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HEMATOPOIESIS
From the Department of Hematology and Oncology and the
Department of Laboratory Medicine and Clinical Sciences, Graduate
School of Medicine, Kyoto University, Kyoto, Japan; the Department of
Biochemistry, Kobe University School of Medicine, Kobe, Japan; and the
Departments of Oral Biology and Microbiology, State University of New
York at Buffalo, Buffalo, NY.
The roles of the protein tyrosine kinases Pyk2 (also called RAFTK
or CAK Several signaling molecules play important roles in
the differentiation and execution of unique functions of hematopoietic cells. As a useful model of myeloid differentiation, the HL-60 cell
line has been used extensively to study these roles. HL-60 cells can be
differentiated with various inducing agents, and HL-60 cells treated
this way have the properties of mature myeloid cells.1 The
N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) receptor belongs to the family of G protein-coupled, heptahelical receptors and is one of the first markers to appear during
differentiation of myeloid cells. The ligand, fMLP, mediates effects by
means of its receptor, which leads to intracellular signaling followed by cell adhesion and migration.
The well-characterized cell-surface receptors for extracellular matrix
proteins on neutrophils belong to the Various protein tyrosine kinases (PTKs) have been reported
to be involved in the differentiation and functions of myeloid cells. The roles of Src family PTKs have been well
investigated,4-8 but only a few studies of
Syk9,10 and Pyk2 have been done.11 Pyk2,12 which is also called RAFTK13 or CAK
We treated HL-60 cells with dimethyl sulfoxide (DMSO) to induce
differentiation toward granulocytes. The granulocytic HL-60 cells were
then stimulated with fMLP and attached to Fg, one of the ligands for
Reagents and antibodies
Cell culture and differentiation-induction studies
Cell stimulation and adhesion assays Various concentrations of fMLP were added to HL-60 cell suspensions. In some experiments, HL-60 cells were pretreated with either 20 µg/mL mouse anti- 2 integrin monoclonal
antibody IB4 for 45 minutes on ice, 20 µg/mL normal mouse IgG for 45 minutes on ice, or 50 ng/mL PT for 4 hours at 37°C in
polypropylene tubes.
For adhesion assays, 100-mm culture dishes (3020-100; Iwaki Glass Co, Chiba, Japan) were precoated with Fg (100 µg/mL; 10 mL/dish) overnight at 4°C. Nonspecific binding was blocked with 5% BSA for 1 hour at 37°C. After the BSA was removed, the dishes were washed once gently with PBS and 5 × 106 cells were seeded on the dishes in a 10-mL volume of culture medium. After incubation with 1 µmol/L fMLP for 30 minutes at 37°C, the cells were harvested for cell lysate preparation and cell counting. Immunoprecipitation procedures Cells in suspension in polypropylene tubes (5 × 106 cells) were stimulated with fMLP for various times and spun down by centrifugation (10 000g in a flash at 4°C). The incubation medium was removed by aspiration. The pelleted cells were then lysed in 1 mL nonionic lysis buffer (1% Triton X-100, 50 mmol/L Tris-hydrochloric acid [HCl] at pH 7.4, 150 mmol/L sodium chloride (NaCl), 5 mmol/L EDTA, 1 mmol/L sodium vanadate, and 1 mmol/L phenylmethyl sulfonyl fluoride) and kept on ice for 15 minutes. Cells that detached spontaneously or failed to attach after the stimulation were collected and resuspended in polypropylene tubes; these cells were handled in the same manner as cells in suspension. Cells that attached to Fg-coated dishes (5 × 106 cells) on stimulation with fMLP were lysed directly in 1 mL lysis buffer and disrupted by scraping. The lysates were transferred to polypropylene tubes, kept on ice for 15 minutes, and then centrifuged at 15 000g for 10 minutes at 4°C. The supernatants were incubated with 2.5 µg of an antibody for 1 hour at 4°C, and 20 µL protein A-Sepharose diluted in PBS was added. After another hour at 4°C, the immunoprecipitates were washed 3 times with 1 mL lysis buffer. For immunoblotting, the immunoprecipitates were boiled with sodium dodecyl sulfate (SDS) sample buffer (2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.001% bromophenol blue, and 62.5 mmol/L Tris-HCl [pH 6.8]) for 3 minutes.SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analyses For day-course studies to detect expression of proteins, whole cell lysates were prepared by boiling with SDS sample buffer for 3 minutes. Whole cell lysates or immunoprecipitates were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skim milk in low-salt Tween 20-Tris-buffered saline (T-TBS) (10 mmol/L Tris-HCl [pH 7.4] and 100 mmol/L NaCl containing 0.1% Tween 20) for 30 minutes at 37°C. This was followed by incubation with primary antibodies at either 0.1 µg/mL (for anti-Pyk2, anti-Syk, and anti-2 integrin antibodies) or
1 µg/mL (for antiphosphotyrosine monoclonal antibody 4G10) in
low-salt T-TBS for 1 hour at room temperature. The membranes were then
washed and incubated with horseradish peroxidase-conjugated secondary
antibodies at 0.5 µg/mL in low-salt T-TBS for 30 minutes at room
temperature. After washing with low-salt T-TBS and subsequently with
low-salt TBS (10 mmol/L Tris-HCl [pH 7.4] and 100 mmol/L NaCl),
enhanced chemiluminescence (ECL) assays were performed to visualize
positive bands on x-ray films.
In vitro kinase assays Immunoprecipitates were washed 3 times with 1 mL lysis buffer and incubated in a 20-µL reaction mixture (50 mmol/L HEPES-sodium hydroxide [pH 8.0], 10 µmol/L sodium vanadate, 50 mmol/L magnesium acetate, 150 mmol/L NaCl, and 10 µmol/L adenosine triphosphate [ATP]) for 30 minutes at 30°C. The reaction was terminated by adding SDS sample buffer and boiling for 3 minutes. The samples were separated by SDS-PAGE, transferred to PVDF membranes, and subjected to immunoblotting analysis with antiphosphotyrosine antibody. The increase in tyrosine phosphorylation was detected by the ECL assay.
Expression of Pyk2 and Syk during granulocytic differentiation of HL-60 cells HL-60 cells can be induced to differentiate by various agents, including DMSO, which induces these cells to differentiate toward granulocytes.1 We first examined the expression of Pyk2 and Syk during DMSO-induced differentiation of HL-60 cells by using immunoblotting analyses. The expression Pyk2 was low in undifferentiated cells, but an apparent induction occurred on day 1 and reached the peak level in differentiated cells on day 4 (Figure 1A). On the other hand, Syk was already abundant in undifferentiated cells, and the expression level did not change during differentiation (Figure 1B). Morphologic evaluation with May-Grünwald-Giemsa staining indicated that about 90% of the cells differentiated toward granulocytes after exposure to DMSO for 4 days. Our immunoblotting analysis did not detect expression of FAK during granulocytic differentiation of HL-60 cells (data not shown).
Tyrosine phosphorylation of Pyk2 on stimulation with fMLP Previous studies indicated that the number of fMLP receptors and the ability to respond to fMLP increase in DMSO-differentiated granulocytic HL-60 cells.42,43 These results were confirmed in our experiments by immunoblotting and flow cytometric analyses with anti-fMLP receptor polyclonal antibody (data not shown). We then assessed whether Pyk2 is involved in signaling by means of the fMLP receptor in granulocytic cells. Figure 2 shows results of dose-dependence and time-course studies of tyrosine phosphorylation of Pyk2 on stimulation with fMLP. Tyrosine phosphorylation of Pyk2 was detected in the presence of as little as 1 pmol/L fMLP and depended on the amount of fMLP used, with the maximal level reached at an fMLP concentration of 100 nmol/L (Figure 2A). After stimulation with 1 µmol/L fMLP (Figure 2B), an increase in tyrosine phosphorylation of Pyk2 was detected at 30 seconds, reached the maximal level at 1 minute, and then gradually returned to the baseline level. Tyrosine phosphorylation was not detected in the immunoprecipitates with normal goat IgG or those with protein A alone (Figure 2B). To confirm that Pyk2 is involved in fMLP receptor-mediated signaling, an effect of PT, which inhibits the signaling of the Gi-protein subunit by ADP ribosylation in HL-60 cells,44 was examined. Pretreatment with PT for 4 hours,
which was sufficient to inhibit the signal mediated by the fMLP
receptor,45 inhibited tyrosine phosphorylation of
Pyk2 (Figure 2B). These data indicate that Pyk2 is involved in fMLP
receptor-mediated signaling.
fMLP-induced attachment of granulocytic cells to Fg is mediated by
2 integrin, we examined
whether the attachment was mediated by 2 integrin.
First, we examined the expression of 2 integrin in granulocytic cells by using immunoblotting analysis. As shown in Figure
3A, 2 integrin was induced
in the course of granulocytic differentiation of HL-60 cells. Next, we
evaluated the effect of pretreatment with mouse anti-2
integrin monoclonal antibody IB4 on cell attachment to Fg-coated
dishes. We observed that IB4 inhibited fMLP-induced cell attachment by
about 95%. This inhibition was not observed when the cells were
pretreated with normal mouse IgG. These results suggest that
fMLP-induced attachment of granulocytic cells to Fg is mediated by
2 integrin.
Pyk2 and Syk are tyrosine phosphorylated in relation to cell attachment to Fg To determine the effect of cell attachment to Fg on cell signaling, tyrosine phosphorylation of Pyk2 and Syk was examined. On stimulation with fMLP, tyrosine phosphorylation of Pyk2 was detected at 1 minute and reduced at 30 minutes in cells in suspension (Figure 2B), but it was augmented at 30 minutes in cells attached to Fg-coated dishes (Figure 3B). Tyrosine phosphorylation was not detected when the cells detached spontaneously after 180 minutes of stimulation (Figure 3B). In the studies of Syk, tyrosine phosphorylation was not detected at either 1 minute or 30 minutes in cells in suspension after stimulation with fMLP, but it was detected in cells attached to Fg-coated dishes (Figure 3C).To evaluate whether tyrosine phosphorylation of Pyk2 and Syk in the
fMLP-stimulated cells attached to Fg-coated dishes was mediated by
Association of Pyk2 and Syk with 2 integrin in
fMLP-stimulated granulocytic cells. Pyk2 was not detected in anti-2
integrin immunoprecipitates of unstimulated cells or cells stimulated
for 1 minute, but it became detectable both in cells kept in suspension
and in cells attached to Fg-coated dishes after 30 minutes of
stimulation with fMLP (Figure 4A). On the
other hand, Syk was constitutively coprecipitated with anti-2
integrin antibody, regardless of whether or not the cells were
stimulated with fMLP or whether they were in suspension or attached
(Figure 4B). This association of Pyk2 and Syk was not detected in
immunoprecipitates with normal mouse IgG or those with protein A alone
(Figure 4A-B). Conversely, 2 integrin was barely
detectable in anti-Pyk2 immunoprecipitates of cells kept in suspension
(Figure 4A) and cells attached to Fg-coated dishes after 30 minutes of
stimulation with fMLP (Figure 5A). In
anti-Syk immunoprecipitates, 2 integrin was not detected
in similar time-course studies (Figure 4B and 5A).
To examine whether Pyk2 and Syk are functionally associated with Additional kinase assays were performed in the combination of anti-
In the current study, we examined the involvement of Pyk2 and Syk in functional activation of HL-60 cells. During granulocytic differentiation, Pyk2 was induced, but its tyrosine phosphorylation was not enhanced (Figure 1A and data not shown). Pyk2 was also induced in the course of 12-o-tetradecanoyl-phorbol-13-acetate (TPA)-induced monocytic differentiation of HL-60 cells, with apparent tyrosine phosphorylation (data not shown). Because it has been demonstrated that Pyk2 is activated when it is tyrosine phosphorylated,20,32 Pyk2 might not be activated during DMSO-induced granulocytic differentiation, but it may be activated during TPA-induced monocytic differentiation of HL-60 cells. Various cytoplasmic PTKs have been found to be related to differentiation of HL-60 cells. Src family PTKs Lyn and Fgr are expressed and tyrosine phosphorylated during retinoic acid-induced granulocytic differentiation, and inhibition of these PTKs by antisense oligodeoxynucleotide caused apoptosis.8 Induction and tyrosine phosphorylation of Fgr was also confirmed in DMSO-differentiated cells.7 Lyn, Fgr, and Fyn were all shown to be activated in the course of monocytic differentiation, and inhibition of these PTKs by PTK inhibitors modulated differentiation.5,6 We found that Syk was constitutively expressed but apparently not tyrosine phosphorylated during DMSO-induced differentiation (Figure 1B and data not shown). Therefore, PTKs involved in differentiation of HL-60 cells might vary in their differentiation inducers and differentiation lineages. Treatment of HL-60 cells with DMSO resulted in expression of fMLP receptor during granulocytic differentiation (data not shown). To determine whether functional activation of granulocytic cells correlates with activation of Pyk2, we treated the cells with fMLP and detected tyrosine phosphorylation of Pyk2 (Figure 2A-B). This phosphorylation was blocked by PT (Figure 2B), which indicated that Pyk2 is involved in G protein-coupled, receptor-mediated signaling as described in several studies.12,21-26 G protein-coupled, receptor-mediated signaling leads to activation of
some integrins. It was demonstrated that stimulation of neutrophils
with fMLP caused alteration of Stimulation of granulocytic cells with fMLP caused not only alteration
of Cell attachment to ligand-coated dishes often gives rise to
"outside-in" signaling through integrins in an activated form. Because we wondered whether Pyk2 is activated through the ligation of
We also showed that Pyk2 and Syk are associated with In summary, we speculate that the following molecular events occur.
During granulocytic differentiation, fMLP receptor and
We thank Dr Momoyo Asahi (Fukui Prefectural University, Fukui, Japan) for discussions about fMLP signaling.
Submitted March 18, 1999; accepted May 2, 2000.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Kaoru Tohyama, Department of Laboratory Medicine and Clinical Sciences, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan: e-mail: ktohyama{at}kuhp.kyoto-u.ac.jp.
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Top
Abstract
Introduction
receptor I,
cell migration.
EMBO J.
1998;17:5933-5947