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INTRODUCTION |
The human epidermal growth factor receptor
(EGFR)1 family comprise four
members, EGFR/HER1, erbB2/HER2, erbB3/HER3, and
erbB4/HER4, which transduce distinct signals for
proliferation and differentiation (1). EGFR signaling is initiated by
ligand binding, receptor dimerization, and autophosphorylation to
create phosphotyrosine residues that provide coded SH2 domain docking
sites for receptor-specific substrate recruitment (2). Assembly of SH2
containing intracellular effector molecules ensues at the receptor with
subsequent activation of several downstream signaling pathways. Some
SH2 domain proteins serve as adaptor molecules and couple receptors to
phosphorylation dependent signaling cascades such as the Ras/MAP kinase
pathway (3) and phosphoinositide 3-kinase activation (4). Other SH2
domain substrates, such as phospholipase C-
, have intrinsic enzymatic activity that can be stimulated by tyrosine phosphorylation. Subsequent breakdown of phosphoinositides by activated phospholipase C-
leads to the generation of inositol trisphosphate that enhances cytoplasmic Ca2+ flux from intracellular stores (5-7).
Understanding the intrinsic feedback regulatory mechanisms of cell
surface receptor tyrosine kinases (RTKs) such as the EGFR family, which
trigger many of these downstream signaling networks, is of particular
interest as they are implicated in the progression of a variety of
aggressive carcinomas (8, 9).
Ligand-induced desensitization mechanisms are important aspects of the
regulation of transmembrane receptors, and the EGFR has provided an
ideal model system for delineating these events. Activated receptors
are slowly internalized via clathrin-coated pits and are ultimately
delivered to lysosomes for degradation (10, 11). This process of
ligand-induced down-regulation occurs over several hours; however, in
the short term phosphorylation of the receptor on serine and threonine
residues is thought to be one of the primary mechanisms for attenuation
of its kinase activity. Activation of protein kinase C
(PKC)-dependent signaling pathways by phorbol ester leads
to a loss in high affinity EGF binding as well as an inhibition in RTK
activity (12, 13). The latter is linked to site-specific
phosphorylation of threonine 654 in the juxtamembrane region; however,
PKC-mediated modulation of ligand binding is independent of any major
serine/threonine phosphorylation sites in the receptor (13). It has
been suggested that PKC-mediated phosphorylation at threonine 654 may
contribute only partially to EGFR desensitization due to low
stoichiometry of phosphorylation in some cells (14). Phosphorylation at
threonine 669 by MAP kinase may also regulate the EGFR kinase (15, 16); however, substitution with negatively charged glutamic acid residues to
mimic constitutive phosphorylation of both threonine 654 and threonine
669 partially modulated, but failed to block, EGFR signaling (17). In
contrast, agents that increase cytosolic calcium, such as thapsigargin
and ionophore A23187, significantly inhibit EGFR tyrosine kinase
activity, and it has been suggested that this effect might be partially
due to Ca2+/calmodulin-dependent protein kinase
II (CaM kinase II)-mediated phosphorylation at serines 1046/1047 (18,
19).
We have previously mapped a novel CaM kinase II phosphorylation site to
threonine 1172 in erbB2/HER2 and shown that this site can
contribute to the regulation of the RTK activity (20). Here, we have
extended the same experimental approach to the EGFR and have identified
new CaM kinase II sites in the cytoplasmic tail, which lie within a
consensus -S-X-D- phosphorylation sequence. Parallel
analyses of site-specific mutant receptors have revealed pronounced
differences in the potential to transform mouse NIH3T3 fibroblasts and
to up-regulate tyrosine autokinase activity. We identify serine 1142 (-S-L-D-) as a preferred site compared with serines 1046/1047, and phosphorylation of these sites together with
serine 1057 (-S-I-D-) and serine 744 (-S-V-D-) in
the kinase domain provides a clearer explanation of the regulation of
EGFR signaling by CaM kinase II. Furthermore, we present evidence that the mechanism for controlling receptor tyrosine autophosphorylation may
involve inhibition of cytoplasmic tail interactions with the EGFR
kinase domain, thereby preventing an enzyme-substrate interaction.
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EXPERIMENTAL PROCEDURES |
Construction of EGFR Mutants--
Site-directed mutagenesis of
the human EGFR cDNA cloned into the pCMV-1 expression plasmid was
performed as described previously (21). Mutations were confirmed by
dideoxy sequencing and restriction enzyme analysis. Other mutants used
in this study have been described previously; HER-1/2-T1172A (20) and
HER
CT (21).
Preparation and Phosphorylation of GST Fusion
Proteins--
Amino acid residues tyrosine 974 to alanine 1186 of the
human EGF receptor (HERWT, 212 amino acids; HER
15, 197 amino acids), serine 1013 to proline 1084 of the chicken EGF receptor (CER, 72 amino
acids), and serine 1013 to proline 1084 (v-erbB.ES4, 50 amino acids) were amplified from appropriate cDNAs by polymerase chain reactions (numbering of receptor residues is based on equivalent residues in the human EGFR as in Ref. 22). Smaller EGFR GST fusion
proteins were prepared in the same way to generate the following
sequences; GST-1, threonine 1022 to tyrosine 1045; GST-2, phenylalanine
1041 to aspartic acid 1056; GST-3, aspartic acid 1048 to proline 1095;
GST-4, phenylalanine 1062 to histidine 1105; GST-5, proline 1130 to
glycine 1165; GST-III, alanine 731 to leucine 758. Amplified fragments
were subcloned into the bacterial expression vector pGEX-2T (Amersham
Pharmacia Biotech) and GST fusion proteins isolated as described
previously (20). Rat CaM kinase II-
overexpressed in HEK-293 cells
was purified by calmodulin-Sepharose affinity chromatography and used
for in vitro phosphorylation of purified GST proteins as
described (20).
Generation of Recombinant EGFR Retrovirus and Focus Formation
Assays--
EGFR mutants were subcloned into the retroviral pLEN
vector (23). Constructs were transfected into the helper virus-free packaging cell line HEK-293-eco (ATCC) by calcium phosphate
precipitation (24). After 2 days, ecotropic virus-containing
supernatants were collected, and viral titers were normally in the
range of 1-5 × 105 colony-forming units. For focus
formation assays passage 10 NIH3T3 fibroblasts (4 × 104 cells) were seeded into 6-cm dishes 18 h prior to
infection (8 h) with EGFR mutant retroviruses (m.o.i. of 0.01-0.001)
followed by infection (8 h) with
2TGF
virus (m.o.i. of 0.02),
which was kindly provided by David Lee. Cells were seeded into a 6-cm
plate and grown in Dulbecco's modified Eagle's medium containing 4% fetal calf serum. Media were changed every 2 days, and after 12-14 days foci were stained with 0.5% crystal violet.
Kinase Assays--
HEK-293 fibroblasts (1 × 106 cells/10-cm dish) were transfected with receptor
constructs subcloned into pCMV1 (30 µg of DNA) together with 10 µg
of pCMV1 or pCMV1-CaM kinaseII1-290 as described
previously (21). Two days after transfection, cells were stimulated
with 100 ng/ml EGF for 10 min, lysed in 1 ml "lysis buffer" (50 mM HEPES, pH 7.5, containing 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, 0.2 mM phenylmethylsulfonyl fluoride) and immunoprecipitated
with monoclonal antibody 108 (20). Immunoprecipitates were washed five
times and resuspended in 100 µl of "wash buffer" (50 mM HEPES,
pH7.5, 150 mM NaCl, 10% glycerol, 0.1% Triton X-100) containing 5 mM MnCl2, 2 mM CaCl2,
100 µM ATP, and 50 units of calmodulin. Samples that had
previously been transfected with CaM kinase II1-290 were
phosphorylated with 0.1 µg of purified CaM kinase II at 4 °C (30 min) to ensure complete and efficient phosphorylation of receptors.
Immune complexes were washed three times in wash buffer and resuspended
in 100 µl of wash buffer containing 5 mM
MnCl2 and 5 mM MgCl2.
Phosphorylation was initiated by addition of 5 µCi of
[
-32P]ATP and after 5 min at room temperature
terminated by addition of EGTA to a final concentration of 10 mM. Immunoprecipitates were washed once with wash buffer
containing 10 mM EGTA and resuspended in 100 µl of
SDS-sample buffer. Samples were separated by SDS-7.5% PAGE, and gels
were dried and exposed to x-ray film. Radioactive spots corresponding
with the migration of the full-length receptor were excised and
quantitated by scintillation counting. To examine receptor
autophosphorylation by Western blotting, cells were transfected as
above, and immunoprecipitated receptors were separated by 7.5% SDS-PAGE and transferred onto nitrocellulose. Filters were incubated with anti-phosphotyrosine monoclonal antibody (4G10; Upstate
Biotechnology) or anti-EGFR monoclonal antibody (Sigma; catalog number
E3138), and immunoreactive proteins were visualized using the ECL
system (Amersham Pharmacia Biotech).
Association of GST Fusion Proteins with Wheat Germ
Agglutinin-purified HER
CT--
Four plates of HEK-293 cells (1 × 106 cells/10-cm dish) were each transfected with 100 µg of pCMV1-HER
CT. Lysates were incubated by rotation with 50-µl
packed volume of wheat germ agglutinin-Sepharose beads (1 h; 4 °C).
GST-HER-WT (amino acids 974-1186) was either left unphosphorylated or
phosphorylated (1 h; 4 °C) with 0.2 µg of CaM kinase II in the
presence of 100 µM ATP under the same conditions
described above for GST fusion protein phosphorylation. After washing
wheat germ agglutinin-Sepharose-bound protein six times with wash
buffer, pre- or nonphosphorylated GST-HER-WT reactions were added to
beads and incubated by rotation (1 h; 4 °C) followed by a further
six washes with wash buffer. Beads were resuspended in SDS-sample
buffer and bound protein-separated SDS-10% PAGE followed by staining
with Coomassie Blue or immunoblotting against an anti-EGFR monoclonal
antibody (Sigma; catalog number E3138). Immunoreactive proteins were
visualized using the ECL system (Amersham Pharmacia Biotech).
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RESULTS |
Phosphorylation of EGFR-GST Fusion Proteins with CaM Kinase
II--
To test whether the EGFR cytoplasmic tail is a substrate for
CaM kinase II, we constructed a pGEX plasmid in which the DNA coding
sequence representing amino acids 974-1186 (HER-WT) was fused to the
DNA encoding GST. Purified HER-WT was efficiently phosphorylated by CaM
kinase II in vitro in contrast to purified GST alone (Fig.
1a). HER-WT contains a region
corresponding with a synthetic peptide (RRFLQRYSSDPTGAL;
EGFR1041-1053), which has been shown previously to be a
substrate for CaM kinase II and has an optimal consensus
-R-X-X-S/T- phosphorylation sequence at serine
1047 (18). Serines 1046/1047 within this region are also major in
vivo sites of phosphorylation in the EGFR (25).

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Fig. 1.
Phosphorylation of EGFR cytoplasmic tail
fusion proteins by CaM kinase II. a, GST fusion proteins (10 µg of protein) were phosphorylated with CaM kinase II as described
under "Experimental Procedures" and samples separated by SDS-12.5%
PAGE. Gels were either stained with Coomassie Blue or dried and exposed
to x-ray film. HER-WT, residues tyrosine 974 to alanine 1186 of the human EGFR; HER-delta15, tyrosine 974 to alanine 1186 of the human EGFR without residues 1039-1053; CER, serine
1013 to proline 1084 of the chicken EGFR; ES4, serine 1013 to proline 1084 of v-erbB.ES4. b, CaM kinase II
phosphorylated HER-WT was hydrolyzed and separated by two-dimensional
thin layer chromatography followed by autoradiography as described
previously (20). The migration of standard phosphoamino acids is
indicated by arrows. PS, phosphoserine;
PT, phosphothreonine; PY, phosphotyrosine.
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We therefore prepared a GST fusion protein that has a deletion
corresponding with residues 1039-1053 (HER-delta15), and in vitro phosphorylation of this protein with CaM kinase II was only partially reduced relative to HER-WT, indicating that there are additional major sites of phosphorylation in the EGFR cytoplasmic tail
(Fig. 1a). Shorter fusion proteins were also examined using the same approach; however, in this instance peptide sequences were
generated from the highly homologous CER and also the structurally related v-erbBES4 oncogene. The v-erbBES4
oncoprotein is derived from the CER, and as well as having a large
deletion of the ligand binding domain, also has several cytoplasmic
domain mutations, including a deletion of residues equivalent to
1040-1060 in the human EGFR (26).
The short 72-amino acid CER protein comprises residues serine 1016 to
glutamine 1087, and this can also be efficiently phosphorylated in vitro by CaM kinase II in contrast to the short 50-amino
acid ES4 fusion protein that underwent only background phosphorylation (Fig. 1a). These data taken together therefore suggest that
the cytoplasmic tail of the EGFR is an excellent substrate for CaM kinase II in a region that includes serines 1046/1047 as well as at
other unidentified sites. Phosphoamino acid analysis of HER-WT after
CaM kinase II phosphorylation also indicated that these sites were only
at serine residues (Fig. 1b).
Mapping of Novel CaM Kinase II Sites in the EGFR Cytoplasmic
Tail--
Serine 1047 resides within the traditional
-R-X-X-S/T- for CaM kinase II phosphorylation;
however, scanning of residues 974-1186 in the EGFR reveals no other
consensus sites. Several other physiological substrates for CaM kinase
II have been described that do not have recognizable consensus sites
(27). For example, the intermediate filament protein vimentin has a
phosphorylation site in which the critical determinant of site
specificity is an acidic residue in the second position on the
C-terminal side of the phosphorylation site (28). Analysis of vimentin
and other peptide substrates have suggested that the sequence
-S-X-D- may serve as a novel recognition site for
phosphorylation by CaM kinase II with the possibility of additional
preference for a hydrophobic residue at position X (27, 28).
Support for this observation comes from the identification of a CaM
kinase II site at serine 142 of the cAMP response element-binding
protein, which negatively regulates transcriptional activity and fits
this alternative consensus (29).
As a starting point to mapping the other sites in HER-WT, we examined
residues 974-1186 for -S-X-D- sequences. Prospective sites
were found at serine 1057 (-S-I-D-), serine 1096 (-S-R-D-), and serine
1142 (-S-L-D-). Previously reported sites at serine 1046 and serine 1047 lie within -S-X-D- and
-R-X-X-S- consensus sites, respectively. In
addition, serine 1040 (-K-E-D-S-) resides within the
-R-X-X-S- consensus but has a basic lysine
instead of arginine at the third position C-terminal to the serine, and it has been reported that a limited number of substrates have this site
preference for CaM kinase II (27). In an effort to identify any of the
potentially new sites in the EGFR as targets for CaM kinase II,
EGFR-GST fusion proteins were prepared that spanned these serines,
which were also individually mutated to alanine (Fig.
2a). In vitro
phosphorylation identified the major site of CaM kinase phosphorylation
at serine 1142 and confirmed that serines 1046/1047 can also be
phosphorylated, albeit to a much lesser extent, with a clear preference
for serine 1047 (Fig. 2b). Serine 1057 also underwent a
significant degree of phosphorylation comparable with serine 1047;
however, serine 1096 and serine 1040 were very poorly phosphorylated
(Fig. 2b). Quantitation of in vitro
phosphorylation reactions indicated that serine 1142 incorporated about
5-fold more phosphate than serines 1046/1047 and about 10-fold more
than serine 1057 (data not shown). The consensus sequence and location
of individual phosphorylation sites in the cytoplasmic tail of the EGFR
are depicted in Fig. 2c.

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Fig. 2.
Identification of CaM kinase II
phosphorylation sites in the EGFR cytoplasmic tail. Smaller HER
GST fusion proteins, both wild-type and site-specific alanine mutants,
were prepared and phosphorylated with CaM kinase II as described under
"Experimental Procedures." GST-1, threonine 1022 to
tyrosine 1045; GST-2, phenylalanine 1041 to aspartic acid
1056; GST-3, aspartic acid 1048 to proline 1095;
GST-4, phenylalanine 1062 to histidine 1105;
GST-5, proline 1130 to glycine 1165. Samples were separated
by SDS-12.5% PAGE, and gels were either stained with Coomassie Blue
(a) or dried and exposed to x-ray film (b).
c, regions covered are depicted diagramatically, and
proposed consensus phosphorylation sites are
underlined.
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Potentiation of EGFR Transforming Activity by Mutation of CaM
Kinase II Phosphorylation Sites Reveals Their in Vivo Functional
Significance--
Previously we have mapped a single CaM kinase II
site in HER2/c-erbB2 and by two-dimensional mapping showed
that this site could be hyperphosphorylated in
[32P]orthophosphate-loaded cells in vivo (20).
Peptide maps generated in the same way by expression of full-length
EGFR or specific site mutants were complicated and have proven
difficult to interpret due to overlapping migration of a number of
phosphopeptide spots. We have used several proteases to generate
peptides, including trypsin, chymotrypsin, and V8 protease, and due to
the absence of sufficient proteolytic cleavage sites and close
proximity of some of the CaM kinase II phosphorylation sites, we have
been unable to distinguish in vivo sites on this basis.
However, there are several phosphopeptide spots that show significantly
enhanced 32P phospholabeling in the presence of
constitutively active CaM kinase II1-290 (data not shown).
We have therefore assessed the in vivo biological
consequence of serine phosphorylation site-specific mutation by
comparing the ability of EGFR mutants to induce transformed foci in 3T3 fibroblasts. In this regard, mutation of serines 1046/1047 to alanine
in the EGFR increases both signaling strength and oncogenic potential
(19). We have used retroviral co-infection of early passage fibroblasts
with the EGFR and transforming growth factor-
to induce autocrine
activation and the appearance of transformed foci. The number and size
of foci with the wild-type EGFR could be approximately doubled by
mutation of serines 1046/1047, and additional mutation of the major
phosphorylation sites at serines 1057/1142 led to a dramatic
enhancement of the transformed phenotype (Fig.
3).

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Fig. 3.
Focus formation of NIH3T3 fibroblasts
expressing EGF receptor mutants. Passage 10 NIH3T3 fibroblasts
(8 × 104 cells) were infected (4 h) with HER mutant
retroviruses (m.o.i. 0.004) and then infected (4 h) with transforming
growth factor- virus (m.o.i. 0.02) as described under
"Experimental Procedures." Cells were grown in Dulbecco's modified
Eagle's medium, 4% fetal calf serum and after 14 days foci were
stained with crystal violet. Data are representative of two independent
experiments.
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CaM Kinase II Phosphorylation of Serine 744 within an -S-X-D-
Consensus in Kinase Sub-domain III of the EGFR Affects Receptor
Tyrosine Kinase Activity--
The functional significance and
preference of serine 1142 in the EGFR cytoplasmic tail as a site for
CaM kinase II led us to examine the whole of the EGFR intracellular
tail for other -S-X-D- consensus sequences. One additional
site was found at serine 744, which, based on sequence alignment of
known kinases, is located within sub-domain III or helix C of the
tyrosine kinase domain (30). This site is conserved between the type I
RTKs, except HER3, and there is almost complete identity in the whole of sub-domain III between EGFR and HER2/c-erbB2 apart from
substitution of the EGFR equivalent of serine 744 for glycine in
HER2/c-erbB2 (Fig.
4a). A GST fusion protein
comprising sub-domain III of the EGFR can be phosphorylated by CaM
kinase II, and this can be prevented by mutation of serine 744 to
alanine (Fig. 4b). In addition, when the serine 744
alanine mutation is incorporated into the full-length receptor, there
is approximately a 2-fold increase in receptor autophosphorylation
assessed by overexpression in HEK-293 cells and anti-phosphotyrosine
blotting (Fig. 4c). We then attempted to mimic
hyperphosphorylation of serine 744 by mutation to a negatively charged
aspartic acid and found that receptor tyrosine autophosphorylation is
significantly impaired (Fig. 4c). This loss of activity is unlikely to be due to an effect on overall domain structural integrity, since mutation of the same site to alanine up-regulates kinase activity.

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Fig. 4.
CaM kinase II phosphorylation of sub-kinase
domain III in the EGFR. a, amino acid sequence
alignment of sub-domain III in the kinase domains of HER-1 (EGFR),
HER-2/c-erbB2, HER-3, and HER-4 is based on the classification of
conserved regions found in all tyrosine kinases (30). Serine 744 in the
HER-1 is highlighted, and the proposed consensus CaM kinase
phosphorylation site in HER-1, HER-3, and HER-4 is
underlined. b, GST.III-WT comprises residues
alanine 731 to leucine 758 of HER-1, and GST.III-S744A has an alanine
mutation at serine 744. Proteins were phosphorylated by CaM kinase II
as described under "Experimental Procedures" and separated by
SDS-12.5% PAGE. Gels were either stained with Coomassie Blue or dried
and exposed to x-ray film. c, HEK-293 cells expressing
wild-type or mutant receptors were starved in 0.5% fetal calf serum,
Dulbecco's modified Eagle's medium for 24 h and stimulated for
10 min with 100 ng/ml EGF. After lysis, EGF receptors were
immunoprecipitated, separated by SDS-7.5% PAGE, and immunoblotted
against anti-phosphotyrosine antibody (anti-PY) or anti-EGFR
antibody (anti-HER).
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Regulation of EGFR and HER2/c-erbB2 Tyrosine Kinase Activity by CaM
Kinase II--
We examined whether phosphorylation of the EGFR by CaM
kinase II could inhibit receptor tyrosine kinase activity by immune complex kinase assay. The kinase activity of the wild-type EGFR is
reduced to about 40% by co-expression in HEK-293 cells with a
constitutively active form of CaM kinase II lacking a C-terminal auto-inhibitory domain (Fig. 5). Mutation
of serines 1046/1047 increased kinase activity by about 1.5-fold, which
was enhanced slightly by additional mutation of serine 1057, and
significantly increased by approximately 3.5-fold compared with
wild-type EGFR when serine 1142 was also mutated (Fig. 5). Mutation of
serine 744 to aspartic acid, making this site appear constitutively
phosphorylated, severely affected kinase activity, and mutation to
alanine caused a 2-fold increase relative to wild-type EGFR; however,
kinase activity was not significantly inhibited by co-expression with constitutively active CaM kinase II in contrast with wild-type, S1046A/S1047A, S1046A/S1047A/S1057A, and S1046A/S1047A/S1142A (Fig. 5).
The tyrosine autokinase activity of HER2/c-erbB2, in the
form of a chimeric receptor comprising the extracellular domain of
EGFR, was increased about 1.3-fold by mutation of threonine 1172 to
alanine, and this activity could not be significantly inhibited by CaM
kinase II1-290 (Fig. 5; see also Ref. 20).

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Fig. 5.
Autokinase activity of EGFR and HER-2
receptor mutants. Receptor immunoprecipitates from transfected
HEK-293 cells in the presence ( ) or absence ( ) of CaM kinase II
were assessed for autokinase activity as described under
"Experimental Procedures." Data are expressed relative to the
wild-type receptors and are equalized to allow for receptor expression
levels. Results are representative of two separate experiments and
presented as means ± S.D.
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Mechanism of EGFR Kinase Regulation; CaM Kinase II Phosphorylation
of the EGFR Cytoplasmic Tail Inhibits Its Association with the Tyrosine
Kinase Domain--
Since the EGFR cytoplasmic tail is a substrate for
its own tyrosine kinase, we attempted to develop an experimental system for assessing whether this enzyme substrate interaction could be
detected. We overexpressed in HEK-293 cells a mutant EGFR that lacks
the C-terminal residues 974-1186 (HER
CT), semipurified this
receptor using wheat germ agglutinin-Sepharose, and incubated this
complex with purified GST fusion protein HER-WT comprising residues
974-1186 (Fig. 6). The resulting complex
was washed extensively and bound proteins separated by SDS-PAGE. The
major Coomassie Blue-detectable protein migrates at approximately 130 kDa, corresponding with the size of HER
CT, and immunoblotting of
these samples against an antibody specific for the EGFR cytoplasmic
tail (HERCT) indicates that the GST HER-WT can form a stable complex
with HER
CT (Fig. 6b). It is also apparent that this
interaction is significantly inhibited following prephosphorylation of
GST HER-WT with CaM kinase II (Fig. 6b), and this effect was
prevented by mutation of major phosphorylation sites at serines 1046, 1047, 1057, and 1142 to alanine (data not shown).

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Fig. 6.
Effect of CaM kinase II on EGFR kinase domain
and cytoplasmic tail interactions. a, schematic
representation of experimental design to assess interactions between
the EGFR kinase domain and cytoplasmic tail. b, wheat germ
agglutinin precipitates of HER CT were incubated with GST-HERCT (20, 8, or 3 µg), which had been prephosphorylated in the presence or
absence of purified CaM kinase II as described under "Experimental
Procedures." Samples were separated by SDS-10% PAGE and stained with
Coomassie Blue or immunoblotted against anti-EGFR antibody (Sigma;
E3138, anti-HERCT).
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DISCUSSION |
In this study, we provide evidence that CaM kinase II contributes
significantly to the control of EGFR signaling and attenuation of
tyrosine autokinase activity. Two serine residues at positions 1046 and
1047 have been implicated previously in EGF-induced receptor desensitization (18). We now confirm that these are phosphorylated by
CaM kinase II with a marked preference for serine 1047 and find
additional sites at serines 1142 and 1057 that reside within an
alternative -S-X-D- consensus phosphorylation sequence. Serine 1142 is
a preferred site of phosphorylation and combined site-directed mutagenesis of this, together with other CaM kinase II sites in the
EGFR cytoplasmic tail, leads to overt transformation of NIH3T3 fibroblasts and increases receptor tyrosine autokinase activity about
3-fold.
The tyrosine-specific phosphorylation function of RTKs is indispensible
for the engagement of intracellular effector systems that govern
cellular responses such as proliferation, differentiation, and
migration. While receptor-specific substrate recruitment provides some
definition in the transmission of distinct signals, differences in
amplitude and duration of receptor tyrosine kinase activity can make
important contributions to the nature of the biological response (31).
For instance, transient MAP kinase activation downstream of the EGFR
mediates a proliferative response in PC12 cells, while sustained
activity from an activated EGFR mutant results in differentiation (32).
It is feasible that cell surface receptors that fail to down-regulate
efficiently can lead to the relatively prolonged activation of MAP
kinase and other common downstream signaling molecules and that this
may be an important determinant of transforming potential. Therefore,
the greater oncogenic capacity of RTKs such as the type I family member
HER2/c-erbB2, which shares considerable sequence homology
with the EGFR, may be a reflection of impaired mechanisms of
down-regulation and kinase desensitization. Within
HER2/c-erbB2 the position equivalent to the EGFR site for
CaM kinase phosphorylation at serine 1047 is substituted for a glutamic
acid (residue 1114 in HER2/c-erbB2). We have reported
previously that HER2/c-erbB2 contains only a single CaM
kinase II site at threonine 1172 within an
R-X-X-S/T consensus (20). Our findings in this
present study indicate that, in marked contrast to the EGFR, tyrosine
autokinase activity of HER2/c-erbB2 is not affected in cells
which overexpress CaM kinase II1-290, even though mutation
of threonine 1172 to alanine up-regulated tyrosine autokinase activity
about 1.5-fold. Taken together, these observations suggest that an
important aspect of the differential signaling properties between the
EGFR and HER2/c-erbB2 might relate to the number of CaM
kinase II phosphorylation sites and also the absence in
HER2/c-erbB2 of a major regulatory site equivalent to serine
1142 found in the EGFR. The sustained signal emanating from receptors
with fewer CaM kinase II regulatory sites may in turn partly explain
the observation that HER2/c-erbB2 is 100-fold more oncogenic
than the EGFR in cellular transformation assays (33).
The carboxyl tail of the EGF receptor is thought to have a regulatory
function; however, it is not yet clear whether it determines a positive
or negative response. The main sites of autophosphorylation (tyrosines
992, 1068, 1086, 1148, and 1173) are found in the carboxy tail and
provide interaction sites for many of the known SH2 domain-containing substrates. Many studies relating to the signal transducing properties of the carboxy tail have employed consecutive C-terminal deletions that, in most cases, remove many of the substrate binding sites. The
biological effects of C-terminal deletions have yielded conflicting results. Removal of 202 C-terminal residues enhanced fibroblast transformation (34); however, truncation of the C-terminal tail at
residue 973 does not result in a constitutively active kinase (35).
Naturally occurring C-terminal truncations and mutations in the EGF
receptor have been found in a number of oncogenic products of avian
erythroblastosis viruses (37). Sequence alterations responsible for
their increased oncogenic capacity include deletions in the carboxy
tail and point mutations in the kinase domain (26). An internal
deletion of 21 amino acids in v-erbB, corresponding to
residues 1040-1060 in the human EGF receptor, were found to be
essential for transformation (38, 39). This region includes the
negative regulatory CaM kinase II phosphorylation sites at serines
1046/1047 and serine 1057. These observations support an important role
for CaM kinase II site-specific phosphorylation in regulating normal
EGFR function. Deletion of these three regulatory sites in
v-erbB could contribute to defects in desensitization followed by an accumulation of an active tyrosine kinase within the
cell that is manifest as a transformed phenotype.
Activation and regulation of the EGFR kinase is clearly complex and
involves covalent modification as well as a number of critcal
protein-protein interactions. Binding of EGF leads to receptor
dimerization and simultaneous activation of the tyrosine kinase domain
(40). Interactions within the kinase domains then contribute to the
stabilization of an active dimeric conformation (21). The recent
crystal structure of the fibroblast growth factor receptor tyrosine
kinase domain has been determined, and a comparatively large buried
dimeric interface was found between two conserved regions comprising
helix C of the kinase domain (41). The crystal structure of the EGFR
kinase domain is not known; however, a recent model predicts a dimeric
contact between helices C that is responsible for maintaining an active
conformation in both a symmetric and asymmetric dimer (42).
Interestingly, we find an additional CaM kinase II phosphorylation site
at serine 744 (-S-V-D- consensus), which is located at the C-terminal
end of helix C in the kinase domain (Fig. 4). This represents a region of the kinase domain that is well conserved in the type I RTK family
with the exception of HER-3, which displays no kinase activity. Substitution of Serine 744 to a negatively charged aspartic acid residue to mimic phosphorylation at this site abolishes activity of the
kinase, and conversely, mutation to alanine up-regulates kinase about
2-fold (Figs. 4 and 5). It is also notable that both HER2/c-erbB2 and the EGFR have identical sequences in helix
C, with the exception of a substitution to glycine in
HER2/c-erbB2 of the position equivalent to serine 744 in the
EGFR (Fig. 4a). The absence of this site in
HER2/c-erbB2 may therefore also contribute to it being
refractory to regulation by CaM kinase II (Fig. 5).
There are several plausible explanations for the mechanism by which
serine/threonine phosphorylation mediates inhibition of RTK activity.
Receptors are known to be activated by dimerization (21, 43); however,
no marked effect of PKC or CaM kinase II on EGF receptor aggregation
was observed in experiments using covalent cross-linking analysis (18,
44). Alternative possibilities include the recruitment of tyrosine
phosphatases that is sensitive to serine/threonine phosphorylation of
the receptor, or that activation of serine/threonine kinases leads
directly to cellular phosphatase activation and thereby indirectly
modulates RTK activity (17). A model involving fold-back inhibition by
the carboxyl tail of the EGF receptor has been proposed (45). We have,
for the first time, been able to detect interactions between the EGFR
cytoplasmic tail and its own kinase domain and show that this
interaction can be disrupted by prephosphorylation of the EGFR
cytoplasmic tail with CaM kinase II (Fig. 6). These data raise the
intriguing possibility that the mechanism by which CaM kinase II
regulates EGFR signaling and autokinase activity is through a direct
effect on a substrate interaction with an enzyme active site.
Additional regulation and complete shutdown of the kinase might then be
achieved by the subsequent phosphorylation of serine 744 in helix C of the EGFR kinase domain, which would disrupt the helix C interactions required for stabilization of an active kinase configuration. Serine
744 phosphorylation may also be a dominant regulatory modification, since the S744A mutant EGFR no longer retains sensitivity to kinase regulation in the presence of constitutively active CaM kinase II, in
contrast with the alanine mutants in the EGFR cytoplasmic tail (Fig.
5).
In conclusion, the studies reported here highlight the importance of
CaM kinase II in the feedback regulation and differential control of
RTK signaling. We have shown that the number and location of CaM kinase
sites within important structural domains can account for the degree of
tyrosine kinase regulation and ultimately contribute to the strength of
signal emanating from activated cell surface receptors such as
HER2/c-erbB2 and the EGFR. An important goal for future
research will be to fully understand the structural aspects of these
events and, through the use of phosphopeptide-specific antisera raised
against individual phosphorylation sites, examine spatial and temporal
signal activation of these key receptor regulatory pathways in
vivo.