Department of Biochemistry and Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
The heregulin receptor tyrosine kinase ErbB-4 is constitutively cleaved, in the presence or absence of ligand, by an exofacial proteolytic activity producing a membrane-anchored cytoplasmic domain fragment of 80 kD. Based on selective sensitivity to inhibitors, the proteolytic activity is identified as that of a metalloprotease. The 80-kD product is tyrosine phosphorylated and retains tyrosine kinase activity. Importantly, the levels of this fragment are controlled by proteasome function. When proteasome activity is inhibited for 6 h, the kinase-active 80-kD ErbB-4 fragment accumulates to a level equivalent to 60% of the initial amount of native ErbB-4 (~106 receptors per cell). Hence, proteasome activity is essential to prevent the accumulation of a significant level of ligand-independent, active ErbB-4 tyrosine kinase generated by metalloprotease activity. Proteasome activity, however, does not act on the native ErbB-4 receptor before the metalloprotease-mediated cleavage, as no ErbB-4 fragments accumulate when metalloprotease activity is blocked. Although no ubiquitination of the native ErbB-4 is detected, the 80-kD fragment is polyubiquitinated. The data, therefore, describe a unique pathway for the processing of growth factor receptors, which involves the sequential function of an exofacial metalloprotease and the cytoplasmic proteasome.
WHEN growth factor ligands bind to their cognate
receptors, tyrosine kinase activity is activated,
and results in the initiation of multiple signal
transduction pathways. Coincidentally, activated ligand-
receptor complexes are subject to less defined processes that alter their activity and cell surface distribution, and/or number. Most all ligand-occupied growth factor receptor
tyrosine kinases are rapidly internalized by receptor-mediated endocytosis through clathrin-coated pits (Sorkin and
Waters, 1993 Within the ErbB family of receptor tyrosine kinases
(Earp et al., 1995 This article focuses on a protein kinase C-independent
basal or constitutive mechanism that generates a similar
hydrolysis of ErbB-4. This hydrolysis is due to a metalloprotease and produces an active tyrosine kinase, whose
levels are, in turn, controlled by proteasome activity.
Materials
EGF was prepared from mouse submaxillary glands as previously described (Savage and Cohen, 1972 Cell Culture
T47-14 cells, transfected NIH 3T3 cells that overexpress human ErbB-4
(~106 receptors per cell), have been described elsewhere (Baulida et al.,
1996 Immunoprecipitation and Immunoblotting
Cell lysates were obtained as previously described (Vecchi et al., 1996 In Vitro Kinase Assay
T47-14 cells overexpressing ErbB-4 were washed and the cell monolayers
solubilized at 4°C in TGH buffer without Na3VO4. Equal aliquots of cell
lysates (100 µg protein) were immunoprecipitated by adding 0.5 µg of antibody to ErbB-4. After a 2-h incubation at 4°C, protein A-Sepharose was
added for 1 h. The immunocomplexes were then washed twice with TGH
buffer without Na3VO4 and twice with kinase buffer (20 mM Hepes, pH
7.4, 3 mM MnCl2, 20 mM MgCl2, 50 mM NaCl, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, and 100 µM Na3VO4). The immunocomplexes were resuspended in 50 µl of kinase buffer containing 20 µM cold ATP, and 4 µg of
recombinant PLC- Lectin Fractionation
WG agarose was used to selectively adsorb the highly glycosylated native
ErbB-4 receptor, but did not react with the 80-kD ErbB-4 fragment expected to contain little, if any, carbohydrate. Lysates (100 µg) in TGH
buffer were incubated for 4 h at 4°C with 50 µl WG agarose (cross-linked
4% beads) that had been prewashed in TGH buffer. After incubation, the
mixture was centrifuged and the supernatant recovered. Anti-ErbB-4 was
added to the supernatant for analysis of kinase activity and ErbB-4 protein as described above. The WG agarose beads were also analyzed directly for adsorbed kinase activity and ErbB-4 protein.
Metalloprotease Cleavage of ErbB-4
Previous data demonstrated a PMA-stimulated proteolytic cleavage of ErbB-4, which produces a soluble extracellular domain and a membrane-anchored fragment that
includes the tyrosine kinase cytoplasmic domain (Vecchi
et al., 1996 Table I.
Influence of Protease Inhibitors on ErbB-4 Proteolysis
). Tyrosine-kinase activity, as well as internalization sequences in the receptor carboxyl terminus, are
essential for this step in receptor trafficking. Internalized
ligand-receptor complexes subsequently are sorted to lysosomes where both receptor and ligand are degraded.
This process is thought to represent an attenuation mechanism necessary for the proper biological response, as it
produces a dramatic decrease or downregulation in the
number of surface receptors. It has been reported that
growth factor binding to internalization-defective receptors leads to increased transforming potential, presumably due to persistent signaling at the cell surface (Wells et al., 1990
; Masui et al., 1991
).
), the activated EGF receptor or ErbB-1
is rapidly and extensively downregulated by this pathway
(Carpenter and Cohen, 1976
). However, other members of
this family, which bind heregulin, are not subject to rapid
internalization and downregulation (Baulida et al., 1996
;
Pinkas-Kramarski et al., 1996
). This includes the two receptors, ErbB-3 and ErbB-4, which bind heregulin directly (Plowman et al., 1993
; Carraway et al., 1994
; Tzahar et al.,
1994
), as well as the ErbB-2/ErbB-3 heterodimer, which
also constitutes a high affinity heregulin receptor (Sliwkowski et al., 1994
). As heregulin is not trafficked to the
internalization pathway by receptor-mediated endocytosis, it seems likely that other mechanisms of receptor regulation at the cell surface may control the function of these
receptors. A recent study found that protein kinase C activation brings about the rapid and extensive proteolytic
cleavage of ErbB-4, producing a soluble fragment (120 kD)
representing the extracellular ligand-binding domain and
a membrane-anchored fragment (80 kD) composed of the
entire cytoplasmic and transmembrane domain (Vecchi
et al., 1996
).
Materials and Methods
). Heregulin
1 was a generous gift of M. Sliwkowski (Genentech Inc., San Francisco, CA). Betacellulin, heparin-binding EGF, and heregulin
were obtained from R & D Systems Inc.
(Minneapolis, MN). Neuregulin-2 was a gift from K. Carraway, III (Harvard Medical School, Cambridge, MA). Polyclonal IgG to the carboxyl
terminus (residues 1291-1308) of ErbB-4 were purchased from Santa
Cruz Biotechnology, Inc., Santa Cruz, CA). Serum raised against the carboxyl-terminal sequence 1108-1264 of ErbB-4 was supplied by M. Kraus
(Instituto Europeo di Oncologia, Milan, Italy) (Vecchi et al., 1996
). Antisera to PLC-
1 was described previously (Arteaga et al., 1991
). Anti-phosphotyrosine purified IgG and HRP-conjugated protein A were purchased from Zymed Labs, Inc., South San Franscisco, CA). Polyclonal
antibodies to Shc proteins were products of Transduction Laboratories
(Lexington, KY). Rabbit anti-ubiquitin serum, protein A-Sepharose, and
enhanced chemiluminescence (ECL)1 reagents were obtained from Sigma
Chemical Co. (St. Louis, MO). 125I-protein A was a product of ICN Biomedicals, Inc. (Irvine, CA) and Immunobilon-P membranes were from
MCI. PMA and wheat germ (WG) agarose were from Sigma Chemical
Co., bisindolylmaleimide (GF109203X) and N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal (ALLN) were from Calbiochem-Novabiochem Corp.
(La Jolla, CA). The metalloprotease inhibitor BB-3103 was provided by
A. Drummond (British Biotech Pharmaceuticals Limited, Oxford, England). The metalloprotease inhibitors N-(DL-[2-9{hydroxy-aminocarbonyl}methyl]-4-methypentanoyl)-L-3-terbutyl-L-alanine, 2-aminoethylamide
(TAPI-2), and Batimastat (BB-94) were gifts of L. Matrisian and P. Dempsey (Vanderbilt University, Nashville, TN). The proteasome inhibitor lactacystin was purchased from J.E. Corey (Harvard University, Cambridge, MA), and N-carboxybenzyl-leucyl-leucyl-leucine (MG-132) and carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone (Z-Leu3-VS) were gifts of S. Cohen (Vanderbilt University, Nashville, TN; through H. Ploegh, Harvard University). PMA, metalloprotease inhibitors, and proteasome inhibitors ALLN, Z-leu3-VS, and MG-132 were dissolved in DMSO.
; Vecchi et al., 1996
). These cells were routinely grown in 5% CO2 at
37°C in DME containing 20 mM Hepes, pH 7.4, 50 µM Gentamycin
(GIBCO BRL, Grand Island, NY), and 10% calf serum. Atrial tumor myocytes, AT-1 cells, derived from T antigen transgenic mice (Steinhelper et al.,
1990
; Delcarpio et al., 1991
), were provided by D.M. Roden (Vanderbilt
University). These cells, maintained as transplanted tumors, were prepared and grown in culture as previously described (Yang et al., 1994
).
Under these conditions, the cells maintain the phenotype properties of cardiac myocytes (Yang and Roden, 1996
; Lanson et al., 1992
). Experimental
cultures were generally grown in 60- or 100-mm-diam culture dishes.
).
Briefly, after overnight starvation in DME and 0.5% serum, monolayers
were incubated for the indicated times at 37°C in basal medium (DME,
0.1% BSA, and 20 mM Hepes, pH 7.2) with indicated additions, i.e., inhibitors, growth factors. The cells were next washed with calcium and magnesium-free PBS and then solubilized for 20 min at 4°C in TGH buffer (1%
Triton X-100, 10% glycerol, 20 mM Hepes, pH 7.2, 100 mM NaCl, 1 mM
phenylmethylsulphonylfluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin,
and 1 mM Na3VO4). Lysates were clarified by centrifugation (14,000 g, 10 min) at 4°C and protein concentration was determined by the modified
method of Bradford (Bio-Rad Laboratories, Hercules, CA). The ErbB-4
protein was immunoprecipitated by adding ~1 µg of ErbB-4 antibody per 200 µg of cell lysate for 2 h at 4°C and then incubating (1 h, 4°C) with protein A-Sepharose CL-4B. Subsequently, the immunocomplexes were extensively washed with TGH buffer and resuspended in 1x Laemmli
buffer. After boiling, proteins in the samples were electrophoretically separated on 7.5% SDS-PAGE gels and transferred to nitrocellulose membranes for Western blotting. Membranes were blocked with 5% milk in
PBS containing 0.05% Tween for 1 h before blotting with antibodies to
anti-ErbB-4, -Shc, -PLC-
1, and -ubiquitin. Before anti-phosphotyrosine
blotting, membranes were blocked by incubating for 1 h with 3% BSA in
TBST buffer (0.05% Tween, 150 mM NaCl, 50 mM Tris, pH 7.4). Membranes were then incubated with the appropriate antibody for 2 h at room
temperature and washed with PBS or TBST buffer, incubated with 125I-protein A for 1 h at room temperature, and after five washes with PBS or
TBST buffer, visualized by autoradiography (X-Omat AR film; Eastman
Kodak, Rochester, NY). Where indicated, bound antibody was detected
with HRP-protein A and ECL.
1 (Horstman et al., 1995
) was added. The reaction
mixtures were then incubated at room temperature for the indicated times
before stopping the reaction by adding 50 µl of 2x Laemmli buffer and boiling for 5 min. Proteins were subsequently separated on a 7.5% SDS-PAGE gel and analyzed for phosphotyrosine content by Western blotting
with antibody to phosphotyrosine. The amount of tyrosine phosphorylated PLC-
1 was quantitated by densitometric scanning. For each phosphorylation sample, a parallel aliquot of lysate was analyzed for ErbB-4
protein by immunoprecipitation and Western blot, as described above.
Results
). To identify the type of protease involved in
this cleavage, cells expressing ErbB-4 were preincubated
with various protease inhibitors before the addition of
PMA. The cleavage of ErbB-4 was then detected by assaying the 80-kD cytoplasmic domain with anti-ErbB-4. The
results, shown in Table I, revealed that three metalloprotease inhibitors reduced the PMA-stimulated cleavage by
90%. Inhibitors of other types of proteases were relatively
ineffective in this assay. Hence, these data implicate a metalloprotease as the likely enzyme that modulates ErbB-4
structure in response to PMA. Previously, a low level of a
similarly sized cytoplasmic domain fragment was detected in the absence of PMA (Vecchi et al., 1996
). As the amount
of this 80-kD fragment constitutively released from ErbB-4
is small (~4%) relative to native ErbB-4 and PMA-stimulated fragment levels (Table I), one cannot accurately
judge inhibitor effects on the constitutive hydrolysis under
these conditions.
Proteasome Function in ErbB-4 Cleavage
Previous studies (Vecchi et al., 1996) showed that the
PMA-stimulated cleavage of ErbB-4 was independent of
heregulin binding. Various ErbB-4 ligands (including heregulin-
and -
isoforms, betacellulin, heparin-binding
EGF, and neuregulin-2), were assayed for their capacity to
alter the basal level of receptor cleavage. However, none
had a significant influence (data not shown).
However, when ErbB-4-expressing cells are exposed to
the proteasome inhibitors ALLN or lactacystin, the intracellular level of the 80-kD ErbB-4 fragment is slowly but
dramatically increased (Fig. 1 A, lanes 4-7). The level of
accumulation of 80-kD fragment under these conditions
equals that previously reported (Vecchi et al., 1996) after
PMA treatment (Fig. 1, lane 2). Lactacystin is considered
the most specific inhibitor of proteasome activity (Fenteany et al., 1995
). In addition, two peptide aldehyde proteasome inhibitors, MG-132/Z-Leu 3-H and Z-Leu 3-Vs
(Bogyo et al., 1997
) also enhanced accumulation of the 80-kD
fragment (data not shown). Hence, proteasome activity
normally limits the level of ErbB-4 80-kD fragment accumulation. Additional experiments have shown that, similar to the 80-kD fragment generated in the presence of
PMA (Vecchi et al., 1996
), the 80-kD fragment that accumulates in the absence of proteasome function is tyrosine
phosphorylated (Fig. 1 B, lanes 3-7) and is membrane-
localized, as determined by cell fractionation (data not
shown).
To determine the rate and extent of this basal level of
ErbB-4 hydrolysis, the proteasome inhibitor ALLN was
added to stabilize the 80-kD fragment and accumulation
of the fragment was measured at various times thereafter.
As shown in Fig. 2 A, accumulation occurred slowly for
several hours, until 4-6 h when the amount of 80-kD fragment was readily detectable. At this time, the cellular pool
of 80-kD ErbB-4 fragment was equal to ~60% of the initial level of native ErbB-4 receptor. Since the cells used in
this experiment overexpress ErbB-4 (~106 receptors per
cell), it is estimated that the basal rate of ErbB-4 cleavage
is ~105 receptors per hour. In a separate experiment, cells
were incubated with ALLN for 6 h to accumulate an 80-kD
ErbB-4 fragment. The ALLN was then removed by washing, which restores proteasome function within 60 min
(Rock et al., 1994), and the half-life of the 80-kD fragment
was determined (Fig. 2 B). Under these conditions, the fragment was degraded with a half-life of ~4 h.
Ubiquitination is a frequent posttranslational marker
for proteasome substrates. Therefore, we used antibody to
ubiquitin to determine whether the 80-kD molecule is
modified in this manner. Cells were treated with PMA, heregulin, ALLN, or lactacystin for the indicated times and
then lysates were precipitated with antibody to the ErbB-4.
Subsequent Western blotting demonstrates the specific polyubiquitination of the ErbB-4 fragment in cells treated with ALLN or lactacystin (Fig. 3 A, lanes 4 and 5). Heterogeneous ubiquitinated products were detected from 80 kD to
the top of the gel. Using normal rabbit IgG, control precipitations of lysates from cells treated with proteosome
inhibitors showed that the detection of ubiquitinated proteins is specific for anti-ErbB-4. Interestingly, no ubiquitination of the 80-kD ErbB-4 fragment generated by PMA treatment was detected (Fig. 3 A, lane 2) even though
equivalent levels of 80-kD fragments were produced by
PMA, ALLN, and lactacystin (Fig. 3 B, lanes 2, 4, and 5).
Also, no ubiquitination of the native ErbB-4 receptor
could be detected in the absence or presence of heregulin
(Fig. 3 A, lanes 1 and 3). Addition of ALLN for 1 h also
failed to reveal the presence of ubiquitinated native receptor (data not shown).
The results described above were obtained with NIH
3T3 cells that overexpress the transfected ErbB-4 receptor. To ascertain that the proteolytic activities toward
ErbB-4 are not a consequence of the exogenous expression of this receptor, AT-1 cardiac myocytes, which express endogenous ErbB-4, were assayed. As shown in Fig. 4, addition of PMA (lane 8), or the addition of proteasome
inhibitors (lanes 2-7) produced accumulation of an 80-kD
ErbB-4 fragment in these cells. It is probable that ErbB-4
is physiologically important in these cells, as targeted disruption of the ErbB-4 gene in mice produces, in null homozygotes, embryonic lethality due to abnormal heart development (Gassmann et al., 1995).
Interrelationship of Metalloprotease and Proteasome Functions
The results cited above suggest both metalloprotease and proteasome activities are involved in a constitutive pathway of ErbB-4 cleavage. However, these data only suggest that the 80-kD fragment detected in the presence of proteasome inhibitors represents accumulation of the metalloprotease cleavage product of ErbB-4. It is plausible that the constitutive level of 80-kD ErbB-4 fragment is derived from cleavage of native ErbB-4 by another protease or splicing of ErbB-4 mRNA so as to produce an amino-terminal truncation of the native protein. Therefore, we tested whether the accumulation of 80-kD fragment in the presence of a proteasome inhibitor depends on metalloprotease activity.
As shown in Fig. 5 A, cells were pretreated with the metalloprotease inhibitor BB-94 for 30 min before the addition of the proteasome inhibitor ALLN for an additional 4 h.
The amount of 80-kD fragment was then compared to that
detected in cells treated for the same time with ALLN
only. The results show that inhibition of metalloprotease
activity reduces by ~80% the ALLN-dependent accumulation of the 80-kD ErbB-4 fragment (Fig. 5, compare
lanes 4 and 5). This result indicates that the metalloprotease-generated 80-kD fragment is the substrate for proteasome activity.
Although the PMA-stimulated accumulation of the 80-kD
ErbB-4 fragment is dependent on protein kinase C activity
(Vecchi et al., 1996), it is unclear whether the constitutive
hydrolysis may also depend on endogenous protein kinase
C activity. This has been tested by adding the specific protein kinase C inhibitor GF109203X to cells and then measuring the accumulation of the fragment after the addition
of either PMA or ALLN. As shown in Fig. 5 B, the protein
kinase C inhibitor completely blocked the hydrolysis stimulated by PMA (Fig. 5, lane 3) but had no influence on accumulation of the 80-kD fragment in the presence of
ALLN (Fig. 5, lane 5). This result indicates that the basal
level of ErbB-4 proteolysis is not dependent on protein kinase C activity.
Tyrosine Kinase Activity of the 80-kD Fragment
The 80-kD ErbB-4 fragment that accumulates in the presence of proteasome inhibitors represents the entire cytoplasmic domain of ErbB-4 and may, therefore, retain tyrosine kinase activity. This possibility has been tested by
using in vitro kinase assays. After cell incubation in the absence or presence of ALLN, cell lysates were immunoprecipitated by antibody to the ErbB-4 carboxyl terminus.
The immunoprecipitates were then incubated with kinase reaction components, including unlabeled ATP and recombinant tyrosine kinase substrate PLC-1 (Horstman
et al., 1995
). Phosphotyrosine blotting was used to detect
phosphorylated PLC-
1 and ErbB-4 blotting was used to
assess the levels of native ErbB-4 and the 80-kD ErbB-4 fragment. Control experiments showed that the ErbB-4
receptor is equally active in vitro regardless of whether the
cells are treated with or without heregulin (data not shown).
The results, shown in Fig. 6 A, indicate that the native
ErbB-4 (Fig. 6, lanes 3-5) and the ALLN-dependent 80-kD
ErbB-4 fragment (Fig. 6, lanes 6-8) phosphorylate PLC-
1
to an equivalent extent and at approximately the same
rate under these conditions. In this experiment, ErbB-4 blots (Fig. 6 B) show comparable levels of native ErbB-4
from control cells and 80-kD fragment from ALLN-treated cells. Control assays showed that no kinase activity
was detected in the absence of ATP or when an unrelated
IgG was used for immunoprecipitation (Fig. 6, lanes 6-8).
After incubation in the presence of ATP, the 80-kD fragment migrates at a slightly higher molecular mass indicative
of autophosphorylation (Fig. 6, lanes 6-8).
When lysates from ALLN-treated cells are immunoprecipitated with anti ErbB-4, a small amount of native ErbB-4
is detected together with the 80-kD fragment (Fig. 6 B).
Although the amount of native ErbB-4 in the ALLN samples is low, ~20% of that in untreated cells, it is plausible
that it might significantly contribute to the observed phosphorylation activity in vitro. Two approaches have been
used to ascertain the possible contribution of native ErbB-4
to the kinase activity measured in immunoprecipitates
from ALLN-treated cells. First, to approximate the low
level of native ErbB-4 in ALLN lysates, control lysates were
diluted (1:3) to determine if that reduced level of receptor
is sufficient to phosphorylate PLC-1 under these conditions. As shown in Fig. 7, dilution did reduce by ~75%
both the amount of native ErbB-4 (Fig. 7, lane 8) and the
kinase activity toward PLC-
1 (Fig. 7, lane 1) and does approximate the level of native ErbB-4 protein present in immunoprecipitates of ALLN lysates (Fig. 7, compare
lanes 8 and 12). Nevertheless, this low level of native
ErbB-4 does contribute to the kinase activity measured in
ALLN lysates.
To measure the kinase activity of the 80-kD fragment only, lysates from ALLN-treated cells were precleared with WG lectin to adsorb the highly glycosylated native ErbB-4. The 80-kD fragment is expected to contain little, if any, carbohydrate. These WG supernatants were then immunoprecipitated with anti ErbB-4 and assayed for kinase activity (Fig. 7 A) and ErbB-4 reactive protein (Fig. 7 B). The results show that the 80-kD fragment retains phosphorylation activity in the absence of detectable native ErbB-4 protein. WG agarose effectively removed, respectively, the native ErbB-4 protein (Fig. 7, lane 10) and its in vitro kinase activity (Fig. 7, lane 2) from control lysates. The kinase activity (Fig. 7, lane 4) and protein (Fig. 7, lane 11) of the native ErbB-4 from the control lysates were recovered on the WG beads. Hence, WG fractionation effectively removes detectable levels of native ErbB-4 protein and kinase activity before immunoprecipitation.
When ALLN samples were precleared with WG agarose, neither the amount of kinase activity (Fig. 7, lane 6)
nor 80-kD protein (Fig. 7, lane 13) was significantly decreased. In the WG agarose supernatant from the ALLN
lysate, the presence of even a small amount of native
ErbB-4 was no longer detectable (Fig. 7, compare lanes 12 and 13). The low level of native ErbB-4 kinase activity and
protein originally present in ALLN lysates was retained
on the WG agarose beads (Fig. 7, lanes 7 and 14). These
results demonstrate that the ErbB-4 80-kD fragment is, in
fact, an active tyrosine kinase. We have used densitometric scanning of the antiphosphotyrosine signal, representing PLC-1 phosphorylation, and the anti-ErbB-4 signal,
indicating the level of ErbB-4 native or 80-kD fragment, to
approximate a kinase-specific activity. This analysis included shorter exposures of the data shown in Figs. 6 and
7. In all samples, the specific phosphorylation activity of
the 80-kD fragment was equal to that of the native ErbB-4
receptor (data not shown).
Numerous studies have demonstrated the ligand-dependent degradation of growth factor receptors through a
mechanism involving internalization of coated pits and endosome sorting to the lysosome (Sorkin and Waters,
1993). In certain instances, recycling of growth factor receptors and ligands from endosomes to the cell surface has
been noted. The cell surface ligand-dependent trafficking of EGF receptor-like ErbB receptors is novel in that the
endocytic pathway is not used to rapidly internalize occupied receptors after ligand binding (Baulida et al., 1996
;
Pinkas-Kramarski et al., 1996
). This may suggest alternate
mechanisms exist to desensitize these surface ligand-receptor complexes as the mitogenic potency of EGF and heregulin are similar (Baulida et al., 1996
).
A previous study has demonstrated that protein kinase
C activation rapidly produces a cleavage of the ErbB-4
ectodomain (Vecchi et al., 1996). The ectodomain cleavage
of several growth factor receptor tyrosine kinases has been
observed in cells treated with PMA (Downing et al., 1989
;
Yee et al., 1993
; Brizzi et al., 1994
; Yee et al., 1994
; O'Bryan
et al., 1995
; Cabrera et al., 1996
; Vecchi et al., 1996
; Jeffers
et al., 1997
); however, the PMA-sensitive protease(s) has
not been identified. Based on sensitivity to several metalloprotease inhibitors, we conclude that protein kinase C activation directly or indirectly enhances the activity of a
member of this protease family toward the ErbB-4 extracellular domain. Several reports indicate that PMA increases expression of metalloprotease genes (Birkedal-Hansen et al., 1993
), but protein synthesis is not required
for the PMA-induced cleavage of ErbB-4 (Vecchi et al.,
1996
). Hence, the action of protein kinase C in this system is most likely at the secretion and/or activation step of the metalloprotease latent precursor, known sites of regulation (Coussens and Werb, 1996
). However, the latter seems
unlikely given the intracellular localization of protein kinase C and the extracellular location of metalloproteases,
which function mainly in the degradation of extracellular
matrix components. A transmembrane disintegrin molecule could provide a means of communication, as recently
reported with the cleavage of tumor necrosis factor-
precursor (Black et al., 1997
; Moss et al., 1997
).
The data in this report show that a constitutive cleavage
of ErbB-4 also occurs in the absence of exogenous protein
kinase C activation and produces an 80-kD transmembrane and cytoplasmic domain fragment of ErbB-4 that is
very similar to the ErbB-4 fragment observed after PMA
addition to cells (Vecchi et al., 1996). Most likely this basal
receptor degradation represents a low level of metalloprotease activity in the extracellular environment. Although
this ErbB-4 fragment is generally found at low levels in the
cell, proteasome activity is crucial to prevent its accumulation. Blocking proteasome activity with specific proteasome inhibitors results in accumulation of this receptor
fragment to significant levels, approaching the cellular levels of native ErbB-4. Since no ErbB-4 80-kD fragment accumulates in cells treated with both proteasome and metalloprotease inhibitors, the proteasome seems to degrade
the 80-kD fragment but not the intact ErbB-4 receptor. This is reinforced by the finding that the 80-kD fragment is
ubiquitinated, but no ubiquitin is detectable on the intact
ErbB-4 molecule under several conditions including those
comparable to the detection of ubiquitin on the PDGF (Mori
et al., 1992
; Yarden et al., 1986
), EGF (Galcheva-Gargova
et al., 1995
; Mori et al., 1995
), stem cell factor/c-kit
(Miyazawa et al., 1994
; Yee et al., 1994
), and colony stimulating factor-1/c-fms (Mori et al., 1995
) receptor tyrosine kinases. In each of these instances, however, ubiquitination occurred subsequent to growth factor binding. ErbB-4
receptor metabolism, therefore, represents a novel coupling of two protease activities acting in series to allow
ligand-independent degradation of ErbB-4 and to prevent
accumulation of cytoplasmic domain receptor fragment. If, in fact, the proteasome recognizes the 80-kD fragment
but not the intact ErbB-4 receptor, this implies that lack of
an extracellular domain in some way, perhaps involving
topological distribution, leads to recognition of the ErbB-4
cytoplasmic domain by the ubiquitination system.
Recently the hepatocyte growth factor receptor, Met,
has been shown to be ubiquitinated and subject to proteosome-mediated degradation in the absence or presence of
its ligand (Jeffers et al., 1997). In this system, prior cleavage of the intact receptor was not reported to be required
for ubiquitination. After proteasome inhibition a 50-kD
fragment of Met was detected that represented the cytoplasmic tyrosine kinase domain. Interestingly, PMA also stimulated the accumulation of a similarly sized Met fragment. In this system, however, the 50-kD fragment accumulated to only a low level relative to the level of intact
Met. Phosphotyrosine was detectable on the 50-kD fragment though kinase activity of the fragment was not assayed given the low amount of fragment produced. Analogously, PMA induces ectodomain cleavage of the NGF
receptor TrkA resulting in a tyrosine phosphorylated cytoplasmic domain fragment with unreported kinase activity
(Cabrera et al., 1996
).
There are several reported instances where removal, by
proteolysis or deletion mutagenesis, of the ectodomain of
receptor tyrosine kinases has produced an active or activated kinase domain. This includes the insulin receptor
(Ellis et al., 1987; Goren et al., 1987
; Wang et al., 1987
;
Shoelson et al., 1988
; Hsuan et al., 1989
; Lebwohl et al.,
1991
), insulin-like growth factor I receptor (Liu et al., 1992
,
1993
), and the Drosophila Sevenless receptor (Basler et al.,
1991
). Oncogenic forms of the avian EGF receptor involve
deletion of the extracellular domain as well as other more
subtle changes, all of which contribute to its transforming potential (Carter et al., 1994).
Using an in vitro assay with recombinant SH2 domain-containing protein (PLC-1) as a substrate, we have compared the kinase activity of the 80-kD ErbB-4 fragment
accumulated in proteasome-inhibited cells to that of the
intact receptor recovered from control cells. The data
show that both molecules tyrosine phosphorylate PLC-
1 at comparable efficiencies. These data indicate that this
ErbB-4 fragment has intrinsic tyrosine kinase activity.
Therefore, it is possible that the fragment has ligand-independent biological activity, increasing the importance of
proteasome degradation of the fragment. Unfortunately, it
is not possible to test the kinase activity of the 80-kD fragment in intact cells due to its proteasome sensitivity and
the toxicity of proteosome inhibitors. Regardless of kinase
activity, the tyrosine phosphorylated fragment is constitutively generated in ErbB-4-expressing cells and may act
not only as a kinase, but as a membrane-localized docking molecule for signaling molecules with SH2 domains. Coprecipitation data have shown the association of PLC-
1
and Shc with the 80-kD fragment produced in PMA-treated cells (Vecchi et al., 1996
). In some instances, docking at the cytoplasmic face of the plasma membrane may
be more critical than tyrosine phosphorylation for the
function of certain signaling molecules that associate with receptor autophosphorylation sites, for example PI-3 kinase and Grb-2 (Pawson and Schlessinger, 1993
).
There are now examples of endogenous ectodomain cleavage of several growth factor receptor tyrosine kinases. If these results with ErbB-4 are generally applicable, proteasome function may also limit the accumulation of active tyrosine kinase fragments in other receptor systems.
Received for publication 11 August 1997 and in revised form 12 September 1997.
This research was supported by a National Cancer Institute grant (CA24071).The authors thank S. Carpenter for the manuscript preparation, L. Rudolph-Owen for reading the manuscript, S. Ermini for technical assistance, D. Horstman for the recombinant PLC-1, and H. Waldrop, and D. Roden for AT-1 cells (all from Vanderbilt University).
ECL, enhanced chemiluminescence;
PLC-1, phospholipase c-
1;
WG, wheat germ.
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