(Received for publication, August 25, 1995; and in revised form, October 9, 1995)
From the
The signaling molecules insulin receptor substrate (IRS)-1 and the newly described IRS-2 (4PS) molecule are major insulin and interleukin 4 (IL-4)-dependent phosphoproteins. We report here that IL-2, IL-7, and IL-15, as well as IL-4, rapidly stimulate the tyrosine phosphorylation of IRS-1 and IRS-2 in human peripheral blood T cells, NK cells, and in lymphoid cell lines. In addition, we show that the Janus kinases, JAK1 and JAK3, associate with IRS-1 and IRS-2 in T cells. Coexpression studies demonstrate that these kinases can tyrosine-phosphorylate IRS-2, suggesting a possible mechanism by which cytokine receptors may induce the tyrosine phosphorylation of IRS-1 and IRS-2. We further demonstrate that the p85 subunit of phosphoinositol 3-kinase associates with IRS-1 in response to IL-2 and IL-4 in T cells. Therefore, these data indicate that IRS-1 and IRS-2 may have important roles in T lymphocyte activation not only in response to IL-4, but also in response to IL-2, IL-7, and IL-15.
Interleukin 2 (IL-2) ()and IL-4 are lymphokines that
have potent mitogenic effects on peripheral blood
lymphocytes(1, 2) . These cytokines also have a number
of overlapping biochemical effects. They bind a common hematopoietin
receptor subunit termed
c (3, 4) and activate
Janus family protein-tyrosine kinases JAK1 and
JAK3(5, 6) . After IL-2-induced activation of these
protein-tyrosine kinases, tyrosine phosphorylation of many signaling
components occurs, including the receptor proteins
themselves(7) , the adaptor molecule Shc(8) , STAT
(signal transducers and activators of transcription)
proteins(9, 10, 11, 12) , and
phosphatidylinositol 3-kinase (PI 3-kinase)(13) .
Binding of IL-2 to its receptor results in rapid tyrosine phosphorylation of the receptor cytoplasmic domain(7) . A number of SH2-containing signaling molecules such as the adaptor molecule Shc and the STATs are thought to bind directly to tyrosine residues on the intracellular regions of the receptor(10, 14) . IL-2 also activates PI 3-kinase, but it is not clear whether this substrate binds to the IL-2 receptor. In contrast, it is clear that the IL-4 receptor can recruit PI 3-kinase via the tyrosine phosphorylation of the cytosolic docking protein IRS-1 or the newly cloned IRS-2 (4PS) molecule(15, 16, 17) . These large cytosolic docking molecules have many phosphotyrosine residues which provide binding sites for important SH2-containing molecules such as Crk, Grb-2, Nck, PI 3-kinase, and SH-PTP2 to the receptor complex (18, 19, 20, 21) .
The
phosphorylation of IRS-1 is thought to be important for glucose
transport (22) and for both insulin- and IL-4-induced
proliferation (23) . Indeed, 32D cells expressing an IL-4
receptor mutant that does not phosphorylate IRS-1 will not proliferate
in response to IL-4(24) . Collectively, these data suggest that
IRS-1 and perhaps IRS-2 play an important role in the signaling
response to growth factors such as insulin and IL-4. More recently,
other cytokines and growth factors such as IFN, IFN
, IL-9,
and leukocyte inhibitory factor have also been shown to induce the
tyrosine phosphorylation of
IRS-1(25, 26, 27, 28, 29) .
These reports suggest that IRS-1 may be an important signaling
component utilized by many growth factors.
Although IL-2 signal transduction has been investigated extensively, IL-2 has not been reported to induce phosphorylation of IRS-1 or IRS-2. Therefore, it was important to assess whether IL-2 could induce phosphorylation of IRS-1 and IRS-2 and thus provide a means of explaining PI 3-kinase recruitment in response to IL-2. We report here that IL-2, IL-7, and IL-15, as well as IL-4, rapidly stimulate the tyrosine phosphorylation of IRS-1 and IRS-2 in human T cells. In addition, we show that the Janus kinases JAK1 and JAK3 can associate with and tyrosine-phosphorylate IRS-1 and IRS-2. Moreover, we demonstrate a rapid IL-2-induced association of PI 3-kinase with IRS-1. These results suggest that IRS-1 and IRS-2 may have significant roles in T lymphocyte activation in response to not only IL-4 but also IL-2, IL-7, and IL-15.
Recombinant
human IL-2 was kindly provided by Cetus Oncology Corp. IL-4 and IL-7
were obtained from PeproTech (Rocky Hill, NJ). IL-12 was received from
Dr. Stanley Wolf (Genetics Institute, Cambridge MA). IL-15 was obtained
from Immunex (Seattle, WA) and R & D Systems (Minneapolis, MN).
IFN was obtained from Hoffman-La Roche Inc. Monoclonal anti-JAK1
was obtained from Transduction Laboratories (Lexington, KY). Rabbit
polyclonal anti-JAK1, JAK2 antibodies, and monoclonal
antiphosphotyrosine, 4G10, were purchased from Upstate Biotechnology
Industries (Lake Placid, NY). Polyclonal rabbit anti-JAK3 was made in
our laboratory(30) , rabbit anti-IRS-1 and anti-IRS-2 sera were
made against recombinant baculovirus-purified IRS-1 protein and against
a unique COOH-terminal murine IRS-2(23) .
Figure 1: Tyrosine phosphorylation of IRS-1 and IRS-2 by IL-2, IL-4, IL-7, and IL-15. A, IL-2 and IL-4 induce tyrosine phosphorylation of IRS-1 in T cells. PHA-activated T cells were treated with IL-2, IL-4, or IL-12 for 5 or 15 min, lysed, and immunoprecipitated with anti-IRS-1 and immunoblotted with antiphosphotyrosine. B, IL-2 and IL-4 induce IRS-1 and IRS-2 tyrosine phosphorylation in T cells. T cells either unstimulated or treated with IL-2 or IL-4 for 15 min, immunoprecipitated with anti-IRS-1 or anti-IRS-2 as indicated, and immunoblotted with antiphosphotyrosine. C, IL-2 and IL-4 phosphorylate IRS-1 Natural Killer (NK) cells; D, the NK 3.3 cell line. NK and NK 3.3 cells were untreated or treated with IL-2, IL-4, or IL-12 for 15 min, were lysed and immunoprecipitated with anti-IRS-1 antisera. E, T cells were treated with IL-2, IL-4, IL-7, and IL-15, immunoprecipitated with anti-IRS-1, and blotted with antiphosphotyrosine.
Recently, the IRS-1 homologue, IRS-2 (4PS), has been cloned from myeloid cells(17) . IRS-1 and IRS-2 exhibit considerable overall sequence identity and can be used interchangeably by both insulin and IL-4 to mediate mitogenesis in the 32D cell system(17, 23) . We therefore examined whether IL-2 could also induce the tyrosine phosphorylation of IRS-2. Fig. 1B shows that tyrosine phosphorylation of both IRS-1 and IRS-2 was detected in response to IL-2 and IL-4 in human T cells. The antibodies used to identify IRS-1 and IRS-2 are specific for each of the molecules; this is confirmed since these two proteins can be distinguished by their migration on SDS-PAGE. As shown in Fig. 1B, consistent with the predicted molecular masses, IRS-2 (180 kDa) migrates slower than IRS-1 (165 kDa) (17) . To confirm that equivalent levels of IRS-1 and IRS-2 were immunoprecipitated, the filter was reblotted with the respective antibodies, and the levels were identical in each lane (results not shown).
IL-2 is an important regulator of Natural Killer (NK) cell and B cell function. We therefore examined whether IL-2-dependent IRS-1 tyrosine phosphorylation could be detected in human peripheral blood NK cells or B cells. Increased IRS-1 phosphorylation was readily observed in response to IL-2 and IL-4 in NK cells, but again IL-12 did not induce tyrosine phosphorylation of IRS-1 in these cells (Fig. 1C). Marked increases in the levels of IRS-1 phosphotyrosine were also observed in the NK cell line, NK 3.3, in response to IL-2 and IL-4 treatment (Fig. 1D). IL-2-induced IRS-1 phosphorylation was also observed in an Epstein Barr virus-transformed B cell line from a normal donor and, in the T cell line, Kit 225 (results not shown). These data further suggest that IL-2-induced phosphorylation of IRS-1 may be an important signaling event in all human lymphocyte subpopulations.
IL-7 and IL-15 also
bind the IL-2 receptor c subunit and activate JAK1 and JAK3 in T
cells(12, 33) . We therefore examined whether tyrosine
phosphorylation of IRS-1 would occur in response to IL-7 and IL-15. T
cells were treated with IL-2, IL-4, IL-7 (100 units/ml), and IL-15
(5000 units/ml), lysed, and immunoprecipitated as above. As with IL-2
and IL-4, tyrosine phosphorylation of IRS-1 was detected in response to
IL-7 and IL-15 in T cells (Fig. 1E). These findings
establish that tyrosine phosphorylation IRS-1 occurs in T cells in
response not only to IL-4 but also to IL-2, IL-7, and IL-15.
Figure 2:
IRS-1 and IRS-2 associate with JAK1 and
JAK3. A, PHA-activated T cells were untreated or treated with
IL-2 (1000 units/ml) or IL-15 (5000 units/ml) for 15 min, lysed, and
immunoprecipitated with anti-IRS-1, and blotted with
antiphosphotyrosine (upper panel) and anti-JAK3 (lower
panel). B, T cells were untreated or treated with IL-2
(1000 units/ml) or IL-4 (100 units/ml) for 15 min, lysed, and
immunoprecipitated with anti-IRS-1 or anti-IRS-2 and blotted with
anti-JAK1. C, IRS-2 alone or IRS-2 with c, JAK3, or JAK1
was expressed in Cos cells and lysates from these cells were
immunoprecipitated with IRS-2, resolved on SDS-PAGE, and blotted for
phosphotyrosine.
To determine if JAK1 or JAK3 could interact directly with the IRS
molecules and to examine whether these Janus kinases could induce
tyrosine phosphorylation of the IRS proteins, we co-expressed IRS-2
with the Janus family kinases in Cos cells and analyzed the IRS-2
immunoprecipitates. IRS-2 did not exhibit basal tyrosine
phosphorylation when expressed alone or with the IL-2 receptor chain
c (Fig. 2C, lanes 1 and 2,
respectively). In contrast, IRS-2 tyrosine phosphorylation was readily
detected in cells expressing IRS-2 in combination with JAK1 or JAK3 (Fig. 2C, lanes 3 and 4,
respectively). Tyrosine-phosphorylated JAK1 and JAK3 were detected in
the IRS-2 immunoprecipitates from these cells indicating again that
these kinases associate with IRS-2 (Fig. 2C).
Interestingly, in cells expressing JAK1, higher levels of IRS-2
tyrosine phosphorylation were observed than in cells expressing JAK3,
even though JAK1 and JAK3 were expressed at comparable levels (data not
shown). Likewise, higher levels of JAK1 protein were observed in the
IRS-2 immunoprecipitates. Taken together, these data indicate that
IRS-1 and IRS-2 can interact directly with the JAKs and may be
substrates for these kinases, suggesting that JAK/IRS association may
be important for IRS-1 and IRS-2 tyrosine phosphorylation in response
to IL-2 and IL-4.
Figure 3: IL-2 and IL-4 induce association of p85 with IRS-1. T cells were unstimulated or stimulated with IL-2 or IL-4 for 5 or 15 min, lysed, and immunoprecipitated with anti-p85.
The recent identification and cloning of the IRS-1-like molecule, IRS-2, has confirmed that both proteins exhibit substantial structural homology and indicates that they may have similar functional roles (17) . Although, mice lacking IRS-1 have a reduced ability to uptake glucose and are diminished in size, they are otherwise normal(22, 37) , suggesting that IRS-2 may compensate for the absence of IRS-1 in many signaling pathways. Also, data reported from 32D cells showing that IRS-1 and IRS-2 can be used interchangeably by IL-4 and insulin indicate that they have similar roles in mediating a mitogenic response (35) . Our data establish that IRS-1 and IRS-2 are both tyrosine-phosphorylated by IL-2, IL-4, and IL-15 in primary human lymphocytes. This suggests that IRS-1 and IRS-2 may have similar functions; that they both are utilized for cytokine signaling and that they may have important roles in the IL-2- and IL-4-induced response.
The functional importance of
IL-2-induced IRS-1 and IRS-2 phosphorylation has yet to be defined.
However, IRS-1 has been shown to be important for mediating insulin and
IL-4-induced proliferative effects(23) . IRS-1 phosphorylation
has been shown to enhance insulin-stimulated MAP kinase activation. It
has also been suggested that the binding of IRS-1 to Grb-2 may enhance
insulin-stimulated mitogenesis via the activation of the Ras/Raf
pathway(16) . However, as the adaptor molecule Shc is recruited
by IL-2 in T cells(8) , and as ShcGrb-2 complexes are
known to form(16) , it is possible that Shc and IRS-1 have
synergistic roles in the IL-2 response. Therefore, IRS-1
phosphorylation is likely to represent an alternative pathway for
IL-2-induced proliferation in T cells. In addition, as the tyrosine
phosphorylation of IRS-1 by cytokines such as growth hormone (GH),
IL-9, IFN
, insulin, and now IL-2 induce association of IRS-1 with
PI 3-kinase, this may be a commonly used mechanism for PI 3-kinase
activation. In summary, IRS-1 probably acts conjointly with other
pathways of IL-2-mediated signaling, thereby amplifying the IL-2
proliferative response.
The mechanism by which cytokines that bind
hematopoietin family receptors (i.e. IL-2 and IL-4) induce
tyrosine phosphorylation of IRS-1 has been unclear(34) . Our
findings suggest that JAK1 and JAK3 can bind to and phosphorylate
IRS-1. In addition, it is clear that growth hormone, which activates
JAK2(36) , also phosphorylates IRS-1(27) , and that a
number of cytokines, such as IFN, IFN
, and IL-13
phosphorylate IRS-1 but do not activate JAK3 (26, 27) . Therefore, while the tyrosine
phosphorylation of IRS-1 may result from Janus kinase activity, it
appears to be independent of the particular JAK activated. However, the
finding that the JAKs can directly phosphorylate and are physically
associated with the IRS-1 and IRS-2 proteins suggests that the
activation of JAK kinases may be a common mechanism by which many
growth factors phosphorylate IRS-1 and IRS-2.
Although IL-4-induced
IRS-1 phosphorylation has been reported previously(23) ,
IL-2-induced IRS-1 phosphorylation has not. This is surprising since
IL-2 signal transduction is an intensively investigated area. A
possible explanation for this is that IRS-1 and IRS-2 are not expressed
in certain T cell lines commonly used to examine IL-2 signaling. ()However, it should be re-emphasized that the present study
employed normal peripheral blood lymphocytes, thus reinforcing the
physiological relevance of the observations.
The findings described in this paper strongly suggest that IRS-1 and IRS-2 may be important docking molecules recruited in response to IL-2, IL-4, and IL-15 in human T cells. The results further indicate that the JAKs associate with and phosphorylate IRS-1 and IRS-2, and that IL-2 and IL-4 induce association of PI 3-kinase with these docking molecules and thus provide a means of amplifying the cytokine-dependent signal in a manner that may not rely on recruitment of the signaling molecules to the phosphorylated receptor. Therefore, IL-2-dependent IRS-1 and IRS-2 phosphorylation is likely to have an important role in T lymphocyte activation.