(Received for publication, December 18, 1995; and in revised form, February 2, 1996)
From the
We have isolated a second human Stat5 cDNA, Stat5B, and
demonstrated that the genes encoding both Stat5A and Stat5B are located
at chromosome 17q11.2. Both genes were constitutively transcribed in
peripheral blood lymphocytes. By using specific antisera, we
demonstrated that both Stat5A and Stat5B are activated by interleukin-2
(IL-2) in peripheral blood lymphocytes, natural killer-like YT leukemia
cells, and human T cell lymphotropic virus type I-transformed MT-2 T
cells. In COS-7 cells, which constitutively express the Janus family
tyrosine kinase Jak1, reconstitution of IL-2-induced Stat5A and Stat5B
DNA binding activities was dependent on the coexpression of Jak3 along
with the IL-2 receptor chain and the common cytokine receptor
-chain. This IL-2-induced Stat5 activation was dependent on the
presence of either of two tyrosines (Tyr-392 or Tyr-510) in the IL-2
receptor
chain, indicating that either of these two tyrosines can
serve as a docking site. Moreover, we demonstrated that human Stat5
activation is also dependent on Tyr-694 in Stat5A and Tyr-699 in
Stat5B, indicating that these tyrosines are required for dimerization.
The COS-7 reconstitution system described herein provides a valuable
assay for further elucidation of the IL-2-activated JAK-STAT pathway.
The interaction of interleukin-2 (IL-2) ()with IL-2
receptors (IL-2R) on lymphocytes, natural killer cells, and monocytes
induces pleiotropic biological effects on the immune system. Functional
IL-2Rs contain IL-2R
and the common cytokine receptor
-chain
(
) (1, 2, 3) , two members
of the cytokine receptor superfamily(4) . Intermediate affinity
receptors contain only IL-2R
and
, whereas high
affinity receptors additionally contain IL-2R
; both of these forms
of IL-2 receptors are capable of transducing IL-2
signals(2, 3) . IL-2R
is also a component of the
IL-15 receptor(5, 6) , while
is
shared by the receptors for IL-2(1) ,
IL-4(7, 8) , IL-7(9, 10) ,
IL-9(11, 12) , and IL-15(5) . In humans,
mutation of the
gene can result in X-linked severe
combined immunodeficiency(13, 14, 15) .
IL-2 stimulation of lymphocytes rapidly activates the Janus family
tyrosine kinases Jak1 and Jak3(11, 16, 17) .
Jak1 associates with IL-2R, and Jak3 primarily associates with
(11, 18, 19) . JAK family
kinases play critical signaling roles for interferons and for many
other cytokines by activating STAT (signal transducers and activators
of transcription) proteins that then dimerize, rapidly translocate into
the nucleus, and modulate gene expression(20, 21) . At
least six different human STAT genes have been identified (20, 21) . They encode an even greater number of
protein products due to alternative mRNA splicing. Many cytokines are
known to activate more than a single STAT protein, allowing for
additional complexity due to the formation of both homo- and
heterodimers between different STAT proteins. For example, IL-2 rapidly
activates Stat5 in freshly isolated peripheral blood lymphocytes (PBL)
and both Stat3 and Stat5 in PBL preactivated for 72 h with
phytohemagglutinin (PHA) (22) and in natural killer-like YT
leukemia cells(22, 23) .
We now report the cloning
of a second human Stat5 cDNA (Stat5B) and the chromosomal localization
of both human Stat5A and Stat5B. We also provide evidence that both
Stat5A and Stat5B are activated in normal PBL by IL-2 in vivo.
Using a transient transfection system in COS-7 cells, we have
reconstituted IL-2-induced activation of both Stat5A and Stat5B, shown
that either Tyr-392 or Tyr-510 of IL-2R is required for
IL-2-dependent Stat5 activation, and identified a tyrosine residue in
each human Stat5 protein that is essential for its activation by Jak1
and Jak3.
The NaeI-XhoI fragment of human Stat5A was subcloned between the EcoRV and XhoI sites of pSX, a eukaryotic expression vector; the Stat5B cDNA was subcloned between the EcoRI and NotI sites of pSX. The resulting plasmids are denoted pSXStat5A and pSXStat5B, respectively. For chromosomal localization, fluorescent in situ hybridization was performed using human leukocytes from normal donors as described (27) with pSXStat5A and pSXStat5B as probes.
Figure 1: Alignment of amino acid sequences of human Stat5A and Stat5B, murine Stat5A and Stat5B, and ovine Stat5. Residue numbers are on the right. Dots indicate identical nucleotides; hyphens are gaps introduced to optimize alignment. The putative DNA-binding(38, 39) , SH3(25) , and SH2 domains (25) and the phosphorylated tyrosines (23, 25, 34) are boxed. The sequences for Stat5 proteins are from the following sources: Stat5A ( (23) and this study; GenBank accession numbers L41142 and U43185), Stat5B (this study; GenBank accession number U47686), murine Stat5A ((34) ; GenBank accession number Z48538), murine Stat5B ((34) ; GenBank accession number Z48539), and ovine Stat5 ((25) ; GenBank accession number X78428). Hu, human; Mu, murine; MGF, mammary gland transcription factor.
Figure 2: Human Stat5A and Stat5B genes map to chromosome 17. Shown are metaphase cells following fluorescent in situ hybridization with human Stat5A (A) and Stat5B (B) cDNAs. 35 of 72 cells examined for Stat5A and 23 of 58 cells examined for Stat5B exhibited paired hybridization signals at 17q11.2 (arrows), and an additional seven (for Stat5A) and eight (for Stat5B) cells showed one hybridization signal at the same locus. No significant background was noted at any other chromosomal location. Chromosomes were identified using Quinacrine Fluorescence Hoechst (QFH) banding by simultaneous Hoechst 33258 staining (not shown).
Figure 3:
Stat5A and Stat5B transcripts differ in
size. Total RNA from fresh PBL was separated on a 1%
formaldehyde-agarose gel, blotted onto a nylon membrane, and hybridized
sequentially with P-labeled probes specific for human
Stat5A (lane 1) and Stat5B (lane 2). Although the
major Stat5A mRNA detected by Northern blotting is 3.8 kb, we have
isolated one Stat5A cDNA of 5.5 kb in a YT cDNA library, indicating
that longer transcripts also exist. It is therefore conceivable that
alternative polyadenylation may explain this
finding.
Figure 4:
Both Stat5A and Stat5B are activated by
IL-2 and can bind DNA. A, specificity of Stat5A and Stat5B
antisera. [S]Methionine-labeled rabbit
reticulocyte lysates not programed (lanes 1 and 4) or
programed with human Stat5A (lanes 2 and 5) or Stat5B (lanes 3 and 6) were immunoprecipitated (IP)
by R1216 anti-Stat5A (lanes 1-3) or R1219 anti-Stat5B (lanes 4-6) antisera and analyzed on 8% SDS gels (Novex,
San Diego, CA). B, Stat5A and Stat5B are activated by IL-2 in
PBL and YT cells. PBL preactivated with PHA for 3 days and rested
overnight (lanes 1, 2, 5, and 6)
and YT cells (lanes 3, 4, 7, and 8)
were not treated (lanes 1, 3, 5, and 7) or were treated with IL-2 (lanes 2, 4, 6, and 8). Cellular lysates were immunoprecipitated
with anti-Stat5A (lanes 1-4) or anti-Stat5B (lanes
5-8); subjected to Western blotting with 4G10 (upper
panels), anti-Stat5A (lower left panel), or anti-Stat5B (lower right panel); and developed by ECL. C and D, activation by IL-2 of Stat5A and Stat5B in nuclear extracts
from fresh PBL (lanes 1), PBL preactivated by PHA (lanes
2), MT-2 cells (lanes 3), and YT cells (lanes
4). STAT proteins were purified by GAS motif DNA affinity columns
and subjected to Western blotting with anti-Stat5A (C) or
anti-Stat5B (D). In each panel, molecular masses (in
kilodaltons) are indicated on the left, based on the mobilities of
Seeblue markers (Novex).
Figure 5:
Reconstitution of Stat5 binding activity
in COS-7 cells: demonstration that Tyr-392 and Tyr-510 of IL-2R,
Tyr-694 of Stat5A, and Tyr-699 of Stat5B are required for IL-2-induced
Stat5 activation. A, COS-7 cells were transfected with pSX (lanes 1 and 2); IL-2R
and
cDNAs (lanes 3 and 4); IL-2R
,
, and Jak1 cDNAs (lanes 5 and 6);
IL-2R
,
, and Jak3 cDNAs (lanes 7 and 8); IL-2R
,
, Jak1, Stat5A, and Stat5B
cDNAs (lanes 9 and 10); IL-2R
,
, Jak3, Stat5A, and Stat5B cDNAs (lanes 11 and 12); and IL-2R
,
, Stat5A, and
Stat5B cDNAs (lanes 13 and 14). Two days later, cells
were either not treated (lanes 1, 3, 5, 7, 9, 11, and 13) or treated with
IL-2 (lanes 2, 4, 6, 8, 10, 12, and 14), and nuclear extracts were
prepared. The specific complexes formed with endogenous Stat1 and
transfected Stat5 proteins are indicated. Although some COS-7 cells
have higher levels of Stat1 than others, the pattern for IL-2-dependent
Stat5 activation was consistent (data not shown). B, Stat5A (lanes 1 and 2), Stat5B (lanes 3 and 4), or both Stat5A and Stat5B (lanes 5 and 6) cDNAs were cotransfected into COS-7 cells with IL-2R
,
, and Jak3 cDNAs. Two days later, nuclear extracts
were prepared from cells not treated (lanes 1, 3, and 5) or treated with IL-2 (lanes 2, 4, and 6). Similar levels of Stat5B expression (lanes 3 and 4 versus lanes 5 and 6) were confirmed by Western
blotting (data not shown). C, wild-type IL-2R
(WT; lanes 1 and 2) or IL-2R
mutated at
Tyr-338, Tyr-355, Tyr-358, and Tyr-361 (IL-2R
FFFFYY; lanes 3 and 4), or Tyr-392 (IL-2R
YYYYFY; lanes 5 and 6), or Tyr-510 (IL-2R
YYYYYF; lanes 7 and 8), or both Tyr-392 and Tyr-510 (IL-2R
YYYYFF; lanes 9 and 10) was cotransfected with
, Jak3, Stat5A, and Stat5B into COS-7 cells. Two days
after transfection, nuclear extracts were prepared from cells not
treated (lanes 1, 3, 5, 7, and 9) or treated with IL-2 (lanes 2, 4, 6, 8, and 10). D, COS-7 cells were
transfected with IL-2R
and Jak1 (lane 1); IL-2R
,
Jak1, and Stat5B (lane 2); Stat5B and Jak1 (lane 3);
or IL-2R
and Stat5B (lane 4). E, COS-7 cells
were transfected with
and Jak3 (lane 1);
, Jak3, and Stat5B (lane 2); Stat5B and Jak3 (lane 3); or
and Stat5B (lane 4). F, Stat5A (lanes 1 and 2), Stat5A Y694F (lanes 3 and 4), Stat5B (lanes 5 and 6), and Stat5B Y699F (lanes 7 and 8) were
cotransfected into COS-7 cells with IL-2R
,
, and
Jak3 cDNAs. 48 h later, COS-7 cells were not treated (lanes 1, 3, 5, and 7) or treated with IL-2 (lanes
2, 4, 6, and 8), and nuclear extracts
were prepared. G, Stat5A (WT; lane 1),
Stat5A Y694F (lane 2), Stat5B (WT; lane 3),
and Stat5B Y699F (lane 4) cDNAs were cotransfected into COS-7
cells with IL-2R
,
, and Jak1 cDNAs. For F and G, expression levels of wild-type and mutant Stat5
proteins were similar (data not shown). For D-G, 2 days
after transfection, nuclear extracts were prepared, and electrophoretic
mobility shift assays were performed using the
-casein
probe.
The above
experiments were performed using a combination of Stat5A and Stat5B. We
found that either Stat5A or Stat5B alone could also result in DNA
binding to the -casein probe (Fig. 5B, lanes
1-4), suggesting that both Stat5A and Stat5B homodimers
could bind to the probe. The greater binding activity seen with Stat5B
compared with Stat5A (lane 4 versus lane 2) was likely due to
the greater expression of Stat5B in the transfected COS-7 cells (data
not shown). Coexpression of Stat5A and Stat5B consistently resulted in
a greater DNA binding activity than the sum of the binding seen with
Stat5A and Stat5B alone (lane 6 versus lanes 4 and 2), suggesting that Stat5A-Stat5B heterodimers can also bind
DNA.
Although Stat5 DNA binding activity was greatly diminished in
COS-7 cells transfected with the double mutation of Tyr-392 and Tyr-510
of IL-2R, it was still detectable (Fig. 5C, lanes 9 and 10). This result appears to be somewhat
different from our previous observation that a truncated form of the
IL-2R
chain lacking Tyr-392 and Tyr-510 is not able to mediate
STAT protein activation by IL-2 in 32D cells(22) . We reasoned
that the difference might reflect the higher levels of JAK kinases
and/or Stat5 proteins in COS-7 transfectants than in 32D cells. Indeed,
either Jak1 or Jak3 can activate Stat5B in the absence of receptor
chains, although IL-2R
facilitates Stat5B activation (Fig. 5, D and E). Transfection of COS-7 cells
with IL-2R
and Jak1 (without Stat5B) activated endogenous Stat1 (Fig. 5D, lane 1). Although overexpression of
Jak1 activated transfected Stat5B (lane 3), coexpression of
Jak1 with IL-2R
caused a higher level of activation of Stat5B than
that seen with Jak1 alone (lane 2 versus lane 3). In contrast,
coexpression of
with Jak3 did not augment Stat5B
activation more than Jak3 alone (Fig. 5E, lane 2
versus lane 3). This is consistent with the presence of STAT
protein docking sites on IL-2R
, but not on
(22, 31, 32) . These results
demonstrate that overexpression of Stat5 with either Jak1 or Jak3 is
sufficient to cause Stat5 activation, suggesting that when the level of
expression and/or activities of JAK kinases and/or STAT proteins are
disregulated, STAT proteins may directly associate with JAK kinases
without requiring receptor docking sites for the activation. However,
under physiological conditions, the presence of docking sites on
IL-2R
is clearly required for Stat5 activation by
IL-2(22, 31, 32) .
We have isolated cDNAs encoding two closely related human Stat5 proteins. Whereas only a single human Stat5 cDNA was previously identified, we have now shown that Stat5 DNA binding activity results from two closely related human proteins, Stat5A and Stat5B, analogous to the murine system. Both genes are located at chromosome 17q11.2, suggesting that they arose by tandem gene duplication. Interestingly, Stat5A and Stat5B are more similar to their murine homologues (34, 35) than to each other (Fig. 1).
Using antisera specific for Stat5A and Stat5B, we demonstrated that both Stat5A and Stat5B are activated by IL-2 in normal PBL, YT cells, and human T cell lymphotropic virus type I-transformed MT-2 cells and that both Stat5 proteins are involved in DNA binding. Stat5A and Stat5B migrate at different molecular masses, with Stat5B existing in at least two forms. These findings at least partially explain the heterogeneity observed previously in the mobility of Stat5(22, 23) .
Dimerization of IL-2R and
was previously
shown to be essential for transducing IL-2-induced
signals(28, 44) . We now demonstrate that in COS-7
cells, which constitutively express endogenous Jak1, we can
reconstitute IL-2-induced Stat5 activation by transfecting Jak3 in
addition to receptor chains, Stat5A, and/or Stat5B. Unexpectedly, high
constitutive levels of Stat5 binding activity were observed with Jak1
(in both the presence and absence of Jak3); this implies that with
supernormal levels of Jak1, there is direct activation of Stat5 by
Jak1. Using this COS cell reconstitution system, we have established
that activation of both Stat5A and Stat5B requires either Tyr-392 or
Tyr-510 of IL-2R
as a docking site. Moreover, we show that Tyr-694
of Stat5A and Tyr-699 of Stat5B are essential for the activation of
these proteins.
Although the specific roles of Jak1 and Jak3 in IL-2
signaling remain unclear, in vitro experiments indicate that
Jak1 can potently phosphorylate the IL-2R docking sites Tyr-392
and Tyr-510 (29) , whereas the importance of Jak3 is
underscored by the lack of Stat6 activation in an Epstein-Barr
virus-transformed B cell line derived from a Jak3-deficient patient
with severe combined immunodeficiency (45) and in
Jak3-deficient mice(46) . We therefore hypothesize that Jak3
may play a critical role in the phosphorylation of Stat5A and Stat5B,
particularly since Stat5 and Jak3 can be coprecipitated in human T cell
lymphotropic virus type I-transformed T cell lines, consistent with the
possibility that they physically interact(47) .
Stat5 proteins can be activated by a variety of different cytokines or growth factors including prolactin, IL-2, IL-3, IL-7, IL-15, granulocyte-macrophage colony-stimulating factor, erythropoietin, growth hormone, and thrombopoietin. A number of these cytokines can reconstitute Stat5 binding activity in COS cells. Interestingly, however, although prolactin can activate transcription of a STAT-dependent reporter construct in these cells(36) , IL-2 cannot, even when both Stat3 and Stat5 are cotransfected (data not shown). Thus, cytokine-specific and/or in some cases tissue-specific signaling pathways may be involved in transcriptional activation by Stat5 proteins. Nonidentical sets of genes are induced by at least some of the cytokines that activate Stat5, indicating that Stat5 alone does not determine the specific biological effects of each cytokine.
It has recently been shown that serine phosphorylation of Stat3 is also required for the formation of a stable Stat3 dimer-DNA complex in a cell type- and binding site-dependent manner(48) , and both tyrosine phosphorylation and serine phosphorylation of Stat1 and Stat3 are essential for maximal interferon-induced transcription(49) , indicating important roles for both JAK kinases and serine kinases. In addition to activating a variety of tyrosine kinases(2) , IL-2 also activates other signaling molecules, including serine kinases (50) and Ras(51) . The COS-7 reconstitution system reported herein may be valuable in screening transfected gene products to help identify other signaling molecules required for mediating IL-2-dependent, Stat5-mediated transcription and in clarifying whether Stat5A homodimers, Stat5B homodimers, and Stat5A-Stat5B heterodimers regulate the same or different genes.