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Tyrosine Phosphorylation is Essential for Microfilament Assembly in B Lymphocytes I. Melamed, G.P. Downey, and C.M. Roifman*
Division of Immunology and Allergy, Departmentsof Pediatrics,TheHospital for Sick Children, Toronto, Ontario, Canada,M5G 1X8 Received
April
3,
1991
Summary: The B cell antigenreceptorregulatesthe tyrosine kinasesignaltransductionpathway andit mediatesa variety of morphologicalchangessuchascapping andmembraneruffling. The relationshipbetweenthesetwo events is unclear. We showhere that cross-linkingthe antigen receptor on humanB lymphocytes, in addition to increasingtyrosine phosphorylationof specific substrates,inducesthe conversionof G-actin to F-actin. Preincubationof B lymphocytes with two different tyrphostins blocked anti-IgM-induced tyrosine phosphorylationand actin polymerization. The ability of the ty-rphostinsto block anti-IgM inducedconversionof G-actin to F-actin indicatesthat a tyrosine kinaseactsasan essentiallink betweenthe B cell antigenreceptorthe early changesin cytoskeletalreorganization. 0 1991Academic Press,Inc. Cross-linkingthe antigen-receptorrapidly resultsin a variety of contractileeventsin B cells including receptor capping and endocytosis(1,2). Immunofluorescentandultrastructural studies have suggestedthat microfilamentshave a central role in theseevents (3). Immunocytcchemical studieshave demonstratedthat microfilaments,composedprimarily of actin, arelinked to the inner surfaceof the plasmamembraneand form a complex network, the membraneskeleton(4). Interior to this submembraneous region, the microfilamentsform an intracellular array of interconnected filament that provides structural supportto the cell. Furthermore, the stateof organizationof the microfilamentscan influence the mitogeniceffects of a variety of stimuli (5). One of the earliest changesin the cytoskeleton after ligand bindsto its receptor is the conversionof monomeric globular (G)-actin to filamentous(F)-actin, a processknown asactin polymerization or assembly (6). Although the associationbetweenreceptor-bindingandrapid polymerization of actin hasbeen demonstratedin severalreceptor systemson various cell types (7), the mechanismthat controlsthe stateof actin reorganization remainsunknown. Recently, regionsof homology to the domainof the src family (SH-3) have beendetected in actin-binding proteinsincluding myosin and spectrin,raising the possibility that this domainis important for associationof the SH-3 domainwith the cytoskeleton (8). Further, the c-abl protein, * To whom correspondenceshouldbe addressed. Abbreviations: G, globular, F, fiiamentous; NBD, 7-nitrobenz-2-oxa-1,3diazole; RFI, relative fluorescenceindex; SDS-PAGE, sodiumdcdecyl sulfatepolyacrylamide gel electrophoresis;SDS, sodiumdodecyl sulfate; PIP2, phosphoinositolbisphosphate. 0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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a substrate for activated protein-tyrosine kinases was found to be associated with actin filaments (9). These recent data suggest the possibility that receptor-mediated increase in tyrosine-kinase activity may be involved in the rearrangement of actin filaments. In B lymphocytes, cross-linking
the antigen receptor results in a rapid phosphorylation on
tyrosine residues of a host of substrates (10-12). However, the identity, localization, and function of most of these substrates of protein tyrosine kinase remain largely unknown. In the present study, we have established that cross-linking
the antigen receptor results in a
rapid polymerization of actin in human B cells. By using specific tyrosine kinase blockers, tyrphostins, we show that actin assembly is dependent on antigen-receptor-mediated tyrosine phosphorylation.
MATERIALS
AND
METHODS
Polyclonal goat anti-human-~ chain antibodies were obtained from Tago Inc (Burlingame, CA), and covalently coupled to 200 mg of micronized hydrophilic polycarbamide beads (Bio-Rad, Boston, MA) in the presence of 1-ethyl-3-13dimethy1, according to the manufacturer’s instructions (17). 7-nitrobenz-2-oxa-1,3diazole (NBD)-phallacidin was obtained from Molecular Probes (Eugene, OR); lysophosphatidyl choline from Avanti Polar Lipids (Pelham, AL); [3H] Thymidine from DuPont-New England Nuclear Co.(Boston, MA); flatbottomed 96-well microtiter plates from Costar (Cambridge, MA); RPMI 1640 and phosphatebuffered saline (pH 7.4) from the Ontario Cancer Institute (Toronto, Canada). RPMI 1640 culture medium supplemented with 10% fetal calf serum (Gibco, Grand Island N.Y.) was used for lymphocyte culture; 125I-protein A was obtained from Amersham (Chicago, IL). Polyclonal antiphosphotyrosine antibodies were a kind gift from Dr. T. Pawson, Mt. Sinai Research Institute,Toronto, Canada. Tyrphostins were kindly provided by Dr. Levitzki (Hebrew University, Jerusalem, Israel). CeZZPreparation: Human mononuclear cells were isolated by Ficoll-Hypaque (Pharmacia, Uppsala Sweden) density-gradient centrifugation. T-cell depletion was accomplished with FicollHypaque centrifugation, which removed the cells that rosetted with 2-aminoethyl-isothiouronium bromide-treated sheep erythrocytes (Boehringer Manheim, Dorval, PQ. The resulting B-cellenriched population was less than 2% CD2+ (T cell marker) and greater than 96% CD20+ (B cell marker), as measured by immunofluoresence staining on a flow cytometer (Epics V, Coulter Electronics, Hialeah,Fl.). F-a&z Detemination: B-cell content of polymerized actin (F-a&n) was determined by NBD-phallacidin staining of fixed and permeabilized cells by the method of methanol extraction described by Howard and Gresajo (13). The results were quantified by fluorescence spectrophotometry. The relative fluorescence index (RFI) was calculated from the ratio of the fluorescence of stimulated-cell to control-cell populations. Selected experiments were also analyzed on an Epics Profile fluorescence-activated cell sorter (Coulter, Hialeah, FL). Cells were excited with an argon laser at 488 nm and emissions were recorded at 520 nm with band-pass and short-pass filters. Gating was done on the forward-angle and right-angle light scatter only to exclude debris and cell clumps. A minimum of 10,000 cells were measured for each condition and expressed as RFI relative to control. Elecrrophoresis and Western Blotting: Sodium dodecyl sulfate polyacxylamide gel electrophoresis (SDS-PAGE) was carried out according to the method described by Laemmli (14) and Western blotting analysis was performed essentially as described (15). B cells were stimulated as indicated, sedimented, and lysed with boiling sodium dodecyl sulfate (SDS) sample buffer. Proteins were separated on SDS-PAGE, transferred to nitrocellulose, and blotted with the anti-phosphotyrosine antibodies. Antibody reactivity was detected with t251-labeled protein A and autoradiography. Statistical Analysis: Data are reported as the mean + SEM of the number of experiments indicated. All data were analyzed by Student’s paired t-test. 1425
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RESULTS We used Western blotting with specific polyclonal anti-phosphotyrosine
antibodies to
detect increases in tyrosine phosphorylation mediated through the antigen receptor on B cells. These polyclonal anti-phosphotyrosine
antibodies have been demonstrated to react specifically with
phosphotyrosine and not with phosphoserine or phosphothreonine (16). Identical results were obtained with monoclonal antibodies (not presented). Incubation of B lymphocytes with anti-IgM antibodies induced a rapid increase in tyrosine phosphorylation of eight bands with molecular weights 46,57, 68,74,93, 110, and 145 kD (Fig 1). Tyrosine phosphorylation was detected within 30 s of addition of anti-IgM antibodies with maximal increases observed l-3 min later (not presented). Previous reports have demonstrated that binding of antibody to the antigen receptor on human B cells (11) and murine B cells (12) was associated with increased phosphorylation of tyrosine residues. The antigen-receptor-mediated tyrosine phosphorylation is probably essential for later events such as lymphocyte proliferation because tyrosine kinase inhibitors can block B-cell (17- 18) as well as T cell (19) activation. However, most of the tyrosine phosphorylated substrates in B cells have not yet been identified. Further, the exact subcellular localization of these substrates and their association with other
Anti-IgM AG-30 AG-127
- + + - - + - - -
3
: +
E
2 1
145 110 93 74
Ok-x---
-
[anti-IgM
anthod:]
@g/m?)0
68 -
57 46-
’
h
02 Figure I. Effect of anti-&M antibodies tyrosine residues in B cells. Anti-IgM
and tyrophostins
0- 0
30
60 90 Time (s)
on the phosphorylation
120
150
of proteins
on
antibodies (25 @ml) were added to B-cell cultures in the
presence or absence of the tyrphostins AG-30 (50 PM) and AG-127 (50 PM). Cells were incubated with the tyrphostins 2 h before and terminated 1 minutes after the addition of anti-IgM antibodies. After electrophoresis and blotting, the hosphotyrosine-containing proteins were detected using phosphotyrosine antibodies and 129..I protein A. The resultant autoradiogram is displayed. The data represent 1 of 5 independent experiments with identical results.
2. F-actin content of B cells stimulated with anti-IgM. B cells (1x106) were stimulated The F-actin content in B cells cultured for 3 min with the indicated concentration of anti-IeM.( with anti-IgM (25 pg/ml) measures the kinetics of the response for various periods of time as indicated. (B). The relative fluoresence index (RFI) was calculated as described in Materials and Methods . Each data point represents the mean f SEM of 6 experiments with different donors.
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03
[Tyrphostin (I-W
] Log Fluorescence
Intensity
Figure 3. Effect of tyrophostins on anti-IgM induced change in F-actin content. Doseresponse
of AG-30, AG-127,andAG-183changein F-a&n content.B cellwerepreincubated with various concentration of AG-30, AG-127,andAG-183for 2 h andweresubsequently stimulated with anti-IgM for 3 minfor F-actin. The uptakeof F-actinwasdetermined asdescribed in Materials and Methods. &‘&, Fluorescence (F-a&n ) distriburion histogram of resting hwnan B-cells (A) stimulated with anti-&M antibodies (25 @nl) for 3 min with (B) and without (C) preincubation with tyrphostin AC-30 (50MJ for 2 h. Thecellswerestainedwith NBD-phallacidin andanalyzedby
flow cytometryasdescribed.Computertracesof 3 representative histograms areillustrated.
known cellular structuresremain unknown. Recently, regionshomologousto the SH-3 domainof the src family have beendetectedin actin-bindingproteins suchasspectrinandmyosin (8). Further, deletion of the SH-3 domainof c-ubl protein led to its dissociationfrom actin andresulted in transformation (9). The resultsraisethe possibility of a link betweentyrosine kinasesinvolved in signaltransductionand the cells’ cytoskeleton. One of the earliestcytoskeletal changesafter ligandsbind to their receptorsis the conversionof monomericG-actin to F-actin (6). Fig. 2 illustratesthe time- andconcentrationdependentincreaseof polymerized actin that occurswhen the antigenreceptoris cross-linkingwith anti-IgM antibodies. The increasein F-actin wasdetectedwithin 1 min and hadreacheda maximum by 2-3 mitt after stimulationwith anti-IgM antibodies.The peak responsewasattained at anti-IgM concentrationsof 25 ~&nl. If actin polymerization is dependenton antigen-receptor-mediated increasein tyrosine kinaseactivity, than inhibition of tyrosine kinaseactivity shouldinterfere with actin assembly.We testedthis possibility by measuringF-actin content in anti-IgM-stimulated humanB cellsthat were pretreatedwith the tyrosine kinaseinhibitors AG-30 and AG- 127. Thesecompounds,which were designedafter erbstatin, are specific tyrosine kinaseinhibitors (20) that, unlike quercetin(21) or genestein(22) do not block serineor thereoninekinases. As indicated in Figs. 3 and 4, AG-30 and AG-127 inhibited anti-IgM-induced actin assemblyin a dosedependentmanner. In contrast, AG- 183, which is an effective inhibitor of purified epidetmalgrowth-factor-receptortyrosine kinaseactivity (23) had little or no effect on anti-IgM-induced actin assembly(Fig. 3). Similarly, preincubationwith 50 PM of AG-30 andAG-127 blocked anti-IgM-induced tyrosine phosphorylation (Fig. 1). At least2 h of preincubationwasrequired to consistentlyblock tyrosine phosphorylation(not presented),as hasbeenreportedfor other cell types (24). Once again,AG183had little effect on anti-IgM-induced tyrosine phosphorylation(not shown). Taken together 1427
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F-actin polymerization in B cells is dependent on
ligand-induced increased tyrosine kinase activity.
DISCUSSION In this study, we have shown that tyrphostins AG-30 and AG-127 prevent anti-IgMinduced tyrosine phosphorylation and actin polymerization.
In contrast, AG-183 in similar
concentrations (24) had little effect on anti-IgM-induced changes in any of these processes. The ability of the tyrphostins to block anti-IgM-induced actin assembly indicates that a tyrosine kinase likely acts as an intermediary between the B-cell antigen receptor and the process of actin polymerization.
We have previously shown that these tyrphostins block anti-IgM-induced
phosphoinositol bisphosphate (PIP?) (17). Similar results were obtained in T cells using genestein cm. It is conceivable that phosphoinositides regulate the rearrangement of actin filaments. In fact, PIP2 dissociates profilin from actin monomers (26). Other actin-binding proteins such as myosin (27) and glycophorin (28) also associate with PIP2. These results indicate an intimate association between phosphoinositides and actin-binding proteins, implying that they may regulate the polymerization of actin. Tyrphostins may block actin assembly by inhibiting tyrosine kinase activity of the src-like family. Phosphatidil inositides, as well as diacylglycerol, have been shown to be phosphorylated by activities that copurified with pp60v-s’c (29). It is therefore possible that tyrphostins may block tyrosine kinase activity of enzymes that are localized upstream of the phospholipase C-specific phosphatidyl inositol pathway or the newly described phosphatidyl inositol3-kinase pathway (30). Alternatively, tyrphostines may inhibit actin assembly by interfering directly with the processing of G-actin and its binding proteins. In fact, homologous regions to the src family, SH-3, have been detected in actin-binding proteins including myosin, spectrin, and a yeast cytoskeletal protein (8). Taken together our results show that actin filament rearrangement which is believed to be a hallmark of growth-factor
stimulation and transformation in other cell types (30), is also induced in
human B lymphocytes activated with anti-IgM antibody. The process of actin polymerization seem to be controlled by upstream events of antigen-receptor-mediated
increases in tyrosine kinase
activity.
ACKNOWLEDGMENT This work was supported by a grant from the National Cancer Institute of Canada.
REFERENCES Schreiner, G.F., and Unanue, E.R. (1976) Adv. Immunol. 24, 37-165.. Braun, J. and Unanue, E.R. (1980) Immunol. Rev. 52, 3-28. Weeds, A. (1982) Nature 296, 811-816. Sundqvist, K-G. and Ehrnst, A. (1976) Nature. 264, 226231. Goroff, D., Stall, A., Mond, J.J., and Finkelman, F.D. (1986) J.Immunol. 1428
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6. 7. if lb. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. ii* 24: 25. 26. 2;. Z:
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Mann, G. M., Allen, R.A., Bokoch, G.M., Painter, R.G., Traynor ,A.E., and Sklar, L.A. (1987) Physiol. Rev. 67,285-322. Downey, G.P., Chan, C.K. and Grinstein, S. (1989) B&hem. Biophys. Res. Commun.. 164, 700-705. Drubin, D.G., Mulholland, J., Zhu, Z., and Botstein, D. (1990) Nature. 343, 288-290. Jackson, P. and Baltimore, D. (1989) EMBO J. 8,449-456. Nel, A.E., Landreth, G.E., Goldschmidt-Clermont, P.J., Tung, H.E., and Galbraith, R.M. (1984) Biol. Biophys. Res. Commun. 125, 859-866. Gold, M.R., Law, D.A., and Defranco, A.L. (1990) Nature. (London) 345, 810-813. Campbell, M.-A., and Sefton, B.M. (1990) EMBO J. 9, 2125-2131. Howard, T.H., and Oresajo, C.O. (1985) J.Cell Biol.101, 1078-1085. Laemmli,U.K. (1970) Nature 229,680-685. Kamps, M.P and Sefton, B.M. (1988) Oncogene 2, 305-315. Mills, G.B., May, C., McGill, M., Fung, M., Baker, M., Sutherland, R., and Greene, W.C. (1990) J. Biol. Chem. 265, 3561-3567. Padeh, S., Levitzki, A., Gazit, A., Mills, G.B. and Roifman, C.M. (1991) J.Clin. Invest. (in press). Roifman, C.M., Milles, G.B., Chin, K., Gazit, A., Gilon, C., and Levitzki, A. J. Immunol. (In press). June, C.H., Fletcher, M.C., Ledbetter, J.A., Schiever, G.L., Siegel, J.N., Phillips, A.F., and Samelson, L.E. Proc. Natl. Acad. Sci. (USA). 87,7722-7726. Gazit, A., Yaish, P., Gilon, C., and Levitzki, A. (1989) J. Med. Chem. 32, 2344-2352. Akiyama, T., Ishida, J., Nakagawa, S., Ogawara, H., Watanabe, S.-I., Itoh, N., Shibuya, M. , and Fukani, Y. (1987) J. Biol. Chem. 262,5592-5595. Graziani, Y., Erikson, E., and Erikson, R.L. (1983) Eur. J. B&hem. 135,583-589. Yaish, P., Gazit, A., Gilon, C., and Levitzki, A. (1988) Science. 242, 933-935. Lyall, R.M., Zilberstein, A., Gazit, A., Gilon, C., Levitzki, A., and Schlessinger, J. (1989) J. Biol. Chem. 264, 14503-14509. Lane, P.J.L, Ledetter, J.A., McConnell, F.M., Draves, K., Deans, J., Schieuen, G.L., Clark, E.A. (1991) I. Immunol. 146, 715-722. Goldschmidt-Clermont, P.J., MacLesky, L.M., Baldassare, J.J., and Pollard, T.D. (1990) Science. 247:1575-1578. Adams, R.J. and Pollard, T.D. (1989) Nature. 340, 565-568. Anderson, R.A. and Marchesi, V.T. (1985) Nature. 318,295-298. Fukui, Y. and Hanafusa, H. (1989) Mol. Cell. Biol. 9, 1651-1658. Severinsson, L., Ek, B., Mellstrom, K, Claesson-Welsh, L., and Heldin, C,-H. (1990) Mol. Cell. Biol. 10, 801-809.
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Tyrosine Phosphorylation is Essential for Microfilament Assembly in B Lymphocytes I. Melamed, G.P. Downey, and C.M. Roifman*
Division of Immunology and Allergy, Departmentsof Pediatrics,TheHospital for Sick Children, Toronto, Ontario, Canada,M5G 1X8 Received
April
3,
1991
Summary: The B cell antigenreceptorregulatesthe tyrosine kinasesignaltransductionpathway andit mediatesa variety of morphologicalchangessuchascapping andmembraneruffling. The relationshipbetweenthesetwo events is unclear. We showhere that cross-linkingthe antigen receptor on humanB lymphocytes, in addition to increasingtyrosine phosphorylationof specific substrates,inducesthe conversionof G-actin to F-actin. Preincubationof B lymphocytes with two different tyrphostins blocked anti-IgM-induced tyrosine phosphorylationand actin polymerization. The ability of the ty-rphostinsto block anti-IgM inducedconversionof G-actin to F-actin indicatesthat a tyrosine kinaseactsasan essentiallink betweenthe B cell antigenreceptorthe early changesin cytoskeletalreorganization. 0 1991Academic Press,Inc. Cross-linkingthe antigen-receptorrapidly resultsin a variety of contractileeventsin B cells including receptor capping and endocytosis(1,2). Immunofluorescentandultrastructural studies have suggestedthat microfilamentshave a central role in theseevents (3). Immunocytcchemical studieshave demonstratedthat microfilaments,composedprimarily of actin, arelinked to the inner surfaceof the plasmamembraneand form a complex network, the membraneskeleton(4). Interior to this submembraneous region, the microfilamentsform an intracellular array of interconnected filament that provides structural supportto the cell. Furthermore, the stateof organizationof the microfilamentscan influence the mitogeniceffects of a variety of stimuli (5). One of the earliest changesin the cytoskeleton after ligand bindsto its receptor is the conversionof monomeric globular (G)-actin to filamentous(F)-actin, a processknown asactin polymerization or assembly (6). Although the associationbetweenreceptor-bindingandrapid polymerization of actin hasbeen demonstratedin severalreceptor systemson various cell types (7), the mechanismthat controlsthe stateof actin reorganization remainsunknown. Recently, regionsof homology to the domainof the src family (SH-3) have beendetected in actin-binding proteinsincluding myosin and spectrin,raising the possibility that this domainis important for associationof the SH-3 domainwith the cytoskeleton (8). Further, the c-abl protein, * To whom correspondenceshouldbe addressed. Abbreviations: G, globular, F, fiiamentous; NBD, 7-nitrobenz-2-oxa-1,3diazole; RFI, relative fluorescenceindex; SDS-PAGE, sodiumdcdecyl sulfatepolyacrylamide gel electrophoresis;SDS, sodiumdodecyl sulfate; PIP2, phosphoinositolbisphosphate. 0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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a substrate for activated protein-tyrosine kinases was found to be associated with actin filaments (9). These recent data suggest the possibility that receptor-mediated increase in tyrosine-kinase activity may be involved in the rearrangement of actin filaments. In B lymphocytes, cross-linking
the antigen receptor results in a rapid phosphorylation on
tyrosine residues of a host of substrates (10-12). However, the identity, localization, and function of most of these substrates of protein tyrosine kinase remain largely unknown. In the present study, we have established that cross-linking
the antigen receptor results in a
rapid polymerization of actin in human B cells. By using specific tyrosine kinase blockers, tyrphostins, we show that actin assembly is dependent on antigen-receptor-mediated tyrosine phosphorylation.
MATERIALS
AND
METHODS
Polyclonal goat anti-human-~ chain antibodies were obtained from Tago Inc (Burlingame, CA), and covalently coupled to 200 mg of micronized hydrophilic polycarbamide beads (Bio-Rad, Boston, MA) in the presence of 1-ethyl-3-13dimethy1, according to the manufacturer’s instructions (17). 7-nitrobenz-2-oxa-1,3diazole (NBD)-phallacidin was obtained from Molecular Probes (Eugene, OR); lysophosphatidyl choline from Avanti Polar Lipids (Pelham, AL); [3H] Thymidine from DuPont-New England Nuclear Co.(Boston, MA); flatbottomed 96-well microtiter plates from Costar (Cambridge, MA); RPMI 1640 and phosphatebuffered saline (pH 7.4) from the Ontario Cancer Institute (Toronto, Canada). RPMI 1640 culture medium supplemented with 10% fetal calf serum (Gibco, Grand Island N.Y.) was used for lymphocyte culture; 125I-protein A was obtained from Amersham (Chicago, IL). Polyclonal antiphosphotyrosine antibodies were a kind gift from Dr. T. Pawson, Mt. Sinai Research Institute,Toronto, Canada. Tyrphostins were kindly provided by Dr. Levitzki (Hebrew University, Jerusalem, Israel). CeZZPreparation: Human mononuclear cells were isolated by Ficoll-Hypaque (Pharmacia, Uppsala Sweden) density-gradient centrifugation. T-cell depletion was accomplished with FicollHypaque centrifugation, which removed the cells that rosetted with 2-aminoethyl-isothiouronium bromide-treated sheep erythrocytes (Boehringer Manheim, Dorval, PQ. The resulting B-cellenriched population was less than 2% CD2+ (T cell marker) and greater than 96% CD20+ (B cell marker), as measured by immunofluoresence staining on a flow cytometer (Epics V, Coulter Electronics, Hialeah,Fl.). F-a&z Detemination: B-cell content of polymerized actin (F-a&n) was determined by NBD-phallacidin staining of fixed and permeabilized cells by the method of methanol extraction described by Howard and Gresajo (13). The results were quantified by fluorescence spectrophotometry. The relative fluorescence index (RFI) was calculated from the ratio of the fluorescence of stimulated-cell to control-cell populations. Selected experiments were also analyzed on an Epics Profile fluorescence-activated cell sorter (Coulter, Hialeah, FL). Cells were excited with an argon laser at 488 nm and emissions were recorded at 520 nm with band-pass and short-pass filters. Gating was done on the forward-angle and right-angle light scatter only to exclude debris and cell clumps. A minimum of 10,000 cells were measured for each condition and expressed as RFI relative to control. Elecrrophoresis and Western Blotting: Sodium dodecyl sulfate polyacxylamide gel electrophoresis (SDS-PAGE) was carried out according to the method described by Laemmli (14) and Western blotting analysis was performed essentially as described (15). B cells were stimulated as indicated, sedimented, and lysed with boiling sodium dodecyl sulfate (SDS) sample buffer. Proteins were separated on SDS-PAGE, transferred to nitrocellulose, and blotted with the anti-phosphotyrosine antibodies. Antibody reactivity was detected with t251-labeled protein A and autoradiography. Statistical Analysis: Data are reported as the mean + SEM of the number of experiments indicated. All data were analyzed by Student’s paired t-test. 1425
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RESULTS We used Western blotting with specific polyclonal anti-phosphotyrosine
antibodies to
detect increases in tyrosine phosphorylation mediated through the antigen receptor on B cells. These polyclonal anti-phosphotyrosine
antibodies have been demonstrated to react specifically with
phosphotyrosine and not with phosphoserine or phosphothreonine (16). Identical results were obtained with monoclonal antibodies (not presented). Incubation of B lymphocytes with anti-IgM antibodies induced a rapid increase in tyrosine phosphorylation of eight bands with molecular weights 46,57, 68,74,93, 110, and 145 kD (Fig 1). Tyrosine phosphorylation was detected within 30 s of addition of anti-IgM antibodies with maximal increases observed l-3 min later (not presented). Previous reports have demonstrated that binding of antibody to the antigen receptor on human B cells (11) and murine B cells (12) was associated with increased phosphorylation of tyrosine residues. The antigen-receptor-mediated tyrosine phosphorylation is probably essential for later events such as lymphocyte proliferation because tyrosine kinase inhibitors can block B-cell (17- 18) as well as T cell (19) activation. However, most of the tyrosine phosphorylated substrates in B cells have not yet been identified. Further, the exact subcellular localization of these substrates and their association with other
Anti-IgM AG-30 AG-127
- + + - - + - - -
3
: +
E
2 1
145 110 93 74
Ok-x---
-
[anti-IgM
anthod:]
@g/m?)0
68 -
57 46-
’
h
02 Figure I. Effect of anti-&M antibodies tyrosine residues in B cells. Anti-IgM
and tyrophostins
0- 0
30
60 90 Time (s)
on the phosphorylation
120
150
of proteins
on
antibodies (25 @ml) were added to B-cell cultures in the
presence or absence of the tyrphostins AG-30 (50 PM) and AG-127 (50 PM). Cells were incubated with the tyrphostins 2 h before and terminated 1 minutes after the addition of anti-IgM antibodies. After electrophoresis and blotting, the hosphotyrosine-containing proteins were detected using phosphotyrosine antibodies and 129..I protein A. The resultant autoradiogram is displayed. The data represent 1 of 5 independent experiments with identical results.
2. F-actin content of B cells stimulated with anti-IgM. B cells (1x106) were stimulated The F-actin content in B cells cultured for 3 min with the indicated concentration of anti-IeM.( with anti-IgM (25 pg/ml) measures the kinetics of the response for various periods of time as indicated. (B). The relative fluoresence index (RFI) was calculated as described in Materials and Methods . Each data point represents the mean f SEM of 6 experiments with different donors.
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OH
03
[Tyrphostin (I-W
] Log Fluorescence
Intensity
Figure 3. Effect of tyrophostins on anti-IgM induced change in F-actin content. Doseresponse
of AG-30, AG-127,andAG-183changein F-a&n content.B cellwerepreincubated with various concentration of AG-30, AG-127,andAG-183for 2 h andweresubsequently stimulated with anti-IgM for 3 minfor F-actin. The uptakeof F-actinwasdetermined asdescribed in Materials and Methods. &‘&, Fluorescence (F-a&n ) distriburion histogram of resting hwnan B-cells (A) stimulated with anti-&M antibodies (25 @nl) for 3 min with (B) and without (C) preincubation with tyrphostin AC-30 (50MJ for 2 h. Thecellswerestainedwith NBD-phallacidin andanalyzedby
flow cytometryasdescribed.Computertracesof 3 representative histograms areillustrated.
known cellular structuresremain unknown. Recently, regionshomologousto the SH-3 domainof the src family have beendetectedin actin-bindingproteins suchasspectrinandmyosin (8). Further, deletion of the SH-3 domainof c-ubl protein led to its dissociationfrom actin andresulted in transformation (9). The resultsraisethe possibility of a link betweentyrosine kinasesinvolved in signaltransductionand the cells’ cytoskeleton. One of the earliestcytoskeletal changesafter ligandsbind to their receptorsis the conversionof monomericG-actin to F-actin (6). Fig. 2 illustratesthe time- andconcentrationdependentincreaseof polymerized actin that occurswhen the antigenreceptoris cross-linkingwith anti-IgM antibodies. The increasein F-actin wasdetectedwithin 1 min and hadreacheda maximum by 2-3 mitt after stimulationwith anti-IgM antibodies.The peak responsewasattained at anti-IgM concentrationsof 25 ~&nl. If actin polymerization is dependenton antigen-receptor-mediated increasein tyrosine kinaseactivity, than inhibition of tyrosine kinaseactivity shouldinterfere with actin assembly.We testedthis possibility by measuringF-actin content in anti-IgM-stimulated humanB cellsthat were pretreatedwith the tyrosine kinaseinhibitors AG-30 and AG- 127. Thesecompounds,which were designedafter erbstatin, are specific tyrosine kinaseinhibitors (20) that, unlike quercetin(21) or genestein(22) do not block serineor thereoninekinases. As indicated in Figs. 3 and 4, AG-30 and AG-127 inhibited anti-IgM-induced actin assemblyin a dosedependentmanner. In contrast, AG- 183, which is an effective inhibitor of purified epidetmalgrowth-factor-receptortyrosine kinaseactivity (23) had little or no effect on anti-IgM-induced actin assembly(Fig. 3). Similarly, preincubationwith 50 PM of AG-30 andAG-127 blocked anti-IgM-induced tyrosine phosphorylation (Fig. 1). At least2 h of preincubationwasrequired to consistentlyblock tyrosine phosphorylation(not presented),as hasbeenreportedfor other cell types (24). Once again,AG183had little effect on anti-IgM-induced tyrosine phosphorylation(not shown). Taken together 1427
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F-actin polymerization in B cells is dependent on
ligand-induced increased tyrosine kinase activity.
DISCUSSION In this study, we have shown that tyrphostins AG-30 and AG-127 prevent anti-IgMinduced tyrosine phosphorylation and actin polymerization.
In contrast, AG-183 in similar
concentrations (24) had little effect on anti-IgM-induced changes in any of these processes. The ability of the tyrphostins to block anti-IgM-induced actin assembly indicates that a tyrosine kinase likely acts as an intermediary between the B-cell antigen receptor and the process of actin polymerization.
We have previously shown that these tyrphostins block anti-IgM-induced
phosphoinositol bisphosphate (PIP?) (17). Similar results were obtained in T cells using genestein cm. It is conceivable that phosphoinositides regulate the rearrangement of actin filaments. In fact, PIP2 dissociates profilin from actin monomers (26). Other actin-binding proteins such as myosin (27) and glycophorin (28) also associate with PIP2. These results indicate an intimate association between phosphoinositides and actin-binding proteins, implying that they may regulate the polymerization of actin. Tyrphostins may block actin assembly by inhibiting tyrosine kinase activity of the src-like family. Phosphatidil inositides, as well as diacylglycerol, have been shown to be phosphorylated by activities that copurified with pp60v-s’c (29). It is therefore possible that tyrphostins may block tyrosine kinase activity of enzymes that are localized upstream of the phospholipase C-specific phosphatidyl inositol pathway or the newly described phosphatidyl inositol3-kinase pathway (30). Alternatively, tyrphostines may inhibit actin assembly by interfering directly with the processing of G-actin and its binding proteins. In fact, homologous regions to the src family, SH-3, have been detected in actin-binding proteins including myosin, spectrin, and a yeast cytoskeletal protein (8). Taken together our results show that actin filament rearrangement which is believed to be a hallmark of growth-factor
stimulation and transformation in other cell types (30), is also induced in
human B lymphocytes activated with anti-IgM antibody. The process of actin polymerization seem to be controlled by upstream events of antigen-receptor-mediated
increases in tyrosine kinase
activity.
ACKNOWLEDGMENT This work was supported by a grant from the National Cancer Institute of Canada.
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