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

Translocation of spectrin and protein kinase C to a cytoplasmic aggregate upon lymphocyte activation CAROL 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 for Immunology and Respiratory Medicine, Denver, CO 80206

Communicated by Daniel Branton, February 10, 1992

changes in spectrin distribution. Here, the question of whether the distribution patterns of spectrin are affected by the early events following T-cell activation was addressed. In particular, the localization of spectrin was studied in relation to the distribution and activity of the enzyme protein kinase C (PKC), a protein that has been reported to reorganize within cells in response to triggering of various cell surface receptors and to participate in intracellular signaling events. PKC has been identified as an important component of the inositol phospholipid cascade that is activated by stimulation of the lymphocyte antigen receptor and other cell surface molecules in various tissues. Previous biochemical experiments have reported that PKC translocates from the soluble to the particulate fraction upon activation. The latter is often assumed to be the plasma membrane. However, this positioning within the cell does not explain studies suggesting that PKC is needed to phosphorylate substrates throughout the cell, including the nucleus (for review, see ref. 5). Moreover, biochemical evidence from lymphocytes and other tissues has suggested that PKC may be associated with the detergent-resistant cellular framework (6, 7). However, neither the subcellular distribution ofPKC in lymphocytes nor the nature of its association with cytoskeletal components has been defined. In this paper, we present data concerning the distribution of PKCB1I and its relationship to the spectrin-based cytoskeleton in resting and activated tissue lymphocytes. These data provide information pertaining to the physiological mechanisms controlling lymphocyte spectrin distribution.

We have previously reported that mammaABSTRACT lian tissue lymphocytes exhibit significant heterogeneity with respect to the subcellular distribution of spectrin and that this phenomenon may result from a dynamic behavior of spectrin in response to activation signals. Here, we further characterize the involvement of spectrin in lymphocyte activation by examining its relationship with protein kinase C (PKC). PKC isoenzymes are a family of cytosolic kinases that translocate from the soluble to particulate fraction upon cell stimulation. It is reported here that activation of lymph node T cells through the antigen-specific receptor, or direct activation of PKC by phorbol esters, results in a striking increase in cells expressing a cytoplasmic aggregate of spectrin. Additionally, a concurrent increase in cells expressing aggregates of the PHlI isozyme of PKC is observed. Immunofuorescence staining revealed that spectrin and PKC8II are colocalized in untreated lymphocytes and that these two proteins are coincidently translocated to the same focal aggregate within the cytoplasm following stimulation. This redistribution of spectrin and PKCfi is blocked by pretreatment with calphostin C, a specific inhibitor of PKC. Solubility studies showed that there is an increase of both proteins in the detergent-insoluble fraction of lymphocytes upon activation, and immunoprecipitation studies indicated that the soluble form of these molecules may be associated directly or indirectly as part of a complex of proteins. These data indicate that the positioning of the spectrin-based cytoskeleton is sensitive to activation signals and may play a role in the function or positioning of PKCfiII.

There is increasing evidence that spectrin can play a more dynamic role in various cytoplasmic and plasma membranerelated events in nucleated cells than in the erythrocyte, where it is a major component of the membrane skeleton providing structural support for the plasma membrane (for review, see ref. 1). For example, we have observed that the level and distribution of spectrin at the plasma membrane of untreated lymphocytes can vary considerably (2-4). Although many lymphocytes have the expected membraneassociated "ring"-type immunofluorescence staining pattern, or display diffuse staining, a significant proportion of cells contains numerous focal accumulations or a single large aggregate of the protein that can be seen at some distance from the cell periphery in the trans-Golgi region of the cytoplasm or near the nucleus (3). In other cells, a distinct "cap" of membrane-associated spectrin is visible. Using in vitro T lymphocyte models that constitutively express a cytoplasmic spectrin aggregate, we have demonstrated that spectrin can be rapidly redistributed to the plasma membrane following various stimuli that induce interleukin 2 secretion (2). These initial studies were useful in identifying distinct morphological subsets of lymphocytes and in suggesting that an event(s) relating to lymphocyte function may signal

MATERIALS AND METHODS Cells. T and B lymphocytes were isolated from 6- to 8-week-old BALB/c mice using Ficoll-Paque (Pharmacia LKB). Enriched populations of T lymphocytes were obtained by passage of lymph node cell suspensions over nylon wool (8). Antibodies. The anti-chicken erythrocyte a-spectrin antiserum used in this study has been characterized (2-4). The isotype-specific rabbit anti-PKCPII peptide antiserum was provided by Alan P. Fields (9). Anti-PKCP3-specific monoclonal antibodies were also purchased from GIBCO/BRL. Secondary antisera were purchased from either Miles/ICN or Boehringer Mannheim. Activation Protocols. Nylon wool-enriched T cells were stimulated at a final concentration of 2 x 106 per ml. Phorbol 12-myristate 13-acetate (PMA; Sigma) was added directly to the cell suspension to obtain a final concentration of 10 ng/ml. Solvent was used as a control in each experiment. T cells were activated using an immobilized (H57-597) panreactive anti-mouse T-cell aj3 receptor monoclonal antibody (10). Cells added to uncoated plates and to plates coated with

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Abbreviations: PKC, protein kinase C; TCR, T-cell receptor; PMA, phorbol 12-myristate 13-acetate.

tTo whom reprint requests should be addressed.

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protein A alone were used as controls in each experiment. In some experiments, T cells were preincubated with or without 0.5 AM calphostin C (Kamiya Biomedical, Thousand Oaks, CA) for 30 min prior to 1 hr of stimulation. After various periods of time, the cells were harvested and prepared for immunofluorescence microscopy. Immunolocalization of Spectrin and PKCI3II. Isolated lymphocytes were prepared for staining as described (4). The fixed and permeabilized cells that were adhered onto Alcian blue-coated coverslips were- probed with goat anti-a-spectrin antiserum, followed by fluorescein-conjugated donkey antigoat IgG. The coverslips were then probed with an isotypespecific rabbit anti-PKC/31I peptide antiserum, followed by rhodamine-conjugated goat anti-rabbit IgG. Identical results were obtained using anti-PKCj3 monoclonal antibodies. In some experiments, T cells were treated with a cytoskeletal extraction buffer (CSK buffer) (11), containing 1% Triton X-100, before fixation. In Figs. 1 and 5 the cells were examined on a Bio-Rad MRC-600 confocal microscope and photographs were taken on T-Max (Kodak) film. The cells in Fig. 2 were analyzed on an Olympus BM-2 microscope and photographs were taken on Tri-X (Kodak) film. Identical results were obtained when the staining procedure was performed on cells in suspension or freshly isolated without any purification steps. The percentage of cells with spectrin/ PKC(311 aggregates was quantified by counting a minimum of 200 cells per group. Each value represents the mean ± SEM of three experiments. Immunoprecipitation and Western Blot Analysis. For immunoprecipitation, 2 x 107 T cells were washed in ice-cold phosphate-buffered saline plus 3 mM phenylmethylsulfonyl fluoride, 25 ,ug of aprotinin per ml, 5 mM Na-p-tosyl-Larginine methyl ester, and 10 1LM leupeptin, solubilized for 10 min in 900 ,ul of 15 mM Tris hydrochloride (pH 7.5) containing 1% (vol/vol) Triton X-100, 120 mM NaCI, and 25 mM KCl, pelleted in a microcentrifuge at 16,000 x g for 10 min, and immunoprecipitated as described (12). The immunoprecipitated material was run on a 10% SDS/acrylamide gel. For immunoprecipitation using rabbit anti-a-spectrin antiserum, the gel was transferred to nitrocellulose and probed with goat anti-a-spectrin antiserum (2) or 1251-labeled anti-PKCPII antiserum. The 125I-probed blot was exposed at -800C to preflashed XAR-5 (Kodak) x-ray film using intensifying screens (DuPont). For immunoprecipitation using anti-PKCf3 monoclonal antibodies, the blot was probed with the same unlabeled monoclonal anti-PKCB antibody used for immunoprecipitation or rabbit anti-spectrin antibodies, followed by horseradish peroxidase-labeled secondary antibodies. The reaction product was visualized using an enhanced chemiluminescence Western blotting detection system (Amersham). Solubility Studies. T cells (2 x 107) were stimulated with or without 10 ng of PMA per ml of culture medium for 2 hr, washed, and solubilized as described above for the immunoprecipitation experiments. The resulting pellet was resuspended in hot 2x SDS sample buffer, vortexed, and boiled for 3 min. The samples were probed by Western blot analysis. The reaction product was quantified by densitometry.

RESULTS Initially, the distribution patterns of spectrin and PKCB1I within isolated tissue lymphocytes were compared. The confocal image shown in Fig. 1A reveals several of the distinctive patterns of spectrin distribution that occur naturally among tissue lymphocytes (see also, refs. 3 and 4). In untreated, freshly isolated lymphocytes, spectrin is seen in various patterns, including plasma-membrane-associated rings, cytoplasmic aggregates, membrane-associated caps, and accumulations at the nuclear envelope (a pattern difficult

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

FIG. 1. Coimmunolocalization of spectrin and PKC(II in isolated murine T and B lymphocytes using confocal microscopy. Identical cells were stained by indirect immunofluorescence for spectrin (A) and PKC,3II (B). Murine lymphocytes exhibit a heterogeneous distribution pattern of spectrin and PKCPII. Cells containing a focal accumulation of spectrin also exhibit a coincident staining pattern of PKC3II (arrows). Note that in some cells, staining can be visualized at the nuclear envelope (arrowheads). This observation is consistent with a previous report in which PKC was shown biochemically to be associated with the nuclear fraction in certain populations of B lymphocytes (7). The position of the nucleus was determined by phase-contrast microscopy. (Bar = 10 ,m.)

to discern using conventional microscopy). Unexpectedly, PKCfII was found to be colocalized with spectrin in the same cells (Fig. 1B). Thus, PKC(3II is also heterogeneously distributed in tissue-derived lymphocytes. This heterogeneity among lymphocytes is also observed in situ (data not shown). The coincident localization of spectrin and PKCBII, together with the observation that spectrin distribution patterns can be altered by activation in an in vitro cell model (2), led us to examine the relationship of these two proteins following the activation of tissue-derived lymphocytes. Phorbol esters such as PMA, as well as structural analogs of diacyglycerol such as 1-oleoyl-2-acetylglycerol, were used to stimulate PKC activity directly in T cells. At various times after incubation with these agents, a significant increase in the number of cells containing a large cytoplasmic aggregate of spectrin was observed (compare untreated cells in Fig. 2 Top with those in Fig. 2 Middle; data shown for PMA treatment). Additionally, there was a coincidental increase in cells expressing aggregates of the (311 isozyme of PKC. This striking alternation in the distribution patterns of both proteins was rapid; the percentage of T cells with aggregates increased from approximately 20%o to 50%6 5 min after the addition of PMA and rose to >75% within the first 30 min. When lymphocytes were treated with an inactive PMA epimer that does not activate PKC, no morphological alterations could be seen (data not shown). The morphological data described above indicated an association between the distribution of PKC(311 and that of spectrin and demonstrated a coincidental movement of these two molecules. Since PKC has been shown to play a role in T-cell activation, these observations suggest that spectrin may be functionally linked to activation-induced events. To investigate this hypothesis, we immobilized an artti-mouse a4 T-cell receptor (TCR)-specific monoclonal antibody (H57597) that directly activates a subset of T cells via the TCR complex. Using this method, a rapid and long-lasting increase in the percentage of T cells that express large, coincident aggregates of spectrin and PKCPH1 was again observed (Fig. 2 Bottom). The percentage of T cells with spectrin and PKC(3II aggregates rose from approximately 15% in control cells to 45% within 5 min of treatment, reaching a maximum of 55%. Thus, the translocation of PKC.8 and spectrin to a focal center within the cytosol is temporally linked to T-cell activation mediated by the antigen receptor in a large subset

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

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of lymph node T cells. It should be emphasized that there are several PKC isoenzymes known to occur in lymphocytes (13) and that their subcellular distribution may differ from that reported here for PKC,8II. The above data suggested that those tissue lymphocytes that naturally express large cytoplasmic aggregates of spectrin and PKCf3II might be cells in which activation of PKC has occurred physiologically. To test this hypothesis, the distribution of spectrin and PKCB in lymphocytes was examined after treatment with an inhibitor of PKC activity. We used the highly specific PKC inhibitor calphostin C, which interacts with the regulatory domain of the enzyme and inhibits the binding of diacylglycerol analogues to PKC (14). When freshly isolated lymphocytes were treated with calphostin C alone, a steady decline in the percentage of cells expressing aggregates was observed (to