Mechanisms of Ageing and Development
104 (1998) 41 – 58

Effect of age on mitogen induced protein tyrosine
phosphorylation in human T cell and its subsets:
down-regulation of tyrosine phosphorylation of
ZAP-70
B. Chakravarti a,*, D.N. Chakravarti c, J. Devecis a, B. Seshi b,
G.N. Abraham a
a

Clinical Immunology, Allergy and Rheumatology Unit, Department of Medicine and Center on Aging,
Uni6ersity of Rochester Medical Center, 601 Elmwood A6enue, Box 695, Rochester, NY 14642, USA
b
Department of Pathology and Laboratory Medicine, Uni6ersity of Rochester Medical Center,
601 Elmwood A6enue, Box 626, Rochester, NY 14642, USA
c
Department of Protein and Analytical Chemistry, Wyeth-Lederle Vaccines and Pediatrics,
West Henrietta, NY 14586, USA
Received 5 December 1997; received in revised form 8 April 1998; accepted 14 April 1998

Abstract
Several events of T cell activation have been reported to decline in humans with age. Since
protein tyrosine phosphorylation is an early critical event of T cell activation, we performed
a systematic analysis of the age-associated changes in the mitogen induced protein tyrosine
phosphorylation of human T lymphocytes using SDS-PAGE and Western blotting techniques. Following stimulation with Con A and PHA, an identical pattern of protein tyrosine
phosphorylation was observed in the lysates of T cells prepared from seven healthy young
adults and eight healthy elderly human subjects. Five different high molecular mass proteins
(75, 115, 120, 140 and 170 kDa) were consistently tyrosine phosphorylated in all of the
donors from both age groups and peaked between 3 and 10 min. Tyrosine phosphorylation
of the above substrates was observed in both CD4 and CD8 subsets. When compared for
individual donors from both age groups, variations in the T cell response with regard to net
Abbre6iations: ConA, concanavalin A; PHA, phytohemagglutinin; OD, optical density; PBMC,
peripheral blood mononuclear cell; PBST, phosphate buffered saline containing 0.05% Tween-20.
* Corresponding author. Tel.: + 1 716 2737666; fax: +1 716 2737515.
0047-6374/98/$19.00 © 1998 Published by Elsevier Science Ireland Ltd. All rights reserved.
PII S0047-6374(98)00049-9

42

B. Chakra6arti et al. / Mechanisms of Ageing and De6elopment 104 (1998) 41–58

tyrosine phosphorylation for all the substrates was observed. However, the mitogen induced
level of tyrosine phosphorylation of only p75 was found to be signiﬁcantly lower in
unfractionated T cells as well as CD4 and CD8 subsets of older subjects than that of young
subjects. Using immunoblotting, p75 was identiﬁed as ZAP-70, a member of the syk family
of protein tyrosine kinases. Understanding of the biochemical basis of the reduced level of
tyrosine phosphorylation of ZAP-70 will be helpful in delineating the molecular basis of
age-associated impairment of T cell activation. © 1998 Published by Elsevier Science Ireland
Ltd. All rights reserved.
Keywords: Ageing; T cell; Protein tyrosine phosphorylation

1. Introduction
Age-associated impairment of immune function has been reported in human and
animal model systems. Although several studies have indicated that T cell activity
declines with age, its biochemical basis is not yet fully understood. Distal events of
T cell activation e.g. proliferation, IL-2 production and responsiveness show
age-related impairment (Foad et al., 1974; Hung et al., 1975; Hefton et al., 1980;
Miller and Stutman, 1981; Thoman and Weigle, 1981; Gillis et al., 1981; Jonocourt
et al., 1981; Gilman et al., 1982; Thoman, 1985; Cheung et al., 1985; Nagel et al.,
1988). In recent years, signal transduction events leading to the transmission of a
signal from the cell surface to the nucleus have been studied extensively in various
cell systems, and some have indicated that alterations in signal transduction occur
with aging in lymphocytes (Fulop, 1994). Additionally it is suggested that multiple
steps of the signal transduction cascade are defective which may have cumulative
effects on the age-associated decline in immune-potential of the T cells. Different
early events of transmembrane signaling impaired in T lymphocytes isolated from
aged human and/or mice include protein kinase C translocation (Fong and
Makinodan, 1989; Fulop et al., 1995), phosphoinositide metabolism (Di Pietro et
al., 1993) and Ca2 + mobilization (Miller et al., 1987; Lerner et al., 1988). In mice,
studies have documented age dependent changes in the protein phosphorylation
(Patel and Miller, 1992) particularly tyrosine speciﬁc protein phosphorylation in T
cells (Shi and Miller, 1992, 1993; Ghosh and Miller, 1995; Garcia and Miller, 1997).
However, the alteration of immunological functions that occur with aging may
differ between humans and mice and there are only limited data regarding the role
of protein phosphorylation in aging in humans (Quadri et al., 1996; Whisler et al.,
1996). Although mice have been used widely to study immunosenescence since the
conditions of experiments are more easily controlled and well-deﬁned, it is appropriate to delineate the underlying cause of immune dysfunction using human
subjects.
Since protein tyrosine phosphorylation is known to play a critical role in several
important biological events including T cell proliferation (Samelson and Klausner,
1992), we have carried out the present investigation to evaluate the effects of age on
the induction of protein tyrosine phosphorylation in resting human peripheral T

B. Chakra6arti et al. / Mechanisms of Ageing and De6elopment 104 (1998) 41–58

43

cells and T cell subsets by stimulation with ConA and PHA. The data show
constitutive as well as activation-dependent tyrosine phosphorylation of several
proteins. While we observed that stimulation of peripheral T cells with the above
two mitogens leads to increased tyrosine phosphorylation of at least ﬁve different
intracellular substrates (molecular masses between 75 and 170 kDa) in all of the
young and elderly individuals studied, only the net tyrosine phosphorylation of p75,
which has been identiﬁed as ZAP-70, declined signiﬁcantly in T cells from old
subjects compared to that from young subjects. In addition, we observed that for
all the above substrates, different levels of tyrosine phosphorylation was achieved
by different donors from both age groups indicating the heterogeneous nature of
the activation response. The biochemical basis of the age-associated decline in
mitogen induced tyrosine phosphorylation of ZAP-70 is currently under
investigation.

2. Materials and methods

2.1. Reagents
BSA, ConA and PHA were obtained from Sigma Chemical Company (St. Louis,
MO). Biotinylated anti-phosphotyrosine monoclonal antibody (4G10, IgG 2b) and
ZAP-70 were purchased from Upstate Biotechnology, (Lake Placid, NY). Anti-SLP
76 was a gift from Dr Gary Koretzsky, University of Iowa. Streptavidinhorseradish peroxidase conjugate and nitrocellulose membranes were obtained from
Amersham (Arlington Heights, IL). Ficoll-paque was obtained from PharmaciaLKB (Piscataway, NJ). IsoCell Human T cell isolation kit, IsoCell Human CD4
and CD8 isolation kits, SuperSignal CL-HRP substrate system as well as immunopure elution buffer were obtained from Pierce (Rockford, IL). Horseradish peroxidase conjugated goat anti-mouse antibody was obtained from Bio-Rad
Laboratories.

2.2. Isolation of T cells and CD4 and CD8 subsets
PBMCs were isolated from blood of seven healthy young (six B 30 years, one
aged 43 years) and eight healthy elderly ( \ 70 years) donors following centrifugation (400×g) on Ficoll-Paque (Boyum, 1968). The elderly donors were considered
healthy on the basis of clinical evaluation according to the Senieur protocol. No
donor was on any medication preceding or during the study. Initial T cell
enrichment from the PBMCs was carried out by afﬁnity chromatography using the
IsoCell human T cell isolation kit. CD4 and CD8 cells were enriched from a
portion of the enriched T cells by negative selection using the IsoCell Human CD4
and IsoCell human CD8 isolation kits, respectively. The enriched T cells as well as
CD4 and CD8 subsets were adhered at 37°C for 1 h in a humidiﬁed atmosphere of
95% CO2 to remove the monocytes, the non-adherent cells collected, pelleted by
centrifugation at 200× g for 10 min, resuspended in RPMI-1640 and stored on ice

44

B. Chakra6arti et al. / Mechanisms of Ageing and De6elopment 104 (1998) 41–58

until further use. T cells as well as CD4 and CD8 subsets puriﬁed as described
above showed \ 90% purity when analyzed by FACScan.

2.3. Stimulation and lysis of unfractionated T cells as well as CD4 and CD8
subsets
Cells (1 × 106/ml) resuspended in RPMI-1640 were stimulated with ConA (4
vg/ml) or PHA (1 vg/ml) for 3–15 min. The cells (1× 106) were pelleted by
centrifugation (12000× g, 2 min) at 4°C, and lysed immediately by adding 200 vl
hot lysis buffer (100 mM Tris, pH 8.0 containing 4 M urea, 1% SDS, 20 mM DTT
and 2 mM pervanadate) and heated in boiling water bath for 15 min. Lysates were
then centrifuged at 12000×g for 15 min. The supernatant was used immediately or
stored at − 80°C for SDS-PAGE analysis.

2.4. Identiﬁcation and quantitation of phosphotyrosine containing proteins by
SDS-PAGE, Western blotting and densitometric analysis
Phosphotyrosine containing proteins were detected by SDS-PAGE of the lysates
using a gradient of 5-12.5% acrylamide (w/v) with subsequent electrophoretic
transfer to nitrocellulose membranes followed by immunostaining using biotinylated anti-phosphotyrosine antibody (4G10, IgG 2b). The following procedure was
used: the non-speciﬁc binding sites were blocked by immersing the membrane in
PBST containing 5% BSA for 1 h and then washed 3 times for 15 min with PBST.
The membrane was then incubated with biotinylated anti-phosphotyrosine antibody
in PBST containing 1% BSA for 1 h, washed with PBST as described above,
incubated with streptavidin-horseradish peroxidase conjugate in PBST containing
1% BSA for 1 h, and again washed with PBST. Finally, the membrane was
incubated with Super Signal CL-HRP substrate working solution (prepared according to manufacturer’s instruction) for 5 min, dried in air and exposed to autoradiography ﬁlm. All steps were carried out at room temperature. Chemiluminescence
was detected following development of the ﬁlm.
For molecular characterization of a 75 kDa phosphotyrosine containing protein,
the immunoblot developed using anti-phosphotyrosine antibody was stripped of the
immunodetection system by incubating with immunopure elution buffer for 2 h at
room temperature, washed twice with PBST for 10 min/wash, incubated with
substrate as described above and was exposed against ﬁlm to determine if the blot
was sufﬁciently stripped. The above steps were repeated if signal was detected,
otherwise the blot was reprobed with anti-ZAP-70, a non-speciﬁc murine monoclonal antibody or anti-SLP-76 (Jackman et al., 1995) followed by a horseradish
peroxidase conjugated goat anti-mouse antibody (for anti-ZAP-70 and murine
monoclonal antibody) or horseradish peroxidase conjugated rabbit anti-goat antibody (for anti SLP-76) in lieu of the streptavidin-conjugate using the same
procedure. Chemiluminescence was detected by autoradiography.
For detection as well as quantitation of phosphotyrosine proteins, 25–50 vl of
the supernatant obtained from 1.25 ×105 –2.5×105 cells was loaded onto each lane

B. Chakra6arti et al. / Mechanisms of Ageing and De6elopment 104 (1998) 41–58

45

of a gel. Individual lanes of each autoradiograph were scanned using a laser
densitometer (Molecular Dynamics, Sunnyvale, CA). In order to avoid any potential error from differences of exposure time, ﬁlm sensitivity or differential reactivities of different batches of antibodies, each optical density (OD) value was
normalized with respect to the OD of the 82 kDa tyrosine phosphorylated protein
(p82) in the same lane which was used as an internal standard. In preliminary
experiments, the intensity of p82 was not changed by activation with ConA or
PHA. Since a constitutive level of phosphorylation was observed for the proteins of
interest, the net phosphorylation was measured as the difference of normalized OD
of any particular protein obtained from cells incubated with or without (i.e. in
presence of medium only) stimulating agent.

2.5. Statistical analysis
The two age groups were compared using Wilcoxon rank sum test.

3. Results

3.1. Protein tyrosine phosphorylation in T cells following stimulation with ConA
and PHA
Initially we identiﬁed the proteins which were tyrosine phosphorylated after
mitogen induced T cell activation and also determined the level of tyrosine
phosphorylation at different time intervals following stimulation. As evident from
Fig. 1, ﬁve different proteins with molecular masses (75, 115, 120, 140 and 170 kDa)
which were constitutively expressed (i.e. when incubated with medium only), were
consistently tyrosine phosphorylated at high levels at all three time points tested i.e.
3, 10 and 15 min in young as well as old subjects.
In order to determine a time point for study, we compared the rate of induction
of tyrosine phosphorylation of the above substrates by three different donors (two
young and one old). As shown in Fig. 2, the level of induction of tyrosine
phosphorylation of proteins varied among the donors. In general, the majority of
responses to either PHA or ConA occurred at the same time point of stimulation
as shown in Fig. 2, representative full data for two young and one elderly donor
showed occasional differences as to the time of peak response which are not related
to the age of the donor e.g. when stimulated with PHA, one young donor showed
the maximum tyrosine phosphorylation at 10 min and the others (one young and
one elderly) at 3 min for p75. A similar result was also observed for p140 when
stimulated with ConA. Since the maximum level of tyrosine phosphorylation was
short lived and was not the same for all the substrates stimulated by ConA and
PHA (Fig. 2), we compared the initial rate of tyrosine phosphorylation at 3 min for
further studies.

46

B. Chakra6arti et al. / Mechanisms of Ageing and De6elopment 104 (1998) 41–58

3.2. Protein tyrosine phosphorylation in CD4 and CD8 subsets following
stimulation with ConA and PHA
Fig. 3 shows the pattern of tyrosine phosphorylated proteins present constitutively, and those typically accumulated in CD4 and CD8 T cells from both age
groups following 3 min of stimulation with ConA or PHA. Using the antiphosphotyrosine monoclonal antibody, we observed rapid tyrosine phosphorylation of ﬁve
different proteins i.e. p75, p115, p120, p140 and p170 in CD4 as well as CD8
subsets identical to that observed in unfractionated T cells from 15 different donors
belonging to both age groups (seven from healthy young and eight from healthy
elderly individuals). A representative example from young and elderly subjects is
shown in left and right panels respectively of Fig. 3.

3.3. Comparison of net protein tyrosine phosphorylation in response to ConA and
PHA in T cells as well as CD4 and CD8 subsets: effect of aging
Fig. 4 shows normalized OD values for each inducible tyrosine phosphorylated
protein following stimulation of unfractionated T cells as well as CD4 and CD8
subsets from 15 different donors (as described above) with ConA as well as PHA
for 3 min. Isolation and stimulation of unfractionated T cells as well as CD4 and
CD8 subset from an individual donor were done on the same day.
As evident from Fig. 4A, net tyrosine phosphorylation of p75 in unfractionated
T cells following stimulation with ConA (P= 0.004) as well as PHA (P= 0.043)

Fig. 1. Protein tyrosine phosphorylation in unfractionated T cells from a young donor induced by ConA
and PHA at different time intervals. The ﬁgure is a representative example of the protein tyrosine
phosphorylation pattern observed with young as well as elderly individuals. T cells were incubated at
1 × 106/ml without or with ConA (4 vg/ml) as well as PHA (1 vg/ml) for 3, 10 and 15 min at 37°C. Lane
1, 2, and 3 from each time point represent SDS-PAGE analysis of post-nuclear supernatant made from
the lysed T cells stimulated in presence of medium, ConA and PHA respectively (for details, see Section
2). The proteins which were consistently phosphorylated in all the donors above the constitutive level
following stimulations are indicated by solid arrows on the right whereas p82, used as an internal
standard for quantitation is indicated by a solid line. The molecular weight standards are shown by solid
arrow on the left. The estimated molecular masses of the tyrosine phosphorylated proteins are indicated
on the right. DF represents the dye front.

B. Chakra6arti et al. / Mechanisms of Ageing and De6elopment 104 (1998) 41–58

47

Fig. 2. Estimation of tyrosine speciﬁc phosphorylation of p75, p115, p120, p140 and p170 in response
to ConA and PHA at different time intervals by three different donors (young donor 1, – 2 – ; young
donor 2, – 
 –; old donor 1, –  –). Tyrosine phosphorylation of any particular protein was estimated
as the difference of its OD (normalized to p82) between the lysate of stimulated cells and unstimulated
cells. For details, see Section 2.

48

B. Chakra6arti et al. / Mechanisms of Ageing and De6elopment 104 (1998) 41–58

Fig. 3. Effect of age on tyrosine speciﬁc phosphorylation of proteins. Left and right hand panels are
representative SDS-PAGE proﬁles of tyrosine phosphorylated proteins in unfractionated T cells (represented as CD3 in the ﬁgure) as well as in CD4 and CD8 T cell subsets obtained from young and old
subjects respectively. The cells were incubated at 1 ×106/ml without or with ConA (4 vg/ml) as well as
PHA (1 vg/ml) for 3 min at 37°C. Lanes 1, 2, and 3 from each set represent tyrosine phosphorylated
proteins present in the post-nuclear supernatant made from the lysed T cells stimulated in presence of
medium, Con A and PHA respectively (for details, see Section 2). Only those proteins which were
consistently phosphorylated in all the donors above the constitutive level following stimulations are
indicated by solid arrows on the right whereas p82, used as an internal standard for quantitation is
indicated by a solid line. The molecular weight standards are shown by solid arrows on the left. DF
represents the dye front.

was found to be signiﬁcantly lower in old subjects than young subjects. For p115,
p120, p140 and p170, the net tyrosine phosphorylation in T cells of old subjects was
not statistically different from young subjects (Fig. 4(B–E)). Similar results were
also obtained with CD4 as well as CD8 subsets with regard to tyrosine phosphorylation. As evident from Fig. 4A, a signiﬁcant reduction in net tyrosine phosphorylation of p75 in CD4 as well as CD8 subsets were noted in the elderly subjects
following stimulation with ConA (CD4: P= 0.008 and CD8: P= 0.002) or PHA
(CD4: P =0.024 and CD8: P = 0.002). However, in case of other mitogen inducible
tyrosine phosphorylated proteins, the net tyrosine phosphorylation of the older
subjects was not statistically different from young subjects (Fig. 4(B–E)) in either
T cell subset. From these results, it is concluded that age related decline in the
mitogen induced level of tyrosine phosphorylation most consistently involves the
p75 intracellular substrate in stimulated T cells and both the CD4 and CD8 subsets.

3.4. Molecular characterization of p75
In order to determine whether p75 was a tyrosine phosphorylated substrate that
has been previously shown to play an important role in early events of T cell
activation, the immunoblots were treated with antibodies speciﬁc for ZAP-70 and
SLP-76-speciﬁc as well as a non-speciﬁc murine monoclonal antibody. Immunoblotting of resting as well as stimulated T cell lysate using anti-phosphotyrosine and anti-ZAP-70 antibodies (for details, see Section 2) characterized p75 as
ZAP-70 (Fig. 5) while anti-SLP-76 and the non-speciﬁc murine monoclonal antibody failed to react with p75.

B. Chakra6arti et al. / Mechanisms of Ageing and De6elopment 104 (1998) 41–58

49

4. Discussion
Although phosphorylation of tyrosine residues accounts for only a small percentage of the total phosphate associated with proteins in vivo (Hunter and Cooper,
1985, 1986), it plays a key role in the regulation of cell proliferation and differentiation. In the present study, we observed ﬁve different constitutively expressed
cellular substrates with molecular masses 75, 115, 120, 140 and 170 kDa that were
consistently tyrosine phosphorylated following mitogen stimulation of unfraction-

Fig. 4. Effect of age on tyrosine speciﬁc protein phosphorylation in unfractionated T cells (represented
as CD3 in the ﬁgure) as well as CD4 and CD8 subsets. Con A and PHA were used as the stimulating
agents. Tyrosine phosphorylation of any particular protein was estimated as the difference of its OD
(normalized to p82) between the lysate of stimulated cells and unstimulated cells. The geometric mean
values are represented by squares. To obtain the geometric mean value of phosphorylation of any
protein by a particular group of donors where some of the donors showed dephosphorylation, the
negative OD value was substituted by a low OD value (0.001). Y and O represent young and old donors,
respectively.

50

B. Chakra6arti et al. / Mechanisms of Ageing and De6elopment 104 (1998) 41–58

Fig. 4. (continued)

ated T cells and the CD4 and CD8 subsets from all 15 donors. Although other
tyrosine phosphorylated proteins were present in the T cells of both young and old
subjects (Figs. 1 and 3), they were either not detected in all the donors, remained
unchanged, or changed only marginally following stimulation, irrespective of the
age of the donors. Further, the data demonstrate that there is an age-dependent
decline in the tyrosine phosphorylation of at least one protein, p75 in all cell
samples. Using immunoblotting, p75 was identiﬁed as ZAP-70 and p82, used as
internal standard, as SLP-76 (data not shown). Since we observed at least partial
phosphorylation of all the above substrates, it is apparent that transmembrane
signal transduction is preserved in all donors, regardless of age.
Previous studies on tyrosine phosphorylation in human T cells have involved
both T cell lines and freshly isolated peripheral T cells but thus far no such studies
on the human T cell subsets have been reported. Stimulation of peripheral T cells
with PHA has indicated rapid phosphorylation of a 42 kDa tyrosine and serine
phosphorylated protein (Mire et al., 1986) and a 66 kDa tyrosine phosphorylated

B. Chakra6arti et al. / Mechanisms of Ageing and De6elopment 104 (1998) 41–58

51

Fig. 4. (continued)

protein in human T lymphocytes (Wender and Bass, 1986) in presence of [k32
P]ATP. During the present study while a 66 kDa tyrosine phosphorylated protein
was observed in resting T cells from all the donors, its level was constant or
changed marginally following stimulation (data not shown) and a mitogen inducible
42 kDa tyrosine phosphorylated protein was observed only with limited number of
donors. On the other hand, using antiphosphotyrosine monoclonal antibodies, a
consistent pattern of tyrosine phosphorylation of high molecular mass proteins
(75– 170 kDa) was always noted following mitogenic stimulation (Figs. 1 and 3) of
unfractionated T cells as well as CD4 and CD8 subsets. Tyrosine phosphorylation
of several high molecular mass proteins has been observed following ligation of
different T cell surface molecules e.g. TCR, CD3, and CD28 (Jackman et al., 1995;
Lahesmaa et al., 1995). Ligation of VLA-4 of human T cells stimulates tyrosine
phosphorylation of several high molecular mass substrates e.g. 70, 105, 125 and 140
kDa (Sato et al., 1995) that may be similar to those observed in this study.
Molecular characterization of these mitogen activated tyrosine phosphorylated

52

B. Chakra6arti et al. / Mechanisms of Ageing and De6elopment 104 (1998) 41–58

Fig. 4. (continued)

proteins and precise deﬁnition of their roles in the signal transduction pathway
should provide further information on the biochemical pathways involved in the
early phase of T cell activation.
As evident from Fig. 1 and Fig. 2, we observed tyrosine speciﬁc phosphorylation
within ﬁrst 3 min which is consistent with observations obtained by others with
human and murine T cells (June et al., 1990; Patel and Miller, 1992). We also
observed that maximum tyrosine speciﬁc phosphorylation was attained between 3
and 10 min and in most cases did not remain at the peak level for a prolonged
period of time since dephosphorylation was usually observed at 15 min or earlier.
This is in accordance with the reports of June et al. (1990) of rapid phosphorylation
and dephosphorylation of tyrosine phosphorylated proteins of molecular masses
135 and 100 kDa following stimulation with anti-TCR antibodies. Since the
maximum level of phosphorylation was short lived (Fig. 2), we compared the initial
rate of phosphorylation at 3 min following stimulation. Although in the initial

B. Chakra6arti et al. / Mechanisms of Ageing and De6elopment 104 (1998) 41–58

53

Fig. 4. (continued)

studies using different cellular substrates, phosphorylation was noted within 3 min
(Fig. 2 (A – E)), occasionally absence of phosphorylation or even dephosphorylation
of some of the substrates by a very limited number of subjects from both age groups
was observed and has been shown as zero or negative phosphorylation respectively
in Fig. 4(A – E). However, a longer incubation period always led to phosphorylation
of these substrates by the same donors (data not shown) which suggests that their
tyrosine phosphorylation abilities were not totally impaired. Fig. 4(A–E) also shows
that there is a wide variation among different donors from both age groups with
respect to their tyrosine phosphorylation abilities, i.e. donors may be low,
medium or high responder. Hence, it is quite possible that in spite of substantial
decline in phosphorylation abilities of high responders with age, the phosphorylation abilities of these subjects can remain higher than for young low responders.
Such a dilemma can only be solved by a longitudinal study of phosphorylation
abilities of individual donors at multiple points over a prolonged period of time.

54

B. Chakra6arti et al. / Mechanisms of Ageing and De6elopment 104 (1998) 41–58

Fig. 5. Molecular characterization of p75. The left hand panel represents the tyrosine phosphorylated
proteins present in the post-nuclear supernatant made from unstimulated T cells (lane 1) as well as those
stimulated in presence of Con A (lane 2) and PHA (lane 3). The right hand panel represents the
development of the same membrane with anti-ZAP-70 subsequent to removal of antiphosphotyrosine
antibody (for details, see Section 2). The presence of ZAP-70 in the post-nuclear supernatant made from
unstimulated T cells as well as those stimulated in presence of ConA and PHA is indicated in lanes 4,
5 and 6 respectively.

We also observed that the same donor showed a differential ability with respect to
phosphorylation of a different substrate, i.e. the same high responder donor for one
substrate may be a high, medium or poor responder for another substrate (data not
shown). While the exact cause of the variable abilities among different donors is not
clear, the host genetic background or nutritional status may be possible contributing factors. Further studies are required to correlate such factors to this critical T
cell activation event.
As evident from Fig. 4, tyrosine phosphorylation of p75 was signiﬁcantly reduced
in the old as compared to the young subjects. However tyrosine phosphorylation
levels of p115, p120, p140 and p170 were not statistically different between the
young and old age groups. Quadri et al. (1996) have observed age-associated defects
in tyrosine phosphorylation of several intracellular substrates in unfractionated T
cells following stimulation of different cell surface receptors. However, molecular
identiﬁcation of any of these substrates was not evident from their study. Neither
was it evident whether the observed defects were speciﬁc for CD4 or CD8 subset or
not. Our present data clearly indicate tyrosine phsophorylation of ZAP-70, a key
enzyme in the signal transduction pathway, is down-regulated due to aging. Also
the defect was observed in both CD4 and CD8 subsets indicating possible impairment of immune activation in both subsets due to aging. Unlike human, Shi and
Miller (1992) have indicated that aging impaired tyrosine phosphorylation of all of
the mitogen inducible tyrosine phosphorylated proteins in puriﬁed splenic T cells
and a differential effect was observed in the CD4 and CD8 T cell subsets (Shi and

B. Chakra6arti et al. / Mechanisms of Ageing and De6elopment 104 (1998) 41–58

55

Miller, 1993). Among three of the tyrosine phosphorylated proteins (120, 80 and 40
kDa), impairment of tyrosine phosphorylation of all three substrates was observed
in the CD8 T cells, but only of 80 kDa in the CD4 T cells. In addition, unlike mice,
in the present study, the induced levels of all the above tyrosine phosphorylated
proteins of CD4 and CD8 subsets from same age group showed no statistical
difference (data not shown).
The basal level of tyrosine phosphorylation of ZAP-70 did not vary signiﬁcantly
in unfractionated T cell or its subsets isolated from two different age group donors
(data not shown; for CD3: P = 0.82; CD4: P= 0.64; CD8: P= 0.07). The geometric
mean values of net tyrosine phosphorylation (measured as a ratio of OD with
respect to p82, for details see Section 2), of young and old donors were—CD3: 0.05
and 0.04 respectively; CD4: 0.03 and 0.05 respectively; CD8: 0.03 and 0.09
respectively. Similar results, i.e. no statistically signiﬁcant difference in the basal
level of tyrosine phosphorylation of p170, p140, p120 and p115 were observed in
unfractionated T cells as well as CD4 and CD8 subsets isolated from two different
age group donors (data not shown). High basal level of tyrosine phosphorylation of
the above proteins observed with an old donor (Fig. 3, right) was rather unique and
not the characteristic of any other donor from the same age group.
Three different protein tyrosine kinases, fyn, lck (belonging to src family protein
kinase) and ZAP-70 (belonging to syk family protein kinase) play crucial roles in
early events of T cell activation (Chan et al., 1994). The present investigation clearly
indicates that there is an age-related down-regulation of the level of mitogen
induced tyrosine phosphorylation of ZAP-70 (c.f. Fig. 5). While the role of ZAP-70
in the signal transduction pathway following TCR activation is well established,
temporal course of interaction of this protein tyrosine kinase with other molecules
of the signaling cascade is not fully understood. It is not clear from the present
study how the decline in tyrosine phosphorylation of ZAP-70 can affect signaling
events which will take place following mitogenic stimulation of T cells. However, it
can be speculated that the mechanism by which this will affect the T cell activation
will depend on the location of the defective tyrosine phosphorylation site in ZAP-70
molecule, i.e. whether the defect is in the kinase domain (tyr 492 and 493), the src
homology two domain (tyr 69 and 178) and/or the autophosphorylation sites (tyr
126 and 292). These eventually may lead to altered catalytic activity of ZAP-70 and
lck as well as impaired association of ZAP-70 with lck. All these will ultimately
affect the activation of T cells (Duplay et al., 1994; Watts et al., 1994; Wange et al.,
1995).
While the total amount of ZAP-70 represents tyrosine phosphorylated+ non-tyrosine phosphorylated form of the enzyme, it is the tyrosine phosphorylated
ZAP-70, which we have measured, plays a crucial role in the context of T cell
activation. It appears that there are several possibilities which may lead to the
observed decline of the mitogen induced level of tyrosine phosphorylation of
ZAP-70. It is well established that the function of a protein can be modulated by
reversible phosphorylation and the state of phosphorylation of a protein reﬂects the
relative activities of its kinases and phosphatases. Hence, we anticipate a possible
alteration in the balance of protein tyrosine kinases, e.g. fyn and lck as well as

56

B. Chakra6arti et al. / Mechanisms of Ageing and De6elopment 104 (1998) 41–58

phosphatase, e.g. CD45 activities regulating the level of phosphorylation of this
particular protein. Since we did not observe synchronous down regulation of
phosphorylation of all ﬁve substrates to a signiﬁcant level, it is possible that these
substrates are under the control of different intracellular kinases and/or phosphatases. Apart from the possible defects of kinase and/or phosphatase activities,
the observed decline of tyrosine phosphorylation of ZAP-70 may be due to its lower
expression and/or stability with age. Further studies are in progress in our laboratory to identify the underlying biochemical basis of the current observation.

Acknowledgements
This work was supported by Rochester area Pepper Center Grant P60 AG10463,
and the James P. Wilmot foundation (GNA). The authors would like to thank Prof.
Richard Miller for helpful discussion.

References
Boyum, A., 1968. Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin.
Lab. Invest. 21, 77–89.
Chan, A.C., Desai, D.M., Weiss, A, 1994. The role of protein tyrosine kinases and protein tyrosine
phosphatases in T cell antigen receptor signal transduction. Ann. Rev. Immunol. 12, 555 – 592.
Cheung, H.T., Wu, W.T., Pahlavani, M., Richardson, A., 1985. Effect of age on the interleukin-2
messenger RNA level. Fed. Proc. 44, 573.
Di Pietro, R., Rana, R.A., Sciscio, A., Marmiroli, S., Billi, A.M., Cataldi, A., Cocco, L., 1993.
Age-related events in human active T lymphocytes: changes in the phosphoinositidase C activity.
Biochem. Biophys. Res. Commun. 194, 566 – 570.
Duplay, P., Thome, M., Herve, F., Acuto, O., 1994. p56 lck interacts via its src homology 2 domain with
the ZAP-70 kinase. J. Exp. Med. 179, 1163 – 1172.
Foad, B.S., Adams, L.E., Yamaguchi, Y., Litwin, A., 1974. Phytomitogen responses of peripheral blood
lymphocytes in young and older subjects. Clin. Exp. Immunol. 17, 657 – 664.
Fong, T.C., Makinodan, T., 1989. Preferential enhancement by 2-mercaptoethanol of IL-2 responsiveness of T blast cells from old over young mice is associated with potentiated protein kinase C
translocation. Immunol. Lett. 20, 149 – 154.
Fulop, T., 1994. Signal transduction changes in granulocytes and lymphocytes with ageing. Immunol.
Lett. 40, 259–268.
Fulop, T., Leblanc, C., Lacomb, G., Dupuis, G., 1995. Cellular distribution of protein kinase C
isozymes in CD3-mediated stimulation of human T lymphocytes with aging. FEBS Lett. 375, 69 – 74.
Garcia, G.G., Miller, R.A., 1997. Differential tyrosine phosphorylation of zeta chain dimers in mouse
CD4 T lymphocytes: effect of age. Cell. Immunol. 175, 51 – 57.
Ghosh, J., Miller, R.A., 1995. 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 Dev. 80,
171–187.
Gillis, S., Kozak, R., Durante, M., Weksler, M.E., 1981. Immunological studies of aging: decreased
production of and response to T cell growth factor by lymphocytes from aged humans. J. Clin.
Invest. 67, 937–942.
Gilman, S.C., Rosenberg, J.S., Feldman, J.D., 1982. T lymphocytes of young and aged rats. II.
Functional defects and the role of interleukin-2. J. Immunol. 128, 644 – 655.

B. Chakra6arti et al. / Mechanisms of Ageing and De6elopment 104 (1998) 41–58

57

Hefton, J.M., Darlington, G.J., Cassazza, B.A., Weksler, M.E., 1980. Immunologic studies of aging. V.
Impaired proliferation of PHA responsive human lymphocytes in culture. J. Immunol. 125, 1007 –
1010.
Hung, C.Y., Perkins, E.H., Yang, W.K., 1975. Age-related refractoriness of PHA induced lymphocyte
transformation. II. 125I-PHA binding to spleen cells from young and old mice. Mech. Ageing Dev.
4, 103–112.
Hunter, T., Cooper, J.A., 1985. Protein tyrosine kinases. Ann. Rev. Biochem. 54, 897 – 930.
Hunter, T., Cooper, J.A., 1986. In: Boyer, P.D., Krebs, E.G. (Eds.), The Enzymes, vol. 17. Academic,
Press, Orlando, pp. 192–248
Jackman, J.K., Motto, D.G., Sun, Q., Tanemoto, M., Turck, C.W., Peltz, G.A., Goretzky, G.A.,
Findell, P.R., 1995. Molecular cloning of SLP-76, a 76-kDa tyrosine phosphoprotein associated with
Grb2 in T cells. J. Biol. Chem. 270, 7029 – 7032.
Jonocourt, F., Bettens, F., Krisktensen, F., DeWeck, A.L., 1981. Age-related changes of mitogen
responsiveness in different lymphoid organs from outbred NMRI mice. Immunobiology 158,
439–449.
June, C.H., Fletcher, M.C., Ledbetter, J.A., Samelson, L.E., 1990. Increases in tyrosine phosphorylation
are detectable before phospholipase C activation after T cell receptor stimulation. J. Immunol. 144,
1591–1599.
Lahesmaa, R., Allsup, A., Soderberg, C., Jackman, J., Findell, P., Peltz, G.A., 1995. Modulation of the
Grb2-associated protein complex in human CD4 + T cells by receptor activation. J. Immunol. 155,
3815–3822.
Lerner, A., Philosophe, B., Miller, R.A., 1988. Defective calcium inﬂux and preserved phosphate
generation in T cells from old mice. Aging Immunol. infect. Dis. 1, 149 – 158.
Miller, R.A., Stutman, O., 1981. Decline, in aging mice of the anti-2,4,6 trinitrophenyl (TNP) cytotoxic
T cell response attributable to loss of Lyt2-, interleukin-2 producing helper cell function. Eur. J.
Immunol. 11, 751–756.
Miller, R.A., Jacobson, B., Weil, G., Simons, E.R., 1987. Diminished calcium inﬂux in lectin-stimulated
T cells from old mice. J. Cell. Physiol. 1323, 337 – 342.
Mire, A.R., Wickremasinghe, G., Hoffbrand, A.V., 1986. Mitogen treatment of permeabilized human T
lymphocytes stimulates rapid tyrosine and serine phosphorylation of a 42 kDa protein. FEBS Lett.
206, 53–58.
Nagel, J.E., Chopra, R.K., Chrest, F.J., McCoy, M.T., Schneider, E.L., Holbrook, N.J., Adler, W.H.,
1988. Decreased proliferation, interleukin 2 synthesis, and interleukin 2 receptor expression are
accompanied by decreased mRNA expression in phytohemagglutinin-stimulated cells from elderly
donors. J. Clin. Invest. 81, 1096–1102.
Patel, H.R., Miller, R.A., 1992. Age-associated changes in mitogen-induced protein phosphorylation in
murine T lymphocytes. Eur. J. Immunol. 22, 253 – 260.
Quadri, R.A., Plastre, O., Phelouzat, M.-A., Arbogast, A., Proust, J.J., 1996. Age-related tyrosine
speciﬁc protein phosphorylation defect in human T lymphocytes activated through CD3, CD4, CD8
or the IL-2 receptor. Mech. Ageing Dev. 88, 125 – 138.
Samelson, L.E., Klausner, R.D., 1992. Tyrosine kinase and tyrosine based activation motifs. Current
research on activation via the T cell antigen receptor. J. Biol. Chem. 267, 24913 – 24916.
Sato, T., Tachibana, K., Nojima, Y., D’Avirro, N., Marmot, C., 1995. Role of VLA-4 molecule in T
cell. Identiﬁcation of the tyrosine phosphorylation pattern induced by the ligation of VLA-4. J.
Immunol. 155, 2938–2947.
Shi, J., Miller, R.A., 1992. Tyrosine-speciﬁc protein phosphorylation in response to anti-CD3 antibody
is diminished in old mice. J. Gerontol. 47, B147 – 153.
Shi, J., Miller, R.A., 1993. Differential tyrosine-speciﬁc protein phosphorylation in mouse T lymphocyte
subsets. Effect of age. J. Immunol. 151, 730 – 739.
Thoman, M.L., 1985. Role of interleukin-2 in the age-related impairment in immune function. J. Am.
Ger. Soc. 33, 781–787.
Thoman, M.L., Weigle, W.O., 1981. Lymphokines and aging: interleukin-2 production and activity in
aged animals. J. Immunol. 127, 2102– 2106.

58

B. Chakra6arti et al. / Mechanisms of Ageing and De6elopment 104 (1998) 41–58

Wange, R.L., Guitian, R., Isakov, N., Watts, J.D., Abersold, R., Samelson, L.E., 1995. Activating and
inhibitory mutations in adjacent tyrosines in the kinase domain of ZAP-70. J. Biol. Chem. 270,
18730–18733.
Watts, J.D., Affolter, M., Krebs, D.L., Wange, R.L., Samelson, L.E., Aebersold, R., 1994. Identiﬁcation
by electrospray ionization mass spectrometry of the sites of tyrosine phosphorylation induced in
activated Jurkat T cells on the protein tyrosine kinase ZAP-70. J. Biol. Chem. 269, 29520 – 29529.
Wender, H., Bass, G., 1986. Tyrosine phosphorylation of a 66 000 Mr soluble protein in lectin-activated
human peripheral blood T lymphocytes. J. Immunol. 136, 4226 – 4231.
Whisler, R.L., Newhouse, Y.G., Bagenstose, S.E., 1996. 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, 201 – 210.

.

