 5, 10, or 30 min. For stimulation with PMA, cells were incubated with 10 ng/ml PMA at 37°C for the same time course. Stimulation was terminated by washing the cells with ice cold PBS, followed by lysis in 1% NP-40, 20 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EGTA, 1 mM EDTA, 50 mM NaF, 2 mM Na3VO4, 5 mM sodium pyrophosphate, 2 mg/ml aprotinin, 5 mg/ml leupeptin, 5 mg/ml pepstatin, and 1 mM benzamidine.
G. Gorgas et al. / Mechanisms of Ageing and De6elopment 94 (1997) 71–83
75
2.4. Western blotting Lysates were centrifuged at 14 000 rpm at 4°C for 10 min to remove cellular debris, and then boiled in 2×SDS reducing buffer. Samples were run on 12% (for ERK, using 3 × 106 cell equivalents/lane) or 8% (for RSK, using 1 × 106 cell equivalents/lane) SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked in PBS with 0.1% Tween-20 (PBS-T) containing 5% BSA or 5% nonfat dry milk overnight at 4°C. Anti-ERK antibody (Zymed, S. San Francisco, CA) was used at a dilution of 1:15 000 (70 ng/ml), and anti-RSK (Upstate Biotechnology, Lake Placid, NY) was used at 175 ng/ml in PBS-T containing 1% BSA; some early experiments used anti-ERK antibody produced in the Guan laboratory. Blots were incubated with primary antibody for 1 h at room temperature and then washed 4 times for 15 min each in PBS-T at room temperature. Blots were then incubated with a 1:2000 dilution of horseradish peroxidase conjugated anti-rabbit IgG in PBS-T containing 1% BSA for 1 h at room temperature followed by extensive washing with PBS-T. Blots were developed using enhanced chemiluminescence (ECL) according to the manufacturer’s instructions (Kirkegaard and Perry, Gaithersburg, MD).
2.5. Densitometry and data analysis For analysis of MEK activity, anti-ERK immunoblots were scanned by a laser densitometer (Molecular Dynamics, Sunnyvale, CA), and analyzed using an ImageQuant software package. For analysis of ERK activity, anti-RSK immunoblots were scanned using a Sharp image scanner (model JX-330), and analyzed using SigmaGel software. Experiments comparing naive to memory cells were also analyzed using the Sharp image scanning system. In each case the parameter of interest was the relative amount of enzyme (ERK or RSK) that exhibited a retarded electrophoretic mobility. To control for differences in exposure level and protein loading, OD values for the slower band were expressed as a percentage of the sum of the OD values for both the rapid and slow-migrating species. These normalized values were then subjected to a two-way analysis of variance (ANOVA) in which age (or T cell subset), and experiment were treated as independent variables, and the normalized OD values were treated as dependent variables.
3. Results
3.1. PMA-induced shift in ERK2 electrophoretic mobility To confirm previous reports that PMA-induced activation of MEK would convert ERK2 to a more slowly migrating form, we exposed T cells to PMA (10 ng/ml) for various intervals and then examined ERK2 mobility by an immunoblotting method. The results (Fig. 1) showed that 5 min of exposure to PMA led to a virtually quantitative conversion of the 42 kD form of ERK2 to a form with the
76
G. Gorgas et al. / Mechanisms of Ageing and De6elopment 94 (1997) 71–83
Fig. 1. Detection of ERK2 in PMA stimulated T cells by immunoblotting. T cells (3×106/ml) were incubated with 10 ng/ml PMA at 37°C. Postnuclear supernatants were separated on 12% SDS-PAGE and transferred to nitrocellulose. The membrane was then probed for ERK2 using ERK antisera and ECL detection reagents.
apparent molecular weight of 42.5 kD. This conversion was transient, with partial reversal apparent by 30 min. A series of experiments using T cells derived from mice of differing ages revealed no effect of age on PMA-induced alteration of ERK2 mobility (not shown). Similar experiments (not shown) using anti-ERK1 antibodies demonstrated a PMA-induced shift in ERK1 mobility, which was equally apparent for T cells from old and young mice.
3.2. Age-dependent loss in MEK-mediated ERK2 mobility shift in responses to cross-linked anti-CD3 antibody We next carried out a series of experiments to see if aging altered MEK activation when T cells were activated by cross-linking the CD3o chain of the TCR complex. Fig. 2 shows a representative experiment. In T cells from young mice, cross-linking CD3 leads to the appearance of a more slowly migrating form of ERK2, with peak levels achieved within 2–5 min, and a nearly complete return to
Fig. 2. Effect of age on ERK2 mobility shift in cross-linked anti-CD3 stimulated T cells; a representative experiment. T cells (3×106/ml) were incubated with anti-CD3 for 30 min on ice, and then stimulated by the addition of goat anti-hamster Ig cross-linking antibody at 37°C. Postnuclear supernatants were separated on 12% SDS-PAGE and transferred to nitrocellulose. The membrane was then probed for ERK2 using ERK antisera.
G. Gorgas et al. / Mechanisms of Ageing and De6elopment 94 (1997) 71–83
77
Fig. 3. Age-related decline in ERK2 mobility shift in response to cross-linked anti-CD3 stimulation. Values indicate the percentage of ERK2 in the slower migrating band for each individual lane, presented as means9 S.E.M. from 7 experiments.
baseline within 30 min. Quantitative densitometry suggested that only about 8% of the ERK2 (in young mice) was converted to the slower isoform at the peak of the response. We were unable to see an effect of anti-CD3 stimulation on ERK1 mobility (not shown). T cells from middle-aged and from old mice responded with a similar time course, but with lower levels of the slower moving form. Fig. 3 shows the results of a series of 7 such experiments, each involving three individual mice of differing ages. Analysis of variance showed that there were significant differences between young and old mice in ERK2-converting activity at each of the early time points (1, 2, and 5 min after activation). Differences between young and old mice were about two-fold. Middle-aged mice had intermediate levels of MEK function, not significantly different from either young or old mice. We concluded that CD3-mediated activation of MEK is impaired by aging. Since aging leads to a decrease in the proportion of cells with the surface markers of naive lymphocytes, and a corresponding increase in memory T cells [15,16,49], we wished to determine whether the decline with age in CD3-induced MEK function might reflect merely differences between naive and memory cells in this aspect of the CD3 activation cascade. Fig. 4 presents results of an experiment in which naive (CD44lo) and memory (CD45RBlo) T cells were prepared from the
Fig. 4. ERK2 mobility shift in cross-linked anti-CD3 stimulated naive and memory T cells; a representative experiment. T cells were separated as explained in Section 2. Separated naive and memory cells (2 ×106) were incubated with anti-CD3 for 30 min on ice, and then stimulated by the addition of goat anti-hamster cross-linking antibody at 37°C. Postnuclear supernatants were separated on 12% SDS-PAGE and transferred to nitrocellulose. The membrane was then probed for ERK2 using a polyclonal ERK antibody.
78
G. Gorgas et al. / Mechanisms of Ageing and De6elopment 94 (1997) 71–83
Fig. 5. Effect of age on pp90rsk mobility shift in cross-linked anti-CD3 stimulated T cells; a representative experiment. T cells (1.5 × 106/ml) were incubated with anti-CD3 for 30 min on ice, and then stimulated by the addition of goat anti-hamster cross-linking antibody at 37°C. Postnuclear supernatants were separated on 8% SDS-PAGE and transferred to nitrocellulose. The membrane was probed for pp90rsk using a polyclonal RSK antibody.
CD4 subset of young mice, stimulated with anti-CD3, and subjected to an antiERK immunoblotting procedure. Naive and memory T cells performed equally well in this and in three replicate experiments. We concluded that differences between memory and naive T cells cannot account for the age-dependent loss in MEK activation shown in Figs. 2 and 3.
3.3. Age-related decline in ERK-mediated pp90 rsk mobility shift To see if the anti-CD3 induced generation of the slow-migrating form of ERK2 was accompanied by an increase in ERK function, we employed an assay based upon the ERK-dependent conversion of another downstream protein kinase, pp90rsk, to a slower migrating isoform. Fig. 5 shows the result of one such experiment in which crosslinking surface CD3 led to a rapid appearance of a slower form of RSK, detectable within 60 s, peaking at 5–10 min, and declining to near baseline by 30 min after activation. There is a clear age-dependent decline in this index of ERK activity. Fig. 6 shows averages for a series of six such experiments. T cells from old mice respond less vigorously than cells from young or middle-aged mice. Analysis of variance shows that the differences between young and old mice are significant at the 1, 2, 5, and 10 min time points. The difference (peak minus baseline) for old mice is approximately 60% of the value for young mice.
Fig. 6. Age related decline in pp90rsk mobility shift in response to cross-linked anti-CD3 stimulation. Values indicate the percentage of pp90rsk in the slower migrating band for each individual lane, presented as means9S.E.M. from 6 experiments.
G. Gorgas et al. / Mechanisms of Ageing and De6elopment 94 (1997) 71–83
79
4. Discussion Our data show that CD4 T cells from young mice respond to anti-CD3 stimulation by increases in both MEK and ERK activity, and that T cells from old mice are significantly less able to induce increased function of these two links of the signal transduction pathway. Although the total amount of ERK2 protein detectable by immunoblotting does not change appreciably with age (see Fig. 2), the proportion of ERK2 converted by MEK to the slower-migrating form declines with age about 2-fold at the peak of the response, 2 min after CD3 cross-linking. The proportion of the ERK substrate pp90rsk converted to its slow-migrating form is also diminished about two-fold by aging. The kinetics of MEK and ERK induction are similar in old and young T cells; the age effect is thus a decline in signal strength, rather than a delay in induction. MEK function responds equally well to anti-CD3 activation in naive and memory T cells from young mice. The data are consistent with the idea that diminished induction of IL-2 gene expression by CD4 T cells of young mice might be a consequence of lower activation of MEK and ERK function in these cells. A similar study of MEK and ERK function in T cells from elderly humans has produced data analogous to our own results [50]. Using phosphorylation of myelin basic protein as an assay for ERK function in human T cells stimulated by the plant lectin PHA, the combination of PHA plus PMA, or cross-linked anti-CD3, these authors observed a significant reduction in ERK function in 40–50% of elderly humans, compared to T cells from young controls. MEK function, assessed by the ability to induce ERK activation, also declined significantly (about 35%) with age. PMA, in combination with the calcium ionophore ionomycin, induced increased ERK activity in only 3 of 5 elderly donors tested, and led to higher levels of ERK function in 5 young controls; this result, together with similar data using the phosphatase inhibitor okadaic acid or the G protein activator AlF4 to activate ERK, led the authors to suggest that ERK deficits in T cells from elderly humans were likely to reflect alterations at earlier stages of the activation pathway. Our results show that mice also exhibit an age-related defect in T cell MEK and ERK induction, that the change is demonstrable using ERK2 and pp90rsk mobility shift methods, and that naive and memory T cells show equal levels of MEK induction in responses to cross-linked anti-CD3. In our hands PMA invariably gave a much stronger induction of MEK function than anti-CD3, and we saw no effect of age on PMA-induced MEK function. We do not know if this discrepancy with the data of Whisler et al. [50] reflects a difference between mouse splenic CD4 T cells and human peripheral blood T cells, or instead reflects differences in assay methods. Age-associated decline in MEK function could well result from lowered activation of one or several of the pathways that link MEK to CD3/TCR stimulation. Two-dimensional electrophoretic analysis has shown that many of the phosphoproteins (PPNs) whose level of phosphorylation is rapidly increased after PMA exposure in young T cells are much less responsive in T cells from old mice [9], although some PPNs responded to PMA (or to anti-CD3) only in T cells from old mice, and some responded equally well in mice of any age. These observations
80
G. Gorgas et al. / Mechanisms of Ageing and De6elopment 94 (1997) 71–83
suggested that the pattern of PPNs generated by stimulation of PK-C may be altered by age even though PK-C function is not itself abrogated. The distribution of PK-C isoenzymes between membrane and cytoplasm differs in T cells from young and old humans [51], both in the resting state and after stimulation by anti-CD3. Similar age-related changes in PK-C compartmentalization, if they occur in T cells from mice, could lead to qualitative changes in PPN phosphorylation patterns, and lead to secondary changes in MEK activation. The function of tyrosine-specific protein kinases is also altered in T cells of aging mice, with diminished phosphorylation of several phosphotyrosine-containing PPNs (PY-PPNs) seen in anti-phosphotyrosine Western blots after stimulation by antiCD3, the plant lectin Con A, or antibodies to the ab dimer of the T cell receptor [6]. Anti-CD3 induced phosphorylation of Shc, an SH2-containing PY-PPN that has been suggested to couple TCR stimulation to Ras activation [52], also declines with age in mouse T cells [8]. A decline in Ras activation might also contribute to diminished MEK function after CD3-stimulation; new data on Ras and Raf-1 activity in activated T cells will help to shed further light on this question. It is interesting to note that in contrast to the anti-CD3 results, Shc phosphorylation stimulated by antibodies to mouse CD4 actually increases with age [8]. Since CD4-induced phosphorylation events are typically mediated by pp56lck [53,54], the disparity between the results seen with anti-CD3 and with anti-CD4 suggests that aging may lead to alterations in the compartmentalization of CD4 and/or p56lck that could in turn contribute to defective activation of MEK in old mice. We have also recently noted a decline with age in phosphorylation of the z chain of the CD3/TCR complex (Garcia and Miller, submitted for publication), which might diminish the ability of src-family TPKs to bind to the TCR complex before or during the activation process. It is also possible that changes in other pathways, including that involving activation of Ras through p95vav [55] may be sensitive to aging and influence MEK activation. Changes in phosphatase function, too, could potentially contribute, although we (Ghosh and Miller, unpublished data) do not see any changes with age in the amount or function of CD45, the principal membrane-associated tyrosinespecific protein phosphatase in T lymphocytes. Differences between old and young T cells can in some cases represent differences in function between T cell subsets whose relative proportions vary with age. Aging leads to an increase in memory T cells which, in mice of any age, are less able to produce large elevations in intracellular free calcium concentrations in responses to Con A. Thus the age-related decline in Con A-induced calcium signal can be accounted for by the shift from naive to memory T cells [2,56]. Some of the age-related declines in phosphorylation of PY-PPNs after anti-CD3 stimulation also reflect differences between naive and memory CD4 cells [7], although other age-related changes in PPN pattern seem to affect both naive and memory cells [9]. The data of Fig. 4 show that naive and memory CD4 T cells of young mice are equally able to increase MEK activity in response to CD3 stimulation. Although it remains possible that aging might lead to differential loss of MEK activation in one subset or another, the data of Fig. 4 suggest that the age effect is unlikely to be attributable to the accumulation of memory cells alone.
G. Gorgas et al. / Mechanisms of Ageing and De6elopment 94 (1997) 71–83
81
Acknowledgements This work was supported by NIH Grants AG08808 and AG09801.
References [1] R.A. Miller, B. Jacobson, G. Weil and E.R. Simons, Diminished calcium influx in lectin-stimulated T cells from old mice. J. Cell. Physiol., 132 (1987) 337 – 342. [2] B. Philosophe and R.A. Miller, Diminished calcium signal generation in subsets of T lymphocytes that predominate in old mice. J. Gerontol. Biol. Sci., 45 (1990) B87 – B93. [3] A. Grossmann, J.A. Ledbetter and P.S. Rabinovitch, Aging-related deficiency in intracellular calcium response to anti-CD3 or concanavalin A in murine T-cell subsets. J. Gerontol. Biol. Sci., 45 (1990) B81–B86. [4] A. Lerner, B. Philosophe and R.A. Miller, Defective calcium influx and preserved inositol phosphate generation in T cells from old mice. Aging Immunol. Infect. Dis., 1 (1988) 149 – 157. [5] A. Grossmann, P.S. Rabinovitch, T.J. Kavanagh et al., Activation of murine T-cells via phospholipase-C gamma 1-associated protein tyrosine phosphorylation is reduced with aging. J. Gerontol. A. Biol. Sci. Med. Sci., 50 (1995) B205 – 12. [6] J. Shi and R.A. Miller, Tyrosine-specific protein phosphorylation in response to anti-CD3 antibody is diminished in old mice. J. Gerontol. Biol. Sci., 47 (1992) B147 – B153. [7] J. Shi and R.A. Miller, Differential tyrosine-specific protein phosphorylation in mouse T lymphocyte subsets. Effect of age. J. Immunol., 151 (1993) 730 – 739. [8] J. Ghosh and R.A. Miller, Rapid tyrosine phosphorylation of Grb2 and Shc in T cells exposed to anti-CD3, anti-CD4, and anti-CD45 stimuli: differential effects of aging. Mech. Ageing De6., 80 (1995) 171–187. [9] H.R. Patel and R.A. Miller, Age-associated changes in mitogen-induced protein phosphorylation in murine T lymphocytes. Eur. J. Immunol., 22 (1992) 253 – 260. [10] R.A. Miller and O. Stutman, Decline, in aging mice, of the anti-TNP cytotoxic T cell response attributable to loss of Lyt-2 − , IL-2 producing helper cell function. Eur. J. Immunol., 11 (1981) 751–756. [11] M.L. Thoman and W.O. Weigle, Cell-mediated immunity in aged mice: an underlying lesion in IL 2 synthesis. J. Immunol., 128 (1982) 2358 – 2361. [12] H. Vie and R.A. Miller, Decline, with age, in the proportion of mouse T cells that express IL-2 receptors after mitogen stimulation. Mech. Ageing De6., 33 (1986) 313 – 322. [13] J.J. Proust, D.S. Kittur, M.A. Buchholz and A.A. Nordin, Restricted expression of mitogen-induced high affinity IL-2 receptors in aging mice. J. Immunol., 141 (1988) 4209 – 4216. [14] B. Philosophe and R.A. Miller, T lymphocyte heterogeneity in old and young mice: functional defects in T cells selected for poor calcium signal generation. Eur. J. Immunol., 19 (1989) 695 – 699. [15] A. Lerner, T. Yamada and R.A. Miller, PGP-1hi T lymphocytes accumulate with age in mice and respond poorly to Concanavalin A. Eur. J. Immunol., 19 (1989) 977 – 982. [16] D.N. Ernst, M.V. Hobbs, B.E. Torbett et al., Differences in the expression profiles of CD45RB, Pgp-1, and 3G11 membrane antigens and in the patterns of lymphokine secretion by splenic CD4 + T cells from young and aged mice. J. Immunol., 145 (1990) 1295 – 1302. [17] R. Rajasekar and A. Augustin, Antigen-dependent selection of T cells that are able to efficiently regulate free cytoplasmic calcium levels. J. Immunol., 153 (1994) 1037 – 1045. [18] A. Weiss and D.R. Littman, Signal transduction by lymphocyte antigen receptors. Cell, 76 (1994) 263–274. [19] R.D. Klausner, J. Lippincott-Schwartz and J.S. Bonifacino, The T cell antigen receptor: Insights into organelle biology. Ann. Re6. Cell Biol., 6 (1990) 403 – 431. [20] L.E. Samelson and R.D. Klausner, Tyrosine kinases and tyrosine-based activation motifs. J. Biol. Chem., 267 (1992) 24 913–24 916.
82
G. Gorgas et al. / Mechanisms of Ageing and De6elopment 94 (1997) 71–83
[21] J.C. Cambier, Signal transduction by T- and B-cell antigen receptors: converging structures and concepts. Curr. Opinion Immunology, 4 (1992) 257 – 264. [22] S. Gupta, A. Weiss, G. Kumar, S. Wang and A. Nel, The T-cell antigen receptor utilizes Lck, Raf-1, and MEK-1 for activating mitogen-activated protein kinase. J. Biol. Chem., 269 (1994) 17 349–17 357. [23] S.L. Pelech and J.S. Sanghera, Mitogen-activated protein kinases: versatile transducers for cell signaling. Trends Biochem. Sci., 17 (1992) 233 – 238. [24] J. Blenis, Signal transduction via the MAP kinases: proceed at your own RSK. Proc. Natl. Acad. Sci. USA, 90 (1993) 5889–5892. [25] M.H. Cobb, T.G. Boulton and D.J. Robbins, Extracellular signal-regulated kinases: ERKs in progress. Cell Regul., 2 (1991) 965 – 978. [26] L.B. Ray and T.W. Sturgill, Rapid stimulation by insulin of a serine/threonine kinase in 3T3-L1 adipocytes that phosphorylates microtubule-associated protein 2 in vitro. Proc. Natl. Acad. Sci. USA, 84 (1987) 1502–1506. [27] B. Errede and D.E. Levin, A conserved kinase cascade for MAP kinase activation in yeast. Curr. Opin. Cell Biol., 5 (1993) 254–260. [28] T.G. Boulton, S.H. Nye, D.J. Robbins et al., ERKs: A family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF. Cell, 65 (1991) 663–675. [29] G. Drewes, B. Lichtenberg-Kraag, F. Doring et al., Mitogen activated protein (MAP) kinase transforms tau protein into an Alzheimer-like state. EMBO J., 11 (1992) 2131 – 2138. [30] Y. Gotoh, K. Moriyama, S. Matsuda rt al., Xenopus M phase MAP kinase: isolation of its cDNA and activation by MPF. EMBO J., 10 (1991) 2661 – 2668. [31] H. Gille, A.D. Sharrocks and P.E. Shaw, Phosphorylation of transcription factor p62TCF and MAP kinase stimulates ternary complex formation at c-fos promoter. Nature, 358 (1992) 414 – 417. [32] M. David, III, C. Benjamin, R. Pine, M.J. Weber and A.C. Larner, Requirement for MAP kinase (ERK2) activity in interferon a- and interferon b-stimulated gene expression through STAT proteins. Science, 269 (1995) 1721 – 1723. [33] R. Marais, J. Wynne and R. Treisman, The SRF accessory protein Elk-1 contains a growth factor regulated transcriptional activation domain. Cell, 76 (1993) 1025 – 1037. [34] J.H. Park and L. Levitt, Overexpression of mitogen-activated protein kinase (ERK1) enhances T-cell ctyokine gene expression: role of AP1, NF-AT, and NF-KB. Blood, 82 (1993) 2470 – 2477. [35] R.J. Davis, The mitogen-activated protein kinase signal transduction pathway. J. Biol. Chem., 268 (1993) 14553–14556. [36] A.E. Nel, C. Hanekom, A. Rheeder et al., Stimulation of MAP-2 kinase activity in T lymphocytes by anti-CD3 or anti-Ti monoclonal antibody is partially dependent on protein kinase C. J. Immunol., 144 (1990) 2683–2689. [37] D.M. Payne, A.J. Rossomando, P. Martino et al., Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase). EMBO J., 10 (1991) 885 – 892. [38] N. Gomez and P. Cohen, Dissection of the protein kinase cascade by which nerve growth factor activates MAP kinases. Nature, 353 (1991) 170 – 173. [39] S. Matsuda, Y. Gotoh and E. Nishida, Phosphorylation of Xenopus mitogen-activated protein (MAP) kinase kinase by MAP kinase kinase kinase and MAP kinase. J. Biol. Chem., 268 (1993) 3277–3281. [40] H. Kosako, E. Nishida and Y. Gotoh, cDNA cloning of MAP kinase kinase reveals kinase cascade pathways in yeasts to vertebrates. EMBO J., 12 (1993) 787 – 794. [41] R. Seger, N.G. Ahn, J. Posada et al., Purification and characterization of mitogen-activated protein kinase activator(s) from epidermal growth factor-stimulated A431 cells. J. Biol. Chem., 267 (1992) 14 373–14 381. [42] S. Nakielny, P. Cohen, J. Wu and T. Sturgill, MAP kinase activator from insulin-stimulated skeletal muscle is a protein threonine-tyrosine kinase. EMBO J., 11 (1992) 2123 – 2129. [43] C.M. Crews, A. Alessandrini and R.L. Erikson, The primary structure of MEK, a protein kinase that phosphorylates the ERK gene product. Science, 258 (1992) 478 – 480.
G. Gorgas et al. / Mechanisms of Ageing and De6elopment 94 (1997) 71–83
83
[44] C.F. Zheng and K.L. Guan, Cloning and characterization of two distinct human extracellular signal-regulated kinase activator kinases, MEK1 and MEK2. J. Biol. Chem., 268 (1993) 11 435 – 11 439. [45] D.J. Robbins, E. Zhen, H. Owaki et al., Regulation and properties of extracellular signal-regulated protein kinases 1 and 2 in vitro. J. Biol. Chem., 268 (1993) 5097 – 5106. [46] J. Chung, S.L. Pelech and J. Blenis, Mitogen-activated Swiss mouse 3T3 RSK kinases I and II are related to pp44mpk from sea star oocytes and participate in the regulation of pp90rsk activity. Proc. Natl. Acad. Sci. USA, 88 (1991) 4981 – 4985. [47] R.H. Chen, C. Sarnecki and J. Blenis, Nuclear localization and regulation of erk- and rsk-encoded protein kinases. Mol. Cell. Biol., 12 (1992) 915 – 927. [48] T.W. Sturgill, L.B. Ray, E. Erikson and J.L. Maller, Insulin-stimulated MAP-2 kinase phosphorylates and activates ribosomal protein S6 kinase II. Nature, 334 (1988) 715 – 718. [49] L. Nagelkerken, A. Hertogh-Huijbregts, R. Dobber and A. Drager, Age-related changes in lymphokine production related to a decreased number of CD45RBhi CD4 + T cells. Eur. J. Immunol., 21 (1991) 273–281. [50] R.L. Whisler, Y.G. Newhouse and S.E. Bagenstose, Age-related reductions in the activation of mitogen-activated protein kinases p44mapk/ERK1 and p42mapk/ERK2 in human T cells stimulated via ligation of the T cell receptor complex. Cell. Immunol., 168 (1996) 201 – 210. [51] T. Fulop, Jr., C. Leblanc, G. Lacombe and G. Dupuis, Cellular distribution of protein kinase C isozymes in CD3-mediated stimulation of human T lymphocytes with aging. FEBS Lett., 375 (1995) 69–74. [52] K.S. Ravichandran, K.K. Lee, Z. Songyang, L.C. Cantley, P. Burn and S.J. Burakoff, Interaction of Shc with the z chain of the T cell receptor upon T cell activation. Science, 262 (1993) 902 – 905. [53] A. Veillette, M.A. Bookman, E.M. Horak and J.B. Bolen, The CD4 and CD8 T cell surface antigens are associated with the internal membrane tyrosine-protein kinase p56lck. Cell, 55 (1988) 301–308. [54] N. Abraham, M.C. Miceli, J.R. Parnes and A. Veillette, Enhancement of T-cell responsiveness by the lymphocyte-specific tyrosine protein kinase p56lck. Nature, 350 (1991) 62 – 66. [55] E. Gulbins, K.M. Coggeshall, G. Baier, S. Katzav, P. Burn and A. Altman, Tyrosine kinase-stimulated guanine nucleotide exchange activity of Vav in T cell activation. Science, 260 (1993) 822–825. [56] R.A. Miller, Accumulation of hyporesponsive, calcium extruding memory T cells as a key feature of age-dependent immune dysfunction. Clin. Immunol. Immunopath., 58 (1991) 305 – 317.
.
Diminished activation of the MAP kinase pathway in CD3-stimulated T lymphocytes from old mice Gayle Gorgas a, Elizabeth R. Butch b, Kun-Liang Guan b,c, Richard A. Miller a,c,d,* a
Department of Pathology, Uni6ersity of Michigan School of Medicine, Ann Arbor, MI 48109, USA b Department of Biological Chemistry, Uni6ersity of Michigan School of Medicine, Ann Arbor, MI 48109, USA c Institute of Gerontology, Ann Arbor, MI 48109, USA d Ann Arbor DVA Medical Center, Ann Arbor, MI 48109, USA
Received 2 November 1996; received in revised form 14 December 1996; accepted 14 December 1996
Abstract Stimulation of the ERK family of protein kinases (‘extracellular signal regulated kinases’, also known as MAP kinases) plays an important role in the activation of many cell types, including T lymphocytes. ERKs are activated when they are phosphorylated by an upstream activator, the dual-specific protein kinase MEK. To see if aging leads to an impairment of MEK activation in mouse T cells, we used a mobility shift assay in which activation of MEK leads to phosphorylation and altered mobility of ERK-2 kinase. Similarly, we monitored mobility of pp90rsk, a known ERK substrate, as an indication of ERK function. We found an age-related decline in the ability of mouse T cells to activate both MEK and ERK function in response to stimulation by antibodies to the CD3 chain of the T cell receptor. Aging did not alter the kinetics of enzyme activation, but did diminish (by about 2-fold) the maximal level of substrate converted into the slower migrating form. Naive and memory CD4 T cells from young mice were equally able to convert ERK2 to its slower migrating form, suggesting that the decline in MEK function is not likely to be attributable to the shift, with age, from naive to memory T cell predominance. Our data suggest that age-dependent declines in gene activation, including genes for key cytokines like IL-2, may be due to * Corresponding author. Present address: University of Michigan, MSRB-3 Room 6301, Box 0642, 1150 W. Medical Center Drive, Ann Arbor, MI 48109-0642, USA; Tel.: +1 313 9362122; fax: + 1 313 9369220; e-mail: [email protected] 0047-6374/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 0 4 7 - 6 3 7 4 ( 9 6 ) 0 1 8 5 7 - X
72
G. Gorgas et al. / Mechanisms of Ageing and De6elopment 94 (1997) 71–83
declines in the upstream signals that lead to activation of the MEK/ERK protein kinase cascade. © 1997 Elsevier Science Ireland Ltd. Keywords: ERK; Immune senescence; MAP kinase; MEK; T lymphocytes; Signal transduction
1. Introduction T lymphocytes from old mice, when compared to cells from young mice, show abnormalities in the activation process within the first few min of exposure to mitogenic lectins or antibodies. These defects include lower levels of free calcium ion concentration [1 – 3] and trans-membrane calcium influx [4], declines in tyrosinespecific protein kinase function [5–8], and lower levels of phosphorylation of a wide range of proteins regulated by calcium and protein kinase C levels [9]. These changes early in the activation cascade seem likely to contribute to the age-related decline in expression of the growth-promoting cytokine interleukin 2 (IL-2) [10,11] and its receptor [12,13]. Indeed, enrichment of those T cells in old mice that are able to generate calcium signals also strongly enriches for cells that can produce and respond to IL-2 [14]. The accumulation in old age of memory T cells, and the parallel decline in naive T cells [15,16] can account for many of these changes, in that memory cells tend to show smaller changes in calcium signal generation [2,17] than naive cells, and are also hyporesponsive in tests for mitogen induction of tyrosine-specific protein phosphorylation [7]. Some age-related changes in protein kinase activity, however, are demonstrable in both naive and memory T cell subsets [9]. Stimulation of the T-cell receptor (TCR) initiates a signal transduction cascade that results in the release of numerous cytokines [18], including IL-2. The TCR complex has no intrinsic protein tyrosine kinase (PTK) activity, but induction of PTK activity as a result of TCR stimulation initiates two principal signal transduction pathways, involving activation of at least three PTKs, Fyn, Lck and ZAP-70 [18 – 21]. Both of these pathways begin with PTK dependent induction of phospholipase C (PLC) activity[18]. The hydrolysis of phosphatidylinositol 4,5-bisphosphate by PLC results in the production of inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). This PLC hydrolysis forms a branch point for the two divergent signaling pathways, the first of which depends on increased intracellular calcium (Ca + + ) resulting from IP3 production and leading to an increase in Ca + + / calmodulin dependent serine phosphatase (calcineurin). The calcineurin in turn activates the transcription factor NF-AT thus leading to increased IL-2 gene expression [18]. The DAG produced by PLC is an activator of protein kinase C (PKC) which initiates the second signal transduction pathway. Increased PKC activity results in activation of Ras which in turn activates Raf-1, a protein kinase that can in turn directly activate MEK (‘MAPK or ERK kinase’) [18,22]. MEK
G. Gorgas et al. / Mechanisms of Ageing and De6elopment 94 (1997) 71–83
73
activation results in phosphorylation and increased function of a family of extracellular signal-regulated kinases (ERKs), also known as mitogen activated protein kinases (MAPKs). Activation of the Ras/MEK pathway, in synergy with the Ca + + signal, leads ultimately to increased cytokine production [22]. Members of the ERK family are important components of various signaling pathways in many cell types. They can phosphorylate numerous substrates and may be a key component in linking growth factor receptor activation to serine/ threonine protein phosphorylation [23–25]. ERK activation can be induced by a variety of mitogenic stimuli, including phorbol esters, cytokines, and T-cell antigens, as well as by tyrosine-kinase coupled receptors to growth factors including insulin, epidermal growth factor, fibroblast growth factor and platelet-derived growth factor [25 – 28]. Stimulation of ERK in various non-lymphoid cell types leads to a multitude of cellular responses including phosphorylation of microtubule-associated proteins involved in microtubule rearrangement [29,30] and phosphorylation resulting in subsequent activation of transcription factors TCF and STAT [31 – 33]. In lymphoid cells, activation of ERK results in stimulation of T-cells to produce IL-2 and other cytokines [34–36]. ERK is itself activated by another protein kinase, MEK. MEK is a dual specific kinase which can phosphorylate ERK2 on both the threonine 183 and tyrosine 185 [37]. Two mammalian isoforms of MEK (MEK1 and MEK2), identified by molecular cloning techniques, can phosphorylate and activate ERK [38 – 44]. ERK is the only known substrate for MEK with all other proteins tested thus far being phosphorylated at least 1000-fold less efficiently by MEK [45]. Thus assays based upon the phosphorylation of ERK provide a useful gauge of MEK function. One of the known physiological targets of activated ERK is a serine/threonine specific protein kinase, pp90rsk, that can phosphorylate the 40S ribosomal protein S6[46 – 48]. Previous studies revealed that a Xenopus homolog of the pp90rsk family, which had been inactivated by a protein phosphatase, could be partially reactivated by insulin stimulated ERK2 [48]. Thus, phosphorylation of RSK can be used as a physiological indicator of ERK activity in cells. Since induction of the MEK–ERK system plays a critical role in T cell activation, we undertook a series of experiments to see if aging led to alterations in the ability of cross-linked anti-CD3 antibody to induce MEK and ERK activity in murine splenic CD4 + T lymphocytes. We monitored MEK function using a mobility shift assay, in which altered mobility of ERK2 in lysates of activated T cells provides an index of MEK function. Similarly, ERK activity was monitored by altered mobility of one of its substrates, the ribosomal S6 protein kinase pp90rsk. We report here that aging leads to a decline, of approximately 2-fold, in both MEK and ERK induction by anti-CD3 signals. We also show that both naive and memory T cells from young mice are equally susceptible to induction of MEK function, and thus conclude that the age-related decline in MEK function is unlikely to result simply from an accumulation of memory T cells in old age.
74
G. Gorgas et al. / Mechanisms of Ageing and De6elopment 94 (1997) 71–83
2. Materials and methods
2.1. Mice Specific pathogen-free CB6F1 male mice were purchased from the National Institute of Aging colonies maintained by the Charles River Laboratories (Kingston, NY). After shipment, they were housed under specific-pathogen free conditions for at least one week before use. The mice received rodent chow and water ad libitum. The terms ‘young’, ‘middle-aged’, and ‘old’ refer to mice aged 3 – 6 months, 12 – 14 months, and 18–22 months, respectively. Mice with splenomegaly or visible tumors were not used.
2.2. Cell preparations Spleens were removed aseptically and rubbed between frosted glass slides in Hanks balanced salt solution containing 0.2% BSA (BSS–BSA) to obtain a cell suspension. Debris and clumps of tissue were eliminated by passage of the suspension through nylon mesh. Erythrocytes were then removed by centrifugation over a Lympholyte-M cushion (Cedar lane Laboratories, Hornby, Ontario, Canada). T cell enrichment was accomplished by panning the cell suspension on petri dishes coated with anti-mouse IgG to remove B cells. This procedure regularly yields at least 90% pure T cells. CD4 + memory and CD4 + naive T cells were prepared by negative selection. To prepare CD4 + memory (i.e. CD45RBlo) cells, T cells were incubated with a mixture of anti-CD8 ascites (clone 53-6.7) at 1:200 and 5 mg/ml anti-CD45RB (clone 16A; Pharmingen, San Diego) for 40 min at 4°C. After removal of unbound antibody, cells were incubated for 40 min at 4°C with magnetic beads coated with anti-rat Ig at a bead:cell ratio of 50:1. A magnetic field was then applied at least 3 times to remove all cells bound to beads and free beads. CD4 naive cells (CD44lo) were prepared in the same way, substituting 5 mg/ml anti-CD44 (clone IM7; Pharmingen) for the anti-CD45RB antibody. The resulting preparations were respectively 85% CD4 + /CD44hi and 95% CD4 + /CD45RBhi as judged by FACS analysis. These cells are referred to as CD4 + ‘naive’ and CD4 + ‘memory’ T cells, respectively.
2.3. Cell stimulation Cells (3 ×106) were resuspended in 1 ml of RPMI-1640 supplemented with 10% FCS. Partially purified anti-CD3 ascites was added at a concentration of 6.6 mg/ml and incubated on ice for 30 min. The unbound antibody was removed and the anti-CD3 was crosslinked by the addition of goat anti-hamster Ig at a concentration of 5 mg/ml at 37°C for 0, 1, 2, 5, 10, or 30 min. For stimulation with PMA, cells were incubated with 10 ng/ml PMA at 37°C for the same time course. Stimulation was terminated by washing the cells with ice cold PBS, followed by lysis in 1% NP-40, 20 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EGTA, 1 mM EDTA, 50 mM NaF, 2 mM Na3VO4, 5 mM sodium pyrophosphate, 2 mg/ml aprotinin, 5 mg/ml leupeptin, 5 mg/ml pepstatin, and 1 mM benzamidine.
G. Gorgas et al. / Mechanisms of Ageing and De6elopment 94 (1997) 71–83
75
2.4. Western blotting Lysates were centrifuged at 14 000 rpm at 4°C for 10 min to remove cellular debris, and then boiled in 2×SDS reducing buffer. Samples were run on 12% (for ERK, using 3 × 106 cell equivalents/lane) or 8% (for RSK, using 1 × 106 cell equivalents/lane) SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked in PBS with 0.1% Tween-20 (PBS-T) containing 5% BSA or 5% nonfat dry milk overnight at 4°C. Anti-ERK antibody (Zymed, S. San Francisco, CA) was used at a dilution of 1:15 000 (70 ng/ml), and anti-RSK (Upstate Biotechnology, Lake Placid, NY) was used at 175 ng/ml in PBS-T containing 1% BSA; some early experiments used anti-ERK antibody produced in the Guan laboratory. Blots were incubated with primary antibody for 1 h at room temperature and then washed 4 times for 15 min each in PBS-T at room temperature. Blots were then incubated with a 1:2000 dilution of horseradish peroxidase conjugated anti-rabbit IgG in PBS-T containing 1% BSA for 1 h at room temperature followed by extensive washing with PBS-T. Blots were developed using enhanced chemiluminescence (ECL) according to the manufacturer’s instructions (Kirkegaard and Perry, Gaithersburg, MD).
2.5. Densitometry and data analysis For analysis of MEK activity, anti-ERK immunoblots were scanned by a laser densitometer (Molecular Dynamics, Sunnyvale, CA), and analyzed using an ImageQuant software package. For analysis of ERK activity, anti-RSK immunoblots were scanned using a Sharp image scanner (model JX-330), and analyzed using SigmaGel software. Experiments comparing naive to memory cells were also analyzed using the Sharp image scanning system. In each case the parameter of interest was the relative amount of enzyme (ERK or RSK) that exhibited a retarded electrophoretic mobility. To control for differences in exposure level and protein loading, OD values for the slower band were expressed as a percentage of the sum of the OD values for both the rapid and slow-migrating species. These normalized values were then subjected to a two-way analysis of variance (ANOVA) in which age (or T cell subset), and experiment were treated as independent variables, and the normalized OD values were treated as dependent variables.
3. Results
3.1. PMA-induced shift in ERK2 electrophoretic mobility To confirm previous reports that PMA-induced activation of MEK would convert ERK2 to a more slowly migrating form, we exposed T cells to PMA (10 ng/ml) for various intervals and then examined ERK2 mobility by an immunoblotting method. The results (Fig. 1) showed that 5 min of exposure to PMA led to a virtually quantitative conversion of the 42 kD form of ERK2 to a form with the
76
G. Gorgas et al. / Mechanisms of Ageing and De6elopment 94 (1997) 71–83
Fig. 1. Detection of ERK2 in PMA stimulated T cells by immunoblotting. T cells (3×106/ml) were incubated with 10 ng/ml PMA at 37°C. Postnuclear supernatants were separated on 12% SDS-PAGE and transferred to nitrocellulose. The membrane was then probed for ERK2 using ERK antisera and ECL detection reagents.
apparent molecular weight of 42.5 kD. This conversion was transient, with partial reversal apparent by 30 min. A series of experiments using T cells derived from mice of differing ages revealed no effect of age on PMA-induced alteration of ERK2 mobility (not shown). Similar experiments (not shown) using anti-ERK1 antibodies demonstrated a PMA-induced shift in ERK1 mobility, which was equally apparent for T cells from old and young mice.
3.2. Age-dependent loss in MEK-mediated ERK2 mobility shift in responses to cross-linked anti-CD3 antibody We next carried out a series of experiments to see if aging altered MEK activation when T cells were activated by cross-linking the CD3o chain of the TCR complex. Fig. 2 shows a representative experiment. In T cells from young mice, cross-linking CD3 leads to the appearance of a more slowly migrating form of ERK2, with peak levels achieved within 2–5 min, and a nearly complete return to
Fig. 2. Effect of age on ERK2 mobility shift in cross-linked anti-CD3 stimulated T cells; a representative experiment. T cells (3×106/ml) were incubated with anti-CD3 for 30 min on ice, and then stimulated by the addition of goat anti-hamster Ig cross-linking antibody at 37°C. Postnuclear supernatants were separated on 12% SDS-PAGE and transferred to nitrocellulose. The membrane was then probed for ERK2 using ERK antisera.
G. Gorgas et al. / Mechanisms of Ageing and De6elopment 94 (1997) 71–83
77
Fig. 3. Age-related decline in ERK2 mobility shift in response to cross-linked anti-CD3 stimulation. Values indicate the percentage of ERK2 in the slower migrating band for each individual lane, presented as means9 S.E.M. from 7 experiments.
baseline within 30 min. Quantitative densitometry suggested that only about 8% of the ERK2 (in young mice) was converted to the slower isoform at the peak of the response. We were unable to see an effect of anti-CD3 stimulation on ERK1 mobility (not shown). T cells from middle-aged and from old mice responded with a similar time course, but with lower levels of the slower moving form. Fig. 3 shows the results of a series of 7 such experiments, each involving three individual mice of differing ages. Analysis of variance showed that there were significant differences between young and old mice in ERK2-converting activity at each of the early time points (1, 2, and 5 min after activation). Differences between young and old mice were about two-fold. Middle-aged mice had intermediate levels of MEK function, not significantly different from either young or old mice. We concluded that CD3-mediated activation of MEK is impaired by aging. Since aging leads to a decrease in the proportion of cells with the surface markers of naive lymphocytes, and a corresponding increase in memory T cells [15,16,49], we wished to determine whether the decline with age in CD3-induced MEK function might reflect merely differences between naive and memory cells in this aspect of the CD3 activation cascade. Fig. 4 presents results of an experiment in which naive (CD44lo) and memory (CD45RBlo) T cells were prepared from the
Fig. 4. ERK2 mobility shift in cross-linked anti-CD3 stimulated naive and memory T cells; a representative experiment. T cells were separated as explained in Section 2. Separated naive and memory cells (2 ×106) were incubated with anti-CD3 for 30 min on ice, and then stimulated by the addition of goat anti-hamster cross-linking antibody at 37°C. Postnuclear supernatants were separated on 12% SDS-PAGE and transferred to nitrocellulose. The membrane was then probed for ERK2 using a polyclonal ERK antibody.
78
G. Gorgas et al. / Mechanisms of Ageing and De6elopment 94 (1997) 71–83
Fig. 5. Effect of age on pp90rsk mobility shift in cross-linked anti-CD3 stimulated T cells; a representative experiment. T cells (1.5 × 106/ml) were incubated with anti-CD3 for 30 min on ice, and then stimulated by the addition of goat anti-hamster cross-linking antibody at 37°C. Postnuclear supernatants were separated on 8% SDS-PAGE and transferred to nitrocellulose. The membrane was probed for pp90rsk using a polyclonal RSK antibody.
CD4 subset of young mice, stimulated with anti-CD3, and subjected to an antiERK immunoblotting procedure. Naive and memory T cells performed equally well in this and in three replicate experiments. We concluded that differences between memory and naive T cells cannot account for the age-dependent loss in MEK activation shown in Figs. 2 and 3.
3.3. Age-related decline in ERK-mediated pp90 rsk mobility shift To see if the anti-CD3 induced generation of the slow-migrating form of ERK2 was accompanied by an increase in ERK function, we employed an assay based upon the ERK-dependent conversion of another downstream protein kinase, pp90rsk, to a slower migrating isoform. Fig. 5 shows the result of one such experiment in which crosslinking surface CD3 led to a rapid appearance of a slower form of RSK, detectable within 60 s, peaking at 5–10 min, and declining to near baseline by 30 min after activation. There is a clear age-dependent decline in this index of ERK activity. Fig. 6 shows averages for a series of six such experiments. T cells from old mice respond less vigorously than cells from young or middle-aged mice. Analysis of variance shows that the differences between young and old mice are significant at the 1, 2, 5, and 10 min time points. The difference (peak minus baseline) for old mice is approximately 60% of the value for young mice.
Fig. 6. Age related decline in pp90rsk mobility shift in response to cross-linked anti-CD3 stimulation. Values indicate the percentage of pp90rsk in the slower migrating band for each individual lane, presented as means9S.E.M. from 6 experiments.
G. Gorgas et al. / Mechanisms of Ageing and De6elopment 94 (1997) 71–83
79
4. Discussion Our data show that CD4 T cells from young mice respond to anti-CD3 stimulation by increases in both MEK and ERK activity, and that T cells from old mice are significantly less able to induce increased function of these two links of the signal transduction pathway. Although the total amount of ERK2 protein detectable by immunoblotting does not change appreciably with age (see Fig. 2), the proportion of ERK2 converted by MEK to the slower-migrating form declines with age about 2-fold at the peak of the response, 2 min after CD3 cross-linking. The proportion of the ERK substrate pp90rsk converted to its slow-migrating form is also diminished about two-fold by aging. The kinetics of MEK and ERK induction are similar in old and young T cells; the age effect is thus a decline in signal strength, rather than a delay in induction. MEK function responds equally well to anti-CD3 activation in naive and memory T cells from young mice. The data are consistent with the idea that diminished induction of IL-2 gene expression by CD4 T cells of young mice might be a consequence of lower activation of MEK and ERK function in these cells. A similar study of MEK and ERK function in T cells from elderly humans has produced data analogous to our own results [50]. Using phosphorylation of myelin basic protein as an assay for ERK function in human T cells stimulated by the plant lectin PHA, the combination of PHA plus PMA, or cross-linked anti-CD3, these authors observed a significant reduction in ERK function in 40–50% of elderly humans, compared to T cells from young controls. MEK function, assessed by the ability to induce ERK activation, also declined significantly (about 35%) with age. PMA, in combination with the calcium ionophore ionomycin, induced increased ERK activity in only 3 of 5 elderly donors tested, and led to higher levels of ERK function in 5 young controls; this result, together with similar data using the phosphatase inhibitor okadaic acid or the G protein activator AlF4 to activate ERK, led the authors to suggest that ERK deficits in T cells from elderly humans were likely to reflect alterations at earlier stages of the activation pathway. Our results show that mice also exhibit an age-related defect in T cell MEK and ERK induction, that the change is demonstrable using ERK2 and pp90rsk mobility shift methods, and that naive and memory T cells show equal levels of MEK induction in responses to cross-linked anti-CD3. In our hands PMA invariably gave a much stronger induction of MEK function than anti-CD3, and we saw no effect of age on PMA-induced MEK function. We do not know if this discrepancy with the data of Whisler et al. [50] reflects a difference between mouse splenic CD4 T cells and human peripheral blood T cells, or instead reflects differences in assay methods. Age-associated decline in MEK function could well result from lowered activation of one or several of the pathways that link MEK to CD3/TCR stimulation. Two-dimensional electrophoretic analysis has shown that many of the phosphoproteins (PPNs) whose level of phosphorylation is rapidly increased after PMA exposure in young T cells are much less responsive in T cells from old mice [9], although some PPNs responded to PMA (or to anti-CD3) only in T cells from old mice, and some responded equally well in mice of any age. These observations
80
G. Gorgas et al. / Mechanisms of Ageing and De6elopment 94 (1997) 71–83
suggested that the pattern of PPNs generated by stimulation of PK-C may be altered by age even though PK-C function is not itself abrogated. The distribution of PK-C isoenzymes between membrane and cytoplasm differs in T cells from young and old humans [51], both in the resting state and after stimulation by anti-CD3. Similar age-related changes in PK-C compartmentalization, if they occur in T cells from mice, could lead to qualitative changes in PPN phosphorylation patterns, and lead to secondary changes in MEK activation. The function of tyrosine-specific protein kinases is also altered in T cells of aging mice, with diminished phosphorylation of several phosphotyrosine-containing PPNs (PY-PPNs) seen in anti-phosphotyrosine Western blots after stimulation by antiCD3, the plant lectin Con A, or antibodies to the ab dimer of the T cell receptor [6]. Anti-CD3 induced phosphorylation of Shc, an SH2-containing PY-PPN that has been suggested to couple TCR stimulation to Ras activation [52], also declines with age in mouse T cells [8]. A decline in Ras activation might also contribute to diminished MEK function after CD3-stimulation; new data on Ras and Raf-1 activity in activated T cells will help to shed further light on this question. It is interesting to note that in contrast to the anti-CD3 results, Shc phosphorylation stimulated by antibodies to mouse CD4 actually increases with age [8]. Since CD4-induced phosphorylation events are typically mediated by pp56lck [53,54], the disparity between the results seen with anti-CD3 and with anti-CD4 suggests that aging may lead to alterations in the compartmentalization of CD4 and/or p56lck that could in turn contribute to defective activation of MEK in old mice. We have also recently noted a decline with age in phosphorylation of the z chain of the CD3/TCR complex (Garcia and Miller, submitted for publication), which might diminish the ability of src-family TPKs to bind to the TCR complex before or during the activation process. It is also possible that changes in other pathways, including that involving activation of Ras through p95vav [55] may be sensitive to aging and influence MEK activation. Changes in phosphatase function, too, could potentially contribute, although we (Ghosh and Miller, unpublished data) do not see any changes with age in the amount or function of CD45, the principal membrane-associated tyrosinespecific protein phosphatase in T lymphocytes. Differences between old and young T cells can in some cases represent differences in function between T cell subsets whose relative proportions vary with age. Aging leads to an increase in memory T cells which, in mice of any age, are less able to produce large elevations in intracellular free calcium concentrations in responses to Con A. Thus the age-related decline in Con A-induced calcium signal can be accounted for by the shift from naive to memory T cells [2,56]. Some of the age-related declines in phosphorylation of PY-PPNs after anti-CD3 stimulation also reflect differences between naive and memory CD4 cells [7], although other age-related changes in PPN pattern seem to affect both naive and memory cells [9]. The data of Fig. 4 show that naive and memory CD4 T cells of young mice are equally able to increase MEK activity in response to CD3 stimulation. Although it remains possible that aging might lead to differential loss of MEK activation in one subset or another, the data of Fig. 4 suggest that the age effect is unlikely to be attributable to the accumulation of memory cells alone.
G. Gorgas et al. / Mechanisms of Ageing and De6elopment 94 (1997) 71–83
81
Acknowledgements This work was supported by NIH Grants AG08808 and AG09801.
References [1] R.A. Miller, B. Jacobson, G. Weil and E.R. Simons, Diminished calcium influx in lectin-stimulated T cells from old mice. J. Cell. Physiol., 132 (1987) 337 – 342. [2] B. Philosophe and R.A. Miller, Diminished calcium signal generation in subsets of T lymphocytes that predominate in old mice. J. Gerontol. Biol. Sci., 45 (1990) B87 – B93. [3] A. Grossmann, J.A. Ledbetter and P.S. Rabinovitch, Aging-related deficiency in intracellular calcium response to anti-CD3 or concanavalin A in murine T-cell subsets. J. Gerontol. Biol. Sci., 45 (1990) B81–B86. [4] A. Lerner, B. Philosophe and R.A. Miller, Defective calcium influx and preserved inositol phosphate generation in T cells from old mice. Aging Immunol. Infect. Dis., 1 (1988) 149 – 157. [5] A. Grossmann, P.S. Rabinovitch, T.J. Kavanagh et al., Activation of murine T-cells via phospholipase-C gamma 1-associated protein tyrosine phosphorylation is reduced with aging. J. Gerontol. A. Biol. Sci. Med. Sci., 50 (1995) B205 – 12. [6] J. Shi and R.A. Miller, Tyrosine-specific protein phosphorylation in response to anti-CD3 antibody is diminished in old mice. J. Gerontol. Biol. Sci., 47 (1992) B147 – B153. [7] J. Shi and R.A. Miller, Differential tyrosine-specific protein phosphorylation in mouse T lymphocyte subsets. Effect of age. J. Immunol., 151 (1993) 730 – 739. [8] J. Ghosh and R.A. Miller, Rapid tyrosine phosphorylation of Grb2 and Shc in T cells exposed to anti-CD3, anti-CD4, and anti-CD45 stimuli: differential effects of aging. Mech. Ageing De6., 80 (1995) 171–187. [9] H.R. Patel and R.A. Miller, Age-associated changes in mitogen-induced protein phosphorylation in murine T lymphocytes. Eur. J. Immunol., 22 (1992) 253 – 260. [10] R.A. Miller and O. Stutman, Decline, in aging mice, of the anti-TNP cytotoxic T cell response attributable to loss of Lyt-2 − , IL-2 producing helper cell function. Eur. J. Immunol., 11 (1981) 751–756. [11] M.L. Thoman and W.O. Weigle, Cell-mediated immunity in aged mice: an underlying lesion in IL 2 synthesis. J. Immunol., 128 (1982) 2358 – 2361. [12] H. Vie and R.A. Miller, Decline, with age, in the proportion of mouse T cells that express IL-2 receptors after mitogen stimulation. Mech. Ageing De6., 33 (1986) 313 – 322. [13] J.J. Proust, D.S. Kittur, M.A. Buchholz and A.A. Nordin, Restricted expression of mitogen-induced high affinity IL-2 receptors in aging mice. J. Immunol., 141 (1988) 4209 – 4216. [14] B. Philosophe and R.A. Miller, T lymphocyte heterogeneity in old and young mice: functional defects in T cells selected for poor calcium signal generation. Eur. J. Immunol., 19 (1989) 695 – 699. [15] A. Lerner, T. Yamada and R.A. Miller, PGP-1hi T lymphocytes accumulate with age in mice and respond poorly to Concanavalin A. Eur. J. Immunol., 19 (1989) 977 – 982. [16] D.N. Ernst, M.V. Hobbs, B.E. Torbett et al., Differences in the expression profiles of CD45RB, Pgp-1, and 3G11 membrane antigens and in the patterns of lymphokine secretion by splenic CD4 + T cells from young and aged mice. J. Immunol., 145 (1990) 1295 – 1302. [17] R. Rajasekar and A. Augustin, Antigen-dependent selection of T cells that are able to efficiently regulate free cytoplasmic calcium levels. J. Immunol., 153 (1994) 1037 – 1045. [18] A. Weiss and D.R. Littman, Signal transduction by lymphocyte antigen receptors. Cell, 76 (1994) 263–274. [19] R.D. Klausner, J. Lippincott-Schwartz and J.S. Bonifacino, The T cell antigen receptor: Insights into organelle biology. Ann. Re6. Cell Biol., 6 (1990) 403 – 431. [20] L.E. Samelson and R.D. Klausner, Tyrosine kinases and tyrosine-based activation motifs. J. Biol. Chem., 267 (1992) 24 913–24 916.
82
G. Gorgas et al. / Mechanisms of Ageing and De6elopment 94 (1997) 71–83
[21] J.C. Cambier, Signal transduction by T- and B-cell antigen receptors: converging structures and concepts. Curr. Opinion Immunology, 4 (1992) 257 – 264. [22] S. Gupta, A. Weiss, G. Kumar, S. Wang and A. Nel, The T-cell antigen receptor utilizes Lck, Raf-1, and MEK-1 for activating mitogen-activated protein kinase. J. Biol. Chem., 269 (1994) 17 349–17 357. [23] S.L. Pelech and J.S. Sanghera, Mitogen-activated protein kinases: versatile transducers for cell signaling. Trends Biochem. Sci., 17 (1992) 233 – 238. [24] J. Blenis, Signal transduction via the MAP kinases: proceed at your own RSK. Proc. Natl. Acad. Sci. USA, 90 (1993) 5889–5892. [25] M.H. Cobb, T.G. Boulton and D.J. Robbins, Extracellular signal-regulated kinases: ERKs in progress. Cell Regul., 2 (1991) 965 – 978. [26] L.B. Ray and T.W. Sturgill, Rapid stimulation by insulin of a serine/threonine kinase in 3T3-L1 adipocytes that phosphorylates microtubule-associated protein 2 in vitro. Proc. Natl. Acad. Sci. USA, 84 (1987) 1502–1506. [27] B. Errede and D.E. Levin, A conserved kinase cascade for MAP kinase activation in yeast. Curr. Opin. Cell Biol., 5 (1993) 254–260. [28] T.G. Boulton, S.H. Nye, D.J. Robbins et al., ERKs: A family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF. Cell, 65 (1991) 663–675. [29] G. Drewes, B. Lichtenberg-Kraag, F. Doring et al., Mitogen activated protein (MAP) kinase transforms tau protein into an Alzheimer-like state. EMBO J., 11 (1992) 2131 – 2138. [30] Y. Gotoh, K. Moriyama, S. Matsuda rt al., Xenopus M phase MAP kinase: isolation of its cDNA and activation by MPF. EMBO J., 10 (1991) 2661 – 2668. [31] H. Gille, A.D. Sharrocks and P.E. Shaw, Phosphorylation of transcription factor p62TCF and MAP kinase stimulates ternary complex formation at c-fos promoter. Nature, 358 (1992) 414 – 417. [32] M. David, III, C. Benjamin, R. Pine, M.J. Weber and A.C. Larner, Requirement for MAP kinase (ERK2) activity in interferon a- and interferon b-stimulated gene expression through STAT proteins. Science, 269 (1995) 1721 – 1723. [33] R. Marais, J. Wynne and R. Treisman, The SRF accessory protein Elk-1 contains a growth factor regulated transcriptional activation domain. Cell, 76 (1993) 1025 – 1037. [34] J.H. Park and L. Levitt, Overexpression of mitogen-activated protein kinase (ERK1) enhances T-cell ctyokine gene expression: role of AP1, NF-AT, and NF-KB. Blood, 82 (1993) 2470 – 2477. [35] R.J. Davis, The mitogen-activated protein kinase signal transduction pathway. J. Biol. Chem., 268 (1993) 14553–14556. [36] A.E. Nel, C. Hanekom, A. Rheeder et al., Stimulation of MAP-2 kinase activity in T lymphocytes by anti-CD3 or anti-Ti monoclonal antibody is partially dependent on protein kinase C. J. Immunol., 144 (1990) 2683–2689. [37] D.M. Payne, A.J. Rossomando, P. Martino et al., Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase). EMBO J., 10 (1991) 885 – 892. [38] N. Gomez and P. Cohen, Dissection of the protein kinase cascade by which nerve growth factor activates MAP kinases. Nature, 353 (1991) 170 – 173. [39] S. Matsuda, Y. Gotoh and E. Nishida, Phosphorylation of Xenopus mitogen-activated protein (MAP) kinase kinase by MAP kinase kinase kinase and MAP kinase. J. Biol. Chem., 268 (1993) 3277–3281. [40] H. Kosako, E. Nishida and Y. Gotoh, cDNA cloning of MAP kinase kinase reveals kinase cascade pathways in yeasts to vertebrates. EMBO J., 12 (1993) 787 – 794. [41] R. Seger, N.G. Ahn, J. Posada et al., Purification and characterization of mitogen-activated protein kinase activator(s) from epidermal growth factor-stimulated A431 cells. J. Biol. Chem., 267 (1992) 14 373–14 381. [42] S. Nakielny, P. Cohen, J. Wu and T. Sturgill, MAP kinase activator from insulin-stimulated skeletal muscle is a protein threonine-tyrosine kinase. EMBO J., 11 (1992) 2123 – 2129. [43] C.M. Crews, A. Alessandrini and R.L. Erikson, The primary structure of MEK, a protein kinase that phosphorylates the ERK gene product. Science, 258 (1992) 478 – 480.
G. Gorgas et al. / Mechanisms of Ageing and De6elopment 94 (1997) 71–83
83
[44] C.F. Zheng and K.L. Guan, Cloning and characterization of two distinct human extracellular signal-regulated kinase activator kinases, MEK1 and MEK2. J. Biol. Chem., 268 (1993) 11 435 – 11 439. [45] D.J. Robbins, E. Zhen, H. Owaki et al., Regulation and properties of extracellular signal-regulated protein kinases 1 and 2 in vitro. J. Biol. Chem., 268 (1993) 5097 – 5106. [46] J. Chung, S.L. Pelech and J. Blenis, Mitogen-activated Swiss mouse 3T3 RSK kinases I and II are related to pp44mpk from sea star oocytes and participate in the regulation of pp90rsk activity. Proc. Natl. Acad. Sci. USA, 88 (1991) 4981 – 4985. [47] R.H. Chen, C. Sarnecki and J. Blenis, Nuclear localization and regulation of erk- and rsk-encoded protein kinases. Mol. Cell. Biol., 12 (1992) 915 – 927. [48] T.W. Sturgill, L.B. Ray, E. Erikson and J.L. Maller, Insulin-stimulated MAP-2 kinase phosphorylates and activates ribosomal protein S6 kinase II. Nature, 334 (1988) 715 – 718. [49] L. Nagelkerken, A. Hertogh-Huijbregts, R. Dobber and A. Drager, Age-related changes in lymphokine production related to a decreased number of CD45RBhi CD4 + T cells. Eur. J. Immunol., 21 (1991) 273–281. [50] R.L. Whisler, Y.G. Newhouse and S.E. Bagenstose, Age-related reductions in the activation of mitogen-activated protein kinases p44mapk/ERK1 and p42mapk/ERK2 in human T cells stimulated via ligation of the T cell receptor complex. Cell. Immunol., 168 (1996) 201 – 210. [51] T. Fulop, Jr., C. Leblanc, G. Lacombe and G. Dupuis, Cellular distribution of protein kinase C isozymes in CD3-mediated stimulation of human T lymphocytes with aging. FEBS Lett., 375 (1995) 69–74. [52] K.S. Ravichandran, K.K. Lee, Z. Songyang, L.C. Cantley, P. Burn and S.J. Burakoff, Interaction of Shc with the z chain of the T cell receptor upon T cell activation. Science, 262 (1993) 902 – 905. [53] A. Veillette, M.A. Bookman, E.M. Horak and J.B. Bolen, The CD4 and CD8 T cell surface antigens are associated with the internal membrane tyrosine-protein kinase p56lck. Cell, 55 (1988) 301–308. [54] N. Abraham, M.C. Miceli, J.R. Parnes and A. Veillette, Enhancement of T-cell responsiveness by the lymphocyte-specific tyrosine protein kinase p56lck. Nature, 350 (1991) 62 – 66. [55] E. Gulbins, K.M. Coggeshall, G. Baier, S. Katzav, P. Burn and A. Altman, Tyrosine kinase-stimulated guanine nucleotide exchange activity of Vav in T cell activation. Science, 260 (1993) 822–825. [56] R.A. Miller, Accumulation of hyporesponsive, calcium extruding memory T cells as a key feature of age-dependent immune dysfunction. Clin. Immunol. Immunopath., 58 (1991) 305 – 317.
.