The CD4/CD8:p56lck complex in T lymphocytes: a potential mechanism to regulate T-cell growth

The CD4 and CD8 antigens on the surface of T cells appear to bind to major histocompatibility complex (MHC) class II and I antigens, respectively. These receptors have also been found to regulate T cell growth in a manner independent of MHC recognition. In this report, we describe recent work showing that the CD4 and CD8 receptors are coupled to a protein-tyrosine kinase, p56lck, from T lymphocytes. The p56lck protein is a member of the src family, which plays a crucial role in the activation and transformation of various mammalian cells. The CD4/CD8:p56lck complex is catalytically active as shown by its ability to phosphorylate at 55-60 kDa. Two-dimensional, nonequilibrium gel electrophoresis demonstrated the similarity of p56lck associated with the CD4 and CD8 antigens. Detergents were found to vary in their ability to solubilize the CD4:p56lck complex in a catalytically active form. We further demonstrated by in vitro phosphorylation that members of the CD3 complex including the gamma, delta, and epsilon chains, as well as a putative zeta subunit can be phosphorylated at tyrosyl residues by the CD4/CD8:p56lck complex. Thus, this interaction may play an important role in the activation of T cells, and may mediate the cooperative interaction between the CD4/CD8 antigens and the Ti(TcR)/CD3 complex. This interaction also represents a possible precedent by which other members of the src family (c-src, c-yes, c-fgr, etc.) may be found to interact with mammalian growth receptors.     

Cluster of differentiation antigen 4 (CD4) endocytosis and adaptor complex binding require activation of the CD4 endocytosis signal by serine phosphorylation

Cluster of differentiation antigen 4 (CD4), the T lymphocyte antigen receptor component and human immunodeficiency virus coreceptor, is down-modulated when cells are activated by antigen or phorbol esters. During down-modulation CD4 dissociates from p56(lck), undergoes endocytosis through clathrin-coated pits, and is then sorted in early endosomes to late endocytic organelles where it is degraded. Previous studies have suggested that phosphorylation and a dileucine sequence are required for down-modulation. Using transfected HeLa cells, in which CD4 endocytosis can be studied in the absence of p56(lck), we show that the dileucine sequence in the cytoplasmic domain is essential for clathrin-mediated CD4 endocytosis. However, this sequence is only functional as an endocytosis signal when neighboring serine residues are phosphorylated. Phosphoserine is required for rapid endocytosis because CD4 molecules in which the cytoplasmic domain serine residues are substituted with glutamic acid residues are not internalized efficiently. Using surface plasmon resonance, we show that CD4 peptides containing the dileucine sequence bind weakly to clathrin adaptor protein complexes 2 and 1. The affinity of this interaction is increased 350- to 700-fold when the peptides also contain phosphoserine residues.     

Activated lck tyrosine protein kinase stimulates antigen-independent interleukin-2 production in T cells.

p56lck, a member of the src family of cytoplasmic tyrosine kinases, is expressed predominantly in T cells where it associates with the T-cell surface molecules CD4 and CD8. Mutants of CD4 and CD8 that have lost the ability to associate with p56lck no longer enhance antigen-induced T-cell activation. This suggests that p56lck plays an important role during T-cell activation. In an effort to understand the function of p56lck in T cells, a constitutively activated lck gene (F505lck) was introduced into T-helper hybridoma cell lines by retroviral infection. In four T-cell lines we examined, the activated lck protein stimulated interleukin-2 (IL-2) production, a hallmark of T-cell activation, in the absence of antigenic stimulation. In addition, a marked increase in antigen-independent IL-2 production was apparent when T cells infected with a temperature-sensitive F505lck were shifted to the permissive temperature. Only one cell line expressing F505lck exhibited increased sensitivity to antigenic stimulation. The SH3 domain of p56lck was dispensable for the induction of antigen-independent IL-2 production. In contrast, deletion of the majority of the SH2 domain of p56F505lck reduced its ability to induce spontaneous IL-2 production markedly. Activated p60c-src also induced antigen-independent IL-2 production, whereas two other tyrosine kinases, v-abl and the platelet-derived growth factor receptor, did not. Tyrosine phosphorylation of a 70-kDa cellular protein was observed after cross-linking of CD4 in T cells expressing F505lck but not in cells expressing F527src.     

Tyrosine phosphorylation of a c-Src-like protein is increased in membranes of CD4- CD8- T lymphocytes from lpr/lpr mice.

Mice homozygous for the autosomal recessive lpr gene have a disorder that results in autoimmunity and massive accumulation of T lymphocytes lacking CD4 and CD8 surface markers. These abnormal T cells exhibit constitutive tyrosine phosphorylation of a component of the CD3-T-cell receptor complex. We compared membrane tyrosine phosphorylation in lpr/lpr CD4- CD8- T cells and control T cells, lpr membranes exhibited a 7.3-fold increase (n = 16) in tyrosine phosphorylation of a 60-kilodalton protein. The increase was correlated with the Lpr but not the CD4- CD8- phenotype in that p60 phosphorylation was not increased in membranes from normal CD4- CD8- thymocytes. To identify the p60 in lpr cells, we examined the activity of several T-cell tyrosine-specific protein kinases. p56lck phosphorylation was only slightly increased in lpr membranes (2.2-fold; n = 16). Phorbol ester treatment of intact T cells before membrane isolation caused p56lck to migrate as pp60lck; however, pp60lck could be clearly distinguished from the pp60 in lpr cells by two-dimensional gel electrophoresis. The pp60 from lpr cells exhibited several isoforms at pH approximately 6.3 to 6.5. Although on two-dimensional gels pp60c-src had a pI (6.4 to 6.8) within a similar region, p60c-src mRNA, protein, and kinase activities were not increased in lpr cells. In addition, staphylococcal V8 proteolytic cleavage of the lpr pp60 isolated on two-dimensional gels yielded two major fragments, a pattern distinct from that of pp60c-src. However, by using an antiserum against the C-terminal sequence of c-Src and other related kinases, including p59fyn, the pp60 could be immunoprecipitated in greater amounts from lpr than from control T cells. When pp59(fyn) was selectively immunoprecipitated from T-cell membranes with specific antisera, its molecular weight, proteolytic cleavage pattern, and behavior on two-dimensional gels were identical to those of the pp60 from lpr cells. We conclude that p59(fyn) phosphorylation is increased in membranes from lpr/lpr CD4(-) CD8(-) T cells and that the increase is correlated with constitutive tyrosine phosphorylation and perhaps with the expansion of this unusual T-cell population.     

p56lck protein-tyrosine kinase is cytoskeletal and does not bind to polyomavirus middle T antigen.

p56lck and p60c-src are closely related protein-tyrosine kinases that are activated by similar oncogenic mutations. We have used fibroblast cell lines that express p56lck from introduced DNA molecules to compare the subcellular localizations of p60c-src and p56lck and their abilities to bind polyomavirus middle T antigen (mT). p56lck is associated with the detergent-insoluble matrix, as defined by extraction with solutions containing nonionic detergents, whereas p60c-src is soluble under these conditions. p56lck is also associated with detergent-insoluble structures in a lymphoid cell line, LSTRA. p60c-src binds to mT, but p56lck does not bind detectably. In terms of both solubility and mT interactions, the nononcogenic p56lck more closely resembles oncogenically activated p60c-src mutants than it resembles p60c-src. Because published results show that an intact carboxy terminus is required for p60c-src to bind mT and be soluble, we tested whether the different localization and mT binding properties of p56lck and p60c-src were dictated by their different carboxy termini. A protein consisting largely of p60c-src sequences but carrying a p56lck carboxy terminus was soluble and bound to mT. We suggest that both the solubility and mT-binding properties of p60c-src not only require sequences common to the carboxy termini of p60c-src and p56lck, but also require sequences unique to the body of p60c-src.     

The carboxy-terminal sequence of p56lck can regulate p60c-src.

A chimera containing the coding region for residues 1 to 516 of p60c-src and residues 495 to 509 (the carboxy terminus) of p56lck was constructed and expressed in mouse fibroblasts. The chimeric protein appeared to be phosphorylated and regulated in the same fashion as p60c-src.     

Ganglioside-induced CD4 endocytosis occurs independent of serine phosphorylation and is accompanied by dissociation of P56lck.

Gangliosides induce a selective and complete modulation of CD4 from the surface of T cells. CD4 down-modulation occurs by CD4 endocytosis. This process is independent of serine phosphorylation of the cytoplasmic tail of CD4 and does not require the association between the tyrosine protein kinase p56lck and the cytoplasmic tail of CD4. Ganglioside-induced CD4 endocytosis is accompanied by the loss of p56lck activity associated with CD4. Sequential immunoprecipitation analysis using an anti-CD4 antibody and an anti-p56lck antiserum showed that this is caused by the dissociation of the enzyme from the cytoplasmic tail of CD4. The kinetics of p56lck dissociation after ganglioside treatment is identical to that of CD4 endocytosis, suggesting that p56lck is displaced in the process of endosome formation. The results indicate that CD4 endocytosis alone can cause the dissociation of the p56lck complex without the requirement for CD4 phosphorylation.     

p56lck association with CD4 is required for the interaction between CD4 and the TCR/CD3 complex and for optimal antigen stimulation.

By fluorescence resonance energy transfer, we have previously demonstrated that upon anti-CD3 mAb-mediated activation of a murine T cell hybridoma expressing human CD4, CD4 moves into close association with the TCR/CD3 complex. It was shown that this association between CD4 and the TCR/CD3 complex was dependent upon the presence of an intact CD4 cytoplasmic domain. We have now expressed, in a murine T cell hybridoma, mutated forms of CD4 containing cysteine to serine point mutations at positions 420, 422, or 430. The mutations at positions 420 and 422, but not 430, abolish association with p56lck. By using fluorescence resonance energy transfer, we demonstrate that mutations of CD4 which fail to interact with p56lck are unable to associate with the TCR/CD3 complex under conditions in which wild-type CD4 and the 430 mutant CD4 do associate with the TCR/CD3 complex. In addition, these mutants have a diminished response to CD4-dependent stimuli. We conclude that the association between CD4 and the TCR/CD3 complex during T cell activation plays an important role in CD4-dependent responsiveness and this association requires the interaction of CD4 with p56lck. These results also suggest that a substrate for p56lck may be expressed in the TCR/CD3 complex.     

Phosphatidylinositol (PI) 3-kinase and PI 4-kinase binding to the CD4-p56lck complex: the p56lck SH3 domain binds to PI 3-kinase but not PI 4-kinase.

CD4 serves as a receptor for major histocompatibility complex class II antigens and as a receptor for the human immunodeficiency virus type 1 (HIV-1) viral coat protein gp120. It is coupled to the protein-tyrosine kinase p56lck, an interaction necessary for an optimal response of certain T cells to antigen. In addition to the protein-tyrosine kinase domain, p56lck possesses Src homology 2 and 3 (SH2 and SH3) domains as well as a unique N-terminal region. The mechanism by which p56lck generates intracellular signals is unclear, although it has the potential to interact with various downstream molecules. One such downstream target is the lipid kinase phosphatidylinositol 3-kinase (PI 3-kinase), which has been found to bind to activated pp60src and receptor-tyrosine kinases. In this study, we verified that PI 3-kinase associates with the CD4:p56lck complex as judged by the presence of PI 3-phosphate generated from anti-CD4 immunoprecipitates and detected by high-pressure liquid chromatographic analysis. However, surprisingly, CD4-p56lck was also found to associate with another lipid kinase, phosphatidylinositol 4-kinase (PI 4-kinase). The level of associated PI 4-kinase was generally higher than PI 3-kinase activity. HIV-1 gp120 and antibody-mediated cross-linking induced a 5- to 10-fold increase in the level of CD4-associated PI 4- and PI 3-kinases. The use of glutathione S-transferase fusion proteins carrying Lck-SH2, Lck-SH3, and Lck-SH2/SH3 domains showed PI 3-kinase binding to the SH3 domain of p56lck, an interaction facilitated by the presence of an adjacent SH2 domain. PI 4-kinase bound to neither the SH2 nor the SH3 domain of p56lck. CD4-p56lck contributes PI 3- and PI 4-kinase to the activation process of T cells and may play a role in HIV-1-induced immune defects.     

Human immunodeficiency virus type 1 Nef induces accumulation of CD4 in early endosomes.

We have studied the fate of CD4 in CEM T cells expressing a human immunodeficiency virus type 1 HIV-1 Nef protein. Nef triggered a rapid endocytosis and a degradation of CD4, while most of the p56lck was upheld at the cell membrane. In the presence of Nef, CD4 accumulated in acidic intracellular vesicles that were not stained by antibodies against rab6, a marker of the Golgi apparatus complex. Detection of transferrin in CD4-containing vesicles showed that CD4 was trapped in early endosomes, without significant accumulation of CD4 in late endocytic compartments. Internalization pathways taken by CD4 in Nef+ cells may therefore be different from those observed after treatment with phorbol esters.     

Myxoma virus induces extensive CD4 downregulation and dissociation of p56lck in infected rabbit CD4+ T lymphocytes.

Myxoma virus is a pathogenic poxvirus that induces extensive dysregulation of cellular immunity in infected European rabbits. Infection of a rabbit CD4+ T-cell line (RL-5) with myxoma virus results in dramatic reductions of cell surface levels of CD4 as monitored by flow cytometry. The virus-induced downregulation of CD4 requires early but not late viral gene expression and could not be inhibited by staurosporine, an inhibitor of protein kinase C, which effectively blocks phorbol 12-myristate-13-acetate-induced downregulation of CD4. The decrease in total cellular levels of CD4 during myxoma virus infection could be inhibited by the lysosomotrophic agent NH4Cl, suggesting a lysosomal fate for CD4 during myxoma virus infection. Steady-state levels of the CD4-associated protein tyrosine kinase p56lck remained unchanged during myxoma virus infection, suggesting that p56lck dissociates from CD4 prior to CD4 degradation in virus infected cells. Total p56lck kinase activity was unaffected during myxoma virus infection, although the amount of p56lck physically associated with CD4 declined in parallel with the loss of CD4. Thus, myxoma virus infection of CD4+ T lymphocytes triggers CD4 downregulation via a protein kinase C-independent pathway, causing the dissociation of p56lck and the degradation of CD4 in lysosomal vesicles.     

Second-messenger regulation of receptor association with clathrin-coated pits: a novel and selective mechanism in the control of CD4 endocytosis.

CD4, a member of the immunoglobulin superfamily, is not only expressed in T4 helper lymphocytes but also in myeloid cells. Receptor-mediated endocytosis plays a crucial role in the regulation of surface expression of adhesion molecules such as CD4. In T lymphocytes p56lck, a CD4-associated tyrosine kinase, prevents CD4 internalization, but in myeloid cells p56lck is not expressed and CD4 is constitutively internalized. In this study, we have investigated the role of cyclic AMP (cAMP) in the regulation of CD4 endocytosis in the myeloid cell line HL-60. Elevations of cellular cAMP were elicited by 1) cholera toxin, 2) pertussis toxin, 3) forskolin and IBMX, 4) NaF, or 5) the physiological receptor agonist prostaglandin E1. All five interventions led to an inhibition of CD4 internalization. Increased cAMP levels did not inhibit endocytosis per se, because internalization of insulin receptors and transferrin receptors and fluid phase endocytosis were either unchanged or slightly enhanced. The mechanism of cAMP inhibition was further analyzed at the ultrastructural level. CD4 internalization, followed either by quantitative electron microscopy autoradiography or by immunogold labeling, showed a rapid and temperature-dependent association of CD4 with clathrin-coated pits in control cells. This association was markedly inhibited in cells with elevated cAMP levels. Thus these findings suggest a second-messenger regulation of CD4 internalization through an inhibition of CD4 association with clathrin-coated pits in p56lck-negative cells.     

The lymphocyte-specific tyrosine protein kinase p56lck is endocytosed in Jurkat cells stimulated via CD2.

Incubation of the human T cells, Jurkat, with two sets of activating anti-CD2 mAb (T11(2) + T11(3), D66 + T11(1)) induced delocalization of p56lck and CD2 receptors from the plasma membrane and increased the tyrosine kinase activity of p56lck. The anti-CD2 mAb combination (T11(2) + T11(3)) that produced the most rapid increase in p56lck kinase activity also induced the most rapid delocalization of the kinase. In stimulated cells, both p56lck and CD2 receptors are detected in cytoplasmic vesicles. The internalization of p56lck in endocytic vesicles was established by confocal microscopy. By double staining it was shown that only part of the p56lck colocalized with the internalized CD2 receptor suggesting distinct sorting processes. Internalization of p56lck appeared to be specific of CD2 stimulation as: 1) in Jurkat cells triggered with an anti-CD3 mAb, p56lck was not internalized whereas CD3 receptors were completely endocytosed; 2) when cells were stimulated via CD4, the kinase and CD4 receptors remained associated with the plasma membrane. In addition, internalization of p56lck upon stimulation of CD2 receptors was not modified in CD2+/CD3-Jurkat cells indicating that CD3 is not involved in this process. The identification of different subcellular localizations of p56lck in resting and stimulated T cells should represent an important step in the definition of its functional activity.     

Genetic and immunochemical evidence for CD4-dependent association of p56lck with the alpha beta T-cell receptor (TCR): regulation of TCR-induced activation.

Recent observations suggest that the tyrosine kinase p56lck is involved in the transduction of transmembrane signals through the antigen specific T cell receptor (TCR) in CD4+ T cells. By means of in vitro kinase assays, we have found that p56lck coprecipitated with the TCR from lysates of a murine CD4+ T cell line in the absence of TCR-mediated stimuli. Analysis of CD4- mutants and CD4-transfected cells shows that p56lck-TCR association occurred only when CD4 was present. The functional importance of CD4:p56lck-TCR association was demonstrated by low activating potential of rare clonotypic antibodies which did not coprecipitate CD4:p56lck, as well as by total or partial loss of anti-TCR or antigen induced stimulation in CD4- cells, which could be recovered by CD4 transfection. Complementation assays using different anti-TCR antibodies suggest that cross linking of TCR-p56lck:CD4 plus structural changes in the complex are needed for efficient transduction of activating signals through the TCR in these cells.     

A lymphocyte-specific protein tyrosine kinase, p56lck, regulates the PMA-induced internalization of CD4.

p56lck, a member of the src family of non-receptor protein tyrosine kinases (PTKs), is expressed predominantly in T-lymphocytes. Association of p56lck with CD4 and CD8 T-cell receptor (TcR) accessory molecules suggests that p56lck may play a specialized role in antigen-induced T-cell activation. CD4 and CD8 molecules are known to stabilize the interaction between TcR and the major histocompatibility complex during T-cell activation. To examine the role of p56lck in the dynamics of the CD4 molecule, p56lck-expressing transfectant cell clones were prepared by the transfection of an lck-gene plasmid containing an inducible promoter into a CD4+lck- human monocytoid cell line. When these transfectant cells were stimulated with phorbol ester, CD4 internalization on these p56lck-expressing cell lines was selectively and markedly retarded, as compared to p56lck-negative control cell lines. When cell-surface CD4 and intracellular CD4 were selectively precipitated after stimulation, the intracellular CD4 molecules were dissociated from p56lck whereas the surface-retained CD4 molecules were still associated with p56lck. Moreover, the dissociation of p56lck from CD4 appeared to occur prior to the PMA-induced internalization of CD4. These data indicate that p56lck regulates the PMA-induced internalization of CD4 possibly via its association with CD4. Treatment with genistein, a PTK inhibitor, revealed that the PTK activity of p56lck might not be involved in this regulatory effect of p56lck on CD4 internalization.     

A Raf-1-related p110 polypeptide associates with the CD4-p56lck complex in T cells.

The CD4 and CD8 antigens on T cells have been shown to associate with the Src family member p56lck and a GTP-binding protein, p32. The identification of receptor interactions with intracellular mediators is essential in the elucidation of downstream signals mediated by engagement of these receptor complexes. In this study, we report the detection of an additional 110-kDa polypeptide (p110) associated with the CD4-p56lck complex in human peripheral blood T lymphocytes and leukemic T-cell lines. p110 bound preferentially to CD4-p56lck as an assembled complex and poorly, if at all, to the individual components. p110 was recognized directly by an antiserum to the C-terminal region of the serine/threonine kinase Raf-1 and is related to a p110 polypeptide detected in anti-Raf-1 immunoprecipitates. Despite its association with the CD4-p56lck complex, p110 was found to be phosphorylated predominantly on serine residues. Furthermore, phorbol ester treatment of cells resulted in a transient increase in the detection of p110 associated with CD4-p56lck, concomitant with the modulation of CD4-p56lck from the cell surface. This Raf-1-related p110 is therefore likely to play a role in signals generated from the CD4-p56lck complex. p110 may serve as a bridge between the CD4-p56lck complex and the serine/threonine kinase pathways of T-cell activation.     

Interaction of the unique N-terminal region of tyrosine kinase p56lck with cytoplasmic domains of CD4 and CD8 is mediated by cysteine motifs

p56lck, a lymphocyte-specific member of the src family of cytoplasmic protein-tyrosine kinases, is associated noncovalently with the cell surface glycoproteins CD4 and CD8, which are expressed on functionally distinct subpopulations of T cells. Using transient coexpression of p56lck with CD4 or CD8 alpha in COS-7 cells, we show that the unique N-terminal region of p56lck binds to the membrane-proximal 10 and 28 cytoplasmic residues of CD8 alpha and CD4, respectively. Two cysteine residues in each of the critical sequences in CD4, CD8 alpha, and p56lck are required for association. Our results suggest a novel role for cysteine-mediated interactions between unrelated proteins and provide a model for the association of other src-like cytoplasmic kinases with transmembrane proteins.     

Palmitylation of an amino-terminal cysteine motif of protein tyrosine kinases p56lck and p59fyn mediates interaction with glycosyl-phosphatidylinositol-anchored proteins.

Cross-linking of glycosyl-phosphatidylinositol (GPI)-anchored membrane proteins on T cells can trigger cell activation. We and others have shown an association between GPI-anchored proteins and the protein tyrosine kinases (PTKs) p56lck and p59fyn, suggesting a pathway for signaling through GPI-anchored proteins. Studies of decay-accelerating factor (DAF) or CD59 in either the C32 cell line or the HeLa cell line transfected with PTK cDNA demonstrated that the GPI-anchored proteins associated noncovalently with p56lck and p59fyn but not with p60src. Nonmyristylated versions of p56lck and p59fyn also failed to associate with the GPI-anchored proteins. Mutational analysis of the PTK demonstrated that the association with the GPI-anchored proteins mapped to the unique amino-terminal domains of the PTK. A chimeric PTK consisting of the 10 amino-terminal residues of p56lck or p59fyn replacing the corresponding amino acids in p60src was sufficient for association with DAF, but the converse constructs containing the first 10 amino acids of p60src plus the remainder of p56lck or p59fyn did not associate with DAF. Mutation of cysteine to serine at positions 3 and 6 in p59fyn or positions 3 and 5 in p56lck abolished the association of these kinases with DAF. Mutation of serine to cysteine at positions 3 and 6 in p60src conferred on p60src the ability to associate with DAF. Direct labeling with [3H]palmitate demonstrated palmitylation of this amino-terminal cysteine motif in p56lck. Thus, palmitylation of the amino-terminal cysteine residue(s) together with myristylation of the amino-terminal glycine residue defines important motifs for the association of PTKs with GPI-anchored proteins.     

Human immunodeficiency virus type 1 glycoprotein precursor retains a CD4-p56lck complex in the endoplasmic reticulum.

The cell surface glycoprotein, CD4, is the receptor for human immunodeficiency virus (HIV) in T lymphocytes. Following HIV infection, there is reduced expression of CD4 on the cell surface, and this downregulation probably results, at least in part, from the formation of complexes containing the HIV type 1 (HIV-1) glycoprotein precursor (gp160) and CD4 that are not transported from the endoplasmic reticulum (ER). At the plasma membrane of T cells, CD4 is tightly associated with a cytoplasmic tyrosine kinase (p56lck) that is involved in T-cell activation. Using a transient expression system with HeLa cells, we show by pulse-labeling and immunoprecipitation that newly synthesized CD4 can associate with p56lck before CD4 is transported from the ER. In the presence of HIV-1 gp160, a ternary complex of gp160-CD4 and p56lck forms in the ER. Using confocal immunofluorescence microscopy, we observed complete retention of p56lck in the ER. Such mislocation of a tyrosine kinase to the cytoplasmic face of the ER could play a role in lymphocyte killing caused by HIV infection or expression of gp160 alone.     

Cross-linking of T-cell surface molecules CD4 and CD8 stimulates phosphorylation of the lck tyrosine protein kinase at the autophosphorylation site.

p56lck, a lymphocyte-specific tyrosine protein kinase, binds to the cytoplasmic tails of the T-cell surface molecules CD4 and CD8. Cross-linking of CD4 expressed on the surface of murine thymocytes, splenocytes, and CD4+ T-cell lines induced tyrosine phosphorylation of p56lck dramatically. Cross-linking of CD8 stimulated tyrosine phosphorylation of p56lck strongly in murine L3 and GA4 cells, slightly in splenocytes, but not detectably in thymocytes. Differing effects of cross-linking on in vitro tyrosine kinase activity of p56lck were observed. An increase in the in vitro kinase activity of p56lck, when assayed with [Val5]-angiotensin II as an exogenous substrate, was found to accompany cross-linking of CD4 in three cell lines. No stimulation of the in vitro kinase activity, however, was observed after cross-linking of CD8 in L3 cells. The phosphorylation of p56lck at Tyr-394, the autophosphorylation site, was stimulated by cross-linking in all cell lines examined. Tyr-394 was the predominant site of increased tyrosine phosphorylation in two leukemic cell lines. In the other two cell lines, the phosphorylation of both Tyr-394 and an inhibitory site, Tyr-505, was found to increase. In contrast to cross-linking with antibodies, no striking increase in the tyrosine phosphorylation of p56lck was stimulated by antigenic stimulation. Therefore, the effect of antibody-induced aggregation of CD4 and CD8 on the tyrosine phosphorylation of p56lck differs, at least quantitatively, from what occurs during antigen-induced T-cell activation.