Download Protein kinase Ca activation by RET: evidence for a negative

Oncogene (2003) 22, 2942–2949
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Protein kinase Ca activation by RET: evidence for a negative feedback
mechanism controlling RET tyrosine kinase
Francesco Andreozzi1,2,3, Rosa Marina Melillo1,2, Francesca Carlomagno1,2, Francesco Oriente1,2,
Claudia Miele1,2, Francesca Fiory1,2, Stefania Santopietro1,2, Maria Domenica Castellone1,2,
Francesco Beguinot1,2, Massimo Santoro1,2 and Pietro Formisano*,1,2
1
Dipartimento di Biologia e Patologia Cellulare e Molecolare ‘L. Califano’, Università degli Studi di Napoli ‘Federico II’, via S.
Pansini 5, 80131 Napoli, Italy; 2Istituto di Endocrinologia ed Oncologia Sperimentale del C.N.R. Università degli Studi di Napoli
‘Federico II’, via S. Pansini 5, 80131 Napoli, Italy
We have studied the role of protein kinase C (PKC) in
signaling of the RET tyrosine kinase receptor. By using a
chimeric receptor (E/R) in which RET kinase can be
tightly controlled by the addition of epidermal growth
factor (EGF), we have found that RET triggering induces
a strong increase of PKCa, PKCd and PKCf activity and
that PKCa, not PKCd and PKCf, forms a liganddependent protein complex with E/R. We have identified
tyrosine 1062 in the RET carboxyl-terminal tail as the
docking site for PKCa. Block of PKC activity by
bisindolylmaleimide or chronic phorbol esters treatment
decreased EGF-induced serine/threonine phosphorylation
of E/R, while it caused a similarly sized increase of EGFinduced E/R tyrosine kinase activity and mitogenic
signaling. Conversely, acute phorbol esters treatment,
which promotes PKC activity, increased the levels of E/R
serine/threonine phosphorylation and significantly decreased its phosphotyrosine content. A threefold reduction
of tyrosine phosphorylation levels of the constitutively
active RET/MEN2A oncoprotein was observed upon
coexpression with PKCa. We conclude that RET binds
to and activates PKCa. PKCa, in turn, causes RET
phosphorylation and downregulates RET tyrosine kinase
and downstream signaling, thus functioning as a negative
feedback loop to modulate RET activity.
Oncogene (2003) 22, 2942–2949. doi:10.1038/sj.onc.1206475
Keywords: protein kinase C; tyrosine; phorbol; oncogene; thyroid; familial; papillary; signaling; carcinoma
Introduction
The GDNF family of neurotrophins comprises four
members, collectively designated GFLs (GDNF family
ligands): GDNF, Neurturin, Persephin and Artemin
(Manie et al., 2001). GFLs function as ligands of a
multi-component receptor complex consisting of the
*Correspondence: P Formisano; E-mail: [email protected]
3
Current address: Dipartimento di Medicina Sperimentale e Clinica
‘‘G. Salvatore’’, Università degli Studi di Catanzaro ‘‘Magna Graecia’’
Received 8 August 2002; revised 11 February 2003; accepted 12 February
2003
proto-oncogenic receptor tyrosine kinase, RET and a
GPI-anchored protein named GFRa. The existence of
four different GFRa proteins (GFRa1, GFRa2, GFRa3
and GFRa4) gives different ligands the specificity of
binding to RET-GFRa complex. The GFRa receptors
are linked to the cell membrane via glycosyl-phosphatidylinositol (GPI)-anchors. This signaling complex is
essential for the development of the kidney and some
structures of central and peripheral nervous systems
(Manie et al., 2001).
The RET gene has been associated with several
human diseases and genetic syndromes (Jhiang, 2000).
Loss-of-function mutations in RET, which result either
in the lack of expression or in the expression of a
nonfunctional protein, cause defective intestinal innervation, impaired peristalsis and congenital megacolon
(Hirschsprung’s disease) (Manie et al., 2001). In
papillary carcinomas of the thyroid, chromosomal
inversions or translocations cause the fusion of the
kinase domain of RET with heterologous proteins
containing dimerization motifs. This results in constitutively activated and tyrosine autophosphorylated chimeric RET oncoproteins called RET/PTC (Fagin,
2002). Finally, germline point mutations in RET result
in inherited multiple endocrine neoplasia type 2A and
2B (MEN2A, MEN2B) and familial medullary thyroid
carcinoma (FMTC). These mutations convert RET into
a dominantly transforming oncogene. While cysteine
mutations in MEN2A and FMTC activate the RET
kinase by inducing disulfide-linked homodimerization,
the MEN2B-associated Met918Thr substitution directly
targets the RET kinase domain (Ponder, 1999).
Signal transduction properties of wild-type and
oncogenic RET variants are being elucidated. The
cytoplasmic domain of RET contains 14 tyrosines and
a longer form, that is generated by alternative splicing,
contains two additional tyrosines. Phosphorylated
Y1015 and Y1062 are docking sites for several signaling
molecules. Phospholipase Cg(PLCg) interacts with
activated RET via pY1015. RETpY1062 functions as a
multiple docking site being involved in RET binding
to the phosphotyrosine-binding (PTB) domains of
Shc, IRS1 (insulin receptor substrate 1), FRS2
(FGF receptor substrate 2), RAI and Dok1. The
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Y1062-associated adaptor proteins strongly contribute
to activation of several downstream signaling pathways
such as the ras/MAPK or PI3K/AKT. Accordingly,
Y1062 is essential for mitogenic, transforming and
survival RET signaling (Manie et al., 2001). Eventually,
most recently, serine phosphorylation of RET kinase
induced by cAMP has been shown to be important for
lamellipodia formation, resulting from Rac1 activation
(Fukuda et al., 2002).
Protein kinase C (PKC) enzymes have emerged as key
regulators of receptor tyrosine kinases. PKCs are
subdivided into three subfamilies, based on structural
properties and requirement of specific cofactors (Jaken,
1996; Newton, 1997; Mellor and Parker, 1998). Conventional PKCs (cPKCs, e.g. a, b, and g) are activated
by diacylglycerol (DAG) and calcium; novel PKCs
(nPKCs e.g. d, e, Z and y) do not respond to calcium but
require DAG for activation; and atypical PKCs (aPKCs
e.g. l, z and i) are neither activated by DAG nor by
calcium. PKCs might also be regulated by the stimulation of cell surface receptors, such as the epidermal
growth factor (EGF) and platelet-derived growth factor
(PDGF) receptors (Nishizuka, 1988; Seedorf et al.,
1995a, b). Several PKC isoforms physically interact with
tyrosine kinase receptors, including epidermal growth
factor (EGF) (Chen et al., 1996), insulin-like growth
factor-I (IGF-I) (Li et al., 1998) and insulin (Formisano
et al., 1998; Caruso et al., 2001; Oriente et al., 2001)
receptors. In turn, PKC-mediated serine/threonine
phosphorylation has been shown to inhibit EGFR
(Decker, 1984; Downward et al., 1984; Chen et al.,
1996), insulin receptor (Takayama et al., 1984; Caruso
et al., 2001), and hepatocyte growth factor receptor
(Met) (Gandino et al., 1994) by attenuating their
intrinsic tyrosine-kinase enzymatic activity and their
autophosphorylation on tyrosine.
In this work we show that RET stimulation causes
activation of PKC enzymes representative of the
different PKC families (a, d and z) PKCa physically
associates with RET, this association requiring Y1062;
in turn, PKCa activation negatively modulates RET
kinase and mitogenic signaling.
Materials and methods
Materials
Media, sera and antibiotics for cell culture were from Life
Technologies, Inc. (Grand Island, NY, USA). The lipofectamine reagent, rabbit polyclonal antibodies toward specific
PKC isoforms and the PKC assay system (cat. #13161-013)
were also purchased from Life Technologies. PKC peptide
substrates were from Calbiochem-Novabiochem (La Jolla,
CA, USA). Bisindolylmaleimide was from Alexis, Inc. (San
Diego, CA, USA). Protein electrophoresis reagents were from
Bio-Rad (Richmond, VA, USA). Western blotting and ECL
reagents were from Amersham (Arlinghton Heights, IL, USA).
All other chemicals were from Sigma (St Louis, MO, USA).
Plasmids containing RET/MEN2A (e.g. the Cys634Arg RET
mutant), RET/MEN2A-Y1062F (Melillo et al., 2001b), PKCa,
PKCd and PKCz have been previously described (Formisano
et al., 2000). All RET plasmids encode for RET9 isoform.
Cell culture and transfection
NIH3T3 and 293 cells were grown in DMEM supplemented
with 10% fetal calf serum, 10 000 U/ml penicillin, 10 000 mg/ml
streptomycin and 2% l-glutamine, in a humidified CO2
incubator. NIH3T3 cell lines expressing EGFR-RET (E/R)
or E/R-Y1062F- and RET/GFRa1 have been previously
characterized (Santoro et al., 1994; Carlomagno et al., 1998).
Transient transfection of 293 cells was performed by the
lipofectamine method according to the manufacturer’s instruction. By using pCAGGS-b-gal as a reporter, transfection
efficiency was consistently between 45 and 60%. The expression of the plasmid constructs was routinely controlled by
Western blot with specific antibodies.
PKC activity assay
PKC activity was measured as previously described
(Formisano et al., 1998). Briefly, cells were solubilized in
20 mm Tris, pH 7.5, 0.5 mm EDTA, 0.5 mm EGTA, 0.5%
Triton X-100, 25 mg/ml aprotinin, 25 mg/ml leupeptin (extraction buffer), and then clarified by centrifugation at 5000 g for
20 min. Lysates were supplemented with the lipid activators
(10 mm phorbol 12-myristate 13-acetate, 0.28 mg/ml phosphatidyl serine and 4 mg/ml diolein) and phosphorylation reactions initiated by addition of the substrate solution: 50 mm
Ac-MBP(4-14), 20 mm ATP, 1 mm CaCl2, 20 mm MgCl2,
4 mm Tris, pH 7.5 and 10 mCi/ml (3000 Ci/mmol) [g-32P]ATP.
The reaction mixtures were further incubated for 10 min at
room temperature, rapidly cooled on ice and spotted on
phosphocellulose discs. Bound radioactivity was quantitated
by liquid scintillation. Activity of the specific PKC isoforms
was assayed by using precipitates with specific antibodies.
Determination of diacylglycerol intracellular content
Diacylglycerol (DAG) content was determined as described by
Farese et al. (1988). Briefly, cellular lipids were extracted by
chloroform/methanol/water (2 : 1 : 1, by volume) and centrifuged (2000 g for 10 min). DAG mass in lipid extracts was
determined by using DAG kinase and [g-32P]ATP to convert
DAG into [32P]phosphatidic acid. The latter was purified by
thin layer chromatography and DAG mass was estimated by
comparing the samples with diolein standards (Preiss et al.,
1986).
Western blot analysis and immunoprecipitation procedure
Cells were solubilized in lysis buffer (50 mm HEPES, pH 7.5,
150 mm NaCl, 10 mm EDTA, 10 mm Na4P2O7, 2 mm Na4VO4,
100 mm NaF, 10% glycerol, 1% Triton X-100, 1 mm PMSF,
10 mg/ml aprotinin) for 2 h at 41C. Lysates were clarified at
5000 g for 20 min and protein concentration was determined by
a modified Bradford assay (BioRad). Solubilized proteins were
separated by SDS–PAGE and transferred on 0.45 mm Immobilon-P membranes (Millipore, Bedford, MA, USA). Upon
incubation with the primary and secondary antibodies,
immunoreactive bands were detected by enhanced chemiluminescence (ECL) (Amersham) according to the manufacturer’s
instructions. Immunoprecipitation of specific PKC isoforms
(Formisano et al., 1998) and RET (Santoro et al., 1994) were
accomplished as previously described.
Phosphorylation in intact cells
NIH3T3-E/R cells were incubated in phosphate-free medium
containing 100 mCi/ml [32P]orthophosphate for 8 h. EGF
(10 nm) and/or TPA (100 ng/ml) were subsequently added for
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F Andreozzi et al
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15 and 30 min, respectively. Phosphorylation reactions were
rapidly quenched on ice and the cells solubilized with 1 ml/dish
of lysis buffer. Comparable amounts of proteins were
immunoprecipitated with RET antibodies, resuspended in
Laemmli buffer, separated by SDS–PAGE and analysed by
autoradiography. In order to discriminate between phosphotyrosine and phospho-threonine/serine residues, the gels were
incubated with 1 m KOH for 2 h at 551C. The bands
corresponding to the chimeric E/R protein were excised from
the gels and the radioactivity was quantitated at the b-counter.
The amount of phospho-serine/threonine was estimated based
on the difference of counts before and after alkali treatment. A
densitometric comparison of the bands was also utilized to
estimate E/R phosphorylation.
Thymidine incorporation
The thymidine incorporation assay was accomplished as
previously reported (Santoro et al., 1994). Briefly, NIH3T3E/R cells were seeded in six-well plates and, after 18 h, fed with
DMEM supplemented with 0.25% BSA. The cells were further
incubated for 16 h in the absence or presence of 10 nm EGF,
followed by addition of 500 nCi/ml of [3H]thymidine. After 4 h
the cells were washed with ice-cold 0.9% NaCl and then with
ice-cold 20% TCA followed by solubilization with 1 n NaOH.
Radioactivity was quantitated by liquid scintillation counting.
Results
PKC stimulation by RET
NIH3T3 cells expressing a chimeric construct formed by
the extracellular ligand-binding domain of the EGFR
and the cytosolic portion of RET (E/R) (Santoro et al.,
1994) were treated with 10 nm EGF for 15 min and
assayed for PKC activity. EGF exposure induced a
fivefold increase of PKC activity (Figure 1a). In
comparison, untransfected NIH3T3 cells, which express
negligible levels of EGFR, displayed no significant
increase in PKC activity upon EGF treatment. Acute
(60 min) exposure to 100 nm TPA induced a 10–11-fold
increase of PKC activity in both cell types. We generated
E/R-Y1062F mutant by replacing RET tyrosine 1062
with phenylalanine in the E/R background and stably
expressed it in NIH3T3 cells. E/R, E/R-Y1062F
displayed comparable expression levels in the selected
mass populations (see Figure 3a). EGF-stimulated (but
not TPA-stimulated) PKC activity was reduced by
>80% in E/R-Y1062F cells compared to E/R
(Figure 1a). Pre-exposure to 100 nm bisindolylmaleimide
(a concentration able to block PKCa, while having
slight, if any, effect on PKCd and PKCz) blunted both
EGF- and TPA-induced PKC activity in all cell types
(Figure 1a).
PKC activity was assayed in isozyme-specific (PKCa,
PKCd and PKCz) immunoprecipitates. No PKCbI and
bII were detectable in this cell type (data not shown). In
ligand-stimulated E/R cells, PKCa, PKCd and PKCz
activities were increased by 5-, 4- and 3.5-fold,
respectively. The mutation of Y1062 strikingly obstructed the activation of the various PKCs tested
(Figure 1b).
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Figure 1 PKC activity in NIH3T3 cells expressing E/R. (a) PKC
activity was measured in crude membrane preparations (50 mg/
sample) of parental, and E/R and E/R-Y1062F expressing NIH3T3
cells. Cells were analysed in basal conditions and upon stimulation
with 10 nm EGF for 15 min, 100 ng/ml TPA for 30 min, either in the
presence or in the absence of pretreatment with 100 nm bisindolylmaleimide for 30 min. Bars represent means7s.d. of four
triplicate experiments. (b) PKC activity was assayed in isoformspecific precipitates (200 mg protein/sample) derived from E/R and
E/R-Y1062F cells in basal conditions and upon stimulation with
EGF, in the absence or in the presence of 15 min pretreatment
with 25 mm U73122. Bars represent means7s.d. of four triplicate experiments. (c) NIH3T3 cells expressing GFRa were
stimulated with GDNF (10 nm) for 15 min. PKC activity was
assayed in isoform-specific precipitates of the cell lysates as described above. Bars represent means7s.d. of four triplicate
experiments
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Similar to EGF in cells expressing the chimeric
RET, GDNF (10 nm) induced activation of PKCa, d
and z, respectively, by 3-, 2.5- and 2.2-fold in NIH3T3
cells transfected with GDNF receptor a (GFRa)
(Figure 1c).
A phospholipase C inhibitor, U73122, reduced PKCa
activation by >80% in E/R cells and had no effect on
PKCd and PKCz activities (Figure 1b). To further study
the molecular mechanisms of PKCa activation, we have
evaluated the ability of E/R and E/R-Y1062F to interact
with PLCg. Both chimeric proteins coprecipitated with
PLCg upon EGF stimulation (Figure 2a). In addition,
EGF treatment of the cells increased intracellular
diacylglycerol (DAG) content by about threefold in
E/R and E/R-Y1062F cells and not in mock-transfected
NIH3T3 cells (Figure 2b).
RET binding to PKC isoforms
Figure 2 Activation of PLCg in cells expressing E/R and E/RY1062F. (a) Lysates of parental, E/R and E/R-Y1062F cells
(400 mg protein/sample), unstimulated or stimulated with EGF for
15 min, as indicated, were precipitated with anti-RET and blotted
with PLCg antibody. The autoradiograph is representative of three
independent experiments. (b) Parental, E/R and E/R-Y1062F cells
were stimulated with 10 nm EGF for 15 min. Cellular lipids were
extracted by chloroform/methanol/water, and DAG mass was
measured as described in Materials and methods. Bars represent
means7s.d. of four triplicate experiments
NIH3T3-E/R cells were stimulated with EGF or left
untreated. Cells were harvested and protein lysates were
precipitated with anti-RET and immunoblotted with
isoform-specific PKC antibodies. PKCa coprecipitated
with E/R in EGF-stimulated cells (Figure 3a); conversely, neither PKCd nor PKCz was detectable in E/R
precipitates (data not shown). Similarly, E/R was
detected in PKCa immunoprecipitates, while absent in
PKCd and PKCz precipitates (Figure 3b). Interestingly,
EGF treatment failed to induce coprecipitation of
E/R-Y1062F with PKCa, demonstrating that Y1062 is
essential for RET-PKCa association (Figure 3a). Comparable amounts of chimeric proteins were precipitated
by RET antibodies in E/R and E/R-Y1062F cells
(Figure 3a) and no difference in PKCa, PKCd and
PKCz expression levels was observed in the different cell
lines (Figure 3c). PKCa coprecipitation with E/R was
evaluated in cells which have been either chronically
(24 h) treated with TPA, in order to provoke cPKCs
downregulation, or treated with 100 nm bisindolylmaleimide for 30 min. In both cases, EGF exposure of
the cells did not induce any significant increase of
Figure 3 Co-precipitation of E/R and PKCa. (a) Lysates of parental, E/R and E/R-Y1062F cells (400 mg protein/sample) were
precipitated with anti-RET and blotted with either PKCa (top) or RET antibody (bottom). (b) The same lysates were precipitated with
isoform-specific PKC antibodies and blotted with RET antibody. (c) Control lysates (50 mg) were blotted with PKC antibodies. (d) E/R
cells were treated with either 100 nm bisindolylmaleimide for 30 min or with 100 ng/ml TPA for 24 h; lysates were precipitated with RET
Ab and blotted with PKCa Ab. Aliquots of the lysates were directly blotted with PKCa Ab, without prior RET precipitation, to ensure
TPA-induced PKC downregulation. These pictures are representative of at least four independent experiments
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PKCa coprecipitation with E/R (Figure 3d), thus
indicating that PKCa activation is required for its
docking to RET.
To confirm the physical association between RET and
PKCa we used RET/MEN2A, a naturally occurring
oncogenic RET mutant responsible for the familial
transmission of the MEN2A cancer syndrome. In the
MEN2A mutant, the substitution of an extracellular
cysteine (C634) with an arginine causes a ligandindependent dimerization mediated by disulfide bonds
and constitutive activation (Santoro et al., 1995). 293
cells were transiently transfected with RET/MEN2A or
its Y1062F and Y1015F mutants. Cells expressing
comparable protein levels (Figure 4a) were harvested
and RET/MEN2A-PKCa association was evaluated. As
shown in Figure 4a, PKCa coprecipitated with RET/
MEN2A and only very faintly with Y1062F and Y1015
mutants. PKCd and PKCz, although detectable in total
cell lysates, were not revealed in RET/MEN2A precipitates (data not shown), thus confirming that RET
selectively binds to PKCa.
Interestingly, expression of RET/MEN2A, but not of
its mutated counterparts, caused a 5.6-fold increase of
PKC activity (Figure 4b).
Figure 4 RET/MEN2A binding to and activation of PKC. (a)
Lysates (400 mg protein/sample) of 293 cells, mock-transfected or
transiently transfected with either RET/MEN2A, RET/MEN2AY1062F or -Y1015F, as indicated, were precipitated with RET Ab
and blotted with either PKCa (top) or RET Ab (bottom). Also,
blotting with antibodies to P-Y1062 confirmed lack of RET/
MEN2A-Y1062F phosphorylation (not shown). (b) PKC activity
was measured in crude membrane preparations (50 mg/sample) of
mock-, RET/MEN2A-, RET/MEN2A-Y1062F- or RET/MEN2AY1015F-transfected 293 cells. Bars represent means7s.d. of four
triplicate experiments
Negative control exerted by PKC on RET kinase
Tyrosine autophosphorylation of E/R, a measure of its
activity, was assessed in NIH3T3-E/R cells by phosphotyrosine blotting. As expected, E/R tyrosine phosphorylation increased upon EGF treatment in a
dose-dependent fashion (Figure 5a). Pretreatment of
the cells with bisindolylmaleimide to inhibit PKCa
caused a threefold increase of E/R tyrosine phosphorylation in the absence of EGF stimulation and
potentiated E/R response to EGF. For example, in the
Figure 5 PKC effects on RET tyrosine phosphorylation. (a) E/R cell lysates were immunoprecipitated with RET and blotted with
either anti-RET or antiphosphotyrosine. Where indicated, bisindolylmaleimide was added for 30 min and EGF for additional 15 min.
Densitometric analysis of five experiments7s.d. is shown. (b) E/R cells were treated with 100 ng/ml TPA prior to 15 min stimulation
with 10 nm EGF. Cell lysates were immunoblotted as indicated. Densitometric analysis of five experiments7s.d. is shown
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presence of bisindolylmaleimide, 1 nm EGF induced a E/
R phosphotyrosine content comparable to that obtained
with 10 nm EGF in the absence of bisindolylmaleimide.
Conversely, acute exposure to TPA, to stimulate PKC,
decreased by threefold both basal and EGF-induced
E/R tyrosine phosphorylation (Figure 5b).
NIH3T3-E/R cells were metabolically labeled with
32
P-orthophosphate. E/R precipitates were separated by
Figure 6 PKC effects on RET serine/threonine phosphorylation.
(a) 32P-labeled E/R cells were treated with 10 nm EGF and 100 ng/ml
TPA as indicated. Cell lysates were immunoprecipitated with RET
Ab, separated by SDS–PAGE and autoradiographed. The autoradiograph is representative of four independent experiments. The
bar graph represents a densitometric quantitation of 32P content of
the bands. (b) E/R cells were exposed to bisindolylmaleimide, EGF
or TPA, as indicated. Phospho-serine/threonine content was
quantitated as described in Materials and methods, upon alkali
treatment of the gels. The bands have been excised from the gel and
the amount of phospho-serine/threonine has been estimated based
on the difference of counts before and after alkali treatment. Bars
represent means7s.d. of four independent experiments
SDS–PAGE and analysed by autoradiography to detect
overall (both tyrosine and serine/threonine) phosphorylation. EGF and 100 ng/ml TPA increased E/R
phosphorylation (Figure 6a). Bands were excised from
SDS–PAGE gels before and after treatment with alkali
to hydrolyse phosphoserine and phosphothreonine and
counted. The amount of phospho-serine/threonine was
estimated based on the difference of counts before and
after alkali treatment. As shown in Figure 6b, EGF
exposure increased serine/threonine phosphorylation of
the chimeric protein by about fourfold. Acute treatment
with 100 ng/ml TPA (30 min) also induced a sixfold
increase of E/R phosphorylation. In the absence of
other stimuli, bisindolylmaleimide treatment decreased
E/R serine/threonine phosphorylation by twofold, and it
returned EGF- and TPA-effects to levels comparable to
the basal ones (Figure 6b).
EGF stimulation induces DNA synthesis of NIH3T3E/R cells (Santoro et al., 1994). E/R cells were incubated
with either 1 or 10 nm EGF in the presence or the
absence of 100 nm bisindolylmaleimide for 15 h prior to
measuring 3H-thymidine incorporation. As shown in
Figure 6, EGF stimulation led to a dose-dependent
increase of thymidine incorporation in E/R cells. PKC
inhibition by bisindolylmaleimide significantly potentiated the mitogenic response to EGF. Depletion of
endogenous cPKCs by 24 h exposure to TPA was
similarly accompanied by a 1.6-fold increase of mitogenic response to EGF (Figure 7).
Finally, to directly prove RET kinase inhibition by
PKCa, 293 cells were transiently cotransfected with
RET/MEN2A and either PKCa, PKCd or PKCz
expression vectors. Each PKC isoform was overexpressed by 8–10-fold over the basal endogenous levels
(Figure 8a). PKC activity assayed in PKCa, PKCd and
PKCz transfectants was increased by 5–8-fold with
respect to untransfected cells (data not shown). Transfectants expressing similar RET/MEN2A protein levels
Figure 7 Effects of PKC inhibition on thymidine incorporation in
E/R cells. Thymidine incorporation was assayed in E/R cells
treated with either 1 or 10 nm EGF for 16 h as indicated, in the
absence or in the presence of preincubation with bisindolylmaleimide (100 nm) or TPA (100 ng/ml) for 24 h. Bars represent
means7s.d. of four triplicate experiments
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Figure 8 PKCa effects on RET/MEN2A phosphorylation. Panels
a and b: Lysates were harvested from 293 cells, either mocktransfected (lane 1), transfected with RET/MEN2A alone (lane 2)
or transiently cotransfected with RET/MEN2A and PKCa (lane 3),
PKCd (lane 4) or PKCz (lane 5) and immunoblotted as indicated.
Panel a shows a control of the expression of individual PKC
isoforms with specific antibodies. In panel b, cell lysates have been
immunoprecipitated with RET antibodies. The upper autoradiograph shows the control of RET/MEN2A expression with specific
antibodies and the lower autoradiograph has been obtained by
blotting RET precipitates with antiphosphotyrosine antibodies.
The autoradiographs shown are representative of three independent experiments. Panel c shows the densitometric analysis of
phosphoRET/RET ratio. Bars represent means7s.d. of data
obtained from three independent experiments
were selected (Figure 8b). Antiphosphotyrosine blotting
revealed that overexpression of PKCa reduced RET/
MEN2A phosphotyrosine content by at least threefold,
as compared to the cells expressing RET/MEN2A alone
(Figure 8c). PKCz had only modest effects; PKCd had
virtually no effect (Figure 8c).
Discussion
Growth factor binding triggers RET dimerization,
kinase activation and tyrosine autophosphorylation.
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Phosphorylation of Y905 in the catalytic core stabilizes
the active conformation of the kinase and facilitates
the autophosphorylation of tyrosine residues mainly
located in the C-terminal tail, including Y1062 (Iwashita
et al., 1996). Once phosphorylated, tyrosine 1062
acts as a multiple docking site recruiting several
PTB-domain containing docking proteins (Shc, FRS2,
IRS1 and Dok) which, in turn, bind to adapters,
like Grb2 and Gab1, or enzymes like phosphatidylinositol 3 kinase (PI3K) and the SHP2 phosphatase, thus
activating intracellular signaling events (Manie et al.,
2001).
Here, by using a RET kinase regulatable by the
addition of EGF (E/R) and a constitutively active RET
kinase (RET/MEN2A), we show that protein–protein
interactions at the level of tyrosine 1062 are likely
implicated also in RET signaling termination. Upon
activation, RET tyrosine 1062 binds to PKCa and
induces PKCa activation. This binding can be either
direct or, more likely, indirect and mediated by the
multiple protein scaffolds that associate to this residue.
The lack of PKCa stimulation by the Y1062F RET
mutant can be explained by multiple mechanisms. First
of all, Y1062 is required for PKCa binding. In addition,
Y1062 may also be necesary to trigger signals activating
PKCa. Indeed, by recruiting IRS1, which directly binds
to the p85 regulatory subunit of PI3K (Melillo et al.,
2001a), and Shc and FRS2, which recruit Grb2–Gab1
complexes, thereby associating with PI3K (Besset et al.,
2000; Melillo et al., 2001b), RET Y1062 is crucial for
PI3K activation by RET (De Vita et al., 2000; SegouffinCariou and Billaud, 2000). PI3K-generated phosphoinositides can act as endogenous activators of PKC
isoforms (Nakanishi et al., 1993; Le Good et al., 1998).
It is therefore possible that the impairment of PI3K
recruitment determines the lack of PKCs’ activating
ability of Y1062F RET mutants. This hypothesis is
consistent with our recent finding that IRS1 is involved
in mediating PKCa and PKCb activation by insulin
(Formisano et al., 2000). Phosphorylated RET tyrosine
1015 directly interacts with phospholipase Cg (PLCg) to
enhance DAG production (Borrello et al., 1996). We
have observed that block of PLCg activity by U73122
inhibits RET-induced activation of PKCa, and not of
PKCd and z. However, RET Y1062F mutants retain the
ability to interact with PLCg and nonetheless they fail to
activate PKCs, suggesting that PLCg recruitment,
though necessary, is not sufficient to trigger PKC
activation.
By modulating endogenous PKCa with TPA or
bisindolylmaleimide or by plasmid transfections, we
have found that PKCa promotes RET phosphorylation
on threonine/serine residues, phosphoserine content
being more abundant than phosphothreonine (not
shown). It is likely that PKCa phosphorylates RET
directly. Indeed, inspection (at http://scansite.mit.edu/
(Yaffe et al., 2001)) of the intracellular RET sequence
revealed three sites (threonine 675 in the juxtamembrane
domain, and serines 811 and 819 in the C-terminal lobe
of the kinase) confirming the PKCa phosphorylation
consensus sequence. Inhibition of RET serine/threonine
RET inhibition by PKCa
F Andreozzi et al
2949
phosphorylation by bisindolylmaleimide was accompanied by increased RET autophosphorylation on tyrosine. Conversely, significantly decreased RET tyrosine
phosphorylation was obtained when PKCa was overexpressed or enhanced by acute phorbol esters treatment, thereby indicating that PKCa activity negatively
modulates RET tyrosine kinase activity. In addition,
RET mitogenic signaling was enhanced by PKC
inhibition and/or downregulation.
In conclusion, our findings unveil a negative feedback
circuit controlling RET signaling. According to this
model, RET activation causes phosphorylation of
Y1062 which binds to and leads to the activation of
PKCa. PKCa, in turn, promotes RET phosphorylation
on serine/threonine and inhibits RET kinase activity
thus leading to signaling termination.
Acknowledgements
This study was supported in part by grants from the
Associazione Italiana per la Ricerca sul Cancro (AIRC), by
the EC grants FIGH-CT1999-CHIPS and QLRT-1999-00674,
by the Programma Biotecnologie legge 95/95 (MURST 5%),
by BioGeM s.c.ar.l. (Biotecnologia e Genetica Molecolare nel
Mezzogiorno d’Italia), by the Ministero dell’ Università e della
Ricerca Scientifica. The financial support of Telethon — Italy
(Grant no. 0896 to FB) is gratefully acknowledged. F Fiory is
the recipient of a fellowship of the Federazione Italiana per la
Ricerca sul Cancro (FIRC).
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