(Received for publication, August 9, 1995; and in revised form, October 5, 1995)
From the
Early colony stimulating factor-1 (CSF-1)-induced changes in the
behavior of p120 in mouse BAC1.2F5
macrophages were investigated. p120
is associated with Grb2 in the cytoplasm of unstimulated
cells. Following a 1-min stimulation with CSF-1,
p120
becomes
tyrosine-phosphorylated and associates with tyrosine-phosphorylated Shc
and an unknown phosphotyrosyl protein (pp80). Simultaneously, it is
ubiquitinated and translocated to the membrane. By 10 min of
stimulation, this c-Cbl exhibits substantially decreased tyrosine
phosphorylation and is de-ubiquitinated and relocated in the cytosol.
However, the association of p120
with Shc persists for at least 60 min. These data indicate
that signaling via the CSF-1R involves the transient modification of
p120
and its recruitment as a
complex to membrane.
Colony-stimulating factor-1 (CSF-1) ()regulates the
survival, proliferation, and differentiation of mononuclear phagocytic
cells via its tyrosine kinase receptor, the c-fms protooncogene product (reviewed in (1) ). Incubation of
myeloid cells with CSF-1 activates the CSF-1 receptor (CSF-1R) kinase
leading to its rapid autophosphorylation and the subsequent tyrosine
phosphorylation of certain cellular proteins(2) , including
protein-tyrosine phosphatase 1C (3) and Shc(4) .
The
c-cbl protooncogene product, a 120-kDa protein, was originally
identified as the cellular homologue of the transforming protein of the
murine Cas NS-1 retrovirus that induces pro-B, pre-B, and myeloid
tumors in mice. It is primarily expressed in hematopoietic
cells(5, 6) . Analysis of the sequence reveals that
Cbl has a potential nuclear localization sequence, a region of high
negative charge, a potential RING finger domain, a proline-rich region,
and a leucine zipper-like motif(7) . Several recent studies
have shown that c-Cbl is tyrosine-phosphorylated in T cell receptor
signaling (8, 9) in cells expressing the v-abl and bcr-abl oncogenes(10) , Fc receptor
engagement(11) , and granulocyte-macrophage colony-stimulating
factor and erythropoietin signaling(12) .
p120
is found in macrophages (13, 14) and has recently been shown to be
tyrosine-phosphorylated in response to CSF-1(14) . In this
communication, we show that CSF-1 stimulation of macrophages results in
rapid, transient, tyrosine phosphorylation, membrane translocation, and
ubiquitination of p120
and the
formation of a longer lived p120
complex with Shc.
Figure 1:
CSF-1-stimulated tyrosine
phosphorylation of p120 and its
association with Shc and Grb2 in BAC1.2F5 cells. Cells were stimulated
with 13.2 nM CSF-1 at 37 °C for the times indicated and
lysed in 0.5% Nonidet P-40, and the lysates (lanes 1 and 2) or their IgG (lanes 3 and 4), anti-Cbl (lanes 5-10), anti-Shc (lanes 11 and 12) or anti-Grb2 (lanes 13 and 14)
immunoprecipitates were Western blotted (WB) with anti-Tyr(P) (PY) (A) and reprobed with anti-Cbl (B),
anti-Shc (C), and anti-Grb2 (D). IP,
immunoprecipitation; IgG H, immunoglobulin
heavy.
p120 was
previously shown to associate with Grb2 in Jurkat cells (8) and
cells of the human leukemia cell line UT-7 (12) via the Grb2
SH3 domains. In BAC1.2F5 macrophages, Grb2 co-precipitates with
p120
, independently of CSF-1 stimulation (Fig. 1D, lanes 5-10). A 120-kDa
tyrosine-phosphorylated protein co-precipitated with both Shc and Grb2
after CSF-1 stimulation when they were separately immunoprecipitated
from cell lysates (Fig. 1A, lanes
11-14). However, when these blots were reprobed with
anti-Cbl antibody, p120
was not detected,
possibly because of insufficient amounts and/or reduced detection of
p120
from stimulated cells (see below). In
an additional experiment at 37 °C, p120
also exhibited CSF-1-dependent association with a
tyrosine-phosphorylated protein of 80 kDa (pp80) (data not shown). All
of these associations were more obvious following CSF-1 stimulation at
4 °C, where all three co-precipitated proteins were more
concentrated in the membrane fraction than in cytosol (see below, Fig. 3).
Figure 3:
CSF-1-stimulated ubiquitination of
p120 in BAC1.2F5 cells. Control
cells or cells stimulated with 13.2 nM CSF-1 for 1 min at 37
°C or 120 min at 4 °C were fractionated into cytosolic (Cyt) and Nonidet P-40 solubilized membrane (Mem)
fractions. Anti-Cbl immunoprecipitates of these fractions were Western
blotted (WB) with anti-Tyr(P) (PY) (A) and
reprobed with anti-Cbl (B), anti-ubiquitin (C),
anti-Shc (D), and anti-Grb2 (E). The dark shadow on the left side of C (lane 1) is an
artifact. Controls for the specificity of the ubiquitin staining are
described in the text.
Figure 2:
Subcellular distribution of
p120 before and after CSF-1
stimulation of BAC1.2F5 cells at 37 and 4 °C. Cells were stimulated
with 13.2 nM CSF-1 for the time indicated at 37 °C (A), 4 °C (B), and 4 °C or 4 °C for 120
min and then 37 °C (C). Subcellular fractions and lysates
were prepared as described under ``Materials and Methods.''
In A, portions of the cytosolic and Nonidet P-40-solubilized
membrane fractions at each time point were treated with alkaline
phosphatase, and both untreated and treated fractions were Western
blotted (WB) with 1) anti-Cbl and 2) anti-Tyr(P) (PY). The exposure time of the anti-Cbl immunoblot was 1 min
for the cytosolic fraction and 10 min for the membrane fraction. The
exposure times of the anti-Tyr(P) blots of cytosolic and membrane
fractions were the same. Densitometric analysis of the
p120
band in A was: lanes 2-4, 20, 36, and 45% of zero time (lane
1), respectively; lanes 6-8, 38, 56, and 45% of
zero time (lane 5), respectively. In B, the amounts
of the subcellular fractions electrophoresed were from twice the cell
number used for the amount of SDS cell lysate electrophoresed. Cyt, cytosol; Mem, membrane; Csk,
cytoskeleton; Nuc, nucleus. In C, SDS cell lysates
were prepared as described in the text and immunoblotted with anti-Cbl
antibody.
To obtain information concerning the mechanism of the
apparent CSF-1-induced loss of p120 and to
further study the translocation of p120
,
cells were stimulated for different times at 4 °C, which slows the
rates of early changes, maximizes the extent and yield of
tyrosine-phosphorylated proteins, and blocks internalization of the
ligand-receptor complex (reviewed in (1) ). Whole cell lysates
and cytosolic and membrane fractions were Western blotted with anti-Cbl (Fig. 2B). CSF-1 stimulation at 4 °C resulted in
the generation of additional
p130
-p140
species of Cbl (Fig. 2B, lanes 6, 8, and 10) over a time period (120 min) previously shown to be
equivalent to
30 s of stimulation at 37
°C(2, 15) . When cells stimulated at 4 °C for
120 min were brought back to 37 °C, the higher M
species rapidly disappeared in 3 min, and the amount of
p120
approximated unstimulated levels by 10
min (Fig. 2C). The higher M
forms
were present in both the cytosolic and membrane fractions but
selectively concentrated in the membrane fraction (Fig. 2B, lanes 8 and 10) and
persisted even after alkaline phosphatase dephosphorylation of the
samples (data not shown). As in the case of stimulation at 37 °C
(data not shown), no Cbl was detected in the cytoskeletal (Fig. 2B, lanes 11 and 12) or nuclear (Fig. 2B, lanes 13 and 14) fractions.
In contrast to the results obtained at 37 °C, where CSF-1-induced
translocation to the membrane was apparently <5% (Fig. 2A, lanes 5-8 and 13-16), at 4 °C there was a high degree of
translocation (
50%, Fig. 2B, lanes
7-10). Thus these 4 °C data support the 37 °C data,
indicating that there is a rapid, CSF-1-induced translocation of c-Cbl
to the membrane that is associated with the generation of forms
exhibiting increased apparent M
values that are
barely visible at 37 °C (Fig. 2A, lane
14), possibly because of their instability or further modification
at this temperature. However, cycloheximide (10 µg/ml) pretreatment
of cells at 37 °C failed to block the recovery of
p120
after CSF-1 stimulation despite the
fact that in a parallel experiment it inhibited the incorporation of
[
S]methionine by 40-fold (data not shown). This
observation and the results from the 4-37 °C temperature
shift experiment (Fig. 2C) clearly show that the
transient loss of p120
is not due to protein
degradation but to a reversible modification that results in dispersion
of the c-Cbl protein bands and/or inhibition of its detection by
antibody.
The existence of stable multiubiquitinated proteins (22) and specific deubiquinating enzymes (23) suggests
that ubiquitin may have other functions besides targeting proteins for
degradation. For example, engagement of the high affinity
immunoglobulin E receptor results in its rapid and reversible
multiubiquitination (24) and ubiquitination is involved in
monoamine oxidase B insertion into the mitochondrial outer
membrane(25) . Thus, the reversible ubiquitination of
p120 without degradation is not without
precedent.
Recent studies indicate that p120 is the homologue of the product of the Caenorhabditis elegans gene, sli-1 (suppressor of cell lineage
defect-1)(26) . sli-1 negatively regulates signaling
via the epidermal growth factor receptor homologue LET-23, which
regulates vulval induction. On the basis of genetic studies, it has
been suggested that SLI-1 acts as a negative regulator of the vulval
induction pathway at a step prior to LIN-45 RAF, most likely at the
LET-23/SEM-5 step(26, 27) . We have shown that
p120
is associated with the SEM-5 homologue
Grb2 in the cytoplasm of unstimulated cells and that following
stimulation with CSF-1, it is modified and translocated to the
membrane. Its association with tyrosine-phosphorylated Shc is
consistent with an action of p120
upstream
of Ras, since Shc, via its association with Grb2, mediates activation
of Sos(19) . In support of this, we have recently observed that
the initial membrane association of p120
involves the activated CSF-1R. (
)Of relevance to
a possible negative regulatory role of p120
in
CSF-1 signaling, we have also shown that the rapid CSF-1-induced
modification increasing the M
of the CSF-1R (21) prior to its internalization is ubiquitination,
which may be associated with CSF-1R degradation. It is possible
that one of the functions of p120
is the
targeting of CSF-1R ubiquitination.