Lyn and Fgr are activated in distinct membrane fractions of human granulocytic cells - Nature
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Oncogene (1997) 15, 2021 ± 2029
1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00
Lyn and Fgr are activated in distinct membrane fractions of human
granulocytic cells
Heidi Welch and Isabelle Maridonneau-Parini
Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scienti®que, UPR 9062, 205 route de
Narbonne, 31077 Toulouse Cedex, France
We have previously shown that the src-family protein- lysacharide (Stefanova et al., 1993); (iv) both Lyn and
tyrosine kinase Hck is localized on the azurophil Hck are associated with the immunoglobulin G
granules of human granulocytes, translocates towards receptors (FcgRs) I and II in monocytic cells
the phagosomes during phagocytosis of opsonized (Ghazizadeh et al., 1994; Wang et al., 1994; (v) even
zymosan and is activated during this process. Hck is four src family PTKs are found in a complex with the
also activated upon cell stimulation with the calcium urokinase plasminogen activator receptor (Bohuslav et
ionophore A23187, but not with PMA or the chemotactic al., 1995); and (vi) neutrophils from mice de®cient in
peptide fMLP. Here, we investigated whether the src- both Fgr and Hck show a de®ciency in the immune
family kinases Lyn and Fgr are activated under the same responses against infections with Listeria monocyto-
conditions. Upon stimulation of human neutrophils or genes and defective integrin signalling, whereas cells
retinoic acid-dierentiated NB4 cells with fMLP, only from mice de®cient in only one of these kinases do not
Lyn is activated. Cell stimulation with opsonized (Lowell et al., 1994, 1996).
zymosan or A23187 leads to simultaneous activation of Generation of src-family PTK-de®cient mice has
Lyn and Fgr. In cell fractionation experiments with shown that the loss of one kinase can often be
dierentiated NB4 cells, the kinases show a similar compensated for by other family members (Varmus
subcellular localization: Both co-fractionate quantita- and Lowell, 1994), suggesting partial redundancy in the
tively with plasma-membrane marker in two fractions functions of these kinases. Together with the fact that
that sediment at 11 000 g and 200 000 g. Lyn is several src-family PTKs are expressed within a cell type
selectively activated in the 200 000 g fraction, whereas and involved in the same signal transduction processes,
Fgr is activated in the 11 000 g fraction and distinct sets this observation has even led to the proposal that their
of tyrosine phosphorylated proteins are found in these co-expression might be super¯uous and nonfunctional
fractions. The results suggest that these kinases exert (Erickson, 1993). However, many reports have demon-
their functional roles in distinct cellular compartments in strated speci®city in the functions of src-family PTKs.
human granulocytic cells. In the human neutrophils, Hck can be activated upon
FcgRIIIB ligation, while Fgr is activated via the
Keywords: Fgr; Lyn; Src; Hck; neutrophil; NB4 cells FcgRII (Hamada et al., 1993; Zhou et al., 1995), and
oxygen radicals activate Lyn and Frg but not Hck
(Yan and Berton, 1996; Welch and Maridonneau-
Parini, 1996). Further, in electroporated neutrophils
Introduction stimulated with GTPgS, Hck becomes activated,
whereas Lyn and Fgr do not (Brumell et al., 1996).
Protein-tyrosine kinases (PTKs) of the src family are In human monocytes, engagement of MHC class II
involved in the regulation of a great variety of signal molecules by staphylococcal superantigens activates
transduction processes. Several src-family PTKs are Hck and Fgr, but not Src and Fyn (Morio et al., 1994);
expressed within each cell type, and often more than and in HL60 cells, ionizing radiation activates Lyn, but
one of the kinases associate with a single membrane not Src or Fyn (Kharbanda et al., 1994).
receptor or are activated by the same stimulus (Hardie A further indice for speci®city of function of src-
and Hanks, 1995). Of the ten known src-family family PTKs in myeloid cells can be found in their
members, seven (Fgr, Fyn, Hck, Lyn, Src, Yes and subcellular localization: Fgr which is associated with
Yrk) are expressed in phagocytes (see references in the neutrophil speci®c-granules, translocates towards
Welch et al.,1996), and many reports have shown that the plasma membrane upon cell activation induced by
several of them can be involved in a given signal the chemotactic peptide formyl-methyl-leucyl-phenyla-
transduction process: (i) Both Lyn and Yes are lanine (fMLP) (Gutkind and Robbins, 1989), and we
activated during GM-CSF receptor-mediated neutro- have shown that Hck is mainly associated with the
phil signalling and are recruited into a complex membrane of azurophil granules in human neutrophils
containing phosphatidylinositol-3-kinase (PI3K) (Cor- and granulocytic NB4 cells (MoÈhn et al., 1995; Welch
ey et al., 1993); (ii) inhibition of Lyn and Fgr and Maridonneau-Parini, 1997). Together with the
production by the antisense technique induces apopto- observation that human neutrophil granule exocytosis
sis in granulocytic HL60 cells (Katagiri et al., 1996); can be inhibited by PTK inhibitors (Welch et al., 1996),
(iii) Hck, Lyn and Fgr are activated simultaneously these results suggest that Hck and Fgr might regulate
during stimulation of human monocytes with lipopo- granule mobilization. Moreover, when cells are
stimulated with opsonized zymosan (OZ), Hck
Correspondence: I Maridonneau-Parini translocates with the azurophil granules towards the
Received 31 January 1997; revised 11 June 1997; accepted 19 June phagosomes (MoÈhn et al., 1995, and during this
1997 process, Hck is selectively activated in the azurophilLyn and Fgr in granulocyte activation
H Welch and I Maridonneau-Parini
2022
granule-enriched fraction and in the phagosomal not (Figure 1). Activation of Lyn by fMLP and
fraction (Welch and Maridonneau-Parini, 1997). A23187 was about 1.7-fold, while the eect of OZ
Although Hck is mainly associated with azurophil was slightly weaker. Activation of Fgr was more
granules, it is also present in a granule-free, plasma dicult to detect than that of Lyn, since it was
membrane-enriched fraction and in the cytosol (MoÈhn weaker and variations between dierent experiments
et al., 1995). Since stimulation with the calcium were quite high. On average, it was about 1.5-fold with
ionophore A23187 leads to Hck activation both in the A23187 and 1.3-fold with OZ. The expression levels of
azurophil granule-enriched and the granule-free mem- Lyn and Fgr did not change during these experiments
brane fraction (Welch and Maridonneau-Parini, 1997), as assessed by Western blotting (data not shown). The
this has suggested that dierent subcellular fractions of results suggest that, in human neutrophils, Lyn might
Hck might regulate distinct signalling processes. Under be involved in signal transduction events elicited by the
identical conditions, we investigated here whether, and stimuli fMLP, OZ and A23187, while Fgr might play a
in which subcellular fraction, Lyn and Fgr can be role in OZ and A23187-induced signalling, but possibly
activated, with the aim to identify new functional to a minor extent than Lyn.
dierences between phagocyte src-family PTKs. As we have reported earlier, OZ and A23187 lead,
under identical conditions, also to activation of Hck to
about 1.7-fold over its basal activity (Welch and
Maridonneau-Parini, 1997). Therefore, we have identi-
Results
®ed two stimuli, OZ and A23187, that simultaneously
activate all three kinases in human neutrophils, and we
Activation of Lyn and FGr in human neutrophils
could use them to determine in which subcellular
First, we searched for stimuli that could activate Lyn fraction the increase in kinase activity occurs.
and Fgr. For this, human neutrophils were stimulated
with the receptor agonists OZ or fMLP, or with agents
Expression and localization of Lyn and Fgr in human
that bypass receptors, phorbol-myristate acetate
granulocytic NB4 cells
(PMA) or A23187. These stimuli elicit to various
degrees the granulocyte responses of NADPH oxidase In our previous study, we have shown that the pro®le
and degranulation, and OZ induces also phagocytosis. of Hck activation is similar in human neutrophils and
Lyn and Fgr became activated during stimulation of dierentiated NB4 cells (Welch and Maridonneau-
neutrophils with OZ or A23187, but not during Parini, 1997). To study Hck activation in subcellular
stimulation with PMA. Lyn was, in addition, activated fraction, we have used NB4 cells, fractionated by
by the chemotactic agent fLMP as previously described dierential centrifugation, rather than neutrophils,
(Gaudry et al., 1995; Ptasznik et al., 1995), but Fgr was since Hck is highly susceptible to degradation by
67 — 67 —
Lyn Fgr
Enol. Enol.
43 — 43 —
Figure 1 Activation of Lyn and Fgr in human neutrophils. Human neutrophils were stimulated with 4.5 mg/ml OZ, 5 mM A23287,
5 mM fMLP or 100 ng/ml PMA for the indicated periods of time, and then Lyn and Fgr were solubilized, immunoprecipitated from
the whole-cell lysates and submitted to in vitro PTK activity assay as described in Materials and methods. The autoradiograms show
the autophosphorylation of immunoprecipitated Lyn or Fgr (16107 cell equivalents/lane) and the phosphorylation of the exogenous
substrate enolase (Enol.) of one representative experiment. The histograms show the quanti®cation by b-counting of three
experiments for each kinase (plotted is the mean amount +s.e.m. of [32P] incorporated into enolase)Lyn and Fgr in granulocyte activation
H Welch and I Maridonneau-Parini
2023
neutrophil proteases (MoÈhn et al., 1995; Welch and localized on azurophil granules (MoÈhn et al, 1995). In
Maridonneau-Parini, 1997). To be able to compare the cell fractionation experiments, about 60% of the total
results on Lyn and Fgr with those obtained for Hck, Hck per cell are found in the azurophil-granule
we used here the same cell line and fractionation enriched fraction, while the remainder of Hck is
protocol. Therefore, we studied next the expression equally distributed between the granule-free mem-
and subcellular localization of Lyn and Fgr in NB4 brane fraction and the cytosol (MoÈhn et al., 1995;
cells. Welch and Maridonneau-Parini, 1997). Therefore, the
Upon dierentiation with all-trans retinoic acid subcellular localization of Lyn and Frg is similar, but
(Lanotte et al., 1991), NB4 cells acquire the major diers from that of Hck.
neutrophil-typical responses, NADPH oxidase activity,
degranulation and phagocytosis (Testa et al., 1994;
Lyn is selectively activated in the 200 000 g membrane
Welch and Maridonneau-Parini, 1996). Lyn and Fgr
fraction of NB4 cells
activities were found in non-dierentiated NB4 cells,
but increased 2.2-fold and 3.8-fold, respectively, during Next, we examined in which of the subcellular
dierentiation (Figure 2a). This increase in kinase fractions Lyn becomes activated during stimulation
activity re¯ected a rise in Lyn and Fgr protein of retinoic-acid dierentiated NB4 cells with fMLP,
expression (Figure 2a, upper panel). At day 6, the and whether translocation events can be detected.
kinase activity decreased and the cells were used at day Lyn was exclusively activated in the 200 000 g
5 in the following experiments. membrane fraction, but not in the 11 000 g
Lyn and Fgr were both mostly membrane-bound membrane fraction or in the cytosol (Figure 3). Lyn
594%). They were both present to about the same activation was rapid (evident within 30 s of cell
extent in the 11 000 g membrane fraction and in the stimulation) and transient (decrease after 2 min of
200 000 g membrane fraction. Their localization cell stimulation). There was little translocation of Lyn
correlated largely with the distribution of the plasma between any of the three subcellular fractions upon
membrane marker HLA class I (Figure 2b). In cell stimulation with fMLP as shown on the Western
contrast, we have reported earlier that Hck is majorily blots (Figure 3).
a
0 1 3 4 5 0 1 3 4 5
67 —
67 —
Lyn Fgr
Enol.
43 — Enol.
43 —
0 1 2 3 4 5 6
0 1 2 3 4 5 6
Day of NB4-cell differentiation
Day of NB4-cell differentiation
b
67 — 67 —
Lyn Fgr
43 — 43 —
M11 M200 C
M11 M200 C
% 47 47 6
% 49 45 6
Figure 2 Expression and localization of Lyn and Fgr in NB4 cells (a) The expression and activity of Lyn and Fgr were determined
by Western blotting using 125I-labelled protein A for detection (upper panels; 26105 cells/lane) and by in vitro PTK activity assay
(lower panels; 16106 cells/lane) in whole-cell lysates of human promyelocytic NB4 cells during dierentiation with 1 mM all-trans
retinoic acid for the indicated period of time. (b) The subcellular localization of Lyn and Fgr were examined in retinoic acid-
dierentiated NB4 cells (at day 5). Cells were disrupted in a nitrogen bomb, and the post-nuclear supernatant was fractionated by
dierential centrifugation into the granule-enriched 11 000 g membrane fraction (M11), the granule-free 200 000 g membrane
fraction (M200) and the cytosol (C), as detailed in Materials and methods. The fractionation was monitored by ELISA using MPO
as azurophil-granule marker and HLA class I as plasma-membrane marker. Data are mean+s.e.m. of three experiments. The
presence of Lyn and Fg in the NB4 cell fractions was determined by Western blotting (2.56106 cell equivalents/lane).
Autoradiograms are from one experiment representative of threeLyn and Fgr in granulocyte activation
H Welch and I Maridonneau-Parini
2024
When the cells were stimulated with A23187 or OZ of Lyn in any of the NB4 cell fractions. Also, none of
instead of fMLP, Lyn was also selectively activated in the stimuli had an eect on the minor portion of Lyn
the 200 000 g membrane fraction and not in the that is cytosolic (data not shown). Therefore, all the
11 000 g membrane fraction (Figure 4). As with stimuli that lead to the activation of Lyn selectively
fMLP, the activation of Lyn by A23187 or OZ in the aect the 200 000 g membrane fraction of the kinase.
200 000 g membrane fraction was rapid, transient, and
between two- and two-and-a-half-fold. We checked
Fgr is selectively activated in the 11 000 g membrane
further, whether the lack of eect of PMA on Lyn
fraction of NB4 cells
activity observed in the whole-cell lysates of neutro-
phils (Figure 1) might have been due to simultaneous The PTK activity of Fgr was measured under the same
activation and inhibition of Lyn in dierent fractions. conditions as that of Lyn, in samples issued from the
However, PMA neither altered signi®cantly the activity same experiments. Unlike in the case of Lyn, when the
M11 M200 C
67 —
PTK activity
Lyn
Enol.
43 —
0 0.5 1 2 5 0 0.5 1 2 5 0 0.5 1 2 5
Western blot
67 —
Lyn
43 —
0 0.5 1 2 5 0 0.5 1 2 5 0 0.5 1 2 5
Time (min)
Figure 3 Activation of Lyn in subcellular fractions of fMLP-stimulated NB4 cells. Retinoic acid-dierentiated NB4 cells were
stimulated with 5 mM fMLP for the indicated periods of time, and then fractionated by dierential centrifugation into the 11 000 g
membrane fraction (M11), the granule-free 200 000 g membrane fraction (M200), and the cytosol (C), as detailed in Materials and
methods. Lyn was then solubilized and immunoprecipitated from the fractions, and its in vitro PTK activity was measured (56106
cell equivalents/lane). The quantitiy of Lyn in each fraction was monitored by Western blotting. The autoradiograms are from one
experiment representative of three
Figure 4 Activation of Lyn in subcellular fractions of OZ, A23187, fMLP or PMA-stimulated NB4 cells. Retinoic acid-
dierentiated NB4 cells were stimulated with 4.5 mg/ml OZ, 5 mM A23187, 5 mM fMLP or 100 nM PMA for the indicated periods of
time, and then further processed to determine the activity of Lyn as described in the legend to Figure 3. Data are mean+s.e.m. of
three experiments for each stimulusLyn and Fgr in granulocyte activation
H Welch and I Maridonneau-Parini
2025
cells were stimulated with A23187 or OZ, Fgr was the quantitated Western blot data. It demonstrates
exclusively activated in the 11 000 g membrane fraction that, in all cases, Lyn and Fgr activation was largely
and not in the 200 000 g membrane fraction (Figure 5). due to an increase in speci®c activity.
Activation of Fgr in the 11 000 g membrane fraction
both with A23187 or OZ was about 1.8-fold, and, like
Lyn and Fgr activation is paralled by increased tyrosine
in neutrophils, the activation of Fgr in NB4 cells was
phosphorylation of proteins in the 11 000 g and
weaker than that of Lyn. The expression level of Fgr
200 000 g membrane fractions
did not change during these experiments, a typical
immunoblot on membrane fractions from A23187 Stimulation of dierentiated NB4 cells with opsonized
stimulated cells is shown (Figure 5). As the absence zymosan led to a strong increase in tyrosine
of Fgr activation by fMLP and PMA in neutrophils phosphorylation of a large number of cellular proteins
had suggested (Figure 1), neither of these two stimuli within minutes. In the membrane fractions, distinct sets
had an eect on Fgr activity in any of the NB4 cell of proteins become phosphorylated on tyrosine (Figure
fractions (Figure 5). Further, as with Lyn, none of the 7, arrows) : in the 11 000 g fraction, phosphorylation
stimuli had an eect on the minor cytosolic portion of of a doublet at 32 kDa (molecular mass estimated from
Fgr (data not shown). Therefore, the stimuli that gel electrophoresis data) is especially prominent and a
activate Fgr selectively aect the 11 000 g membrane minor band was found at 45 kDa; in the 200 000 g
fraction of the kinase. These results demonstrate that fraction, proteins of 55, 60 and 69 kDa became
Lyn and Fgr can be simultaneously activated by the tyrosine phosphorylated. Since the increases in
same stimuli, but their activation occurs in dierent tyrosine phosphorylation were as rapid as the
subcellular compartments. activation of Fgr and Lyn, it is likely that Fgr is
among the kinases that phosphorylate the proteins of
the 11 000 g membrane fraction, while Lyn could be
Lyn and Fgr activation is due to increased speci®c
one of the kinases responsible for the tyrosine
activity rather than protein translocation
phosphorylation observed in the 200 000 g fraction.
As shown in Figure 3 by Western blotting, there was
little translocation of Lyn between the two membrane
fractions upon cell stimulation with fMLP. Densito- Table 1 Quantity and speci®c activity of Lyn and Fgr in peak
fractions of stimulated NB4 cells
metric scanning of the Western blots showed that
changes in the quantity of Lyn protein in each of the Total Speci®c
PTK Stimulus Fraction Time Quantity activity activity
fractions at any timepoint of cell stimulation never
(min) (X-fold over basal)
exceeded 20% over the basal level in resting cells (see
also Table 1). We investigated whether this was also Lyn fMLP M200 1 0.91 2.40+0.68 2.64
A23187 M200 1 0.94 2.10+0.78 2.23
true for its activation by the other stimuli and for the OZ M200 1 1.03 1.55+0.16 1.50
activation of Fgr (Figure 6). Densitometric scanning of Fgr A23187 M11 2 1.13 1.74+0.45 1.54
the Western blots con®rmed that the translocation of OZ M11 10 1.12 1.70+0.32 1.52
Lyn and Fgr protein between dierent membrane Quanti®cation of Lyn and Fgr was performed by densitometric
fractions did not exceed 20% over the basal level in scanning of the bands from the Western blots shown in Figures 3 and
resting cells, whatever stimulus was utilised. 6 and comparison of each timepoint to the respective zero-time
control. Relative speci®c PTK activity in the fractions showing peaks
Table 1 shows the relative speci®c activity of Lyn of activation was calculated, dividing the mean activity (shown in
and Fgr in the fractions showing peaks of activation, Figures 4 and 5) by the relative quantity of the Lyn or Fgr in the
calculated from the total activity (Figure 4 and 5) and respective samples
Figure 5 Activation of Fgr in subcellular fractions of OZ, A23187, fMLP or PMA-stimulated NB4 cells. Retinoic acid-
dierentiated NB4 cells were stimulated with 4.5 mg/ml OZ, 5 mM A23187, 5 mM fMLP or 100 nM PMA for the indicated period of
time, and then further processed to determine the activity of Fgr as described in the legend to Figure 3. Data are mean+s.e.m of
four experiments for each stimulus. Samples in Figures 4 and 5 were derived from the same experiments. The autoradiograms shown
are from one representative A23187- experiment (upper panel=M11, lower panel=M200). The bands correspond to the timepoints
indicated at the X-axis except that timepoint 0.5 min is omittedLyn and Fgr in granulocyte activation
H Welch and I Maridonneau-Parini
2026
M11 M200
67 — 67 —
Fgr
OZ
Lyn
43 — 43 —
0 0.5 1 10 30 0 1 5 10 30
Time (min) Time (min)
67 — 67 —
A23187
Fgr Lyn
43 — 43 —
0 0.5 1 2 10 0 0.5 1 2 10
Time (min) Time (min)
Figure 6 Western blots of Lyn and Fgr in subcellular fractions of OZ or A23187-stimulated NB4 cells. Samples (56106 cell
equivalents/lane) of the 11 000 g membrane fraction (M11) or the 200 000 g membrane fraction (M200) that showed activation of
Fgr and Lyn, respectively, were chosen from representative experiments of those shown in Figure 4 and 5 for the stimuli A23187 or
OZ and subjected to Western blotting with anti-Lyn or anti-Fgr antibody as detailed in Materials and methods
Discussion
94 — We have shown here that Lyn and Fgr are both
expressed in human granulocytic NB4 cells, that they
67 — have a similar subcellular localization, and that they
become simultaneously activated during cell stimula-
tion with OZ or A23187, but that their activation takes
place in dierent membrane fractions.
43 —
Expression and localization of Lyn and Fgr
Lyn and Fgr were already expressed in non-
dierentiated NB4 cells, whereas we found in our
previous study that Hck is not present at that stage of
cell maturation (Welch and Maridonneau-Parini,
— 31 1997). Therefore, Lyn and Fgr are expressed earlier
than Hck. Their expression moderately increased
— 27.5
during cell dierentiation with retinoic acid, which is
Time (min) 0 1 10 0 1 10 in line with the generally accepted notion that PTKs of
M11 M200
the Src family exerce their functional roles in mature
cells.
Figure 7 Tyrosine phosphorylation of proteins in the 11 000 g
and 200 000 g membrane fractions of OZ-stimulated NB4 cells.
Retinoic acid-dierentiated NB4 cells were stimulated with OZ for Both Lyn and Fgr were mostly membrane-bound,
1 or 10 min, fractionated by dierential centrifugation, and the which is consistent with other reports on their
proteins from the 11 000 g membrane fraction (M11) and subcellular localization (Notario et al., 1989; Parolini
200 000 g membrane fraction (M200) were subjected to Western et al., 1996). They were present to about the same
blotting (26106 cell equivalents/lane) with anti-phosphotyrosine extent in the granule-enriched 11 000 g membrane
antibodies (PY72). Proteins were separated on 8% (upper panel)
or 15% (lower panel) polyacrylamide gel electrophoresis. fraction and in the granule-free 200 000 membrane
Molecular weight markers are shown on the left of the upper fraction of dierentiated NB4 cells. In contrast, we
panel and on the right of the lower panel have shown earlier that Hck, which is mainlyLyn and Fgr in granulocyte activation
H Welch and I Maridonneau-Parini
2027
associated with azurophil granules, is found to about underestimated the importance of Fgr in cell activation
60% in the granule-enriched fraction, while the by studying the kinase in NB4 cells. However, Fgr
remainder is equally distributed between the granule- activation was also weaker than that of Lyn when
free membrane fraction and the cytosol (MoÈhn et studied in neutrophils (See Figure 1). Several reports
al.,1995; Welch and Maridonneau-Parini, 1997). It is have shown that Fgr is involved in the activation of
unlikely that Fgr or Lyn present in the 11 000 g adherent neutrophils (Berton et al., 1994; Yan et al.,
membrane fraction are associated with azurophil- 1995b; Lowell et al., 1996). When adherent cells are
granule membranes in NB4 cells, since (i) Lyn and stimulated by fMLP or PMA, Fgr is activated but this
Fgr both co-distributed quantitatively with the plasma was abrogated when the cells were treated with anti-
membrane marker HLA class I, whereas Hck does not integrin antibodies that inhibited cell adhesion (Berton
(Welch and Maridonneau-Parini, 1997), and (ii) OZ et al., 1994). This ®nding is consistent with our results
induced the degranulation of the azurophil granules in that PMA and fMLP did not modify the activity of
NB4 cells and a concomitant decrease of Hck protein Fgr in non-adherent cells.
in the granule-enriched membrane fraction (Welch and Having identi®ed stimuli that can induce the
Maridonneau-Parini, 1997), whereas neither the quan- simultaneous activation of Lyn and Fgr in human
tity of Lyn nor Fgr changed signi®cantly in any granulocytes, we used them to determine in which
fraction under these conditions. Therefore, it is likely subcellular fractions the kinases are activated, in order
that Lyn and Fgr are found in the 11 000 g membrane to compare the results to those obtained for Hck in our
fraction due to the presence of plasma membrane in previous study (Welch and Maridonneau-Parini, 1997)
this fraction. We have tested the possibility that a and so to show potential functional dierences between
portion of the plasma membrane might sediment at these kinases. Upon stimulation of NB4 cells with any
11 000 g because of its association with the cytoskele- of the eective agents, Lyn and Fgr were activated in
ton. However, treatment of NB4 cells with cytochalasin distinct fractions: Lyn was exclusively activated in the
D did not alter the fractionation pro®le (data not 200 000 g membrance fraction, whereas Fgr was
shown), so that this hypothesis seems unlikely, and the selectively activated in the 11 000 g membrane frac-
reason why the plasma membrane sediments in the tion and, concomitantly, distinct sets of proteins
11 000 g fraction remains to be identi®ed. became tyrosine-phosphorylated in these fractions.
A partial association of Lyn and Fgr with Triton X- This is in contrast to the situation with Hck, which,
100 insoluble cytoskeleton-associated proteins has been under identical experimental conditions, is activated in
suggested (Parolini et al., 1996; Yan et al 1995a,b). distinct subcellular fractions depending on the stimulus
However, under our solubilization conditions with utilized: OZ activates Hck selectively in the azurophil
Nonidet-P40, Fgr and Hck are solubilized to comple- granule-enriched faction and in an OZ-containing
tion. A small portion of Lyn is detergent resistant, but phagosomal fraction, whereas A23187 activates Hck
it does not change during cell stimulation. both in the granule-enriched and the granule-free
membrane fraction (Welch and Maridonneau-Parini,
1997).
Activation of Lyn and Fgr
Furthermore, neither of the agents that we utilized
We have determined the eects of the stimuli OZ, to stimulate the cells caused major translocation of Lyn
A23197, fMLP and PMA on the PTK activity of Lyn and Fgr between any of the NB4 cell fractions. In
and Fgr, and compared them to the results obtained contrast, OZ caused a major translocation of Hck from
for Hck in our previous study (Welch and the azurophil granules to the phagosomal fraction and
Maridonneau-Parini, 1997). As observed for Hck, A23187 caused an increase of Hck in the granule-
PMA did not modify the kinase activity of Lyn or enriched fraction (Welch and Maridonneau-Parini,
Fgr. fMLP strongly activated Lyn but no Fgr or Hck, 1997). In consequence, the activation of Hck induced
suggesting a certain degree of speci®city of Lyn for by OZ and A23187 was a combination of translocation
fMLP-signalling, whereas OZ and A23187 simulta- events and increases in kinase activity, whereas the
neously activated Hck (Welch and Maridonneau- activation of Lyn and Fgr was almost exclusively due
Parini, 1997), Lyn and Fgr (this report). All three to increases in the kinase activity.
kinases are activated rapidly and transiently, suggest- In conclusion, the three src-family kinases Lyn, Fgr
ing that each might be involved in early transduction and Hck are co-expressed in the same cell type and
signals elicited by these stimuli. The activation of Lyn simultaneously activated by the agents OZ and A23187
by fMLP is a con®rmation of earlier reports (Gaudry during the stimulation of human granulocytes. How-
et al., 1995; Ptasznik et al., 1995), whereas the ever, they show distinct spatial patterns of activation,
activation of Lyn and Fgr by OZ and A23187 has and this could give them access to dierent sets of
not been described before. The level of Lyn and Fgr substrates and eectors. Therefore, our results provide
activation is of similar magnitude as that previously new evidence for the functional speci®city of src-family
observed for Hck, and those found for Lyn and Fgr PTKs that are co-expressed and co-activated within a
in other reports (Welch and Maridonneau-Parini, cell type.
1997; Wang et al., 1994; Berton et al., 1994; Ptasnik
et al., 1995).
We found the activation of Fgr was generally
weaker than that of Lyn. Since Fgr is partially Materials and methods
localized on the speci®c granules in normal neutro-
phils (Gutkind and Robbins, 1989), and NB4 cells lack Reagents and antibodies
this granule population (Khannagupta et al., 1994; All-trans-retinoic acid was dissolved and stored at 10 mM
Lanotte et al., 1991), it is possible that we have in ethanol at 7808C. Cell stimulating agents (from Sigma)Lyn and Fgr in granulocyte activation
H Welch and I Maridonneau-Parini
2028
were prepared and stored as described (Welch and enriched in azurophil granules was obtained by centrifuga-
Maridonneau-Parini, 1996). Sodium orthovanadate (from tion of the post-nuclear supernatant at 11 000 g for
Amersham) was prepared according to (Krypta et al., 10 min, followed by separation of the granule-free
1988) and stored in the dark at 48C for maximally two membranes and the cytosol at 200 000 g for 45 min.
months. Rabbit anti-Fgr antiserum was prepared against Marker proteins, human leukocyte antigen class I (HLA;
the N-terminal residues 15 ± 27 speci®c for Fgr. In plasma membrane marker) and myeloperoxidase (MPO;
immunoprecipitation, it recognized speci®cally a PTK of azurophil-granule marker), were assayed by ELISA (MoÈ hn
55 kDa. The signal was abolished when the serum was et al., 1995).
depleted of reactive antibody by preincubation with the
immunogen peptide, or when pre-immunization serum was
Solubilization and immunoprecipitation of Hck and in vitro
used for immunoprecipitation. Rabbit anti-Fgr antiserum
PTK-activity assay
for Western blotting was a gift from G Berton (Berton et
al., 1994). Anity-puri®ed anti-Lyn IgG was from Santa Lyn and Fgr were solubilized from NB4 cell fractions with
Cruz Biotechnology and anti-phosphotyrosine antibodies a buer containing 1% Nonidet P40, then immunopreci-
(PY72) from Transduction Labs. pitated and assayed for their in vitro PTK activies in the
presence of acid-treated enolase as exogenous substrate,
10 mM MnCl2, 10 mM MgATP and 10 mCi [32P]gATP
Cells (6000 mCi/mmol) essentially as described (MoÈhn et al.,
Human promyelocytic NB4 cells were cultured in suspen- 1995; Welch and Maridonneau-Parini, 1997; Welch et al.,
sion as described (Welch et al., 1996). For dierentiation 1996). For solubilization and immunoprecipitation of Lyn
into neutrophil-like cells, they were maintained in the and Fgr from human neutrophils, the 200 000 g super-
presence of 1 mM all-trans-retinoic acid for 5 days, unless natant of a sonicate of non-dierentiated NB4 cells was
otherwise indicated (Welch and Maridonneau-Parini, used (at 500 mg protein/ml) instead of water in order to
1997). Non-adherent cells were used for experiments. compete-out potential proteolysis of the PTKs as described
Human neutrophils from healthy donors were isolated by (MoÈhn et al., 1995; Welch et al., 1996).
Dextran sedimentation and centrifugation through Ficoll-
Hypaque as described (Maridonneau-Parini and de Western blotting
Gunzburg, 1992), and were resuspended in mimimal
essential medium buered with 20 mM HEPES, pH 7.4 For Western blotting of Lyn and Fgr, proteins were
separated by 8% SDS ± PAGE, transferred to nitrocellu-
lose, blotted with anity-puri®ed rabbit anti-Lyn IgG or
Cell activation rabbit anti-Fgr antiserum, and revealed with ECL
Before cell activation, neutrophils were incubated for 5 min (Amersham) or with 125I-labelled protein A (Maridon-
at 48C with 1 mM diisopropyl-¯uorophosphate and then neau-Parini and de Gunzburg, 1992), as indicated.
washed once. For activation, retinoic acid-dierentiated Quanti®cation of Lyn and Fgr on ECL-Western blots
NB4 cells or neutrophils were taken up at 16107 cells/ml was done by densitometric scanning of autoradiograms
in mimimal essential medium buered with 20 mM HEPES, using the Elecphor program (CRIS, Labege, France).
pH 7.4, prewarmed for 20 min at 378C and then stimulated Quanti®cation of 125I-protein A blots was performed by g-
for various timepoints with either 4.5 mg/ml OZ counting.
(opsonized by incubation with human serum) 5 mM
fMLP, 100 ng/ml PMA or 5 mM A23187 (®nal concentra-
tions). Activation was terminated by transferring the tubes
to a melting-ice bath and immediate addition of 5 vols Abbreviations
icecold HEPES-buered mimimal essential medium con- FcgR, receptor for the Fc-region of IgG. fMLP, formyl-
taining 1 mM EDTA. The cells were then sedimented to a methyl-leucyl-phenylalanine. MPO, myeloperoxidase. PTK,
300 g for 10 m at 48C, and either whole-cell lysates were protein-tyrosine kinase. OZ, serum-opsonized zymosan.
prepared or the cells were fractionated as described below. PMA, phorbol-myristate acetate.
For all experiments, cell activation was monitored in
parallel by measurement of the NADPH oxidase and
degranulation activities as described (Welch and Maridon- Acknowledgements
neau-Parini, 1997). We thank Giorgio Berton for the gift of the anti-Fgr
antibody that we used for Western blotting. HW has
successively received scholarships from the Boehringer
Cell fractionation
Ingelheim Foundation, Germany, and the Ligue Nationale
Retinoic acid-dierentiated NB4 cells were fractionated by contre le Cancer, France. This study was funded in part by
dierential centrifugation as described (Welch and Mar- grants from the Ligue Nationale contre le Cancer,
idonneau-Parini, 1997). Brie¯y, the cells were sonicated, Association pour la Recherche sur le Cancer, Assocation
and the nuclei, cell debris and resting intact cells were de Recherche sur la Polyarthrite and the Association
sedimented at 300 g for 10 min. A membrane fraction Recherche er Partage.
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