(Received for publication, November 28, 1995; and in revised form, February 8, 1996)
From the
The epidermal growth factor (EGF) receptor activates several signaling cascades in response to the ligands EGF and amphiregulin (AR). One of these signaling events involves the tyrosine phosphorylation of STATs (signal transducers and activators of transcription), a process believed to require the activation of a tyrosine kinase of the JAK family. In this report we demonstrate that EGF- and AR-induced STAT activation requires the intrinsic kinase activity of the receptor but not the presence of Jak1. We show that both wild type (WT) and truncated EGF receptors lacking all autophosphorylation sites activate STAT 1, 3, and 5 in response to either EGF or AR. Furthermore, relative to cells expressing WT receptor, ligand-induced tyrosine phosphorylation of the STATs was enhanced in cells expressing only the truncated receptor. These results provide the first evidence that (i) EGF receptor-mediated STAT activation occurs in a Jak1-independent manner, (ii) the intrinsic tyrosine kinase activity of the receptor is essential for STAT activation, and (iii) tyrosine phosphorylation sites within the EGF receptor are not required for STAT activation.
The activated epidermal growth factor (EGF) ()receptor mediates a number of important biological
responses in mammalian cells including the stimulation of cell
division, migration, and differentiation(1) . Six different
polypeptide ligands, which derive from distinct genes, are capable of
binding to the extracellular domain of the EGF receptor. These ligands
include EGF, transforming growth factor-
, amphiregulin (AR),
heparin binding EGF-like growth factor, epiregulin, and
betacellulin(2) . All of these growth factors contain a
characteristic EGF-like domain, which is defined by 6 evenly spaced
cysteine residues that generate 3 loops through the formation of
disulfide bonds. In the case of AR it has been demonstrated that
heparan sulfate proteoglycans are necessary for activation of the EGF
receptor and a cellular response(3) . Upon ligand binding, the
intrinsic tyrosine kinase activity of the EGF receptor is augmented,
resulting in autophosphorylation as well as the phosphorylation of
specific intracellular substrates(4) . Among the target
proteins that become phosphorylated in response to EGF are
mitogen-activated protein kinase, the adapter protein SHC, the
GTPase-activating protein of p21
, phospholipase
C-
, the phosphotyrosine phosphatase 1D, as well as the
non-receptor tyrosine kinase Jak1 (5) and members of the STAT
(signal transducers and activators of transcription) family of
transcription factors(6, 7, 8) . Several
reports have shown that the intrinsic receptor kinase activity is
necessary for activation of a subset of these proteins, whereas others
can be activated also via a kinase-inactive EGF
receptor(9, 10, 11) . Additional mutations
have allowed for the identification of receptor domains that are needed
for cell motility, mitogenesis, and activation of mitogen-activated
protein kinase or phospholipase C-
(12, 13) . In
the case of the STAT proteins it has been suggested that the
phosphorylation of the receptor itself on tyrosine residues is a
requirement for their activation(14) . Based on mutational
analysis of several cytokine receptors and the Src homology 2 (SH2)
domains of the STAT proteins, it is believed that the specificity of
STAT activation is conferred by an interaction between the distinct
tyrosine phosphorylation sites of a certain receptor and the SH2
domains of a STAT(15, 16) . In this report we show
that the EGF receptor kinase activity is needed for STAT tyrosine
phosphorylation, whereas the presence of Jak1 is not necessary.
Furthermore, we provide evidence, using a truncated receptor lacking
all autophosphorylation sites, that receptor phosphorylation is not
required for STAT activation by either AR or EGF.
Figure 1:
Requirement for the intrinsic EGF
receptor kinase activity but not for Jak1 in STAT activation. A, wild type HeLa cells (WT) or HeLa cells lacking
Jak1 (JAK1-) were stimulated for 20 min with either AR
(10 nM), EGF (10 nM), or IFN (10 ng/ml). Cell
lysates were prepared and EMSAs were performed using an SIE probe. CTL, control. B, NR6 fibroblasts expressing either
the wild type (WT) or a kinase-inactive EGF receptor (M721)
were stimulated as described in A, and the lysates obtained
were analyzed using EMSAs. The SIE binding complexes induced by AR/EGF
or IFN
are denoted by SIF-a or SIF-
,
respectively.
Figure 2: Tyrosine phosphorylation sites in the EGF receptor are not required for activation of STAT proteins. NR6 fibroblasts expressing the wild type EGF receptor (WT) or receptors truncated at amino acid 1000 (C`1000), at amino acid 991 (C`991), or at amino acid 973 (C`973) were stimulated as described in Fig. 1A, and the lysates obtained were analyzed using EMSAs. The SIE binding complexes induced by AR and EGF are denoted by SIF-a. CTL, control.
Figure 3: Ligand-induced tyrosine phosphorylation of STATs in cells expressing wild type or C`973 EGF receptors. NR6 fibroblasts expressing either the wild type (WT) (A, C, and E) or the C`973 truncated EGF receptor (B, D, and F) were stimulated with 0, 0.1, 0.5, 5, or 20 nM AR or EGF as described in Fig. 1A, and the lysates obtained were subjected to immunoprecipitations (IP) using affinity-purified polyclonal antibodies against STAT 1, 3, or 5. The immune complexes were resolved by SDS-polyacrylamide gel electrophoresis and transferred to Immobilon, and Western blotting with anti-phosphotyrosine (anti-PY) antibodies (upper panels) was performed. To verify that equal amounts of protein were immunoprecipitated from both cell lines, the blots were reprobed with monoclonal antibodies to the corresponding STAT (lower panels).
In summary, our results demonstrate that the EGF receptor tyrosine kinase is necessary and sufficient to activate several members of the STAT family of transcription factors and that this activation can occur even in the absence of Jak1. As it has been previously shown that the EGF receptor tyrosine kinase can phosphorylate STAT proteins on the correct site in vitro(22) , these findings suggest that STAT activation does not require a member of the Jak family of tyrosine kinases. However, we cannot rule out the possibility that an as yet undiscovered JAK-like kinase is involved in EGF receptor-mediated activation of the STATs. We also provide evidence that autophosphorylation sites in the EGF receptor are not required for STAT activation, which is in contrast with the interpretation of results obtained by Silvennoinen et al.(14) . Although we have been able to reproduce the impaired STAT activation observed with EGF receptors truncated at amino acid 991, which lacks all known autophosphorylation sites, we found that this hindrance is based on the presence of amino acids 973-991 rather than on a loss of receptor phosphorylation sites.
Our results demonstrate that the STATs do not need SH2 domain docking sites within the EGF receptor to become tyrosine-phosphorylated in response to ligand. We have previously shown that both AR and EGF induce in vivo tyrosine phosphorylation of the EGF receptor-related tyrosine kinase erbB2(21) , and similar results have been obtained in NR6 WT, C`973, and M721 cells (data not shown). Therefore, it is theoretically possible that other EGF receptor-associated proteins such as erbB2 may provide the docking sites for the SH2 domain-containing STATs. However, the inability of the kinase-inactive M721 receptor to generate an SIE binding complex indicates that erbB2 cannot substitute for the requisite EGF receptor kinase activity. The fact that truncation of the EGF receptor at residue 973 results in increased ligand-dependent phosphorylation of the STATs is consistent with the previously proposed concept that the COOH-terminal region of the EGF receptor performs an inhibitory function with regard to tyrosine phosphorylation of cellular substrates(24) .