[CANCER RESEARCH 52, 566-570. February 1, 1992]

Surface Immunoglobulin-mediated Signal Transduction Involves RapidPhosphorylation and Activation of the Protooncogene Product Raf-lin Human B-Cells1

Toshiharu Tamaki, Yuzuru Kanakura,2 Akira Kuriii, Hirokazu Ikeda, Hideki Mitsui, Hirosuke Yagura,

Itaru Matsumura, Brian Druker, James D. Griffin, Yoshio Kanayama, and Takeshi YonezawaSecond Department of Medicine, Osaka university Medical School, 1-1-50, Fukushima-ku, Osaka 553, Japan [T. T., Y. K., A. A'., H. I., H. M., H. Y., 1. M., Y. K.,

T. Y.], and Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts 02115 ¡B.D., J. D. G.]

ABSTRACT

The protooncogene product, Raf-l, is a serine/threonine kinase andhas been implicated as an intermediate in signal transduction mechanisms. We examined neoplastic and normal B cells for phosphorylationand activation of Raf-l protein in response to anti-immunoglobulin antibody (anti-Ig). Anti-Ig induced rapid phosphorylation of Raf-l protein inboth neoplastic B-cells of hairy cell leukemia and normal tonsillar B-cells which proliferated well in response to anti-Ig. The increase inphosphorylation was due primarily to an increase in phosphoserine. Theimmune complex kinase assay using Histone V-S as an exogenoussubstrate also showed an increase in Raf-l -associated kinase activity. Aninhibitor of protein kinase C, H7, inhibited the proliferation as well asthe Raf-l phosphorylation in response to the proliferate signal of anti-Ig. Further, downregulation of protein kinase C by the treatment with12-phorbol 13-myristic acid significantly abrogated the induction of Raf-l phosphorylation. These results suggest that, in human B-cells, Raf-lprotein may be involved in the signal transduction pathway mediated bysurface immunoglobulin, and that it may be, at least partially, phosphor-ylated by activated PKC.

INTRODUCTION

Anti-Ig3 have been used to prepare models of activation ofB-cells (1). Cross-linkage of the s-Ig under appropriate conditions results in the delivery of an activation signal to the B-cells, which induces them to enter into cell cycle. As for thesignal transduction system of B-cells through s-Ig, the breakdown of inositol phospholipids and generation of inositol tri-phosphate and DAG have been reported (2). The inositol tri-phosphate causes an increase of intracellular Ca2+ concentra

tion (2), and the DAG causes PKC activation (3, 4). Recently,several groups reported that protein tyrosine phosphorylationwas observed in murine B-cells after stimulation with anti-Ig

(5, 6). The tyrosine phosphorylation was suggested to be mediated through the s-Ig-associated tyrosine kinase which playsa role in signal transduction via s-Ig (5, 6). However, the post-s-Ig signal transduction pathways are not fully understood.Identification of substrates activated by anti-Ig is likely to be

Received 2/8/91; accepted 11/13/91.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' This work was supported in part by grants from the Ministry of Education,

Science, and Culture and by the Osaka Cancer Research Foundation (Y. K.).2To whom requests for reprints should be addressed, at The Second Depart

ment of Internal Medicine, Osaka University Medical School, 1-1-50, Fukushima,Fukushima-ku, Osaka 553, Japan.

3The abbreviations used are: anti-Ig, anti-immunoglobulin antibodies; s-Ig,surface immunoglobulin; DAG. diacylglycerol; PKC, protein kinase C; HCL,hairy cell leukemia; B-CLL, chronic lymphocytic leukemia of B-cell type; SDS,sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PDGF, platelet-derived growth factor; PMSF, phenylmethylsulfonyl fluoride; GM-CSF, gran-ulocyte/macrophage colony-stimulating factor; IL-3. interleukin 3; anti-Ig Poly,anti-human immunoglobulin i.. •¿�.«heavy and light chains; polyTalent); BSA,bovine serum albumin; DMSO, dimethyl sulfoxide; IgG, immunoglobulin G;PMA, 12-phorbol 13-myristic acid.

essential in elucidating the mechanisms of s-Ig signaltransduction.

In a previous report, we examined the mitogenic response ofneoplastic B-cells to anti-Ig (7). Neoplastic cells from twopatients with HCL showed a prominent proliferative responseto anti-Ig, but cells from the patients with B-CLL showed poorresponse (7). The neoplastic cells which proliferate in responseto anti-Ig may serve as a monoclonal model of B-cells in thestudy of s-Ig mediated signal transduction.

In murine fibroblasts, Raf-l has been reported to be rapidlyphosphorylated on serine and tyrosine in response to PDGF(8, 9) and, thus, has been linked to the signal transduction ofseveral growth factor receptors (10). In hematopoietic cells, weand other authors reported activation of Raf-\ protein in response to various hematopoietic growth factors such as GM-CSF, IL-3, and colony-stimulating factor 1 (11-14). Raf-l isitself a serine/threonine kinase and is known to be activated byphosphorylation. Thus, it is considered that the phosphorylation of Raf-l may be a common intermediate step in thetransduction of proliferative signals (10). However, in B-cells,it has not been reported whether the phosphorylation of Raf-lis involved in the s-Ig-mediated signal transduction pathway.

In this study, we examined the expression and phosphorylation state of Raf-l in normal and neoplastic B-cells in responseto anti-Ig. Anti-Ig was found to induce rapid phosphorylationof Raf-l in HCL cells and normal B-cells, and phosphorylationand activation of Raf-l seemed to be mediated, at least partially,through PKC.

MATERIALS AND METHODS

Reagents. F(ab')2 fragments of goat anti-Ig Poly and anti-IgG (heavy

chain specific) were purchased from N. L. Cappel Laboratories, Inc.(Cochranville, PA). Alkaline phosphatase from calf intestine was purchased from Boehringer Mannheim (Indianapolis, IN). Protein A-Sepharose CL4B was obtained from Pharmacia (Uppsala, Sweden).Rabbit anú-Raf-l antiserum was generated against a synthetic peptideas previously described (14). Anti-phosphotyrosine monoclonal antibody was generated by using phosphotyramine as the immunogen. Thisantibody is specific for phosphotyrosine as described previously (15,16). H7 was purchased from Seikagaku Kogyo (Tokyo, Japan).

Cells. Two patients of 19 with B-CLL and two patients with HCL,described in the previous report (7), were included in this study. Thediagnoses were confirmed as reported (7). Types of s-Ig in the two casesof B-CLL were n, it, and Xand ^ and X,respectively; they were 7 and Xin both HCL cases. Cell suspensions from tonsils resected from patientswith chronic tonsillitis were obtained by gentle teasing. Mononuclearcells were separated from the heparinized venous blood or cell suspensions by flotation in Ficoll sodium diatrizoate (LSM; Litton Bionetics,Kensington, M D) and washed 3 times in Hanks' balanced salt solution.

The cells were divided into aliquots, frozen with programmable freezer,and cryopreserved in liquid nitrogen as reported previously (7). Foruse, the cells were thawed rapidly at 37°C,diluted slowly, and washed3 times with Hanks' balanced salt solution. The viability of the re-

566

Research. on October 19, 2020. © 1992 American Association for Cancercancerres.aacrjournals.org Downloaded from

Raf-\ IN HUMAN B-CELLS

covered cells was greater than 90% as determined by the trypan blueexclusion test. In each preparation of mononuclear cells from thepatients, the proportion of neoplastic cells was more than 95%. Fortonsillar cells, B<ells were enriched to more than 95% by deletion ofT-cells resetting with neuraminidase-treated sheep erythrocytes.

Cell Proliferation Assay. The mononuclear cells from HCL Case 1were cultured at 1 x 106/nil in 200 ß\of RPMI 1640 supplementedwith 10% fetal bovine serum in 96-well plates in the presence or absenceof anti-IgG or anti-Ig Poly (100 Mg/ml). In some experiments, variousconcentrations of PKC inhibitor, H7, were used to examine the effectsof PKC on anti-Ig-induced proliferation and phosphorylation of Raf-l.Cells were cultured for 72 h, and proliferation was assessed by ['H]

thymidine incorporation as described previously (7, 16).Stimulation with Anti-Ig and Cell Lysis. The mononuclear cells from

patients and the B-cell-enriched fraction of tonsillar cells were washedfree of serum and incubated in serum-free RPMI 1640 containing 0.5%BSA for 2 h. The cells were then exposed to anti-IgG or anti-Ig Poly(100 Mg/ml) at 37°Cfor 0 to 120 min. For downregulation of PKC,

HCL cells (Case 1) were cultured in RPMI 1640 supplemented with2% fetal calf serum for 24 h in the presence of PMA (10 ng/ml; Sigma)or control medium containing 0.01% DMSO, washed free of serum,and then stimulated with anti-IgG for 15 min. After stimulation, cellswere washed with cold phosphate-buffered saline and lysed in (20 HIMTris-HCI (pH 8.0): 137 mM NaCI:0.5% sodium deoxycholate:l% Non-idet P-40 containing protease and phosphatase inhibitors [1 mM PMSF(Sigma, St Louis, MO), 0.15 unit/ml of aprotinin (Sigma), 10 mMEDTA, 10 Mg/ml of leupeptin (Sigma), 100 mM sodium fluoride, and2 mM sodium orthovanadate] at 4"( ' for 20 min. Insoluble materialswere removed by centrifugation at 4°Cfor 15 min at 10,000 x g. Total

protein content of the lysate was determined by the Bio-Rad proteinassay.

Gel Electrophoresis and Immunoblotting. Lysates ( 120 ¿ig)were mixed1:1 with 2x SDS sample buffer with 2-mercaptoethanol and heated at100°Cfor 5 min prior to SDS-polyacrylamide gel electrophoresis.

Proteins were electrophoretically transferred from the gel onto a 0.2-mm nitrocellulose filter (Schleicher & Schuell, Dassel, Germany) andimmunoblotted with anti-Raf-l antibody as described previously (14).Specificity of the anti-Raf-l antibody was evaluated by blocking withthe immunizing peptide (CTLTTSPRLPVF) as described previously(14).

Phosphatase Treatment of Ruf-l Protein. Phosphatase treatment ofRaf-l protein was performed as described previously (14). Briefly, HCLcells were treated with anti-IgG for 15 min, and the lysates fromstimulated or unsi iundated cells (107/cells) were used for Raf-\ immu-noprecipitation. For phosphatase treatment, the Ruf \ immunoprecip-itates were washed 5 times with 100 mM Tris (pH 9.5): 100 mM NaCl:5mM MgCl2 (alkaline phosphatase reaction buffer) containing proteaseinhibitors (1 mM PMSF, 10 Mg/ml of leupeptin, and 0.15 unit/ml ofaprotinin) and then incubated with calf intestine alkaline phosphatase(600 units/ml) for 1 h at 37°C.After washing, treated and untreated

immunoprecipitates were electrophoresed by SDS-PAGE and transferred to nitrocellulose filters. Raf-l proteins were detected by probingthe blot with anti-Raf-l antibody.

Labeling of the Cells with |32P|Orthophosphate and PhosphoaminoAcid Analysis. HCL cells were washed 3 times with phosphate-freeRPMI 1640 containing 0.5% BSA and cultured for 2 h in the phosphate-free medium. After 2 h of incubation, the cells (IO7)were washedonce with the phosphate-free medium and then incubated for a further2 h in this medium containing 1 mCi (37 MBq) of carrier-free [12P]

orthophosphate (Amersham, Arlington Heights, IL). Labeled cells werecultured with or without anti-IgG (100 Mg/ml) for 15 min and lysed inNonidet P-40 buffer with protease and phosphatase inhibitors. Raf-lproteins were immunoprecipitated, separated by 7.5% SDS-PAGE, andvisualized by autoradiography. The phosphoamino acid composition of32P-labeled bands excised from the SDS-PAGE gel was determined asdescribed previously (14). Phosphoserine, phosphothreonine, and phos-photyrosine controls (Sigma) were detected by reaction with ninhydrin,and the radioactive amino acids were detected by autoradiography.

Immune Complex Kinase Assay. Raf-l was immunoprecipitated withanti-Raf-l antibody from lysates of control (without anti-Ig) or anti-Ig-

stimulated cells. The immune complexes were collected on Protein A-Sepharose beads, washed, and incubated in a kinase buffer [25 mM 4(2-hydroxyethyl)-l-piperazineethanesulfonicacid (pH 7.4): 1 mM dithi-othreitolMO mM MgCl2:25 MMATP] containing 10 fid/ml of [y-"P]ATP for 20 min at 37°C.The immune complexes were then again

washed and separated by SDS-PAGE. In some experiments, Histonetype V-S (5 Mg/50-Mlsample; Sigma, St. Louis, MO) was added as anexogenous kinase substrate.

RESULTS

Anti-Ig Induces Altered Migration of Raf-l in Neoplastic B-Cells. Neoplastic B-cells from HCL Case 1 were analyzed as amonoclonal model of cells which proliferate in response to anti-IgG. The cells were treated with medium alone or anti-IgG ata concentration of 100 Mg/ml, which was shown to be sufficientto induce proliferation of the HCL cells (7), and lysed; then aequal aliquot of protein was analyzed by SDS-PAGE. Raf-lwas detected by immunoblotting with a polyclonal antibodygenerated against a Raf-l synthetic peptide. This antibodydetected a major band at a molecular weight of approximately74,000. After treatment with anti-Ig, more slowly migratingforms of Raf-l were detected as early as 1 min. The change of

the migration peaked at 10 to 30 min and slightly diminishedat 2 h (Fig. 1). Reactivity of the anti-Raf-l antibody with theM, 74,000 and slowly migrating forms could be completelyblocked by the addition of Raf-l synthetic peptide, indicatingthat those bands contained authentic Raf-l. In agreement withprevious results in myeloid cells (14), the slower migratingforms of Raf-l were eliminated by treatment with alkalinephosphatase (Fig. 2). These results suggested that the slowermigration of Raf-l is due to an increased phosphorylation.

In Vivo Labeling and Phosphoamino Acid Analysis. In orderto directly demonstrate the increase in phosphorylation of Raf-\ in response to anti-Ig, HCL cells were labeled in vivo with[32P]orthophosphate and treated media alone or anti-IgG for

15 min. The cells were then lysed, and Raf-l was detected by¡mmunoprecipitation, SDS-PAGE, and autoradiography. Thetreatment with anti-IgG increased 12Pincorporation into Raf-l

(Fig. 3/4). In addition, phosphoamino acid analysis was performed to determine which amino acids were phosphorylatedin Raf-l (Fig. 3ß).Phosphoserine was the predominant phosphoamino acid in Raf-l before stimulation with anti-IgG. Afteranti-Ig stimulation, increased phosphorylation was observed

Time (min)84-

Raf-1

0 1 5 10 30 120

5

47-Fig. I. Altered migration of Raf-\ in HCL cells induced by anti-Ig stimulation.

HCL cells from Case 1 were stimulated with anti-IgG (100 yg/ml) for 0 to 120min at 37'C. The cells were then lysed, and equal aliquots of protein (120 ng)were analyzed by SDS-PAGE. Raf-\ was detected by immunoblotting with anti-Raf-\ antibody. Molecular standards were BSA (M, 84,000) and ovalbumin (M,47,000).

567

Research. on October 19, 2020. © 1992 American Association for Cancercancerres.aacrjournals.org Downloaded from

Kaf-ìIN HUMAN B-CELLS

Anti-Ig

Phosphatase

Raf-1

Fig. 2. Effects of phosphatase treatment on electrophoretic mobility of Raf-limmunoprecipitated from anti-IgG-stimulated HCL cells (Case 1). Rat \ wasimmunoprecipitated from lysates of stimulated or unstimulated cells and thenincubated with calf intestine alkaline phosphatase (600 units/ml) for l h at 37'C.After washing, the immunoprecipitates were electrophoresed by SDS-PAGE andtransferred to nitrocellulose filters. Raf-l proteins were detected by anti-Aa/-lantibody. HCL cells were treated with anti-IgG (100 fig/ml) for 15 min.

primarily on serine residues. This was further confirmed bylong-time exposure of the film.

Anti-Ig-induced Phosphorylation of Raf-l in Normal andOther Neoplastia B-Cells. In order to determine if Raf-l phos-phorylation is induced in normal and neoplastic B-cells, thecells were treated with anti-IgG in HCL cells or anti-Ig Poly inB-CLL cells and tonsillar B-cells for 15 min and examined forphosphorylation of Raf-l by immunoblot as described above.The concentration of anti-Ig was sufficient to induce proliferation in HCL cells and normal B-cells as reported in previousstudy (7). After treatment with anti-Ig, the slower migratingforms of Raf-l were distinctly observed in HCL cells from twopatients, in tonsillar B-cells used as a source of normal B-cells,and in a case (Case 1) of two B-CLL patients, albeit to a lesserdegree (Fig. 4). By contrast, the change of Raf-l migration wasbarely observed in B-CLL cells (Case 2) following treatmentwith anti-Ig (Fig. 4). This result roughly coincides with theprevious findings (7) that B-CLL cells showed lower prolifera-tive response to anti-Ig stimulation than did HCL cells.

Immune Complex Kinase Assay. Raf-l was immunoprecipitated from leukemic cells of HCL Case 1 before and 1,15, and30 min after treatment with anti-IgG, and then kinase activitywas assayed with histone type V-S as an exogenous kinasesubstrate (Fig. 5). An increase of kinase activity was detectedat 15 min and reached its highest point at 30 min, despite aslight decrease after 1-min stimulation. Autophosphorylationof Raf-l was not detected before or after stimulation with anti-

IgG (data not shown).PKC Inhibitor Inhibits Anti-Ig-induced Proliferativi- Response

and Phosphorylation of Raf-l. Leukemic cells from HCL (Case1) were cultured with anti-IgG or anti-Ig Poly in the presenceor absence of the PKC inhibitor, H7, and the effect of H7 onanti-IgG-induced proliferation was determined by [3H]thymi-dine incorporation. As shown in Table 1, anti-IgG and anti-IgPoly had a similar effect on induction of HCL cell proliferation,and anti-IgG-induced proliferation of HCL cells was partiallyor completely inhibited by H7 at a concentration of 10 ^M or20 fiM, respectively.

In addition, in order to assess whether or not PKC is involvedin the phosphorylation of Raf-l, the cells were pretreated for 2h with H7 and stimulated with anti-Ig; Raf-l immunoblot wasconducted (Fig. 6). H7 reduced the density of the slower migrating bands at concentrations of 10 and 20 ¿¿M.Further, HCLcells were treated with PMA for 24 h to downregulate PKC,and anti-Ig-induced phosphorylation of Raf-l was examined.Anti-IgG induced Raf-l phosphorylation after cultivation with

control medium, whereas Raf-l phosphorylation was not detectable in PMA-treated cells (Fig. 7). These results suggestthat PKC may be involved, at least partially, in phosphorylationof Raf-l as well as in anti-Ig-induced proliferation.

DISCUSSION

Raf-l, the product of c-raf-l protooncogene, is known to

encode a A/r 74,000 cytoplasmic serine/threonine protein kinase (8, 9, 10). Raf-l protein is now considered to be phos-phorylated and activated by kinases, which have been activatedby early signals, and to transduce further signals which lead tothe cellular responses (10). However, the expression, function,and phosphorylation of Raf-l have not been studied in B-cellsin any detail.

In the present studies, we have shown that Raf-l protein isexpressed in human B-cells and that it is rapidly phosphorylatedby the proliferative signal through s-Ig in human B-cells. Wepreviously examined the proliferative response of neoplastic B-cells to anti-Ig and showed a striking proliferative response ofneoplastic B-cells from two patients with HCL and poor response of those from 19 B-CLL patients (7). The neoplasticcells from these patients will serve as a monoclonal model ofthe B-cell subset which proliferates in response to anti-Ig. Inmost experiments, therefore, we used neoplastic cells of HCLas a monoclonal model of a B-cell subset which proliferate inresponse to anti-Ig.

Raf-1

B

Fig. 3. Anti-Ig induced Raf-\ phosphorylation in vivo. In A, HCL cells (CaseI) were labeled with [32P]orthophosphate and stimulated with or without anti-IgG (100 nu mil for IS min. Rat \ was immunoprecipitated, separated by SDS-PAGE, and visualized by autoradiography. In B, the "P-labeled bands wereexcised from the SDS-PAGE gel, and phosphoamino acid analysis was conducted.Phosphoserine (PSÕ),phosphothreonine (PT), and phosphotyrosine (PY) controlswere detected by reaction with ninhydrin, and the radioactive amino acids weredetected by autoradiography.

568

Research. on October 19, 2020. © 1992 American Association for Cancercancerres.aacrjournals.org Downloaded from

Raf-\ IN HUMAN B-CELLS

Fig. 4. Effects of anti-Ig stimulation onphosphorylation of Raf-\ in normal and neo-plastic B-cells. Neoplastic B-cells (HCL, 2cases; B-CLL, 2 cases) and normal tonsillar B-cells were stimulated with anti-Ig (100 un mlof anti-IgG in HCL cells or anti-Ig Poly in B-CLL and tonsillar cells) for IS min at 37'C.The cells were then lysed, and Raf-\ was detected by immunoblot.

HCL 1 HCL 2 B-CLL1 B-CLL2 TonsilB

Raf-1

Time (min) O 1 15 30Q)

H¡stoneControl

Anti-Ig Poly

Fig. 5. Immune complex kinase assay for Kaf-l-associated kinase activity. Raf1 was immunoprecipitated from HCL cells (Case 1) treated with anti-Ig (100 »ig/ml) for 0 to 30 min. The immune complexes were collected on Protein A-Sepharose beads, washed, and incubated with histone type VS (5 ^g/sample in50 ii\) and 10 fiCi of [-y-"P!ATP for 20 min at 37'C. The immune complex-histone mixture was separated by SDS-PAGE. Incorporation of ''I' in histone

was visualized by autoradiography. An increase of kinase activity was detected atIS min and reached its highest point at 30 min. Autophosphorylation of Raf-lwas not detected up to 30 min.

Table 1 Effects ofH7 on anti-immunoglobulin-induced proliferation of HCLcells

«»StimulationMedium

Anti-Ig Pol/Anti-IgG'Anti-IgG' +H7(10

MM)Anti-IgG'+ H7

(20(IM)Mean

±SD of triplicate values.One hundred eg ml.cpm143

±43"

16,399 ±1,87318,523 ±1,7543,908 ±468122

±39

The immune complex kinase assay showed that treatment ofHCL cells with anti-Ig resulted in an increase in phosphorylation of Histone V-S which was used as an exogenous substrate.This result is comparable to previous findings in murine 3T3fibroblasts treated with other growth factors (8, 9). How Raf-lmight function in this increase in phosphorylation of HistoneV-S remains to be elucidated at this time, since it is not fullyunderstood yet how Raf-l is activated, what substrate(s) isphosphorylated by activated Raf-l, nor what molecule(s) isassociated with Raf-l. It is possible that Raf-l -associated kinaseimmunoprecipitated with anti-Raf-l antibody may be responsible for the phosphorylation of the exogenous substrate. Nevertheless, the fact that phosphorylation of Raf-l is roughly asso-

Anti-IgG

Anti-IgG+ H7(20/¿M)

Anti-IgG

Fig. 6. Effects of protein kinase C inhibitor, H7, on Raf-l phosphorylation.HCL cells (Case 1) were pretreated with medium alone or with 10 or 20 /IM H7for 2 h and then stimulated with control medium, anti-IgG (100 /^ ml), or anti-Ig Poly ( 100 jig/ml) for 15 min. The cells were then lysed, and Raf-1 was detectedby immunoblot.

Pretreatment

Anti-IgG

PMA Medium

Raf-1

Fig. 7. Effects of protein kinase C depletion on phosphorylation of Raf-l.HCL cells (Case 1) were cultured with or without PMA for 24 h and thenstimulated with anti-IgG (100 «ig/ml)for 15 min. The cells were washed andlysed, and then Raf-l was detected by immunoblot. Also Raf-l was detected fromHCL cells before incubation with PMA or control medium containing 0.01%DMSO.

ciated with an increase in Raf-\ or Raf-l -associated kinasesuggests that Ruf \ might be involved, at least in part, in theslg-induced signal transduction pathway.

Although anti-Ig stimulation can rapidly activate tyrosine

569

Research. on October 19, 2020. © 1992 American Association for Cancercancerres.aacrjournals.org Downloaded from

Raf-\ IN HUMAN B-CELLS

kinase in murine B-cells (5,6), tyrosine residues were not major

sites of phosphorylation. The increase in phosphorylation ofRaf-\ was primarily because of an increase in phosphoserine.

This result is similar to previous findings in other growth factorssuch as GM-CSF and IL-3 (14). Interestingly, the PKC inhibitor, H7, partially inhibited Raf-l phosphorylation as well asthe proliferation induced by anti-Ig stimulation. In addition,anti-Ig could not induce Raf-\ phosphorylation in PKC-de-

pleted HCL cells. These results suggest that PKC might be oneof the kinases responsible for Raf-l phosphorylation in humanB-cells. Recently, similar findings were reported in T-cells, thatthe proliferative signal mediated by T-cell antigen receptorsinduces phosphorylation and activation of Raf-\ via a PKC-

dependent pathway but not via tyrosine kinase associated withT-cell antigen receptors (17). In the signal transduction pathwayof PDGF, however, it was reported that Raf-l is associated

with PDGF receptor and directly phosphorylated by the tyrosine kinase of the PDGF receptor (8, 9). The difference in themechanisms of Raf-l phosphorylation might be attributable to

the characteristics of receptor or cell type.In addition to neoplastic B-cells from HCL patients, normal

B-cells obtained from tonsils also proliferated in response toanti-Ig, and phosphorylation of Raf-l protein was induced inthese cells with anti-Ig treatment. In this study, we used tonsillar

cells obtained from patients with chronic tonsillitis, and consequently some activation of B-cells might already have taken

place. Indeed, a faint phosphorylated band was already seen inthese cells before stimulation with anti-Ig. The weakness in theinduction of Raf-l phosphorylation in tonsillar B-cells may be

attributable to the activation state of the cells before the stimulation with anti-Ig or to the low proportion of cells which reactwith anti-Ig. In contrast to HCL cells, anti-Ig did not stimulateproliferation of B-CLL cells (7), nor induce prominent phosphorylation of Raf-l in these cells following treatment withanti-Ig. The unresponsiveness of B-CLL cells to anti-Ig couldbe explained by their derivation from a B-cell subset which doesnot respond to anti-Ig or by a neoplastic change which madethem be refractory to anti-Ig stimulation because of a defect in

the signal transduction pathway.In summary, our results implicate Raf-l as part of a kinase

cascade involved in signal transduction through s-Ig on humanB-cells. It is not known yet how Raf-l -associated kinase areregulated, or which residues are critical for Raf-l activation.Identification of the in vivo substrates of Raf-l kinase will

provide a clue to further understanding of growth regulationand differentiation of B-cells.

REFERENCES1. De Franco, A. L.. Kung, J. T., and Paul, W. E. Regulation of growth and

proliferation in B-cell subpopulations. Inumino]. Rev., 64: 161-182, 1982.2. Bijsterbosch, M. K., Meade, C. J.. Turner. G. A., and Klaus, G. G. B-

Lymphocyte receptors and phosphoinositide degradation. Cell, 41: 999-1006, 1985.

3. Chen, 2. Z., Coggeshall, K. M., and Cambier, J. C. Translocation of proteinkinase C immunoglobulin-mediated transmembrane signaling in B-lytnpho-cytes. J. Immunol.. 136: 2300-2304, 1986.

4. Nel, A. E., Wooten. M. W., Landreth, G. E., Goldsmidt-Clemont, P. J.,Stevenson, H. C., Miller. P. J., and Galbraith, R. M. Translocation ofphospholipid/Ca2*-dependent protein kinase in B-lymphocytes activated byphorbol ester or cross-linking of membrane immunoglobulin. Biochem. J.,233: 145-149, 1986.

5. Gold, M. R., Law, D. A., and De Franco, A. L. Stimulation of proteintyrosine phosphorylation by the B-lymphocyte antigen receptor. Nature(Lond.), ^5:810-813, 1990.

6. Campbell, M. A., and Sefton. B. M. Protein tyrosine phosphorylation isinduced in murine B-lymphocyte in response to stimulation with anti-im-munoglobulin. EMBO J., 9: 2125-2131, 1990.

7. Tamaki, T., Yonezawa, T., Kanakura, Y., Katagiri, S., Mizutani, H., Kitani,T., and Tarui, S. Mitogenic response of neoplastic B-cells: comparison ofreactivity to Staphylococcus aureus Cowan I and anti-immunoglobulin antibodies. Clin. Exp. Immunol., 64: 181-187, 1986.

8. Morrison, D. K., Kaplan, D. R., Rapp. I .. and Roberts, T. M. Signaltransduction from membrane to cytoplasm: growth factors and membranebound oncogene products increase A'«/I phosphorylation and associatedprotein kinase activity. Proc. Nati. Acad. Sci. USA, 85: 8855-8859, 1988.

9. Morrison, D. K., Kaplan, D. R., Escobedo, J. A., Rapp, U. R., Roberts, T.M., and Williams. L. Direct activation of serine/threonine kinase activity ofRaf-\ through tyrosine phosphorylation by the PDGF0 receptor. Cell, 58:649-657. 1989.

10. Rapp, U. R., Heidecker. G., Huleiheil, M.. Cleveland, J. L.. Choi, W. C,Pawson, T., Ihle, J. N., and Anderson, W. B. Raf family serine/threonineprotein kinase in mitogen signal tranduction. Cold Spring Harbor Symp.Quant. Biol., 53: 173-184, 1988.

11. Carroll, M. P., Clark-Lewis, I.. Rapp, U. R., and May, W. S. Interleukin 3and granulocyte-macrophage colony stimulating factor mediate rapid phosphorylation and activation of cytosolic c-raf. J. Biol. Chem., 265: 19812-19817, 1990.

12. Baccarini, M., Sabatini, D. M.. App, H., Rapp, U. R., and Stanley, E. R.Colony stimulating factor-1 (CSF-1) stimulates temperature dependent phosphorylation and activation of Raf-l proto-oncogene product. EMBO J., 9:3649-3657, 1990.

13. Choudhury, G. G., Sylvia, V. L., Pheifer, A., Wang, L. M., Smith, E. A.,and Sakaguchi. A. A. Human colony stimulating factor-1 activates the c-raf-1 proto-oncogene kinase. Biochem. Biophys. Res. Commun., 772: 154-159,1990.

14. Kanakura, Y., Druker, B., Wood, K. W., Mamón, H. J., Okuda. K., Roberts,T. M., and Griffin, J. D. Granulocyte-macrophage colony-stimulating factorand interleukin-3 induce rapid phosphorylation and activation of the proto-oncogene Raf-l in a human factor-dependent myeloid cell line. Blood, 77:243-248, 1991.

15. Kanakura, Y., Druker, B., Cannistra, S. A., Furukawa, Y., Torimoto, Y., andGriffin, J. D. Signal transduction of the human granulocyte-macrophagecolony stimulating factor and interleukin-3 receptors involves tyrosine phosphorylation of a common set of cytoplasmic proteins. Blood, 76: 706-715,1990.

16. Kanakura, Y., Druker. B., De Carlo. J., Cannistra. S. A., and Griffin, J. D.Phorbol 12-myristate 13-acetate inhibits granulocyte-macrophage colonystimulating factor-induced protein tyrosine phosphorylation in a humanfactor dependent hematopoietic cell line. J. Biol. Chem.. 266:490-495,1991.

17. Siegel, J. N., Klausner, R. D., Rapp, U. R., and Samelson, I. E. T-Cellantigen receptor engagement stimulates c-raf phosphorylation and inducesc-ra/-associated kinase activity via a protein kinase C-dependent pathway. J.Biol. Chem., 265: 18472-18480, 1990.

570

Research. on October 19, 2020. © 1992 American Association for Cancercancerres.aacrjournals.org Downloaded from

1992;52:566-570. Cancer Res   Toshiharu Tamaki, Yuzuru Kanakura, Akira Kuriu, et al.  

-1 in Human B-CellsRafProduct Rapid Phosphorylation and Activation of the Protooncogene Surface Immunoglobulin-mediated Signal Transduction Involves

  Updated version

  http://cancerres.aacrjournals.org/content/52/3/566

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/52/3/566To request permission to re-use all or part of this article, use this link

Research. on October 19, 2020. © 1992 American Association for Cancercancerres.aacrjournals.org Downloaded from