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Lymphoma Thy-1 glycoprotein is linked to the cytoskeleton via a 4.1-like protein.

In this study we have found that the phosphoprotein doublet of 68,000 and 65,000 daltons (68/65 kD) in mouse T-lymphoma cells shares several structural and functional similarities with erythrocyte band 4.1. Our evidence for identifying the 68/65-kD doublet as a lymphoma 4.1-like protein is as follows: it displays an immunological cross-reactivity with anti-erythrocyte band 4.1 antibody; it exhibits a Svedberg unit of sedimentation coefficient of 4 S; it is phosphorylated in the presence of phorbol ester (phorbol-12-O-tetradecanoylphorbol-13-acetate) and its phosphorylation requires Ca2+; it is phosphorylated primarily at serine residues; and it can bind directly to fodrin (a spectrin-like actin- binding protein). In addition, this lymphoma 4.1-like protein can be both colocalized and coisolated with the major T-lymphocyte-specific glycoprotein, Thy-1 (gp 25). Therefore, all of these results strongly suggest that the lymphoma 4.1-like protein (68/65-kD doublet) may play a pivotal role in linking the Thy-1 (gp 25) glycoprotein to fodrin which, in turn, binds to the actin filaments that are responsible for recruiting Thy-1 antigens into cap structures.

T He Thy-1 antigen was first identified as a cell surface alloantigen (having two allotypic forms called Thy-l,1 and Thy-l,2) on thymus and brain cells from mice (55). The Thy-1 glycoprotein (25,000 D) is one of the most abundant membrane components in both thymocytes and neurons of rodents (7,55). Immunoreactive forms of Thy-1 have also been detected in dog and human brain tissue (3,27). Amino acid sequence data indicate that the Thy-1 protein shares a large amount of sequence and structural homology with a single immunoglobulin-variable domain (24).
Although the Thy-1 glycoproteins display properties of integral membrane proteins, no intramembraneous or transmembrane hydrophobic amino acid sequence has been detected (20). Recently, it has been suggested that the Thy-1 antigens are anchored in the plasma membrane via a phospholipid, possibly one with a phosphoinositol domain (44,65). However, this hypothesis has been challenged by cDNA sequence analysis which suggests that a segment of 31 amino acids at the carboxyl terminus of the Thy-1 molecules may be responsible for the integration of this protein into the plasma membrane (21,57). Until these conflicting pieces of evidence can be clarified, the question of whether the Thy-1 antigens penetrate into the plasma membrane remains unresolved.
Antibodies to Thy-1 are known to induce Thy-1 antigens to form surface receptor clusters and aggregates: the so-called patching and capping phenomenon (11,12). This antibodyinduced Thy-1 antigen redistribution appears to be closely correlated with certain physiologically relevant events. For example, proliferation of mouse T-lymphocytes is known to result from the clustering of Thy-1 antigens by anti-Thy-1 antibody (35,45,46); and the outgrowth of neuronal processes in tissue culture is facilitated by anti-Thy-1 antibody binding to their receptors (41). In addition, anti-Thy-1 patching and capping display the following characteristics: (a) the patched and capped structures are closely associated with a number of cytoskeletal proteins (e.g., actin and myosin) (13,17); (b) the capping process is sensitive to microfilament-disrupting agents (cytochalasins B and D), implying that the function of microfilaments is important for Thy-1 antigen movement (B); and (c) actin polymerization occurs concomitantly with Thy-1 cap formation (40). These results provide strong evidence for the direct involvement of the cytoskeleton in the mechanism of Thy-1 antigen movement.
Recently, immunoreactive forms of band 4.1 have been identified in brain tissue (6,33), in fibroblasts (23), and in several blood cell types such as lymphocytes, platelets, and polymorphonuclear leukocytes (62). In these nonerythroid cells, the immunoreactive 4.1 proteins appear as either a singlet or a doublet with apparent molecular masses ranging from 68,000 to 75,000 D after SDS PAGE (8). Synapsin, one of the major components of synaptic vesicles, has been shown to display a number of biochemical properties similar to those described for both erythrocyte 4.1 and brain 4.l-like protein (6). In erythrocytes, band 4.1 has been shown to enhance the interaction between spectrin and actin (22,52,68). In addition, band 4.1 protein also binds to the erythrocyte transmembrane glycoproteins, glycophorin and band 3 protein (the anion transport channel protein) (1,53). Unfortunately, the structural or functional involvement of 4. l-like proteins in specific cellular processes of certain blood cell types such as lymphocytes, platelets, and polymorphonuclear leukocytes is unclear at the present time.
In this study we have found that a 68,000/65,000-D phosphoprotein doublet (designated 68 /65-kD) in T-lymphoma cells also displays immunoreactivity with the erythrocyte membrane skeletal protein, band 4.1. The 68/65-kD doublet shares several structural and functional similarities with the erythrocyte 4.1 protein. Additionally, the lymphoma 4.l-like protein can be colocalized and co-isolated with the major T-lymphocyte-specific glycoprotein, Thy-1 by complementary techniques including double immunofluorescence staining, immunoprecipitation, and sucrose gradient centrifugation. Furthermore, our data indicates that the lymphoma 4.l-like protein (but not Thy-1) binds to fodrin which, in turn, then is able to bind to actin filaments. It is proposed that the lymphoma 4.l-like protein plays a pivotal role in linking Thy-1 to the cytoskeleton which is required for collecting Thy-1 antigens into cap structures.

Cells
The mouse T-lymphoma cell line, BW 5147, an AKR/J lymphoma line (obtained from Dr. R. Hyman, The Salk Institute, San Diego, CA) were grown in DME supplemented with 10% heat-inactivated horse serum (Gibco, Grand Island, NY) at 37°C in 5% CO2/95% air.

Immunofluorescence Microscopy
Surface Labeling. Cells were stained for surface Thy-1 (gp 25) patching and capping by first washing with RPMI 1640 medium and then treating these cells with primary antibodies (monoclonal rat antibodies) raised against Thy-1 (gifts kindly provided by Dr. fan Trowbridge, The Salk Institute) followed by the addition of a secondary antibody such as fluorescein-conjugated goat anti-rat IgG at room temperature or 37°C for 20 min.  (19), and then stained with rabbit anti-erythrocyte 4.1 protein (generously supplied by Dr. Van Bennett, The Johns Hopkins University, Baltimore, MD). These samples were then incubated with rhodamineconjugated goat antibody against rabbit IgG to visualize the distribution of protein 4.1. To detect any nonspecific antibody binding, permeabilized cells were first incubated with antisera that were preabsorbed with soluble, native erythrocyte 4.1 protein to remove anti-protein 4.1 antibodies. These samples were then incubated with rhodamine-conjugated goat anti-rabbit IgG. No label was observed in such control samples.
These fluorescein and rhodamine-labeled samples were examined under a Zeiss photomicroscope using a 63× oil-immersion lens with epiillumination and filter system of CZ 487710 and CZ 487714, respectively. Cells were photographed on Kodak plus-X film (Eastman Kodak Co., Rochester, NY).

Phorbol Ester Treatment
Phorbol ester, such as phorboM2-O-tetradecanoylphorbol-13-acetate (TPA), was purchased from Sigma Chemical Co. (St. Louis, MO). This reagent was then dissolved in DMSO (final concentration of 1%) and used at a final concentration of l0 -7 M. In control samples, cells were incubated in either DMSO plus phosphate-free DME or in phosphate-free DME alone. After incubation, aliquots were removed and cells were processed for further analysis as described below.

Plasma Membrane Isolation
Plasma membrane was isolated by the method described by Johnson and Bourne (37) with slight modifications. Radioactively labeled samples (i.e., J2SI-labeled, [3H]glucosamine-labeled, [35S]methionine-labeled, or 32po4labeled cells) were harvested by low speed centrifugation (500 g~), washed twice with Dulbecco's phosphate-buffered medium and resuspended in a solution of 20 mM Hepes, 2 mM MgCI2, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 lag/ml aprotinin, 1 pg/ml leupeptin, 1 mM 2-mercaptoethanol, pH 7.4, at 0°C. These cells were then lysed by homogenization with a Dounce homogenizer. The lysate was centrifuged at 750 g~ for 5 min and the resulting supernatant centrifuged at 43,000 gay for 20 min. The pellet was resuspended in 10% sucrose (wt/wt) in the same buffer, layered over a discontinuous gradient of 30 and 40% sucrose (wt/wt), and spun at 150,000 g~, for 90 min. The plasma membrane fraction was collected from the 30-40% interface and washed with phosphatebuffered Earle's balanced salt solution, pH 7.3. Enzyme marker assays (5' nucleotidase and/or Na÷/K+-ATPase activities) showed that these plasma membrane preparations were at least 20-fold purified and contained negligible amounts of other membranous organelle contamination (13).

NP-40 Extraction
Isolated plasma membrane was washed and resuspended in phosphatebuffered Earle's balanced salt solution containing 1 mM PMSF and 10 mM iodoacetamide. Detergent extraction was accomplished by adding NP-40 to the phosphate-buffered Earle's balanced salt solution to a final concentration of 1% (vol/vol). Protein concentration in all samples was kept at ,00.5 mg/ml. The samples were incubated at 0°C for 20 min with frequent vortexing and then centrifuged at 100,000 g,v for 45 min. After centrifugation, the supernatant (i.e., the NP-40-soluble fraction) and the pellet (i.e., NP-40-insoluble fraction) were collected for further biochemical analysis as described previously (14,16). Previous studies have shown that the NP-40-insoluble fraction contains the membrane-associated cytoskeleton (13,16).

Immunoblotting and Immunoprecipitation Procedures
Isolated erythrocyte ghost membranes and mouse lymphoma membranes were electrophoresed either on an exponential polyacrylamide gel gradient (6.0-17.0) or a 12% polyacrylamide gel that contained SDS as described below. The polypeptides were transferred to nitrocellulose sheets as described previously (16). Nitrocellulose sheets were subsequently incubated with anti-erythrocyte 4.1 antibody, followed by incubation with 125I-protein A, and analyzed by autoradiography as described below. 32p_, J251_ ' or [3H]Glucosamine-labeled detergent-extracted total mouse lymphoma plasma membranes, cytoskeletal fractions, or isolated proteins were incubated with either rabbit anti-erythrocyte 4.1 protein or anti-erythrocyte 4.1 protein-free (preabsorbed) antiserum at 4°C for 30 min. Anti-4.1 antibody was preabsorbed by an excess amount (at least 1130 ttg/ml) of the erythrocyte band 4.1. Subsequently, goat anti-rabbit IgG was added to the samples at 4°C for 30 min to precipitate the immune complex. The immunoprecipitates were obtained by centrifugation at 700 g~ for 5 min and washed three times with 0.1% NP-40 in phosphate-buffered Earle's balanced salt solution using the same centrifugation conditions. To detect Thy-1 antigens, similar immunoprecipitation procedures were carried out using NP-40-solubilized, ~2SI-labeled plasma membranes. Either monoclonal rat anti-Thy-1 or anti-Tby-l-free (preabsorbed) antibodies followed by rabbit anti-rat and goat anti-rabbit IgG was added to the samples at 4°C for 1 h to generate the immunocomplexes. The immunoprecipitates were subsequently solubilized by SDS, and analyzed by SDS PAGE and autoradiography as described below.

Immunobinding Procedures
Antigenically active molecules were detected by binding with rat anti-Tby-1 (gp 25)-conjugated Sepharose beads. Antibody was coupled to CNBractivated Sepharose 4B beads (Pharmacia Fine Chemicals, Piscataway, NJ) according to the method outlined by Cuatrecasas (26). 125I-labeled membrane proteins were incubated with these antibody-conjugated beads for 2 h. The beads were then poured into small columns and washed extensively with 0.1% NP-40. As a control for nonspecific binding, beads were prepared with preabsorbed anti-Thy-1 antiserum (anti-Thy-I antibodies were removed with excess Thy-1 antigens). Proteins bound to the anti-Thy-l-conjugated beads were detected by counting on a Beckman 5500 Gamma counter (Beckman Instruments Inc., Palo Alto, CA).

SDS PAGE and Autoradiographic Analysis
Electrophoresis was conducted using an exponential polyacrylamide gradient (6.0-17.0%) slab gel and the discontinuous buffer system described by Laemmli (39). All samples were dissolved in a buffer that contained 2% SDS, 0.1 M 2-mercaptoethanol, 0.003% bromophenol blue, 20 mM Tris-HC1, pH 6.8, and then heated to 100°C for 2 rain. Electrophoresis was conducted at a constant current of 15 mA at room temperature for 4 h. The polypeptide banding pattern was revealed by staining with Coomassie Blue. Gels containing samples labeled with ~2sI-, 3~p., [3H]glucosamine ' or [35S]methionine were processed through fluorography (10), vacuum dried, and exposed to Kodak x-ray (X-Omat XAR-5) film at -70°C.

Phosphoamino Acid Analysis
The 32p-labeled 68/65-kD protein isolated from cells that were labeled either in the presence or absence of TPA was recovered from polyacrylamide gel slices by electroelution as described by Hunkapiller et al. (36) with the exception that a Hoeffer cylindrical gel apparatus was used as the elution tank. Hydrolysis of the eluted protein was performed at ll0°C for 2 h by the method of Cooper et al. (25) and the phosphoamino acids were separated by conventional two-dimensional paper chromatography on 3-mm Whatman chromatographic paper (Whatman Inc., Clifton, N J). Separation in the first dimension was achieved at pH 3.5 for 2 h at 2,000 V, and in the second dimension at pH 1.9 for 30 min. The phosphoamino acid standards (i.e., phosphoserine, phosphothreonine, and phosphotyrosine) were mixed with 32P-labeled samples, co-analyzed and identified by ninhydrin staining. The labeled phosphoamino acids were detected by exposing to Kodak x-ray (X-Omat XAR-5) film at -70°C.

Analysis of NP-40-solubilized Plasma Membrane by Sucrose Gradient Centrifugation
Isolated t25I-, [35S]methionine-, or [3H]glucosamine-labeled plasma membrane was extracted with 1% NP-40 and then loaded onto a 36-ml linear sucrose gradient (7-48 % in phosphate-buffered Earle's balanced salt solution, pH 7.2) with a 2-ml cushion of saturated sucrose. The gradient was centrifuged at 70,000 gay for 12 h. Parallel gradients containing protein standards of tetrameric fodrin or spectrin (11 S) (32) and apoferritin (18 S) (61) were used to determine the S value of the gp 25-enriched peak. Thirtysix 1-ml fractions were collected from the bottom of each tube. The individual fractions from the gradient were analyzed by either liquid scintillation counting (for [35S]methionine and [3H]glucosamine labels) or gamma counting (for nSI-surface labeled or nSl-labeled total cellular proteins). Individual or peak fractions (depending upon the radioactive label) were precipitated with 10% TCA, and subsequently analyzed by SDS PAGE and autoradiography. The relative amounts of gp 25 (Thy-1) to other surface proteins in each gradient fraction were determined by scanning densitometry (Quick Scan R & D; Helena Laboratories, Beaumont, TX) of autoradiograms from t25I-surface labeled or [3H]glucosamine-labeled materials. To quantitate the relative molar ratio of the 68/65-kD protein and gp 25 in the 28-S complex, autoradiograms of [35S]methionine-labeled or mI-labeled material were also analyzed by scanning densitometry.

Dissociation of Lymphoma 68/65-kD Protein from 28 S (Thy-1/4.1 Complex) By Urea
Fractions containing ~25I-Thy-1/4.1 or total v-SI-labeled Thy-1/4.1 complex material (gradient fractions [16][17][18][19][20] were pooled, the sucrose was diluted by the addition of phosphate-buffered Earle's balanced salt solution, and centrifuged at 150,000 gay for 3 h. The pelleted material was then resuspended in a small volume of phosphate-buffered Earle's balanced salt solution (100-200 Ixl) containing 2 M urea. The samples were incubated at 20°C for 1 h and then centrifuged in a Beckman airfnge for 40 min at 24 psi (100,000 g~). The supernatants and pellets were then counted on a Beckman 5500 Gamma counter to determine the amount of radioactivity that was extracted into the superuataut during extraction. The extract was then run over a concanavalin A-Sepharose column to remove any Thy-I molecules that were removed along with the 4.l-like proteins during extraction. The extracted material (free of contaminating Thy-1 molecules) was then dialyzed against 20 mM KCI, 10 mM Tris-HCl, pH 8.0, containing 0.5% NP-40. This material was then loaded onto a 16-ml linear sucrose gradient (7-28% in 10 mM Tris-HC1, pH 8.0, 20 mM KCI) with a 0.5-ml cushion of 65% sucrose. The gradient was centrifuged at 70,000 g~ for 22 h. Thirty-two 0.5-ml fractions were collected from the bottom of each tube. The fractions were then counted on a Beckman 5500 Gamma counter to determine the distribution of iodinated protein and analyzed by SDS PAGE and autoradiography. The iodinated peak material from this gradient was then immunoprecipitated with rabbit anti-erythrocyte band 4.1 (as described above) to confirm the presence of the 4.l-like 68/65-kD protein as the major protein in this peak. Protein standards of 3.7-S G-actin (58), 5-S transferrin, and ll-S tetrameric fodrin or spectrin (32) were used to determine the S value of the iodinated peak.

Reassociation of Lymphoma 4.l-like Protein with gp 25 (Thy-l) Molecules
Upon removal of 2 M urea, these unlabeled or t25I-labeled lymphoma 4.l-like proteins (4-S material, 68/65-kD protein prepared according to the same procedures described above) were then incubated with t2~I-labeled and unlabeled Thy-1 molecules (prepared according to the procedures outlined by Williams et al. [69]), respectively for 2 h in 20 mM KCI, 10 mM Tris-HC1, pH 8.0, 0.5% NP-40 at approximately equal molar concentrations. The resulting complex was then analyzed by a sucrose gradient (7-48%) centrifugation as described above or passing over coneanavalin A-Sepharose columns (6.0 x 0.5 cm) equilibrated with 20 mM KC1, 10 mM Tris-HCl, pH 8.0, containing 0.5% NP-40. After binding, the columns were washed with equilibration buffer until no more radioactivity was removed. After washing, columns were elated with 1 M Q-methyl-o-mannoside and 0.5-ml fractions were collected. A parallel concanavalin A-Sepharose column was run as a control for the nonspeciflc binding and/or elution of t25Ilabeled lymphoma 4.1. Fractions from all columns were then counted and plotted vs. fraction number to establish an elution profile for each column. Peak fractions from each column were then processed through anti-Thy-1-conjugated immunobead columns (as described in the immunobeadbinding section).

Actin-binding Analysis
[35S]Methionine-labeled 28-S complex material was either preincubated in the presence or absence of 0.1 p.M of bovine brain fodrin (prepared according to Bennett et at. [9]) in phosphate-buffered Earle's balanced salt solution for 2 h and then added to 4.65 I~M G-actin under polymerizing conditions for 2 h. After 2 h, the material was centrifuged at 20000 g~ for 20 min and assayed for binding by determining the amount of [35S]methionine label that became associated with the actin pellets (54). Controls were carried out to determine the amount of the 28-S material that would pellet in the absence of fodrin and actin.

Fodrin (or Spectrin) Binding Assays
Bovine brain fodrin (or human erythrocyte spectrin) (9, 18) and BSA were each conjugated to CNBr-activated Sepharose beads (Pharmacia Fine Chemicals). t2SI-labeled lymphoma proteins (either the 28-S Thy-1/4.1 complex, 68t65-kD [4-S material], or Thy-1) were then incubated with 200 btl of either fodrin or BSA-conjugated beads overnight at 0°C in phosphatebuffered Earle's balanced salt solution. After binding, the beads were washed extensively with phosphate-buffered Earle's balanced salt solution and then the amount of binding was assayed by gamma counting.

Identification of Thy-1 Antigen and Its Associated Cytoskeletal Proteins
To analyze the major membrane components in the mouse T-lymphoma cell line (BW 5147), cells were either metabolically labeled with [3H]glucosamine or surface-labeled with ~zsI. SDS PAGE and autoradiographic analysis indicates that gp 180 (180 kD), gp 85 (85 kD), and gp 25 (25 kD) are three major glycoproteins in the plasma membrane fraction (Fig.  1 A). It is well known that the Thy-1 alloantigenic determinant is borne on a glycoprotein of •25,000 D in the mouse T-lymphoma cell line (BW 5147) (64). This glycoprotein has been isolated from cultured mouse T-lymphoma cell lines and used to prepare either polyclonal or monoclonal antisera which show a strong cross-reactivity to gp 25 (Fig. 1 B). Control experiments using anti-Thy-l-free serum reveal no detectable gp 25 (Fig. 1 C) confirming the specificity of this immunoprecipitation data.
The availability of anti-Thy-1 antiserum has allowed us to examine Thy-1 (gp 25) patching and capping cytochemically (Fig. 1 D). Treatment of the lymphoma cells with only the primary antibody (monoclonal rat anti-Thy-1 antibody) induces only 10-15 % capped ceils in the total lymphoma cells. The proportion of Thy-1 patched and capped cells is enhanced to 50-60% of the total population if the cells are treated with both primary and secondary antibodies (i.e., rat anti-Thy-1 antibody followed by goat anti-rat antibody) (Fig.  1 D) (12). The Thy-1 patch and cap structures are always accompanied by a localized concentration of intracellular actin and myosin directly under the surface patches and caps (13,17). Therefore, it has been suggested that the cytoskeleton is directly involved in Thy-1 patching and capping (13,17).

Association of CeU Surface Glycoproteins with the Detergent-insoluble Cytoskeleton
One way to gain a better understanding of the interactions occurring between Thy-1 antigens and the cytoskeleton is to isolate and analyze the membrane-cytoskeleton complexes from lymphoma cells. Treatment of isolated lymphoma plasma membrane (t25I-labeled) (Fig. 2, A and B) with the non-ionic detergent, NP-40, solubilizes ~90 % of total membrane proteins (Fig. 2, C and D) and leaves an insoluble residue that can be pelleted by centrifugation at 100,000 gay (Fig. 2, E and F). Our data show that three major surface membrane proteins, gp 180, gp 85, and gp 25 (Fig. 2 F), are preferentially associated with the insoluble fraction which is operationally defined as the cytoskeleton fraction, since it contains actin, myosin, fodrin, and ankyrin-like/72-kD molecules (Fig. 2 E). Recently, we have reported that both gp 180 and gp 85 (via an ankyrin-like molecule) can be linked to the cytoskeletal protein, fodrin (16,18). The nature of the linkage between gp 25 (Thy-1) and the cytoskeleton, however, has not been defined clearly.

Identification of the 68/65-kD Doublet as a 4.l-like Protein
Nonerythroid 4.l-like proteins (ranging in molecular mass from 68,000 to 80,000 D) have been identified in several different cell types (5,23,33,34,62). It has been demonstrated that at least one of these non-erythroid 4.l-like molecules can be phosphorylated by a cAMP-dependent kinase (6,38,50,60). Fig. 3 shows the results of our SDS PAGE and autoradiographic analysis of the phosphoprotein components found in both the total plasma membrane and cytoskeletal fractions (i.e., NP-40-insoluble material) isolated from lymphoma cells that were briefly labeled with H332po4. The 68/65-kD polypeptide doublet is detected in the total plasma membrane (Fig. 3 A) and the cytoskeletal fractions (Fig. 3 B). The membrane-cytoskeleton phosphoprotein of 130 kD has been previously identified as myo- sin light chain kinase (Fig. 3, A and B) (14). We have now identified the 68/65-kD polypeptide doublet in the membrane-cytoskeletal fraction as a phosphorylated lymphoma 4.l-like protein by a standard immunoprecipitation technique (Fig. 4 C) using an antibody shown to be specific for erythrocyte band 4.1 by immunoblotting (Fig. 4 B). The specificity of the immunoreagent was demonstrated by the fact that (a) anti-erythrocyte band 4.1 recognizes only band 4.1 (82,000/78,000 D) in human erythrocyte ghosts (Fig. 4  B); and (b) an anti-4.1-free antiserum (i.e., preabsorbed with purified erythrocyte 4.1 [ Fig. 4 A]) precipitated neither the 68/65-kD polypeptide nor any of the other phosphorylated proteins (Fig. 4 D). Therefore, we propose that the 68/65-kD protein doublet is an immunoreactive form of the erythrocyte band 4.1 found in mouse T-lymphoma cells.
In addition, we have found that the level of phosphorylation of the 4.l-like protein doublet (along with the 130,000-D and 85,000-D proteins but not the 45,000-D and 36,000-D proteins) is significantly enhanced during phorbol ester (TPA) treatment (Fig. 5, A and B). Furthermore, the results of our phosphoamino acid analysis indicate that phosphorylation of the lymphoma 4.l-like protein occurs primarily at serine residues (with very little in threonine and none in tyrosine residues) in both TPA-treated (Fig. 6, a and b) and untreated control samples (Fig. 6, c and d).  lation of the lymphoma 4.l-like protein (primarily at serine residues) (Fig. 6) in a manner similar to that described in erythrocytes (29).

Structural Linkage between the Thy-1 Antigen and the 4.l-like Protein
Double-label immunofluorescence staining shows that during Thy-1 capping (Fig. 4 a), a 4.l-like protein concentrates directly beneath the Thy-1 cap structures (Fig. 4 b). This provides qualitative evidence for the co-redistribution of these two molecules during capping. To demonstrate the possible linkages between these two molecules, we analyzed plasma membrane extracts by immunoprecipitation. The immunoprecipitation of ~25I-labeled plasma membrane with anti-4.1 antibody reveals the co-precipitation of gp 25 (Thy-1 antigen), but not gp 180 or gp 85 (Fig. 4 E). In the presence of anti-4.1-free antiserum, no surface molecules are detected (Fig. 4 F). These data strongly suggest that there is a close association between 4.l-like molecules and the Thy-1 capped structures.
We have used sucrose gradient centrifugation in order to further analyze the nature of the linkage between gp 25 (Thy-1) and the 4.l-like molecule. This approach has recently been found to be very effective in the isolation of membrane-cytoskeleton complexes from lymphoma cells (16,18).  Fig. 7). In contrast, a very large proportion of 125I-labeled gp 25 is detected in the middle of the gradient with an estimated S value of 28 (Fig. 7, curve 3). Svedberg units of sedimentation coefficients were determined relative to ll-S tetrameric spectrin or fodrin (32) and 18-S apoferritin (61) that were used as sedimentation markers. SDS PAGE and au-   [16][17][18][19][20] reveals the presence of only a 68/65-kD doublet and the gp 25 (Thy-1) antigen (Fig. 7, lane 1). Similar results were obtained after identical analysis with ~25I-labeled total cellular extracts (data not shown). Autoradiograms of either ~25I-surface labeled or [3H]glucosamine-labeled material demonstrate that gp 25 is the only surface-labeled glycoprotein present in the 28-S peak (Fig. 7, lanes 2 and 3).
The molar ratio of the 68/65-kD (4.i-like) protein and gp 25 (Thy-1) in the 28-S complex has been examined using autoradiograms of either ~2SI-labeled or [3~S]methionine-la-beled 28-S material (i.e., Fig. 7, lane 1 ). The ratio of 68/65-kD protein to gp 25 was determined by integration of the area under the respective peaks generated by scanning densitometry of the autoradiograms (Fig. 8). For our calculations, we assumed a monomeric form of each subunit of the 68/65-kD protein (68,000-65,000 D per subunit) and a monomeric form of gp 25 (25,000 D). We also assumed equivalent labeling with either 125I or [35S]methionine for both polypeptides, since identical ratios of 68/65-kD to gp 25 were obtained with both isotopes. These preliminary calculations show that the 68/65-kD protein and gp 25 (Thy-1) occur in a stoichiometric ratio of ,-o1:4. Treatment of the 28-S complex material with 2 M urea results in the disruption of the 28-S complex and the release of the 68/65-kD doublet with a Svedberg unit of sedimentation coefficient of 4 S (Fig. 9, A and B) (G-actin [3.7 S], transferrin [5.0 S], and tetrameric fodrin [11 S] were used as standard sedimentation markers [32,58]). A large majority of the 4-S material can be immunoprecipitated with antierythrocyte band 4.1 antibody (Fig. 9, C and D). Upon removal of 2 M urea, the lymphoma 4.l-like protein (4 S; 68/65-kD doublet) is capable of reassociating with Thy-I molecules as a 28-S complex (data not shown). The resulting complex is capable of binding to a concanavalin A-sepharose column and both Thy-1 and 4.l-like material coelute in the presence of ct-methyl-o-mannoside (Fig. 10 A). In addition, the Thy-1/4.1 reassociated material can be quantitatively precipitated by anti-Thy-1 antibody (Fig. 10 B), confirming that both the Thy-1 and 4.l-like polypeptides form a relatively stable complex. Therefore, we believe that the lymphoma 4.l-like protein (4S-68/65-kD molecule) and Thy-1 strongly interact with each other under both in vivo and in vitro conditions.

Interaction between the L y m p h o m a 4.l-like Protein and Fodrin (or Spectrin )
Since previous studies have suggested that the Thy-1 antigen (gp 25) becomes associated with the NP-40-insoluble cytoskeleton in mouse T-lymphoma during receptor capping (16), we have conducted F-actin-binding assays to determine if the isolated 28-S (Thy-1/4.1 complex) material has the ability to bind F-actin filaments in vitro. When 28-S complex material was incubated with actin in the absence of fodrin (or spectrin), no binding with F-actin was observed (data not shown). However, when the 28-S complex was preincubated with fodrin and then assayed for actin binding, it displayed a significant binding capability for F-actin filaments (data not shown). This set of data suggests that the Thy-1/4.1 complex can bind to actin through its interaction with the actinbinding protein, fodrin. To test this hypothesis directly, we isolated the 28-S complex, urea-extracted to dissociate the 4.l-like (4 S -6 8 / 6 5 -k D protein) doublet from the Thy-1 (gp 25) and then assayed for binding to fodrin (or spectrin)conjugated Sepharose beads (Fig. ll, A and B). BSA-conjugated beads were used as a control for the nonspecific adherence of the isolated proteins to protein-conjugated beads. Our data demonstrate that the 28-S complex material has the ability to bind directly to fodrin-(or spectrin-) conjugated beads (Fig. 11 C). The fact that fodrin binding is mediated through the lymphoma 4.l-like protein is demonstrated by the high level of binding between fodrin (or spectrin) and the isolated 68/65-kD doublet (4 S-4.1-1ike material) compared    to the insignificant amount of binding between fodrin (or spectrin) and isolated Thy-1 (Fig. 11). We believe that this fodrin-(or spectrin-) binding assay is specific, since there is only a minimal amount of nonspecific binding of isolated lymphoma proteins (i.e. 68/65 kD, 28 S, or Thy-1) to BSAconjugated Sepharose beads.

Discussion
It has been well established that actomyosin-containing components are closely associated with lymphocyte receptor patch and cap structures (11,12). Recently, analogs of the erythrocyte membrane-cytoskeleton proteins, such as spectrin, ankyrin, and protein 4.1, have also been detected in lymphocytes (12, 16,18,42,48). Most important, lymphocyte surface membranes appear to be selectively associated with the immunoreactive forms of spectrin (so-called fodrin), ankyrin, and protein 4.1, all of which co-cap with surface receptor cap structures (11,12,16,18,42,48). Recently, we have found that two lymphocyte transmembrane glycoproteins, gp 180 and gp 85, are closely associated with fodrin and ankyrin-like proteins, respectively (16,18). Specifically, we have demonstrated that gp 180 (a transmembrane glycoprotein) is preferentially associated with fodrin, in a 1:1 molar ratio (16). Another lymphoma glycoprotein, gp 85, is tightly bound to an ankyrin-like protein in a stable complex that resembles the erythrocyte band 3-ankyrin complex (18). Furthermore, this gp 85-ankyrin complex is capable of binding the actin-binding protein, fodrin (18). Therefore, the functional involvement of fodrin in linking the lymphoma gtycoprotein (gp 180 and gp 85/ankyrin-like complex) to actin filaments is strongly implicated.
As part of our continued interest in exploring the transmembrane interactions which occur between surface receptor molecules and the cytoskeleton, we have investigated whether there is a 4.l-like protein in lymphoma membrane-cytoskeleton preparations. Previously, immunoreactive forms of protein 4.1 have been detected in several different cell types including lymphocytes (62). Spiegel et al. (62) reported that in lymphocytes the immunoreactive form of protein 4.1 appears as a 68,000-D protein by SDS PAGE, im-munoblotting, and autoradiographic techniques. Our immunoprecipitation experiments using a specific erythrocyte 4.1 antibody followed by SDS PAGE and autoradiographic analysis reveals that the lymphoma 4.l-like protein consists of a doublet of 68,000 and 65,000 D. It is not surprising to find a 4.l-like protein in lymphocytes that differs in molecular mass from the erythrocyte band 4.1. Molecular mass variants have been observed in a number of different cell types of both erythroid and non-erythroid origin (5,23,33,34,62). These variations clearly do not necessarily reflect differences in 4.1 function since all of the different molecular mass forms still retain their ability to bind spectrin or fodrin (6).
The . The requirement of Ca 2÷ for phosphorylation suggests the possibility that the enzyme, protein kinase C, might be involved in this phosphorylation reaction.
Recently, Eder et al. (29) have reported that erythrocyte membrane kinases and casein kinase A phosphorylate erythrocyte band 4.1 on both serine and threonine residues. Our phosphoamino acid analysis indicates that lymphoma 4.llike protein is phosphorylated at serine residues (very little phosphorylation can be detected in threonine and none in tyrosine residues) (Fig. 6). This observation suggests that the lymphoma 4.l-like protein might share homology with erythrocyte band 4.1 serving as a substrate for protein kinase(s) (6,38,50,60). At this time, the regulatory mechanism involved in the phosphorylation of this 4.l-like protein is unknown.
Phorbol esters such as TPA are known to stimulate a number of biological activities at nanomolar concentrations (28,47). There is good evidence that phorbol esters can directly activate protein kinase C, an enzyme that normally requires both phospholipid and calcium for its activity (4,49,50,51,63). It is known that protein kinase C is activated specifically by diacylglycerol which is generated during hormonal stimulation of phosphatidylinositol turnover (63). In vitro, TPA can substitute for diacylglycerol and directly activate protein kinase C in the presence of both Ca 2÷ and phospholipid (4,49,50,63). Recently, it has been reported that the phosphorylation of band 4.1 in intact erythrocytes is stimulated by phorbol ester, suggesting the possible involvement of protein kinase C in the phosphorylation of erythrocyte band 4.1 (43). The observations that (a) phorbol ester (TPA) enhances the phosphorylation of lymphoma 4.l-like protein (Fig. 5, A and B) on serine residues (Fig. 6, a-d), and (b) phosphorylation of the lymphoma 4.l-like protein requires the presence of Ca 2÷, in vitro, strongly suggest that the lymphoma 4.llike protein is a substrate for protein kinase C.
Recent studies in erythrocytes indicate that the band 4.1, in addition to being a spectrin-binding protein, may also interact directly with the erythrocyte membrane after the removal of peripheral membrane proteins (1,53,59). At least two membrane-binding sites for 4.1 have been identified: a high affinity site on glycophorin A (1), and a low affinity site associated with band 3 (53). Interestingly, the association between glycophorin and protein 4.1 appears to be regulated by polyphosphoinositides (2). Studies done with phosphatidylserine-containing liposomes have also led to the speculation that phospholipid domains alone could serve as an alternative anchoring site for 4.1, as well as other cytoskeletal proteins (56).
The Thy-1 alloantigenic determinant is a glycoprotein of 25,000 D which, in the mouse, has been termed T-25 (designated as gp 25 in this paper). Both thymocyte and brain Thy-1 glycoproteins display hydrophobic properties which may be responsible for their binding to the plasma membrane (69). However, the initial determination of the sequence of rat brain Thy-1 glycoprotein failed to detect the presence of a hydrophobic segment (20). This has prompted speculation that Thy-1 insertion into the lipid bilayer may be through some hydrophobic components such as a phosphatidylinositol-like molecule that would link the Thy-1 glycoprotein to the membrane (44). Recently, DNA sequence analysis has revealed a 31-amino acid hydrophobic segment that may be important for the integration of the Thy-1 molecule into the plasma membrane (21,57). Like purified glycophorin (31), purified Thy-1 antigen exists as an aggregate of 10-14 Thy-1 monomers (69). This aggregated form of Thy-I (gp 25) may represent pre-existing natural clusters of receptors in the plane of the membrane.
In this study, using complementary techniques such as double-label immunofluorescence staining (Fig. 4, a and b), immunoprecipitation (Fig. 4, A-D), and sucrose gradient centrifugation (Fig. 7), we have established that a 4.l-like protein is closely associated with gp 25 (Thy-1 antigen) in the form of a 28-S complex with a stoichiometric ratio of 1:4 (Fig. 8). The fact that we are able to dissociate and then reassociate the 4.l-like protein and Thy-1 molecule, by adding and then removing 2 M urea (Figs. 9 and 10), suggests that these two proteins specifically interact with each other. Since the binding domain of Thy-1 to the 4.l-like protein has not been determined, we do not preclude the possibility that there may be other minor components (either lipid or protein molecules) responsible for linking the Thy-1 antigen to the 4.l-like protein in lymphoma membranes. Further analysis of the interactions between the lymphoma 4.l-like protein and other transmembrane glycoproteins is currently under investigation in our laboratory.
Most importantly, we have determined that the Thy-1/4.1like protein complex is capable of interacting with actin filaments via fodrin or spectrin (data not shown). This property is one of the best-known characteristics of erythrocyte protein 4.1 that is known to be a spectrin-binding protein (66)(67)(68). Further analysis using a fodrin-(or spectrin-) binding assay confirms that the lymphoma 4.l-like protein (not the Thy-1 antigen) is responsible for the binding of the complex to fodrin or spectrin (Fig. 11).
Our evidence for identifying the 68/65-kD doublet as a 4.l-like protein is summarized as follows: (a) it displays an immunological cross-reactivity with anti-erythrocyte band 4.1 antibody; (b) it exhibits a Svedberg unit of sedimentation coefficient of 4 S; (c) it can be readily phosphorylated in the presence of TPA and Ca 2÷ (but not in the absence of Ca2÷); (d) its major sites for phosphorylation are on serine residues; and (e) it can interact directly with fodrin. In conclusion, we believe our data clearly support the notion that certain linker molecules (e.g., 4.l-like and ankyrin-like proteins) as well as fodrin play a pivotal role in linking surface membrane receptors to the intracellular actin-containing microfilament network whose contraction is required for receptor capping.