Identification and protein kinase C-dependent phosphorylation of a-adducin

in human fibroblasts

AHMAD WASEEM* and H. CLIVE PALFEEYf

Department of Pharmacological and Physiological Sciences, University of Chicago, 947 E 58th St, Chicago, IL 60637, USA

* Present address: Imperial Cancer Research Fund Laboratories, South Minims, Potters Bar, UKt Author for correspondence

Summary

A protein of Afr~120000, related to the human eryth-rocyte membrane skeletal protein a-adducin, hasbeen identified by immunological criteria in humanfibroblasts. Using similar methods, /f-adducin (anAfr~110000 protein that forms a dimeric complexwith a-adducin in the erythrocyte) is not present infibroblasts. Subcellular distribution studies revealthat fibroblast a-adducin is largely associated withthe particulate fraction and is most effectively solu-bilized from that fraction by a combination of non-ionic detergent and high salt. Immunocytochemistryof quiescent fibroblasts shows that a-adducin is clus-tered in large perinuclear arrays that may corre-spond to vesicular structures; weak staining was alsofound in the sub-plasma membrane region. As inerythrocytes, the phosphorylation of fibroblast a-adducin is elevated on exposure of cells to phorbolesters that activate protein kinase C (PK-C). In ad-

dition, various mitogens such as serum, bradykininand vasopressin also stimulate a-adducin phos-phorylation by a PK-C-dependent pathway. The elev-ation in a-adducin phosphorylation is maintained forup to 30 min after mitogen addition. Peptide maps ofphospho-a-adducin from both fibroblasts and eryth-rocytes after PK-C-mediated phosphorylationshowed multiple phosphorylated peptides but withdissimilar migration patterns, suggesting divergenceof structure around the phosphorylation sites. Addu-cin appears to play an important role in the regu-lation of spectrin-actin interactions in the red celland may play a role in cytoskeletal function in thefibroblast.

Key words: protein kinase C, oadducin, fibroblasts.

Introduction

Adducin is the collective name for two membrane skeletalproteins first observed in the human erythrocyte (Palfreyand Waseem, 1985, 1988; Gardner and Bennett, 1986,1987; Mische et al. 1987). The protein exists as a hetero-dimer in solution and forms a doublet on SDS-PAGE withMr values of approximately 120000 (a-adducin) and110000 OS-adducin) (Waseem and Palfrey, 1988). We firstidentified the two adducin subunits as prominent sub-strates for protein kinase C (PK-C) in the erythrocytemembrane (Palfrey and Waseem, 1985) and, simul-taneously, Gardner and Bennett (1986) purified the com-plex and found that it could bind calmodulin (see alsoWaseem and Palfrey, 1988). Subsequent work suggeststhat adducin plays an important role in the regulation ofspectrin-actin association in the red cell membrane skel-eton, though the precise details of these interactions arecurrently being debated (Gardner and Bennett, 1987;Mische et al. 1987). Many protein components of theerythrocyte membrane skeleton are present in other celltypes, though it is at present unclear as to whether astructure resembling the human erythrocyte membraneskeleton exists in non-erythroid cells (for reviews, seeBennett, 1985; Marchesi, 1985). It is of interest, therefore,that proteins exhibiting similarity to adducin have beenfound in other cell types (Palfrey and Waseem, 1988;

Journal of Cell Science 96, 93-98 (1990)Printed in Great Britain © The Company of Biologists Limited 1990

Bennett et al. 1988; Kaiser et al. 1989). For example, usingaffinity-purified antibodies specific for the a-- and/3-subunits of adducin, we have demonstrated the presenceof both proteins in other vertebrate red cells (Waseem andPalfrey, 1988), but only ar-adducin in human platelets andlymphocytes (Palfrey and Waseem, 1988). The purpose ofthe present study was to examine the existence, distri-bution and phosphorylation of adducin in human fibro,-blasts. A preliminary account of this work has beenpresented (Waseem et al. 1988).

Materials and methods

MaterialsAffinity-purified anti-a^ and /S-adducin IgG was prepared aspreviously described (Waseem and Palfrey, 1988). The sources ofother materials were as follows: 126I-labelled Protein A (DuPont-NEN, Boston, MA); [32P]orthophosphate (ICN, Irvine, CA);Staphylococcus aureus cells (Enzyme Center, Boston, MA); affin-ity-purified rhodamine-conjugated goat anti-rabbit IgG (JacksonImmunoresearch, West Grove, PA); phorbol esters (LC ServicesCorp., Boston, MA); bradykinin (Peninsula Labs, Belmont, CA).

Cells and cell cultureHSWP, WI-38 and SV40-transformed WI-38 (SV-WI-38) fibro-blasts were propagated as previously described (Palfrey et al.

93

1987). Down-regulation of PK-C was done by incubating culturesin 1 ^M 12-O-tetradecanoyl phorbol acetate (TPA) for 24-48 h asdescribed elsewhere (Muldoon et al. 1987).

ImmunoblottingThis was performed exactly as previously described (Waseem andPalfrey, 1988) using anti-cv- or /S-adducin antibodies at l^gml"1.For the extraction studies shown in Fig. 2, below, cultures weregrown to confluency on 10 cm dishes and serum deprived (0.1%fetal bovine serum) for 12-96 h. Cultures were washed threetimes in Tris-buffered saline and scraped from the dishes in thesame solution. After pelleting, cells were resuspended (1:5, v/v)in a homogenization buffer (Palfrey et al. 1987) and the mixturewas sonicated briefly. After a low-speed spin to remove debris(1000 £ for 6min), the supernatant was centrifuged at 100 000 gfor 30min to provide crude particulate (pellet) and cytosol(supernatant) fractions. The particulate fraction was resuspendedin homogenization buffer prior to solubilizing treatments. Forimmunoblotting, equivalent volume amounts of different frac^tions were loaded in adjacent lanes.

ImmunofluorescenceFor these studies cells were seeded at low density on 20 mmdiameter glass coverslips 2 days prior to experiment and madequiescent by removal of serum. Coverslips were rinsed thoroughlyin phosphate-buffered saline (PBS) then permeabilized in PBS/0.2% Triton X-100 for 5min. Cells were then fixed by briefimmersion of coverslips in methanol at -70°C. After thoroughwashing in PBS, non-specific binding sites were "blocked' byimmersion in PBS/0.25 % gelatin for 1 h. Coverslips were thenincubated in affinity-purified anti-a-- or anti-/3-adducin or non-immune rabbit IgG (35/jgml"1 in PBS) for 1 h at room tempera-ture, by inversion of the coverslip over a drop of antiserum placedon Parafilm in a humidified box. This was followed by extensivewashing in PBS prior to incubation of the coverslip in secondantibody (affinity-purified rhodamine-conjugated goat anti-rabbitIgG; SO/fgml"1) for 30min at room temperature. Finally, thecoverslips were washed extensively in PBS and mounted in 50 %glycerol/PBS. Specimens were examined by epifluorescence in anOlympus microscope and photographed.

32P-labelling of cultures, immunoprecipitation ofadducinand tryptic fingerprintingFor phosphorylation studies cells were grown to confluence on60 mm plastic dishes and deprived of serum (see above) prior tolabelling and mitogen treatment (Palfrey et al. 1987). Theimmunoprecipitation protocol resembled that previously de-scribed (Palfrey et al. 1987); except that 10-20 ;d anti-a- or anti-£-adducin serum per sample was used. Immunoprecipitates weredissolved in SDS sample buffer and separated on SDS-7.5%polyacrylamide gels, followed by autoradiography. 32P-labelled at-adducin was quantitated by cutting appropriate bands from thegel for liquid scintillation counting. Tryptic fingerprinting of 32P-labelled a-adducin was done as previously described (Waseem andPalfrey, 1988).

Results

Existence of a-adducin in human fibroblastsAffinity-purified anti-human erythrocyte a- and /3-adducinantibodies were used to probe the existence of similarproteins in two normal human fibroblast cell strains(HSWP and WI-38) and one transformed cell line (SV-WI-38) by immunoblotting. As shown in Fig. 1 immunoreac-tivity in all three cell types was only obtained with thecr-adducin antibody. Even much longer autoradiographicexposures did not reveal the presence of protein(s) thatreacted with the ^-adducin antibody, though the erythro-cyte ghost control was clearly positive. An Mr of —120 000was found for fibroblast a--adducin, very similar to that

H10

205-

116-97-

68-

a^Adducin P- Adducin

Fig. 1. Presence of a--adducin but not /3-adducin in humanfibroblasts. Samples of human erythrocyte membranes (5 //gprotein) or crude particulate fractions from WI-38, SV-WI-38and HSWP fibroblasts (50 /<g protein) were separated onSDS-7.5% polyacrylamide gels, transferred to nitrocelluloseand then exposed to affinity-purified anti-a--adducin (left) oranti-/S-adducin (right) antibodies, followed by 128I-labelledprotein A and autoradiography to identify the immunolabelledbands. The bands of lower and higher MT in the erythrocytelanes probably represent breakdown products and aggregates ofadducin subunits, respectively. MT markers to the left in Figs1,2 and 4 are xlO"3.

previously determined for the erythrocyte protein(Waseem and Palfrey, 1988; cf. Gardner and Bennett,1986, where adducin has Afr values of 103 000 and 97000using the Fairbanks gel system). Analysis by two-dimen-sional isoelectric focussing-SDS-PAGE and immunoblot-ting (not shown) showed that fibroblast a--adducin has a piof 5.9, close to its human erythrocyte counterpart (Waseemand Palfrey, 1988).

Subcellular fractionation and solubilization of a-adducinIn order to determine if fibroblast cr-adducin behaved as acytoskeletal/membrane skeletal component we performedsubcellular distribution and extraction studies in conjunc-tion with immunoblotting (Fig. 2). Greater than 70 % ofcr-adducin was found in the crude particulate fraction aftercentrifugation of postnuclear homogenates at 100 000 g.The particulate material was further treated with 0.5%Triton X-100, 1 M NaCl or a combination of these twoagents. Most of the a--adducin was not extracted witheither treatment alone, but when used in combination>90 % of the o--adducin could be solubilized by these twoagents.

Immunofluorescent localisation of a-adducinFibroblasts were permeabilized and incubated with anti-a- or anti-/S-adducin antibodies, or non-immune rabbitIgG, followed by rhodamine-conjugated goat anti-rabbit

94 A. Waseem and H. C. Palfrey

1 2 3 4 5 6 7 8 9

Mr

X1CT3

2 D S -

116 —

6 8 -

* * - — " f

IgG. With the cr-adducm antibody large fluorescent aggre-gates were observed that were concentrated in the perinu-clear cytoplasm but extended towards the cell periphery(Fig. 3A-C). The sub-plasma membrane region also ap-peared to be stained by a--adducin IgG, particularly in theregion of the leading lamella (Fig. 3, and data not shown).No specific staining was seen in samples treated witheither non-immune IgG (Fig. 3D) or /3-adducin antibodies(not shown). No gross alterations in adducin distributionwere found after TPA treatment of cells.

Fig. 2. Subcellular distribution and extraction behaviour of a-adducin in human fibroblasts. Crude particulate and cytosolfractions were prepared from WI-38 fibroblasts as described inMaterials and methods. The crude particulate fraction wasfurther treated with non-ionic detergent, high salt or acombination of the two. Proteins were separated by SDS-7.5 %PAGE, transferred to nitrocellulose, and immunolabelled asdescribed in the legend to Fig. 1. The relative amounts of<¥-adducin in each lane were quantitated by densitometry andare indicated in brackets after the lane designations. Lane 1,homogenate (100%); lane 2, cytosol (26% of lane 1); lane 3,particulate (72 % of lane 1); lane 4, 0.5 % Triton X-100supernatant of particulate (21 % of lane 3); lane 6,0.5% TritonX-100 pellet of particulate (74% of lane 3); lane 6, 1 M NaClsupernatant of particulate (9 % lane 3); lane 7 , 1 M NaCl pelletof particulate (42 % of lane 3); lane 8, 0.5 % Triton X-100+1 MNaCl supernatant of particulate (84% of lane 3); lane 9, 0.5%Triton X - 1 0 0 + I M NaCl pellet of particulate (12% of lane 3).

Phosphorylation of a-adducin in intact fibrnblastsAs adducin is a major substrate for PK-C in erythrocytes,we wished to ascertain if the same was true for fibroblasts.WI-38 and HSWP cultures were labelled to constantspecific activity with [32P]orthophosphate and exposed forvarious times to the PK-C-activating phorbol esters TPAor phorbol dibutyrate (PDBu) or the inactive derivative4ophorbol didecanoate (4o--PDD) (all at 50 nM). Immuno-precipitation of untreated cell extracts with cr-adducinantibodies revealed the presence of a single Mr~120000phosphoprotein, but no phosphoprotein was found withanti-/?-adducin antibodies (not shown). Active phorbolesters (but not 4a--PDD) led to a rapid two- to-fivefoldincrease in the phosphorylation of cr-adducin that wasmaximal 5min after addition of the agents and wasmaintained over the ensuing 30min incubation (Fig. 4).

Fig. 3. Immunofluorescent localisation of a--adducin in fibroblasts. HSWP cultures were processed for immunofluorescence asdescribed in Materials and methods. A-C represent individual cells stained with affinity-purified anti-tr-adducm antibodies, while Dis a cell from a culture stained with non-immune normal rabbit IgG at the same concentration to define the non-specificautofluorescence in the sample.

Fibroblast adducin 95

TPA Serum

1' 2' 5' 10' " 1' 2' 5'

205-

116-

97-

68-

B 200

.- o^Adducin

10 15 20Time (min)

25 30

Fig. 4. Phosphorylation of cr-adducin in fibroblasts and effects of mitogens. A. Autoradiogram of immunoprecipitates of ^P-labelledo--adducin separated by SDS-7.5 % PAGE. Serum-deprived WI-38 cells were prelabelled with [32P]orthophosphate and left untreated(Con) or incubated with 0.1 /uu TPA, 10 % fetal bovine serum for the indicated times or 8-Br-cyclic AMP (2 mM; 10 min). In thisparticular experiment stimulation of phosphorylation of a^adducin was maximal with TPA at 5 min (4.2-fold over control) and withserum at 5 min (3.8-fold over control). Note that 8-Br-cyclic AMP had no effect on a-adducin phosphorylation. B. Time course ofoadducin phosphorylation in response to 0.1 /at TPA and 0.05 /JM bradykinin (BK). a^adducin was immunoprecipitated from HSWPculture extracts as described for A. The a^adducin band was cut from the gel and quantitated by scintillation counting. Similarresults were obtained with WI-38 cells.

Similarly, phy8iological mitogens such as serum andbradykinin (Fig. 4) or vasopressin (not shown) led to anextremely rapid increase in cr-adducin phosphorylation(maximal at 30 s). Again, the level of phosphorylationremained elevated over the ensuing 30 min of incubation(Fig. 4).

In contrast to the effects of serum, bradykinin andvasopressin, epidermal growth factor (EGF) treatment ofcultures did not increase oadducin phosphorylation.Moreover, 8-Br-cyclic AMP also had no effect (Fig. 4A),suggesting that fibroblast ar-adducin may not be a sub-strate for cyclic AMP-dependent protein kinase as it is inthe erythrocyte (Waseem and Palfrey, 1988). a--Adducinphosphorylation was not elevated by either phorbol estersor serum in PK-C-deficient cells produced by long-termincubation of cultures in TPA (see Muldoon et al. 1987).

Two-dimensional tryptic phosphopeptide maps of immu-noprecipitated 32P-labelled ff-adducin from both phorbolester-treated human fibroblasts and erythrocytes werecompared (Fig. 5). Multiple phosphorylation sites werefound in both species, but differences in individual phos-phopeptide migration were apparent that indicate diver-gence between the erythrocyte and fibroblast proteins.

Discussion

It has been proposed that the structural proteins thatcomprise the human erythrocye membrane skeleton havea widespread distribution and may comprise part of themembrane-associated cytoskeleton in many cell types(Bennett, 1985; Marchesi, 1986). Spectrin, ankyrin, actinand band 4.1 have all been found in non-erythroid cells,

but evidence is still lacking that the structural andfunctional interactions between these proteins in othercells resembles that found in the red cell. Adducin wasrecently discovered as a minor component of the humanred cell membrane skeleton and similar proteins havebeen shown to exist in other species and tissues (Palfreyand Waseem, 1988; Bennett et al. 1988; Kaiser et al. 1989).This is the first report of adducin in human fibroblasts. Weused affinity-purified polyclonal antibodies raised againstthe gel-purified a-- and /3-subunits of human erythrocyteadducin (Waseem and Palfrey, 1988) to probe for thepresence of either subunit in fibroblasts. Previous workhas indicated that these antibodies are subunit-specific,i.e. exhibit no cross-reactivity with the other subunit(Waseem and Palfrey, 1988). No jS-adducin could bedetected in the cell types studied here by either: (1) immu-noblotting of homogenate or membrane fractions, (2) im-munofluorescence or (3) immunoprecipitation from 32P-labelled culture extracts. The failure to find a ^-adducinhomologue in fibroblasts (and in human platelets andlymphocytes, see Palfrey and Waseem, 1988) may meanthat tissue-specific or non cross-reactive forms of '/S-adducin' exist or that ff-adducin interacts with (an)otherprotein(s) in non-erythroid cells. Such a hypothesis mustawait the purification of adducin from these tissues andanalysis of interacting proteins. Bennett et al. (1988) havepurified brain adducin and found a major Mr 104 000 bandtogether with minor bands in the Mr 107 000-109 000range by Fairbanks SDS-PAGE. The relationship of thesespecies to erythrocyte a- and /S-adducin is not yet clear.Kaiser et al. (1989) have found adducin-like proteins incertain epithelial and epidermal cells. In some cases

96 A. Waseem and H. C. Palfrey

Erythrocyte a^adducin Fibroblast a^adducin

i

Fig. 5. Both erythrocyte and fibroblast o^adducin are multiply phosphorylated but not identical: tryptic fingerprint analysis. Two-dimensional tryptic fingerprints of 32P-labelled o^adducin derived from human erythrocye membranes isolated from cells treatedwith TPA (1 jiM for lOmin) or from immunoprecipitates of fibroblasts treated with TPA (0.1 ^M for 5min). Separation was byelectrophoresis at pH3.5 (horizontal direction) and chromatography (vertical dimension), as described previously (Waseem andPalfrey, 1988). Note that, although multiple peptides are phosphorylated in both samples, the migration of the individual peptidesdiffers between the two proteins.

doublets that resembled human erythrocyte a- and/J-adducin were found. However, the antibodies used inthat study were prepared against whole brain adducin andthus cannot distinguish between the different subunits ofadducin.

The finding that a- and /3-adducin are not alwayscoordinately expressed can be viewed in the light of ourprevious conclusions regarding the relationship betweenthe two erythrocyte subunits (Waseem and Palfrey, 1988).While a- and /3-adducin exhibited strong similarity aroundtheir sites of phosphorylation, a much more limited overallresemblance was revealed by fingerprinting of the 125I-labelled proteins. Together with the specificity of thepolyclonal antibodies raised against the individual sub-units, these data suggest structural divergence of the oc-and /3-chains and indicate that the two proteins areproducts of different genes. A finding that suggests thatthe two proteins may also perform different functions isthat only the /J-subunit binds a calmodulin (CaM) affinitylabel (Gardner and Bennett, 1986). Thus the function ofadducin in fibroblasts may be considerably different fromthat in erythrocytes.

Though a--adducin was found largely in a particulateform in the present study, the immunocytochemicalstudies reported here suggest that the protein is not onlylocalized in a subplasmalemmal membrane skeletal com-partment in quiescent fibroblasts. A substantial amount ofcr-adducin is found associated with large punctate aggre-gates that are dispersed in the perinuclear cytoplasm.These may correspond to some type of vesicular structure,but as the nature of these aggregates is uncertain atpresent we are currently employing a battery of otherprobes in an attempt to colocalize adducin with othermarkers in a definable structure. The adducin complex canbe effectively solubilized from the erythrocyte membrane

skeleton with high salt (Gardner and Bennett, 1986;Waseem and Palfrey, 1988). In fibroblasts, high salt in thepresence of non-ionic detergent almost completely solubil-ized tf-adducin from the particulate fraction. Given thedifferences in subcellular localization, it is too early to saywhether the relationship of adducin to membrane struc-tures will be similar in the two cell types.

cr-Adducin is a substrate for PK-C in fibroblasts as it isin human erythrocytes (Palfrey and Waseem, 1985;Waseem and Palfrey, 1988). Addition of phorbol esters orphysiological mitogens such as serum and bradykinincaused a two- to fivefold increase in o^adducin phosphoryl-ation that remained elevated for up to 30min afteraddition. The serum and bradykinin responses were abol-ished if cells were rendered PK-C-deficient by long-termincubation in TPA (Muldoon et al. 1987), indicating thatthe stimulation of phophorylation occurring after adminis-tration of such mitogens is mediated by PK-C. Elevatedoadducin phosphorylation was maintained for an ex-tended period after phorbol ester or mitogen addition tothe cells. We have shown elsewhere that the long-lastingPK-C activation (measured as the phosphorylation of anendogenous Afr80000 PK-C substrate) after mitogen ad-dition to human fibroblasts correlates well with elevateddiacylglycerol levels (Etscheid et al. 1988). This contrastswith the relatively short-lived phosphorylation of a sub-strate (elongation factor-2) for a calmodulin-dependentkinase found in the same cell types after mitogen addition(Palfrey et al. 1987). In the latter instance there is also agood correlation between the level of the second messenger(Ca) and the phosphorylation of a cognate substrate. Theseresults suggest that the TK-C signal' considerably out-lasts the 'CaM signal' after mitogen addition to quiescentfibroblasts.

Activation of PK-C is thought to be an important event

Fibroblast adducin 97

in mitogenic signalling in a number of cell types includingfibroblasts (Nishizuka, 1988). Several processes activatedin fibroblasts after stimulation by physiological mitogenscan be duplicated by the addition of PK-C-activatingphorbol esters (such as TPA). These processes includestimulation of Na/H exchange and induction of genetranscription, though we have shown that divergent sig-nal transduction pathways are involved in the responsesto such mitogens in different fibroblast cell types (Muldoonet al. 1987). The complexity of cellular responses to phorbolesters suggests that PK-C has a number of target siteswithin the cell that may be important for realising thepleiotropic response to complex growth factor stimuli. Acommon response to cell stimulation is reorganization ofcytoskeletal and membrane skeletal structures. Indeed,TPA has been reported to cause cytoskeletal alterations incultured fibroblasts, particularly with respect to the redis-tribution of actin filaments (Rifkin et al. 1979; Schliwa etal. 1984; Sobue et al. 1988), but the presumed PK-Csubstrate(s) that might be responsible for such alterationshave not been investigated. A recent report does suggestthat adducin distribution may be dynamic and responsiveto external signals in keratinocytes (Kaiser et al. 1989). Itwill clearly be of interest to investigate the possibility thatadducin is involved in the responses of the fibroblastcytoskeleton to mitogenic signals.

We thank Artelia Watson and Karen Cho for excellent techni-cal assistance, and Drs Hiroshi Iida and Ernest Page for help withthe immunocytochemistry. This work was supported by Biology ofSickle Cell Disease Program Project grant HL 30121.

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WASEEM, A., CHO, K. AND PALFREY, H C. (1988). Human fibroblast a-adducin is phosphorylated by protein kinase C in response to mitogens.Fedn Proc. Fedn Am. Socs. exp. Biol. 2, A349.

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(.Received 27 November 1989 - Accepted 23 January 1990)

98 A. Waseem and H. C. Palfrey