Proc. Nati. Acad. Sci. USAVol. 89, pp. 4947-4951, June 1992Cell Biology

Translocation of spectrin and protein kinase C to a cytoplasmicaggregate upon lymphocyte activationCAROL C. GREG0RIO*, RALPH T. KUBOt, RICHARD B. BANKERT*, AND ELIZABETH A. REPASKY***Department of Molecular Immunology, Roswell Park Cancer Institute, Buffalo, NY 14263; and tDepartment of Medicine, National Jewish Center forImmunology and Respiratory Medicine, Denver, CO 80206

Communicated by Daniel Branton, February 10, 1992

ABSTRACT We have previously reported that mamma-lian tissue lymphocytes exhibit significant heterogeneity withrespect to the subcellular distribution of spectrin and that thisphenomenon may result from a dynamic behavior of spectrinin response to activation signals. Here, we further characterizethe involvement of spectrin in lymphocyte activation by exam-ining its relationship with protein kinase C (PKC). PKCisoenzymes are a family of cytosolic kinases that translocatefrom the soluble to particulate fraction upon cell stimulation.It is reported here that activation oflymph node T cells throughthe antigen-specific receptor, or direct activation of PKC byphorbol esters, results in a striking increase in cells expressinga cytoplasmic aggregate of spectrin. Additionally, a concurrentincrease in cells expressing aggregates of the PHlI isozyme ofPKC is observed. Immunofuorescence staining revealed thatspectrin and PKC8II are colocalized in untreated lymphocytesand that these two proteins are coincidently translocated to thesame focal aggregate within the cytoplasm following stimula-tion. This redistribution of spectrin and PKCfi is blocked bypretreatment with calphostin C, a specific inhibitor of PKC.Solubility studies showed that there is an increase of bothproteins in the detergent-insoluble fraction of lymphocytesupon activation, and immunoprecipitation studies indicatedthat the soluble form of these molecules may be associateddirectly or indirectly as part of a complex of proteins. Thesedata indicate that the positioning ofthe spectrin-based cytoskel-eton is sensitive to activation signals and may play a role in thefunction or positioning of PKCfiII.

There is increasing evidence that spectrin can play a moredynamic role in various cytoplasmic and plasma membrane-related events in nucleated cells than in the erythrocyte,where it is a major component of the membrane skeletonproviding structural support for the plasma membrane (forreview, see ref. 1). For example, we have observed that thelevel and distribution of spectrin at the plasma membrane ofuntreated lymphocytes can vary considerably (2-4). Al-though many lymphocytes have the expected membrane-associated "ring"-type immunofluorescence staining pat-tern, or display diffuse staining, a significant proportion ofcells contains numerous focal accumulations or a single largeaggregate of the protein that can be seen at some distancefrom the cell periphery in the trans-Golgi region of thecytoplasm or near the nucleus (3). In other cells, a distinct"cap" of membrane-associated spectrin is visible. Using invitro T lymphocyte models that constitutively express acytoplasmic spectrin aggregate, we have demonstrated thatspectrin can be rapidly redistributed to the plasma membranefollowing various stimuli that induce interleukin 2 secretion(2). These initial studies were useful in identifying distinctmorphological subsets of lymphocytes and in suggesting thatan event(s) relating to lymphocyte function may signal

changes in spectrin distribution. Here, the question ofwhether the distribution patterns of spectrin are affected bythe early events following T-cell activation was addressed. Inparticular, the localization of spectrin was studied in relationto the distribution and activity of the enzyme protein kinaseC (PKC), a protein that has been reported to reorganizewithin cells in response to triggering of various cell surfacereceptors and to participate in intracellular signaling events.PKC has been identified as an important component of the

inositol phospholipid cascade that is activated by stimulationof the lymphocyte antigen receptor and other cell surfacemolecules in various tissues. Previous biochemical experi-ments have reported that PKC translocates from the solubleto the particulate fraction upon activation. The latter is oftenassumed to be the plasma membrane. However, this posi-tioning within the cell does not explain studies suggesting thatPKC is needed to phosphorylate substrates throughout thecell, including the nucleus (for review, see ref. 5). Moreover,biochemical evidence from lymphocytes and other tissueshas suggested that PKC may be associated with the deter-gent-resistant cellular framework (6, 7). However, neither thesubcellular distribution ofPKC in lymphocytes nor the natureof its association with cytoskeletal components has beendefined.

In this paper, we present data concerning the distributionofPKCB1I and its relationship to the spectrin-based cytoskel-eton in resting and activated tissue lymphocytes. These dataprovide information pertaining to the physiological mecha-nisms controlling lymphocyte spectrin distribution.

MATERIALS AND METHODS

Cells. T and B lymphocytes were isolated from 6- to8-week-old BALB/c mice using Ficoll-Paque (PharmaciaLKB). Enriched populations ofT lymphocytes were obtainedby passage of lymph node cell suspensions over nylon wool(8).

Antibodies. The anti-chicken erythrocyte a-spectrin anti-serum used in this study has been characterized (2-4). Theisotype-specific rabbit anti-PKCPII peptide antiserum wasprovided by Alan P. Fields (9). Anti-PKCP3-specific mono-clonal antibodies were also purchased from GIBCO/BRL.Secondary antisera were purchased from either Miles/ICNor Boehringer Mannheim.

Activation Protocols. Nylon wool-enriched T cells werestimulated at a final concentration of 2 x 106 per ml. Phorbol12-myristate 13-acetate (PMA; Sigma) was added directly tothe cell suspension to obtain a final concentration of 10ng/ml. Solvent was used as a control in each experiment. Tcells were activated using an immobilized (H57-597) pan-reactive anti-mouse T-cell aj3 receptor monoclonal antibody(10). Cells added to uncoated plates and to plates coated with

Abbreviations: PKC, protein kinase C; TCR, T-cell receptor; PMA,phorbol 12-myristate 13-acetate.tTo whom reprint requests should be addressed.

4947

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Dow

nloa

ded

by g

uest

on

May

17,

202

1

4948 Cell Biology: Gregorio et al.

protein A alone were used as controls in each experiment. Insome experiments, T cells were preincubated with or without0.5 AM calphostin C (Kamiya Biomedical, Thousand Oaks,CA) for 30 min prior to 1 hr of stimulation. After variousperiods of time, the cells were harvested and prepared forimmunofluorescence microscopy.

Immunolocalization of Spectrin and PKCI3II. Isolated lym-phocytes were prepared for staining as described (4). Thefixed and permeabilized cells that were adhered onto Alcianblue-coated coverslips were- probed with goat anti-a-spectrinantiserum, followed by fluorescein-conjugated donkey anti-goat IgG. The coverslips were then probed with an isotype-specific rabbit anti-PKC/31I peptide antiserum, followed byrhodamine-conjugated goat anti-rabbit IgG. Identical resultswere obtained using anti-PKCj3 monoclonal antibodies. Insome experiments, T cells were treated with a cytoskeletalextraction buffer (CSK buffer) (11), containing 1% TritonX-100, before fixation. In Figs. 1 and 5 the cells wereexamined on a Bio-Rad MRC-600 confocal microscope andphotographs were taken on T-Max (Kodak) film. The cells inFig. 2 were analyzed on an Olympus BM-2 microscope andphotographs were taken on Tri-X (Kodak) film. Identicalresults were obtained when the staining procedure wasperformed on cells in suspension or freshly isolated withoutany purification steps. The percentage of cells with spectrin/PKC(311 aggregates was quantified by counting a minimum of200 cells per group. Each value represents the mean ± SEMof three experiments.

Immunoprecipitation and Western Blot Analysis. For im-munoprecipitation, 2 x 107 T cells were washed in ice-coldphosphate-buffered saline plus 3 mM phenylmethylsulfonylfluoride, 25 ,ug of aprotinin per ml, 5 mM Na-p-tosyl-L-arginine methyl ester, and 10 1LM leupeptin, solubilized for 10min in 900 ,ul of 15 mM Tris hydrochloride (pH 7.5) containing1% (vol/vol) Triton X-100, 120 mM NaCI, and 25 mM KCl,pelleted in a microcentrifuge at 16,000 x g for 10 min, andimmunoprecipitated as described (12). The immunoprecipi-tated material was run on a 10% SDS/acrylamide gel. Forimmunoprecipitation using rabbit anti-a-spectrin antiserum,the gel was transferred to nitrocellulose and probed with goatanti-a-spectrin antiserum (2) or 1251-labeled anti-PKCPII an-tiserum. The 125I-probed blot was exposed at -800C topreflashed XAR-5 (Kodak) x-ray film using intensifyingscreens (DuPont). For immunoprecipitation using anti-PKCf3monoclonal antibodies, the blot was probed with the sameunlabeled monoclonal anti-PKCB antibody used for immu-noprecipitation or rabbit anti-spectrin antibodies, followedby horseradish peroxidase-labeled secondary antibodies.The reaction product was visualized using an enhancedchemiluminescence Western blotting detection system (Am-ersham).

Solubility Studies. T cells (2 x 107) were stimulated with orwithout 10 ng of PMA per ml of culture medium for 2 hr,washed, and solubilized as described above for the immu-noprecipitation experiments. The resulting pellet was resus-pended in hot 2x SDS sample buffer, vortexed, and boiled for3 min. The samples were probed by Western blot analysis.The reaction product was quantified by densitometry.

RESULTSInitially, the distribution patterns of spectrin and PKCB1Iwithin isolated tissue lymphocytes were compared. Theconfocal image shown in Fig. 1A reveals several of thedistinctive patterns of spectrin distribution that occur natu-rally among tissue lymphocytes (see also, refs. 3 and 4). Inuntreated, freshly isolated lymphocytes, spectrin is seen invarious patterns, including plasma-membrane-associatedrings, cytoplasmic aggregates, membrane-associated caps,and accumulations at the nuclear envelope (a pattern difficult

FIG. 1. Coimmunolocalization of spectrin and PKC(II in isolatedmurine T and B lymphocytes using confocal microscopy. Identicalcells were stained by indirect immunofluorescence for spectrin (A)and PKC,3II (B). Murine lymphocytes exhibit a heterogeneousdistribution pattern of spectrin and PKCPII. Cells containing a focalaccumulation of spectrin also exhibit a coincident staining pattern ofPKC3II (arrows). Note that in some cells, staining can be visualizedat the nuclear envelope (arrowheads). This observation is consistentwith a previous report in which PKC was shown biochemically to beassociated with the nuclear fraction in certain populations of Blymphocytes (7). The position of the nucleus was determined byphase-contrast microscopy. (Bar = 10 ,m.)to discern using conventional microscopy). Unexpectedly,PKCfII was found to be colocalized with spectrin in the samecells (Fig. 1B). Thus, PKC(3II is also heterogeneously dis-tributed in tissue-derived lymphocytes. This heterogeneityamong lymphocytes is also observed in situ (data not shown).The coincident localization of spectrin and PKCBII, to-

gether with the observation that spectrin distribution patternscan be altered by activation in an in vitro cell model (2), ledus to examine the relationship of these two proteins followingthe activation of tissue-derived lymphocytes. Phorbol esterssuch as PMA, as well as structural analogs of diacyglycerolsuch as 1-oleoyl-2-acetylglycerol, were used to stimulatePKC activity directly in T cells. At various times afterincubation with these agents, a significant increase in thenumber of cells containing a large cytoplasmic aggregate ofspectrin was observed (compare untreated cells in Fig. 2 Topwith those in Fig. 2 Middle; data shown for PMA treatment).Additionally, there was a coincidental increase in cells ex-pressing aggregates of the (311 isozyme of PKC. This strikingalternation in the distribution patterns of both proteins wasrapid; the percentage of T cells with aggregates increasedfrom approximately 20%o to 50%6 5 min after the addition ofPMA and rose to >75% within the first 30 min. Whenlymphocytes were treated with an inactive PMA epimer thatdoes not activate PKC, no morphological alterations could beseen (data not shown).The morphological data described above indicated an

association between the distribution of PKC(311 and that ofspectrin and demonstrated a coincidental movement of thesetwo molecules. Since PKC has been shown to play a role inT-cell activation, these observations suggest that spectrinmay be functionally linked to activation-induced events. Toinvestigate this hypothesis, we immobilized an artti-mouse a4T-cell receptor (TCR)-specific monoclonal antibody (H57-597) that directly activates a subset of T cells via the TCRcomplex. Using this method, a rapid and long-lasting increasein the percentage of T cells that express large, coincidentaggregates of spectrin and PKCPH1 was again observed (Fig.2 Bottom). The percentage of T cells with spectrin andPKC(3II aggregates rose from approximately 15% in controlcells to 45% within 5 min of treatment, reaching a maximumof 55%. Thus, the translocation of PKC.8 and spectrin to afocal center within the cytosol is temporally linked to T-cellactivation mediated by the antigen receptor in a large subset

Proc. Natl. Acad. Sci. USA 89 (1992)

Dow

nloa

ded

by g

uest

on

May

17,

202

1

Proc. Natl. Acad. Sci. USA 89 (1992) 4949

C100

C) U

3 0icm 60--

aa2

g 20

5 min 30 min 4 hrTime

C 100

80 -

coc10)

tM 0-_0-

.§ 40jf

o 201 K .

5min 4hrTime

FIG. 2. Phorbol ester treatment or activationthrough the antigen receptor results in an in-crease in the percentage ofT lymphocytes con-taining cytoplasmic aggregates of spectrin andPKCI3. (Top) Untreated T cells, spectrin (A) andPKC3 (B). (Middle) Spectrin (A), PKC,8 (B), andkinetics of spectrin and PKC/3 aggregate forma-tion (C) following treatment with PMA. Openbar, control; closed bar, PMA. (Bottom) Spec-trin (A), PKC6 (B), and kinetics of spectrin andPKC/3 aggregate formation (C) after activation

i' of T cells through the antigen receptor. Open24 hr- bar, control; closed bar, H57-597. Micrographs

show organizational patterns after stimulationfor 5 min. Aggregates remained in treated cellsfor at least 24 hr; the percentage of aggregates insolvent control cells did not change between thetime of plating and the 5-min time point, al-though the percentage of cells containing aggre-gates was observed to decrease slowly over timewith continuous culturing. Arrowheads indicatecells that appear to be forming aggregates. Thisintermediate staining pattern is not seen at latertime points. T-cell activation was confirmed bymeasuring [3H]thymidine uptake (2): control(cells without protein A and H57-597), 269 + 156cpm; control (protein A alone), 563 ± 513 cpm;

24 hr and H57-597-activated cells, 144,810 + 51,935cpm. Each value represents the mean ± SEM.(Bar = 10 Am.)

of lymph node T cells. It should be emphasized that there areseveral PKC isoenzymes known to occur in lymphocytes (13)and that their subcellular distribution may differ from thatreported here for PKC,8II.The above data suggested that those tissue lymphocytes

that naturally express large cytoplasmic aggregates of spec-trin and PKCf3II might be cells in which activation of PKChas occurred physiologically. To test this hypothesis, thedistribution of spectrin and PKCB in lymphocytes was ex-

amined after treatment with an inhibitor ofPKC activity. Weused the highly specific PKC inhibitor calphostin C, whichinteracts with the regulatory domain of the enzyme andinhibits the binding of diacylglycerol analogues to PKC (14).When freshly isolated lymphocytes were treated withcalphostin C alone, a steady decline in the percentage of cellsexpressing aggregates was observed (to <50% of controlvalues within 2 hr of treatment), suggesting that PKC, in aninactive conformation is not maintained as an aggregate. Inaddition, pretreatment with calphostin C significantlyblocked new aggregate formation resulting from PMA treat-ment or TCR activation (Fig. 3), suggesting that access to theregulatory domain ofPKC by an appropriate PKC stimulatoris required for the cytoplasmic accumulation of spectrin andPKC,8.To investigate further the relationship between lympho-

cyte spectrin and PKC/3, experiments were performed todetermine the effects of PMA treatment on the solubility ofboth of these proteins. We found that following PMA treat-

ment, there is a significant increase in the level of spectrin(Fig. 4A) and PKCf3 (Fig. 4B) in the Triton X-100-insolublefraction. This result demonstrates a similarity between thesolubility properties of the two proteins following PMAtreatment and suggests that the spectrin and PKC,8 found incytoplasmic aggregates represent an insoluble pool of theseproteins. These biochemical data were confirmed morpho-

100 -

a 80-CDE0)0 60-

40CD0

20-I0-

StimulationCalphostin C

A

_-_ + +

_-+- +

B

_ _-+ +

+ - +

FIG. 3. Treatment with calphostin C inhibits spectrin and PKCf3redistribution in T lymphocytes induced upon stimulation with PMA(A) or activation through the TCR (B). Open bar, PMA experiment;closed bar, H57-597 activation experiment. Pretreatment of T cellswith calphostin C prior to T-cell activation inhibited proliferation as

measured by [3H]thymidine uptake (not shown).

I I

Cell Biology: Gregorio et al.

Dow

nloa

ded

by g

uest

on

May

17,

202

1

4950 Cell Biology: Gregorio et al.

A C

.51'.j;!"50) --1

r '

owB

80

FIG. 4. Solubility properties and coimmunoprecipitation of spec-trin and PKC,3 from murine T cells. Cells were stimulated with orwithout PMA for 1 hr, followed by extraction in a physiologicalbuffer containing L.0% Triton X-100. The insoluble fractions wereanalyzed by Western blot analysis, which revealed that there was anapproximate 5-fold increase in spectrin and PKC(3 found in the TritonX-100-insoluble fraction following PMA treatment. (A) Blot probedwith anti-spectrin antiserum. Lane 1, untreated insoluble fraction;lane 2, PMA-treated insoluble fraction. The a-spectrin band is at 240kDa. Notice the degradation fragment in lane 2 at =180 kDa. (B) Blotprobed with anti-PKC(8 antiserum. Lane 1, untreated insolublefraction; lane 2, PMA-treated insoluble fraction. The PKCP band isat =80 kDa. Notice the degradation fragment in lane 2 at -65 kDa.(C and D) Spectrin (C) and PKC/3 (D) immunoprecipitates ofuntreated tissue T lymphocytes were analyzed by SDS/PAGEfollowed by Coomassie blue staining of polypeptides immunopre-cipitated (lanes 1) or Western blotting (lanes 2 and 3). (C) Blots ofproteins immunoprecipitated with anti-spectrin antibodies wereprobed for either a-spectrin (lane 2) or PKC,3II (lane 3). (D) Blots ofproteins immunoprecipitated with anti-PKC,8 antibodies wereprobed for a-spectrin (lane 2) or PKC/3 (lane 3). Lanes 2, a-spectrinband at 240 kDa; lanes 3, PKC,3 band at =80 kDa. The arrowheadrepresents immunoreactivity to the heavy chain of the immunoglob-ulin molecules used for immunoprecipitation. The numbers in the leftmargin represent the relative molecular mass standards (kDa). a,a-Spectrin; (3, 13-spectrin. Spectrin immunoprecipitates were ana-lyzed for PKC activity using a PKC activity assay (GIBCO/BRL).Activity was measured by subtracting peptide-incorporated phos-phate (without inhibitor)/min from peptide-incorporated phosphate/min (with inhibitor). Immunoprecipitation with spectrin pre-immuneserum, c0.5 pmol/min/106 cells, immunoprecipitation with spectrinantiserum, 5.4 pmol/min/106 cells, immunoprecipitation withPKC,/11 antiserum (as a positive control), 20 pmol/min/106 cells.Values are the mean of duplicates.

logically when PMA-treated cells were extracted with TritonX-100-containing buffer, prior to fixation, and visualized byimmunofluorescence microscopy. Results from this experi-ment revealed a coincident localization of spectrin and PKC,3(Fig. 5 A and B) in a Triton X-100-insoluble aggregate; theseaggregates were indistinguishable from those found in cellsthat were formaldehyde fixed prior to detergent extractionshown above.

It was also ofinterest to determine whether PKC(3 could becoimmunoprecipitated with spectrin. In general, immunopre-cipitation of cytoskeletal proteins is difficult because of theirrelatively insoluble nature as compared to other proteins.However, although a portion of total cell spectrin remains inthe insoluble pellet under the extraction conditions used, inuntreated lymphocytes at least 40% of total spectrin is solubleunder these Triton X-100 extraction conditions (data notshown) and can be isolated by immunoprecipitation (Fig. 4C,lane 1; ref. 15). When the immunoprecipitate obtained usinganti-a-spectrin antibodies was examined by immunoblotting,spectrin and PKCf3II were detected (Fig. 4C, lanes 2 and 3,respectively). Additionally, both proteins were also presentin immunoprecipitates obtained using anti-PKCf3 antibodies

FIG. 5. PKCP is associated with the Triton X-100-insolubleaggregate induced by PMA treatment in T lymphocytes. PMA-treated T cells were treated with CSK buffer containing 1% TritonX-100 prior to fixation and stained by double immunofluorescencefor spectrin (A) and PKC(3 (B). Treatment of cels with CSK bufferresulted in extraction of most cellular components, whereas thecytoskeleton and cytoskeleton-associated proteins remained on thecoverslip. (Bar = 10 gum.)

(Fig. 4D, lanes 2 and 3). When the gels of the immunopre-cipitates were stained with Coomassie blue (Fig. 4 C and D,lanes 1), other protein bands (particularly ones at approxi-mately 47 kDa and 200 kDa) became apparent, suggestingthat there are additional proteins associated with spectrin andPKCj3 that are not disassociated under the detergent condi-tions used. Furthermore, the 240/235-kDa forms of tissuespectrin were always found in equimolar quantities in theimmunoprecipitates.To confirm the presence of PKC in the spectrin immuno-

precipitates, an in vitro PKC activity assay was used, whichmeasures the incorporation of 32p into a PKC-specific sub-strate. Using this assay, PKC activity was detected in thespectrin immunoprecipitate (Fig. 4 legend). This activity wasblocked by a pseudo-substrate inhibitor peptide and was notdetected in preimmune immunoprecipitates. The immuno-precipitation and the PKC activity assay data suggest thatthese two molecules are noncovalently associated or areindirectly linked by a third protein.

DISCUSSIONIn all mammalian lymphoid organs that have been examined,there is a naturally arising heterogeneity among lymphocyteswith regard to the distribution of spectrin. A major goal ofourresearch is to better understand the physiological factors thatregulate lymphocyte spectrin distribution. Here it was foundthat phorbol ester treatment and TCR stimulation of tissue-derived lymphocytes induce the formation of a large focalaggregate of spectrin within the cytoplasm; similarly, it hasbeen reported that PMA causes a focal accumulation ofPKCwithin lymphocytes (16).

Cellular fractionation studies have demonstrated that ac-tivation of PKC results in translocation of PKCfII activityfrom the cytosol to a "particulate" fraction (for review, seeref. 5). The particulate fraction is often assumed to be theplasma membrane based on its insolubility and on PKC'sdependence on lipids such as diacylglycerol and phosphati-dylserine for activity. However, the reported increase ininsolubility could result from an increased association withTriton X-100-insoluble components of the cell such as thecytoskeleton (6, 7). Our results suggest a mechanism for theobserved phenomenon: the reported increase in insolubilitycould result from an association ofthe (3 PKC isoenzyme withthe spectrin-based cytoskeleton and their redistribution to amore insoluble aggregate. Support for this hypothesis isprovided by the following experimental evidence: (i) thesolubility of PKC(3 and spectrin decreases significantly fol-lowing PMA treatment, (ii) PKC/3 can be visualized withspectrin in a Triton X-100-insoluble aggregate, and (iii) PKC3

Proc. Natl. Acad. Sci. USA 89 (1992)

jC

Dow

nloa

ded

by g

uest

on

May

17,

202

1

Proc. Natl. Acad. Sci. USA 89 (1992) 4951

is associated either directly or indirectly with spectrin, asdetermined by reciprocal immunoprecipitation experiments.The reciprocal immunoprecipitation studies suggest thatthere is a physical association between the two proteins asthey occur in the soluble state in the cytoplasm, although thepresence of other bands in the immunoprecipitates indicatesthat this association may be indirect; for example, a complexof macromolecules may associate with spectrin and PKCP.The other proteins coimmunoprecipitated with our antibod-ies await identification. Possibilities for the 200-kDa bandinclude myosin or a member of the CD45 family of proteins,but our preliminary results are inconclusive. The associationof spectrin and PKCf3II in the soluble pool may reflect asimilar association between the two proteins in the insolublefraction (i.e., in the cytoplasmic aggregate) where they arecolocalized.The finding that the PKC inhibitor calphostin C inhibits the

PMA- and TCR-induced redistribution of spectrin and PKCf3in T lymphocytes suggests that accessibility to the regulatorydomain of PKC by diacylglycerol may be required for themovement of these proteins to a cytoplasmic aggregate. Theobservation that direct activators of PKC induced formationof the aggregate, together with the finding that the PKCinhibitor calphostin C causes aggregate loss, suggests that (i)only PKC/3 in an active conformation is found in the focalaggregate, (ii) active PKC is required for maintaining thecytoskeletal aggregate in the trans-Golgi region, and (iii)freshly isolated lymphocytes containing aggregates are cellsin which PKC activation has occurred physiologically. Thesedata demonstrate that the heterogeneous and dynamic prop-erties of lymphocyte spectrin may, in part, reflect its asso-ciation with PKC activity and movement. We speculate thatspectrin, in accordance with its structural role in erythrocytesand in some nonerythroid models, may provide a frameworkallowing PKC(3 to be distributed or supported at sites whereits substrates are located. Ultrastructural studies (2, 3) haveshown that the focal concentration of spectrin is oftenassociated with the trans-Golgi apparatus (or the nuclearenvelope). Similarly, PKCf3II has been found to be accumu-lated near the Golgi complex of pyramidal cells in rat brainhippocampus (17), suggesting that the presence of PKCf3 andspectrin in the region of the Golgi may not be unique tolymphocytes and may reflect a common function for thesemolecules in some nonerythroid cells. The positioning ofPKC in this area may reflect a requirement for the phosphor-ylation of newly synthesized proteins resulting from trigger-ing of the TCR. Alternatively, spectrin and PKC,3 may beinvolved in some of the Golgi functions, such as regulation ofcell polarity, mobility, and intracellular trafficking (secretionand internalization). In this regard, spectrin and PKC haveindependently been reported to be involved in secretion andin exocytotic processes in other systems (13, 18, 19). It is alsopossible that PKC accumulates in a site enriched for spectrin-associated cytoskeletal proteins since they represent poten-tially important substrates for this enzyme. Therefore, itwould be of interest to determine if other membrane skeletalproteins are associated with spectrin and/or PKCP in lym-phocytes. Previously, we identified vimentin-containing in-termediate filaments enriched in the region of the cytoplasmicaggregate (20). It is not known if lymphocyte spectrin isphosphorylated, but the data presented here make it animportant issue to be addressed.The results reported here regarding cytoskeletal movement

in activated tissue T lymphocytes were unexpected based onour previous findings using an in vitro T-cell hybridomamodel system, DO-11.10 cells. The DO-11.10 cells, whichexhibit transformed or leukemic properties, homogeneouslyexpress cytoplasmic spectrin aggregates. We found that

various stimuli that cause secretion of interleukin 2 in DO-11.10 cells also cause a fragmentation of the aggregate (2).

It is well known that PKC activity is dependent on phos-pholipids. Several studies indicate that various cytoskeletalproteins, including spectrin, have the ability to bind lipids, inparticular, phosphatidylserine; an association of this kindcould provide the necessary source of lipid for PKCB acti-vation in the region ofthe spectrin aggregate (21-23). Anotherpotential source of lipid could be the numerous small mem-branous vesicles known to be present within the aggregate(3). Interestingly, exogenously added phosphatidylserine,but not other phospholipids, has been reported to accumulatein the Golgi apparatus of cultured fibroblasts (24); a similaraccumulation may occur in lymphocytes.

In summary, the natural heterogeneity among isolatedtissue lymphocytes with respect to spectrin may reflectfluctuations in activation potential that occur physiologicallyin response to antigen or other cell surface stimuli. Futurework must focus on identifying the significance of the othernaturally occurring subcellular distributions of lymphocytespectrin.

We thank Drs. Jennifer Black and Adrian Black for many helpfuldiscussions, Dr. Alan P. Fields for his generous gift of anti-PKCPIIpeptide antiserum, and E. Hurley, M. Hochberg, and J. Prendergastfor their technical assistance. This work was supported by grantsfrom the National Institutes of Health (AI30131 and CA22786) andthe American Cancer Society (CH421C) and a Buffalo Foundationgrant. The confocal microscope at Roswell Park Cancer Institute issupported through a grant from the National Cancer Institute(CA16056).

1. Bennett, V. (1990) Curr. Opion. Cell Biol. 2, 51-56.2. Lee, J. K., Black, J. D., Repasky, E. A., Kubo, R. T. &

Bankert, R. B. (1988) Cell 55, 807-816.3. Black, J. D., Koury, S. K., Bankert, R. B. & Repasky, E. A.

(1988) J. Cell Biol. 106, 97-109.4. Repasky, E. A., Symer, D. & Bankert, R. B. (1984) J. Cell

Biol. 99, 350-355.5. Berry, N. & Nishizuka, Y. (1990) Eur. J. Biochem. 189,

205-214.6. Mochly-Rosen, D., Henrich, C. J., Cheever, L., Khaner, H. &

Simpson, P. C. (1990) Cell Regul. 1, 693-706.7. Chen, Z. Z., McGuire, J. C., Leach, K. L. & Cambier, J. C.

(1987) J. Immunol. 138, 2345-2352.8. Julius, M. H., Simpson, E. & Herzenberg, L. A. (1973) Eur. J.

Immunol. 3, 645-649.9. Hocevar, B. A. & Fields, A. P. (1991) J. Biol. Chem. 266,

28-33.10. Kubo, R. T., Born, W., Kappler, J. W., Marrack, P. & Pigeon,

M. (1989) J. Immunol. 142, 2736-2742.11. Greenberg, M. E. & Edelman, G. M. (1983) Cell 33, 767-779.12. Nelson, W. J. & Veshnock, P. J. (1986) J. Cell Biol. 103,

1751-1766.13. Nishizuka, Y. (1988) Nature (London) 334, 661-665.14. Kobayashi, E., Nakano, H., Morimoto, M. & Tamaoki, T.

(1989) Biochem. Biophys. Res. Commun. 159, 548-553.15. Nelson, W. J., Colaco, C. A. L. S. & Lazarides, E. (1983)

Proc. Natl. Acad. Sci. USA 80, 1626-1630.16. Berry, N., Ase, K., Kikkawa, U., Kishimoto, A. & Nishizuka,

Y. (1989) J. Immunol. 143, 1407-1413.17. Nigg, E. A., Schafer, G., Hilz, H. & Eppenberger, H. M.

(1985) Cell 41, 1039-1051.18. Sikorski, A. F., Terlecki, G., Zagon, I. S. & Goodman, S. R.

(1991) J. Cell Biol. 114, 313-318.19. Perrin, D., Langley, 0. K. & Aunis, D. (1987) Nature (London)

326, 498-501.20. Lee, J. K. & Repasky, E. A. (1987) Cell Tissue Res. 247,

195-202.21. Haest, C. W. M. (1982) Biochim. Biophys. Acta 694, 331-352.22. Burn, P. (1988) Trends Biochem. Sci. 13, 79-83.23. Subbarao, N. K., MacDonald, R. I., Takeshita, K. & Mac-

Donald, R. C. (1991) Biochim. Biophys. Acta 1063, 147-154.24. Kobayashi, T. & Arakawa. Y. (1991) J. Cell Biol. 113, 235-244.

Cell Biology: Gregorio et al.

Dow

nloa

ded

by g

uest

on

May

17,

202

1