Proc. Nati. Acad. Sci. USA Vol. 85, pp. 792-7%, February 1988 Cell Biology Mobility of the human T lymphocyte surface molecules CD3, CD4, and CD8: Regulation by a cAMP-dependent pathway (cAMP-dependent protein kinase inhibitor/adenosine/P site/capping) GARY M. KAMMER, CYNTHIA A. BOEHM, STEPHEN A. RUDOLPH, AND LESLIE A. SCHULTZ Departments of Medicine and Pharmacology, Case Western Reserve University School of Medicine, Cleveland, OH 44106 Communicated by Frederick C. Robbins, September 17, 1987

ABSTRACT The present study was undertaken to deter- mobility of CD3, CD4, and CD8 on normal human T lym- mine whether a cAMP pathway mediates the mobility of CD3, phocytes. CD4, and CD8 within the membrane. Crosslinking CD3, CD4, The capping rate of CD3, CD4, and CD8 is significantly and CD8 with monoclonal antibody and anti-antibody induced enhanced by pharmacologic agents that raise intracellular rapid accumulation of intracellular cAMP, occupancy of cAMP concentrations and increase occupancy of cAMP cAMP receptors, and was temporally associated with the receptor sites (9, 10). Moreover, bridging of surface mole- mobilization and directed movement of these molecules to a cules on normal and malignant murine T-cell lines also pole of the cell. This capping process could be partially increases cAMP levels (11). Taken together, these observa- inhibited in a dose-dependent manner by treatment of T cells tions suggested that mobilization of CD3, CD4, and CD8 with 2',5'-dideoxyadenosine, a ribose-modified adenosine an- molecules may activate the cAMP pathway and induce alogue that binds to the P site of the catalytic subunit of directed movement of these molecules to a pole of the cell. adenylate cyclase and reduces adenylate cyclase activity. Fur- The experiments reported herein were undertaken to deter- thermore, inhibition of cAMP-dependent endogenous phos- mine whether capping of the CD3, CD4, and CD8 glycopro- bands in tein molecules on normal human T lymphocytes is mediated phorylation of 17.5-kDa, 23/25-kDa, and 33.5-kDa by a cAMP-dependent pathway. The results demonstrate intact T cells by N-[2-(methylamino)ethyl]-5-isoquinoline- that crosslinking of these molecules induced a rapid increase sulfonamide, a cell-permeable inhibitor of cyclic nucleotide- in intracellular cAMP, occupancy of cAMP receptors, and dependent protein kinase, blocked the capping event. Data the onset of capping. 2',5'-Dideoxyadenosine (ddAdo), a support the conclusion that crosslinking of CD3, CD4, and ribose-modified adenosine analogue that binds to the P site CD8 activates a cAMP-dependent pathway that mediates the on adenylate cyclase and inhibits enzyme activation, par- mobilization and directed movement of these molecules. tially blocked the capping process. However, inhibition of cAMP-dependent protein phosphorylation is an integral step cAMP-dependent phosphorylation completely blocked the leading to the capping process. capping event. These data support the concept that the capping of CD3, CD4, and CD8 is mediated by a cAMP- The mobility of cell membrane glycoproteins is a function of dependent pathway and that protein phosphorylation is an their interaction with the cytoskeleton, the composition of integral step leading to the directed movement of these the membrane phospholipid bilayer, and regulation by par- molecules. ticular intracellular biochemical pathways (1, 2). Modifica- tion of the membrane cholesterol/phospholipid ratio, the MATERIALS AND METHODS content of unsaturated fatty acids, and changes in phospho- lipid methylation (2-4) can extensively alter the mobility of Reagents. The monoclonal antibodies OKT3, OKT4, and membrane Similarly, alterations in actin or OKT8 directed against the cell-surface molecules CD3, CD4, integral proteins. and CD8 were obtained in an azide-free, lyophilized form microtubule polymerization can also affect the mobility of (Ortho Pharmaceuticals, Raritan, NJ). Fluorescein isothio- membrane molecules (5). Thus, membrane glycoprotein cyanate (FITC)-F(ab')2 goat anti-mouse IgG (FITC-anti- mobility appears to reflect, in part, dynamic interactions antibody) and F(ab')2 goat anti-mouse IgG (anti-antibody) between integral membrane proteins, lipids, and the cyto- were obtained from Cappel Laboratories (Cochranville, PA). skeleton-the net result of which determines the membrane 8-[2-3H(N)]Azidoadenosine cyclic monophosphate ([8-3H]- fluidity (1-6). N3-cAMP) was purchased from New England Nuclear. T lymphocytes play a pivotal role in cellular and humoral Adenosine (Ado), 2-chloroadenosine (2-ClAdo), and ddAdo immune responses. The plasma membrane glycoprotein mol- were obtained from P-L Biochemicals. N-[2-(Methylamino)- ecules CD3, CD4, and CD8 are involved in numerous ethyl]-5-isoquinolinesulfonamide (H-8) was obtained from complex functions, including lymphocyte activation and Seikagaku America (St. Petersburg, FL). interactions between accessory cells or target cells and T Cell Isolation. Healthy, fasting adult males and females lymphocytes (7, 8). Movement of these and other surface who were taking neither medications nor any foods contain- molecules appears to be necessary for effective interactions ing methylxanthines for a period of 24 hr served as donors. among cells, capping (the aggregation of crosslinked recep- Peripheral blood mononuclear cells were obtained, and the tors that coalesce into a polar cap) and endocytosis, cell monocytes and B lymphocytes were depleted as previously attachment, and motility. Because the fluidity of the mem- described (10). The resultant cell populations were com- brane determines the rate at which integral molecules move posed of 98% viable T lymphocytes, as determined by within its plane (1, 5, 6), our recent studies have focused on staining with OKT3/FITC-anti-antibody and ethidium bro- the identification of a biochemical pathway that regulates the mide/acridine orange supravital dye. The enriched T-

The publication costs ofthis article were defrayed in part by page charge Abbreviations: 8-N3-cAMP, 8-azidoadenosine 3',5'-cyclic phos- payment. This article must therefore be hereby marked "advertisement" phate; H-8, N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide; in accordance with 18 U.S.C. §1734 solely to indicate this fact. FITC, fluorescein thiocyanate; slg, surface immunoglobulin. 792 Cell Biology: Kammer et al. Proc. Natl. Acad. Sci. USA 85 (1988) 793 lymphocyte subsets were prepared as described (12). After soft laser scanning densitometer, model SL-504-XL, Biomed depletion of the CD8 subset, the proportion of cells bearing Instruments, Fullerton, CA) of the autoradiograph bands. CD4 was 95.8 + 1.3%; contamination was 1.8 0.7%. Endogenous cAMP-Dependent Phosphorylation. T lympho- Similarly, depletion of the CD4 subset yielded 94.3 1.9%o cytes were washed three times in phosphate-free minimal CD8 cells; contamination was 2.0 ± 0.4% (mean ± SD, n = essential medium containing 1% bovine serum albumin 6). Viability remained >95%. (BSA). Cells were then resuspended at 60 x 106 cells per ml Time Course Studies of Capping by Fluorescence Micros- with or without incremental concentrations of H-8, in copy. The capping experiments were done as described (13). [32P]orthophosphate at 0.5 mCi/ml (carrier-free, New En- In experiments utilizing ddAdo or H-8, T lymphocytes were gland Nuclear) for 45 min at 37°C. After being washed in incubated in the appropriate concentrations of the agent for phosphate-free medium without BSA, the cells were resus- 10 min at 37°C, bound with the monoclonal antibody and pended in medium with or without 2.5 mM N6,02'-dibutyryl FITC-anti-antibody at 4°C, washed, and the capping studies cAMP and incremental concentrations of H-8. After incuba- were then done in the presence of the agent. A minimum of tion for 60 min at 37°C, phosphorylation was terminated by 200-300 cells was enumerated for each data point. addition of stop solution and boiling for 10 min. 2- Determination of cAMP by RIA. Enriched T lymphocytes Mercaptoethanol was added to a final concentration of 2% or T-cell subpopulations (1 x 106/ml) were suspended in 150 after cooling of samples to room temperature. Aliquots of mM sodium chloride/10 mM potassium chloride/2% glu- each sample containing the equivalent of 1 x 106 cells were cose/10 mM sodium phosphate/S mM Hepes (pH 7.4) subjected to electrophoresis on NaDodSO4/polyacrylamide (buffer A) at 4°C. The cells were divided into two aliquots gels (12.0%) (16). Gels were stained with Coomassie blue, and were sequentially incubated with monoclonal antibody destained, dried, and autoradiographed with Kodak X-Omat (1 ,ug/ml) and either anti-antibody (100 ,ug/ml) or buffer A at film. Incorporation of 32p was quantified by scanning laser 4°C. After transfer to a 37°C water bath, the cells were densitometry of autoradiograph bands. permitted to cap for specified intervals. The reaction was Estimation of Time Intervals to Half-Maximal Capping. To terminated by the addition of 1 ml of 1 M HCl per 100 ml of compare capping rates of CD3, CD4, and CD8, the time ethanol and freezing in dry ice/acetone. After being dried required to achieve half-maximal capping was estimated as under nitrogen, the residues were dissolved in 1 ml of 50 mM described (9). acetate buffer (pH 6.2). Cyclic AMP was determined by RIA Protein Determinations. Protein concentrations were mea- (14). sured by the Bradford method (17). Photoaffinity Labeling of cAMP Receptors. [8-3H]N3- Statistics. Statistical significance (P < 0.05) was calculated cAMP was used as a photoaffinity label to study cAMP by either the Fisher's exact test or Student's t test. binding sites of enriched T lymphocytes or T-cell subpop- ulations as described (9, 10). T lymphocytes (5 x 106) were RESULTS sequentially incubated with 625 ng of monoclonal antibody or per ml and Crosslinking CD3, CD4, CD8 Induces Turnover ofcAMP 100 ,ug of anti-antibody per ml and were then and Occupancy of cAMP Receptors. capped for varying times between 0 and 30 min at 37°C. As Crosslinking the CD3, CD4, or CD8 surface molecules with monoclonal antibody the cells were harvested, capping was stopped by immediate and anti-antibody results in the formation of randomly incubation on ice. Cells were then washed once at 4°C and dispersed microaggregates throughout the membrane sur- were lysed in medium containing 0.1% Triton-X. The lysates face within 2 min (13). The crosslinking of these molecules were treated with 10 of 1 ,uM /,l [8-3H]N3-cAMP for 45 min and formation of microaggregates induced a rise in intracel- at 4°C. Lysates were irradiated for 10 min with UV light at lular cAMP and occupancy of cAMP receptors (Table 1 and 257 nm, and the contents of each well were then added to 50 Fig. 1). The peak cAMP concentrations were reached within ,ul of stop solution [9% NaDodSO4/15% (vol/vol) glycerol/- 2 min, were between 1.9- and 2.2-fold over baseline, and 30 mM Tris-HCl (pH 7.8)/0.05% bromophenol blue], and the then declined over the ensuing 30 min (Table 1). The mixture was boiled for 5 min. 2-Mercaptoethanol was added accumulation of intracellular cAMP resulted in progressive to a final concentration of 1% after cooling samples to room occupancy of cAMP receptors, as shown by the 65-98% temperature. Aliquots (70 ,u) of each sample were subjected inhibition of [8-3H]N3-cAMP photoaffinity labeling (Fig. 1). to 10.5% NaDodSO4/gel electrophoresis (15). The gels were Some variation in the kinetics of cAMP receptor occupancy stained with Coomassie blue, destained, dried, soaked in may be seen when cells from the same donor are studied on EN3HANCE (New England Nuclear), and autoradiographed different occasions (Fig. 1, CD3 capped, a vs. b). The small with Kodak X-Omat film. 3H incorporation into protein variations seen in the kinetics of cAMP receptor occupancy bands was assessed by scanning laser densitometry (Zeineh are not reflected in any apparent alterations in the capping Table 1. Crosslinking CD3, CD4, and CD8 molecules induces turnover of cAMP Time, cAMP, fmol per 106 T lymphocytes min OKT3 OKT3/a-Ab OKT4 OKT4/a-Ab OKT8 OKT8/a-Ab 0 142 18 143 ± 20 143 ± 17 143 ± 17 142 ± 20 143 ± 16 2 156 23 328 ± 18 160 ± 17 298 ± 21 170 ± 19 274 ± 24 5 139 19 307 ± 25 128 ± 11 220 ± 17 131 ± 16 227 ± 17 10 120 17 170 ± 16 124 ± 16 190 ± 24 129 ± 16 198 ± 21 20 128 24 169 ± 20 130 ± 18 163 ± 21 131 ± 17 187 ± 15 30 119 18 157 ± 17 136 ± 20 163 ± 21 136 ± 15 160 ± 15 T lymphocytes were enriched for cells bearing CD3, CD4, or CD8. Cells were suspended in buffer A at 40C and bound with the appropriate monoclonal antibody (1 gg/ml). After dividing cells into 2 aliquots each, the first aliquot was held at 40C in buffer A for 30 min, whereas each second aliquot was incubated with 100 A&g of anti-antibody (a-Ab) per ml at 40C for 30 min. After being washed, all cells were resuspended in buffer A at 370C for intervals of 0-30 min. Reactions were terminated by washing away extruded cAMP at 40C, addition of 1 ml of 1 M HCI per 100 ml of ethanol, and freezing in dry ice. Cyclic AMP was determined by RIA. 794 Cell Biology: Kammer et aL Proc. Nadl. Acad. Sci. USA 85 (1988)

CELL PREPARATION TIME (min) 1 2 3 4 5 6 7 8 9 10 0 2 5 10 15 20 30

CD3/mAb #f _ m -_ :.

CD/mb/m.:ba .1p CD3/mAb/a-mAb ,:.: ..- .4 lo-510- t 410 310-510-410 310 510 410-3 CD4/mAb _ -mm i_- 4m_ m.W_ Medium Ado 2-CIAdo ddAdo CD4/mAb/a-mAb am [Ado/analogue], M

CD8/mAb dt 4& M&,, FIG. 2. Effect of increasing concentrations of Ado, 2-ClAdo, and ddAdo on cAMP binding to protein kinase. Enriched CD3 cells CD8/mAb/a-mAb _ were divided into two groups; one remained untreated at 40C, whereas the second was bound with OKT3 and anti-antibody. Each FIG. 1. Capping of T-cell surface molecules CD3, CD4, and group was washed, and the group treated with OKT3 and anti- CD8 by crosslinking with monoclonal antibody (mAb) and anti- antibody was further divided into three aliquots. Each aliquot was antibody (a-mAb) induces a time-dependent inhibition of [8-3H]N3- then resuspended in various concentrations of Ado, 2-ClAdo, or cAMP photoaffinity labeling of cAMP receptors. mAb treatment ddAdo (10-' M-10-3 M) and capped with OKT3/anti-antibody for was with specific OKT mAb. The intracellular cAMP receptors are 2 min at 370C. The untreated group was also incubated for 2 min at 3r7C. Simultaneous studies with the regulatory subunits of type I and type II protein kinase A that the monoclonal antibodies OKT4 have Mr of 48,000 and 52,000, respectively. This autoradiograph is and OKT8 using purified subpopulations yielded similar results. representative of ten independent experiments. a and b, Receptor This autoradiograph is representative of four independent experi- occupancy of cells fron' the same donor studied on different ments. occasions. mediates the directed movement of CD3, CD4, and CD8 event; after their formation, microaggregates are quickly molecules. The only known mechanism in eukaryotic cells mobilized to a pole ofthe cell where they form tight caps and by which cAMP conveys its physiologic signal is via binding subsequently undergo endocytosis (13). By contrast, the to and activation of protein kinase A (20). In the T-cell interaction ofT cells with monoclonal antibody alone did not system, treatment of intact cells with either forskolin or induce the formation of microaggregates, the generation of N6,02-dibutyryl cAMP induces phosphorylation of 17.5- cAMP, or cAMP receptor occupancy (Table 1 and Fig. 1). kDa, 23/25-kDa, and 33.5-kDa bands (Fig. 4) (21). Phospho- Partial Inhibition of Capping by ddAdo. Human T lympho- rylation can be blocked in a dose-dependent manner by cytes lack an inhibitory A1 surface adenosine receptor (44). treatment of intact cells with the cell-permeable cyclic However, adenosine agonists that bind to the P site, believed nucleotide-dependent protein kinase inhibitor, H-8 (Fig. 4). to be located on the catalytic subunit of adenylate cyclase H-8 is an isoquinolinesulfonamide derivative that interferes (18, 19), inhibit adenylate cyclase activation and reduce with the function of cyclic nucleotide-dependent protein cAMP formation. To determine whether agonists that bind kinase by competing for the ATP binding site ofthe catalytic to the P site can alter capping, T lymphocytes were incu- subunit but does not alter the binding of cAMP to the bated with the P site a regulatory subunits of protein kinase A (22). To determine agonist, ddAdo. ddAdo is hydropho- whether directed surface mobility of CD3, CD4, and CD8 bic, ribose-modified adenosine analogue that rapidly crosses the cell membrane and binds to the P site. T lymphocytes requires cAMP-dependent phosphorylation, cells were capped in the presence of incremental of were divided into four aliquots; the first was held in culture concentrations H-8 similar to that used to block phosphorylation. This medium at 4°C. The remainder were incubated with varying produced a dose-dependent inhibition ofcapping of each ofthe surface concentrations of Ado, 2-ClAdo, or ddAdo for 5 min at 37°C and then bound with OKT3/anti-antibody at 4°C; capping 100 was subsequently done in the presence of the agents for 2 CONTROL min at 37°C. In the presence ofAdo or 2-ClAdo, capped cells showed a 3-fold rise ± 18 106 approximately (142 fmol per 80 cells to 436 ± 51 fmol per 106 cells) in intracellular cAMP 0 over baseline, resulting in 90-100% inhibition of [8-3H]N3- cAMP photoaffinity labeling of cAMP receptors (Fig. 2, IOOpM ddAdo -60 T lanes 2 to 7). In contrast, T cells capped in the presence of ddAdo showed a maximum 1.3-fold (142 ± 18 fmol per 106 40 jf _. lImM ddAdo cells to 184 ± 20 fmol per 106 cells) increase in cAMP at 10-3 M ddAdo; the reduced cAMP accumulation was re- a- 40 flected in a dose-dependent increase in photoaffinity labeling to 57% of the medium control (Fig. 2, lanes 1 vs. 10). z Moreover, treatment of T cells with ddAdo also resulted in a uJ 20 dose-dependent inhibition of capping; at 1 mM ddAdo, only 0~ half of the cells were able to cap (Fig. 3). This inhibition of capping was significant when compared to untreated cells (% 25 10 15 20 25 30 capped cells at 30 min, 95 ± 2 in medium vs. 53 ± 3 in 1 mM ddAdo, P < 0.0002) and is reflected in a markedly prolonged TIME (mins) time to half-maximal capping = 4 1 (tQQ2)(medium, ti/2 min; FIG. 3. Inhibition of T3 (CD3) capping by ddAdo. Enriched T mM ti,2 = 11 were seen ddAdo, min). Similar findings when lymphocytes were incubated for 5 min at 37rC in culture medium or numbers of cells CD4 and CD8 were tested equal bearing with i0- M or i0-O M ddAdo, bound with OKT3/FITC-anti- (data not shown). antibody at 4°C, washed, and capped with or without the appropriate Inhibition of Capping by H-8. The partial inhibition of concentrations of ddAdo at 3TC for intervals to 30 min. Each data capping by ddAdo suggests that a cAMP-dependent pathway point represents the mean ± SEM of four independent experiments. Cell Biology: Kammer et al. Proc. Natl. Acad. Sci. USA 85 (1988) 795

CULTURE in other species as well. Crosslinking the Thy-1 molecule on MEDIUM [H-81 (M) murine T-lymphoma cells induced accumulation of intracel- IN A' lular cAMP and capping of Thy-1 (11, 23). Furthermore, treatment of these cells with an exogenous cAMP derivative o \0 \0 e~- 4 -. \ increased the proportion of capped cells, a finding identical to that observed in human T cells (9). In contrast, the role of - 97.4 - 66 the cAMP pathway in the mobilization of surface immuno- - 44 globulin (sIg) on B lymphocytes remains controversial. B --33.5 lymphocytes treated with cAMP derivatives failed to dem- -31 onstrate any alteration in the capping rate of sIg (24). Yet, later analyses of sIg cap formation identified an accumula- - 23/25 tion of cAMP beneath the capped sIg, suggesting a role for 4' .. .l#' -17.520. the cyclic nucleotide (25, 26). Nonetheless, there is sufficient -'4.3 evidence to propose that a cAMP pathway may mediate the 60 mobility of specific T-cell surface molecules of different TIME (min) species. The capping event can be partially or completely inhibited FIG. 4. Inhibition of cAMP-dependent endogenous phosphoryl- by pharmacologic agents that interfere with steps along the ation of intact T lymphocytes by H-8. This autoradiograph is cAMP pathway. ddAdo, a hydrophobic, ribose-modified representative of two independent experiments. adenosine analogue that rapidly crosses the cell membrane molecules (Fig. A-C). These observations and binds to the P site on the catalytic subunit of adenylate 5 suggest that the cyclase (18, 19, 27), partially inhibits adenylate cyclase capping process is mediated by a cAMP-dependent pathway activation by a noncompetitive mechanism (27-30). In the and that phosphorylation is an integral biochemical event. T-cell system, ddAdo in concentrations comparable to that used in other systems (100 ,uM-1 mM) induced complete DISCUSSION inhibition of capping in about half of each T-cell subset and a significant slowing of the capping rate in the remainder of Crosslinking the human T-cell surface molecules CD3, CD4, the cells. Parenthetically, because adenosine may also bind or CD8 induced rapid accumulation of intracellular cAMP, to the P site with low affinity (18, 31, 32), this mechanism occupancy of cAMP receptors, and the directed movement could account for its observed inhibitory effect in higher of these molecules to a pole of the cell. The levels of concentrations upon capping (9). Although the precise mech- intracellular cAMP and occupancy of cAMP receptors in- anism by which P-site agonists inhibit adenylate cyclase duced by bridging each of the molecules were quantitatively activation remains uncertain, it has been proposed that similar. However, binding of divalent monoclonal antibody partial inhibition of enzyme activation may be explained by alone to surface molecules failed to generate cAMP turnover the existence of more than a single adenylate cyclase, only or occupancy of cAMP receptor sites (Fig. 1). This inability one of which may be inhibited by ddAdo (18, 19, 31). Other of the divalent antibody alone to activate a cAMP-dependent as-yet-unidentified mechanisms may also be operative. Be- pathway may account for the failure of the antibodies to cause adenylate cyclase activation can alter the capping rate induce capping (13). Thus, crosslinking these molecules (10), diminished enzyme activity probably accounts for both appears to be the initial event required to set the capping the observed inhibition of capping and the reduced capping mechanism in motion. rate. The directed movement of certain T-cell surface mole- Inhibition of cAMP-dependent protein kinase by H-8 also cules appears to be mediated by a cAMP-dependent pathway blocked initiation of the capping event in both T-cell subsets. Because H-8 interferes with the function of protein kinase A by competing for the ATP-binding site of the catalytic 100- A. CD3 subunit (22), cAMP-dependent phosphorylation of intracel- 80 - lular substrates cannot occur in the absence of a functional 60 - catalytic subunit (Fig. 4). Our findings suggest, therefore, that the mobility of CD3, CD4, and CD8 requires effective 40 - phosphorylation of substrate(s). Although the identity of the 20 - molecules phosphorylated after crosslinking of surface mol- z CL ecules remains unknown, possibly certain cytoskeletal ele- a. ments are phosphorylated. cL.0~ 100 B. CD4 Protein kinase A is complexed 0 with several cytoskeletal proteins, including actin, myosin, 80 and microtubule-associated proteins (23, 33-35). Upon z acti- 0 60 - vation of the enzyme by cAMP, the catalytic subunit selec- C13 tively phosphorylates these cytoskeletal proteins and alters 40 - their z FIG. 5. Percent inhibition of functions. cAMP-dependent phosphorylation of a mi- 20 - capping by H-8. Enriched CD3, crotubule-associated protein, MAP-2, disassembles microtu- I-z w CD4, and CD8 lymphocytes were bules and reduces the interactions of microtubules with actin 100 C. CD8 ft incubated with or without incremen- filaments (36, 37). The simultaneous cAMP-dependent w phos- a. tal concentrations of H-8 for 10 min phorylation of discrete cytoskeletal elements may initiate the 80 x/ at 37°(, bound with monoclonal an- contractile process required for mobility of surface mole- tibody and at 6c FITC-anti-antibody cules. That actin, myosin, tubulin, vimentin, and a-actinin 40C, and capped for 30 min at 37°C accumulate beneath the caps formed by CD3, 4C with or without the appropriate con- CD4, and CD8 centrations of H-8. A minimum of (G.M.K., unpublished data) implicates these cytoskeletal 2c 200-300 cells was enumerated for proteins in the directed movement of surface molecules. In each data point. Each point repre- addition to the role of cAMP-dependent phosphorylation in c0 8 7 6 5 4 sents the mean + SEM of four in- capping, the Ca2+ /calmodulin-dependent protein kinases -Log [H-8] (M) dependent experiments. also appear to have an integral role in capping. Ca2+/ 796 Cell Biology: Kammer et al. Proc. Natl. Acad Sci. USA 85 (1988) calmodulin-dependent kinases phosphorylate myosin light 10. Kammer, G. M., Boehm, C. A. & Rudolph, S. A. (1986) Cell chains (23) and microtubule-associated protein 2 (38). The Immunol. 101, 251-258. beneath the capped Thy-1, 11. Butman, B. T., Jacobsen, T., Cabatu, 0. G. & Bourguignon, gathering of myosin light chains L. Y. W. (1981) Cell Immunol. 61, 397-403. T200, and gp69/71 surface molecules of murine T-lymphoma 12. Goldschmidt, L. P., Kresina, T. F. & Kammer, G. M. (1986) cells also associates myosin with the capping process (23). Arthritis Rheum. 29, 646-654. Taken together, the data provide evidence that phosphoryl- 13. Kammer, G. M., Smith, J. A. & Mitchell, R. (1983) J. Immu- atiop is an integral mechanism by which cAMP mediates the nol. 130, 38-4. directed movement of CD3, CD4, and CD8. 14. Steiner, A. L., Kipnis, D. M., Utiger, R. & Parker, C. W. depends (1969) Proc. Natl. Acad. Sci. USA 64, 367-373. The physiologic response of the T lymphocyte 15. Pomerantz, A. H., Rudolph, S. A., Haley, B. E. & Green- upon the signals it receives at surface receptors. Based upon gard, P. (1975) Biochemistry 14, 3858-3862. this data, crosslinking surface molecules by multivalent 16. Laemmli, U. K. (1970) Nature (London) 227, 680-685. antigens may trigger rapid activation of protein kinase A, 17. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. phosphorylation of certain cytoskeletal elements, and di- 18. Londos, C. & Wolff, J. (1977) Proc. Natl. Acad. Sci. USA 74, rected movement of surface molecules to a pole of the cell. 5482-5486. to the T-cell 19. Londos, C., Wolff, J. & Cooper, D. (1983) in Regulatory By contrast, binding of a divalent antibody Function ofAdenosine, eds. Berne, R., Rall, T. & Rubio, R. antigen receptor/CD3 complex, in the presence of interleu- (Nijhoff, Boston), pp. 17-32. kin 1, induces phosphoinositide turnover, protein kinase C 20. Krebs, E. G. & Beavo, J. A. (1979) Annu. Rev. Biochem. 48, activation, interleukin 2 production and interleukin 2- 923-959. receptor expression, and mitogenesis (39). Thus, the extent 21. Schultz, L. A., Rudolph, S. A. & Kammer, G. M. (1987) of crosslinking may be one mechanism that regulates activa- FASEB J. 46, 458. 22. Hidaka, H., Inagaki, M., Kawamoto, S. & Sasaki, Y. (1984) tion of either the cAMP-protein kinase A or phosphoinosi- Biochemistry 23, 5036-5041. tide-protein kinase C pathway and determines the subse- 23. Bourguignon, L. Y. W. & Bourguignon, G. J. (1984) Int. Rev. quent T-cell function. Cytol. 87, 195-224. Over the past several years, both experimental and clinical 24. Schreiner, G. F. & Unanue, E. R. (1975) J. Immunol. 114, protocols have been evaluating the use of monoclonal anti- 802-808. bodies directed against cell-surface antigens as a mode of 25. Earp, H. S., Utsinger, P. D., Young, W. J., Logue, M. & therapy for autoimmune disorders (40), renal allograft rejec- Steiner, A. L. (1977) J. Exp. Med. 145, 1087-1092. Because the efficacy of 26. Curtain, C. C. (1979) Immunology 36, 805-810. tion (41), and T-cell neoplasms (42). 27. Bruns, R. F. (1980) Can. J. Physiol. Pharmacol. 58, 673-691. this therapy depends, in part, upon the binding and contin- 28. Holgate, S., Lewis, R. & Austen, K. F. (1980) Proc. Nat!. ued presence of the antibody on the cell surface (43), loss of Acad. Sci. USA 77, 6800-6804. antibody from the surface by capping and endocytosis or 29. Stiles, G. L. & Lefkowitz, R. J. (1982) J. Biol. 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