Lymphocyte Antigen Receptor Research Articles

A DNA repair defect in Chinese hamster ovary cells affects V(D)J recombination similarly to the murine scid mutation.

Lymphocyte antigen receptor variable regions are encoded by gene segments that are assembled by the site-specific variable (diversity) joining (V(D)J) recombination process. We have assayed the V-3 Chinese hamster ovary cell line, which has a double-strand DNA break repair (dsbr) defect, for the ability to carry out V(D)J recombination following transfection of constructs that encode the RAG-1 and RAG-2 proteins necessary to confer V(D)J recombination activity to non-lymphoid cells. The V-3 cells had substantially impaired ability to undergo V(D)J recombination of transiently introduced test substrates. Although these cells can initiate V(D)J recombination by introducing endonucleolytic scissions at the junctions of V(D)J recombination signal sequences and the flanking coding sequences, they have a greatly impaired ability to rejoin the coding sequences. Detailed characterization of attempted coding joins recovered from these cells indicated that the V(D)J recombination defect in V-3 is similar in phenotype to the murine severe combined immunodeficient (scid) defect and quite distinct from that found in other Chinese hamster ovary dsbr mutant cell lines. Somatic cell complementation analyses between homozygous scid mutant fibroblasts and V-3 cells confirmed that these mutations fall into the same genetic complementation group.

Accessory molecules that influence signaling through B lymphocyte antigen receptors.

The B lymphocyte antigen receptor complex, commonly called the B cell receptor (BCR) complex, consists of surface immunoglobulin (sIg) and heterodimers of the Igα (CD79a, mb1) and Igβ (CD79b, B29) phosphoglycoproteins. Recent reviews detail pertinent findings on the BCR complex.1 The structure of the BCR complex and some cell-surface molecules that influence signaling via the BCR are shown in Figure 1. The clonotypic Ig receptor has only a very short cytoplasmic tail and therefore must rely on the invariant members of the BCR complex to transmit signals to the cytosol after receptor crosslinking. Igα/Igβ associate with sIgM and sIgD and are both necessary and sufficient for the expression of sIg. This heterodimer is analogous to the TCR CD3ε/δ or CD3ε/γ heterodimers,2 which, like Igα/Igβ, (1) contain subunits with a single extracellular Ig-like domain; (2) are phosphorylated on tyrosine (CD3ε and CD3ζ) after crosslinking of antigen receptors; and (3) contain within their cytoplasmic tail a single antigen receptor homology 1 (ARH1) motif, D/E-X7-D/E-X2-Y-X2-L-X7-Y-X2-L/I (X = any amino acid). Regions within the transmembrane domain of sIgM are required for the release of [Ca2+]i or internalization of bound antigen.3 Mutations within the transmembrane domain of sIgM that inhibit the activation of new protein tyrosine phosphorylation (PTP) and release of [Ca2+]i also uncouple sIgM from Igα/Igβ.4 However, even though such a mutant sIgM does not associate with Igα/Igβ, when crosslinked it still induces some new PTP.4 Both Sanchez et al.4 and Kim et al.5 reported that surface chimeric fusion proteins expressing the cytoplasmic tails of Igα vs. Igβ differ in their ability to transmit signals: the Igα but not the Igβ tail can induce new PTP, results consistent with studies suggesting that Igα and not Igβ strongly associates with the protein tyrosine kinase (PTK) p53/56Lyn (Lyn).6 Matsuuchi et al.7 found that sIgM expression could be reconstituted in a pituitary cell line with Igα/Igβ coexpression, but that Igα/Igβ were not sufficient to reconstitute a complete signal through sIgM. Thus, IgM interaction with Igα/Igβ is critical for signaling but other factors may also be required.

Role of the mu immunoglobulin heavy chain transmembrane and cytoplasmic domains in B cell antigen receptor expression and signal transduction.

Membrane immunoglobulins (mIg) serve as the recognition components of B lymphocyte antigen receptors. Binding of antigen to these receptors leads to dramatic effects on B cell growth and viability. We have examined the structural elements of mIgM that are involved in antigen receptor assembly and function. Expression of transfected wild-type mIgM in a B cell line led to assembly with the other two known components of the antigen receptor, Ig-alpha and Ig-beta, expression on the cell surface, and when cross-linked by anti-IgM antibodies, stimulation of signal transductin reactions, including tyrosine protein phosphorylation, inositol phosphate production, and increases in cytoplasmic calcium concentration. Replacement of the highly conserved COOH-terminal 41 amino acids of mIgM heavy chain (mu m) with the transmembrane and cytoplasmic domains of human CD8 alpha resulted in a molecule which was expressed on the B cell surface at levels comparable to wild-type mIgM, but which did not form a complex with Ig-alpha or Ig-beta and did not stimulate any of the signaling reactions mentioned above. Replacement of the basic three-amino-acid cytoplasmic domain of mu m with a different but similarly charged sequence had no effect on cell-surface expression or signaling function. On the other hand, removal of the entire cytoplasmic domain resulted in a molecule which did not bind Ig-alpha and Ig-beta and which did not transduce signals. This effect is probably due to altered post-translational processing of this mutant molecule. Finally, a series of eight single-amino-acid substitutions in the transmembrane domain was constructed. Most of these resulted in the removal of hydroxyl groups from conserved residues postulated to be important for interactions with other components. Each of these mutant molecules was capable of transducing signals when cross-linked by anti-IgM, but one was partially defective. Since alteration of any single residue was not sufficient to disrupt signaling completely, the interactions required for signaling are likely to involve multiple residues, so that elimination of one hydroxyl group does not prevent the interaction. We propose that the cytoplasmic domain of mu m does not play a critical role in receptor function but that the transmembrane domain specifies interactions with other components, probably Ig-alpha and Ig-beta, required for proper antigen receptor signal transduction.

Signaling-defective mutants of the B lymphocyte antigen receptor fail to associate with Ig-alpha and Ig-beta/gamma.

The B cell receptor for antigen, the membrane immunoglobulin (mIg) molecule, is normally expressed in association with at least two other proteins, mb-1 (Ig-alpha) and B29 (Ig-beta/gamma). We have previously described a set of murine B cell clones transfected with wild-type and mutated forms of membrane-bound IgM (mIgM) that vary in their ability to transduce signals to the cell interior. Analysis of the B cell receptor complex in signaling and nonsignaling mutants of mIgM reveals complete concordance between intact signaling ability and ability of the mIgM to associate with Ig-alpha and Ig-beta/gamma. These results point to the importance of the mIgM transmembrane region in mediating binding to accessory molecules, and provide support for a role of Ig-alpha and Ig-beta/gamma that extends beyond surface expression to signal transduction in B lymphocytes.

Molecular Diagnosis of Lymphocytic Infiltrates of the Skin

Advances in our understanding of the molecular genetics of lymphocyte antigen receptors (B-cell immunoglobulin and T-cell antigen receptor), have led to the application of molecular biologic techniques to molecularly characterize lymphocytic infiltrates of the skin. Molecular diagnosis refers to the application of these techniques as a diagnostic aid in the clinicopathologic evaluation of cutaneous lymphocytic infiltrates. Molecular studies have clinical application in the determination of lineage and detection of retroviruses in cutaneous lymphoid neoplasms, distinguishing between lymphoproliferative and reactive infiltrates, and staging and monitoring response to therapy in cutaneous T-cell lymphoma. Southern blot analysis of immunoglobulin and T-cell antigen receptor gene rearrangements may fail to aid the clinician in establishing a diagnosis of a cutaneous malignancy due to the limits of detection sensitivity in minimally infiltrated lesions (eg, parapsoriasis and patch-stage mycosis fungoides) or the still uncertain prognostic significance of clonality in benign cutaneous diseases (eg, follicular mucinosis, pityriasis lichenoides et varioliformis acuta, lymphomatoid papulosis, and cutaneous lymphoid hyperplasia). Molecular studies have enormous research value, providing new means to explore the pathogenesis and clonal evolution of lymphoproliferative skin diseases. Presently, however, they have limited applications as an independent diagnostic tool. As our understanding of the clinical and biologic significance of the molecular detection of clonal lymphocyte populations in the skin expands and as the application of polymerase chain reaction amplification provides us with greater detection sensitivity and specificity, the clinical utility of molecular diagnosis of lymphocytic infiltrates of the skin will be enhanced.

In the groove: the fine detail of antigen presentation to cytotoxic T lymphocytes.

1. A panel of cells designed to express normal or mutated human HLA-A0201 was constructed and used to investigate the interactions between the peptide antigen, class I major histocompatibility complex, CD8 and cytotoxic T lymphocyte antigen receptor. 2. Analysis of the extensive data generated has revealed that the optimum peptide presented to cytotoxic T lymphocytes by HLA-A0201 contains nine amino acids. It lies in the peptide-binding groove of the class I molecule, orientated from N- to C-termini, and its detailed position within the groove has been deduced. The roles of individual residues in the class I molecule in antigen presentation are described, and the site of the ligand on target cells for interaction with CD8 on cytotoxic T lymphocytes is located.

Molecular mimicry of the antigen receptor signalling motif by transmembrane proteins of the Epstein-Barr virus and the bovine leukaemia virus

Background: Many transmembrane proteins of eukaryotic cells have only a short cytoplasmic tail of 10 – 100 amino acids, which has no obvious catalytic function. These tails are thought to be involved either in signal transduction or in the association of transmembrane proteins with the cytoskeleton. We have previously identified, in the cytoplasmic tails of components of B and T lymphocyte antigen receptors, an amino-acid motif that is required for signalling. The same motif is also found in the cytoplasmic tails of two viral proteins: the latent membrane protein, LMP2A, of Epstein Barr virus and the envelope protein, gp30, of bovine leukaemia virus. Interestingly, both viruses can activate infected B lymphocytes to proliferate, as does signalling by the B-cell receptor. Results: In this study, we show that the cytoplasmic tails of the two viral proteins, and the cytoplasmic tail of the B-cell receptor immunoglobulin-α chain, when linked to CD8 in chimeric transmembrane proteins, can transduce signals in B cells. Cross-linking of these chimeric receptors activates B-cell protein tyrosine kinases and results in calcium mobilization. Furthermore, these cytoplasmic sequences are also protein tyrosine kinase substrates and may interact with cytosolic proteins carrying SH2 protein-protein interaction domains. Conclusion: Our findings suggest that viral transmembrane proteins can mimic the antigen-induced stimulation of the B-cell antigen receptor and thus can influence the activation and/or survival of infected B lymphocytes.

Impairment of V(D)J recombination in double-strand break repair mutants.

Cells maintain the integrity of their genome through an intricate network of repair systems that recognize and remove lesions from DNA. The only known site-directed recombination process in vertebrates is the V(D)J recombination of lymphocyte antigen receptor genes. A large panel of cell lines deficient in DNA repair were tested for the ability to perform V(D)J recombination after introduction of the RAG-1 and RAG-2 genes. Two mutants failed to generate normal V(D)J recombination, and further analysis provided evidence for two distinct nonlymphoid-specific genes that encode factors involved in both DNA repair and V(D)J recombination.

Targets of B lymphocyte antigen receptor signal transduction include the p21ras GTPase-activating protein (GAP) and two GAP-associated proteins.

Cross-linking membrane Ig (mIg) on B cells stimulates tyrosine phosphorylation of proteins involved in signal transduction including the mIg-associated proteins Ig-alpha and Ig-beta, the tyrosine kinases p53/p56lyn, p55blk, p59fyn, and PTK72, phosphatidylinositol 3-kinase, phospholipase C gamma 1 and gamma 2, and the mitogen-activated protein kinase. We now show that the p21ras GTPase-activating protein (GAP) is also a substrate for mIg-activated tyrosine kinases. p21ras is a key regulator of cell growth and GAP may act as both a regulator of p21ras activity and as a downstream effector of p21ras. We found that mIg cross-linking caused a rapid increase in tyrosine phosphorylation of GAP in the immature B cell line WEHI-231, the mature B cell lines BAL 17 and Daudi, and the IgG-bearing B cell line A20. In fibroblasts, tyrosine kinase activation causes GAP to associate with two other tyrosine-phosphorylated proteins, p62 and p190, which have homologies to an RNA-binding protein and a transcriptional repressor, respectively. Similarly, mlg cross-linking induced the association of GAP with a 62-kDa tyrosine-phosphorylated protein in BAL 17, WEHI-231, and Daudi cells. Anti-Ig treatment also increased the amount of a 190-kDa tyrosine-phosphorylated protein associated with GAP in WEHI-231 and Daudi cells. After separation by SDS-PAGE and transfer to nitrocellulose, the tyrosine-phosphorylated p62 and p190 present in anti-GAP immunoprecipitates from B cells were capable of binding radiolabeled recombinant GAP, as previously reported for the GAP-associated p62 and p190 from fibroblasts. The amount of p62 that could be detected in this way after immunoprecipitation with antiphosphotyrosine antibodies was much greater from anti-IgM-treated BAL 17 cells than from unstimulated BAL 17 cells. This probably reflects anti-Ig-induced tyrosine phosphorylation of p62. In any case, GAP, p62, and/or p190 may be involved in signal transduction by mIg in B cells.

Signal transduction by T-cell receptors: mobilization of Ca and regulation of Ca-dependent effector molecules.

There have been major advances over the last several years in understanding the molecular basis of signaling by the T lymphocyte (T-cell) antigen receptor. In this article we discuss the early phases of T-cell activation with an emphasis on receptor-associated signaling molecules, mobilization of Ca, and on the possible roles of Ca in signal transduction. Ligation of the extracellular domains of the T-cell receptor activates receptor-associated tyrosine kinases that can phosphorylate the gamma-isoform of phospholipase C, increasing its catalytic activity. This leads to production of inositol 1,4,5-trisphosphate, release of stored intracellular Ca, and activation of Ca-permeable plasma membrane channels. Many of the critical T-cell signal transducing enzymes such as phospholipase C and protein kinase C contain intrinsic Ca-binding domains, but for the most part the rise in cytoplasmic Ca is transduced by specialized Ca-binding proteins that lack catalytic domains. The Ca-binding proteins found in T-cells include members of both the EF-hand and annexin families, as well as other types of Ca-binding proteins. In T-cells, a number of important kinases, phosphatases, and cytoskeleton-modulating enzymes are functionally Ca dependent but have no Ca-binding domains and therefore must sense changes in the cytoplasmic Ca level through interactions with Ca-binding proteins.

A voltage-gated calcium channel is linked to the antigen receptor in Jurkat T lymphocytes

Activation of T lymphocytes results in an increase in intracellular Ca 2+ due in large part to influx of extracellular Ca 2+. Using the patch clamp technique, an inward current in Jurkat T lymphocytes was observed upon depolarization from a holding potential of −90 mV but not from −60 mV. This whole-cell current was insensitive to tetrodotoxin, carried by Ba 2+, and blocked by Ni 2+. Occupancy of the T lymphocyte antigen receptor increased the current's magnitude. These data suggest that antigen receptor-induced Ca 2+ entry in T lymphocytes may be mediated by a voltage-regulated Ca channel.

Molecular biologic studies of Hodgkin's disease.

There has been intense interest in applying the techniques of molecular biology to further our understanding of Hodgkin's disease. Numerous studies of the status of lymphocyte antigen receptor genes have been performed, with conflicting results. There is more uniformity in the results of Epstein-Barr viral (EBV) studies; evidence of EBV can be identified in the Reed-Sternberg cells of approximately 40% to 50% of cases. There is no consensus on whether the t(14;18) is present in tissues involved by Hodgkin's disease.

Immunoglobulin M and D antigen receptors are both capable of mediating B lymphocyte activation, deletion, or anergy after interaction with specific antigen.

A series of immunoglobulin (Ig)-transgenic mice were generated to study the functional capabilities of the IgM and IgD classes of B lymphocyte antigen receptor in regulating both cellular development and responses to specific antigen. B cells from Ig-transgenic mice expressing either hen-egg lysozyme (HEL)-specific IgM or IgD alone were compared with B cells from mice that coexpressed IgM and IgD of the same anti-HEL specificity. In all three types of Ig-transgenic mice, conventional B cells specific for HEL exhibited exclusion of endogenous Ig expression and matured to populate the usual microenvironments in peripheral lymphoid tissues. These peripheral B cells could be stimulated by HEL through either IgM or IgD antigen receptors to generate T cell dependent antibody production in vivo or to enhance T cell independent proliferative responses to lipopolysaccharide in vitro. Conversely, when HEL was encountered in vivo as a self-antigen, B cells expressing HEL-specific IgM or IgD alone were both rendered tolerant. In each case this occurred by clonal anergy in response to soluble autologous HEL, and clonal deletion when HEL was recognized as a membrane-bound self-antigen. Taken together, these findings indicate that IgM and IgD antigen receptors expressed alone on conventional B cells can support normal differentiation, antigen-dependent activation, and induction of self-tolerance, the only overt difference lying in a greater degree of receptor downregulation for IgM relative to IgD after induction of clonal anergy by soluble HEL.

Selective activation of p42 mitogen-activated protein (MAP) kinase in murine B lymphoma cell lines by membrane immunoglobulin cross-linking. Evidence for protein kinase C-independent and -dependent mechanisms of activation

Cross-linking of membrane immunoglobulin (mIg), the B lymphocyte antigen receptor, with anti-receptor antibodies stimulates tyrosine phosphorylation of a number of proteins, including one of 42 kDa. Proteins with a similar molecular mass are tyrosine-phosphorylated in response to receptor stimulation in other cell types and have been identified as serine/threonine kinases, termed mitogen-activated protein (MAP) kinases or extracellular signal-regulated kinases (ERKs). The MAP kinases constitute a family of related kinases, at least three of which have molecular masses of 40-45 kDa. In this paper we show that mIg cross-linking stimulated the myelin basic protein phosphotransferase activity characteristic of MAP kinase in both mature and immature murine B cell lines. This enzyme activity co-purified on three different columns with a 42 kDa protein that was tyrosine-phosphorylated (pp42) in response to mIg cross-linking and which reacted with a panel of anti-(MAP kinase) antibodies. Although immunoblotting with the anti-(MAP kinase) antibodies showed that these B cell lines expressed both 42 kDa and 44 kDa forms of MAP kinase, only the 42 kDa form was activated and tyrosine-phosphorylated to a significant extent. Activation of protein kinase C (PKC) with phorbol esters also resulted in selective tyrosine phosphorylation and activation of the 42 kDa MAP kinase. This suggested that mIg-induced MAP kinase activation could be due to stimulation of PKC by mIg. However, mIg-stimulated MAP kinase activation and pp42 tyrosine phosphorylation was only partially blocked by a PKC inhibitor, the staurosporine analogue Compound 3. In contrast, Compound 3 completely blocked the ability of phorbol esters to stimulate MAP kinase activity and induce tyrosine phosphorylation of pp42. Thus mIg may activate MAP kinase by both PKC-dependent and -independent mechanisms.

The hinge region of the CD8α chain: structure, antigenicity, and utility in expression of immunoglobulin superfamily domains

The lymphocyte surface CD8 antigen is a heterodimer with each chain containing a single Ig-related domain, a hinge-like sequence, a transmembrane segment, and a short cytoplasmic sequence. A soluble form of the rat CD8 alpha chain was produced by introducing a stop codon into the cDNA at the end of the region encoding the extracellular sequence and expressed in Chinese hamster ovary cells. sCD8 alpha was produced at 20 mg/l, and consisted of monomers, dimers, and higher aggregates. The latter could be minimized, but not eliminated, by removal of one of the two cysteine residues in the hinge region by mutation and by growth in serum-free medium. The positions of the N- and O-linked glycosylation sites and the disulphide bond in the Ig-like domain were determined. The MRC OX-8 antibody was shown to react with a region from the CD8 alpha hinge containing 24 amino acids and the antigenic determinant was sensitive to neuraminidase digestion. A construct encoding the Ig-like domain of rat CD8 alpha without the hinge was not expressed in CHO cells, indicating the importance of the hinge region for expression. It seemed possible that the CD8 alpha hinge might facilitate expression of other Ig-related domains and such expression could be detected using the MRC OX-8 antibody. To test the system cDNA constructs were made with the rat CD8 alpha hinge spliced to the V-like domain of mouse CD8 alpha, to the V alpha and V beta domains of a T lymphocyte antigen receptor, and to one or both of the Ig-like domains of the MRC OX-47 membrane antigen. All these forms were expressed as soluble proteins that were detected with the MRC OX-8 antibody. This method may prove useful for the expression of Ig superfamily domains for raising antibodies and other studies.

Gene Analysis and Epstein—Barr Viral Genome Studies of Hodgkin's Disease

Publisher Summary This chapter presents the recent molecular studies of Hodgkin's disease, including analyses of the configurations of the B and T lymphocyte antigen receptor genes, studies on the possible presence of Epstein–Barr viral genomes, studies of interleukin-5 mRNA, and studies of the cytogenetics of Hodgkin's disease. The studies of Hodgkin's disease involved analyzing a set of 16 cases for immunoglobulin heavy- and light-chain gene rearrangements and for rearrangements of the T cell receptor β (TCR-β) gene. The cases chosen for study were of the full spectrum of histologie subtypes and contained differing numbers of Reed–Sternberg cells as assessed by the histologie sections. Because a deficiency in cellular immunity has been found in patients with Hodgkin's disease, a possibility was considered that a virus, such as the Epstein–Barr virus, could be driving such a proliferation. To investigate this possibility, tissues involved by Hodgkin's disease were studied, both with and without detectable immunoglobulin gene rearrangements, for evidence of Epstein–Barr viral genomes.

Lymphocyte antigen receptors: a common design?

A recent flurry of papers has provided detailed information on the components of the T- and B-cell antigen receptors. Although these individual elements, and the subunits they form, clearly differ, Frits Koning argues that the T- and B-cell receptor complexes may share a common underlying design.

Activation of phospholipase C in human B cells is dependent on tyrosine phosphorylation.

Cross-linking of the surface antigen receptor on B lymphocytes has been demonstrated to lead to activation of phospholipase C (PLC) with subsequent increases in production of inositol phosphates and diacylglycerol. In turn, these second messengers increase cytosolic free calcium [( Ca2+]i) and activate the serine threonine phosphotransferase protein kinase C (PKC). These processes are thought to play a major role in B cell activation and proliferation. However, the mechanism linking the B lymphocyte antigen receptor to phospholipase C remains to be identified. We demonstrate herein that activation of the antigen receptor on human lymphocytes, in addition to activation of PLC, increases tyrosine phosphorylation of specific substrates. Tyrphostins, a new class of tyrosine kinase inhibitors which compete for substrate binding site of specific tyrosine kinases have recently been synthesized. Preincubation of B lymphocytes with two different tyrphostins blocked anti-IgM-induced proliferation, oncogene expression, tyrosine phosphorylation, increases in [Ca2+]i, and production of inositol phosphates. The same inhibitors were without effect on B cell proliferation induced by phorbol esters and cation ionophores which directly activate PKC and increase [Ca2+]i thus bypassing PLC. These findings strongly indicate that tyrphostins do not exhibit significant nonspecific toxicity and suggest that they act proximal to PLC. The ability of the tyrphostins to block increases in [Ca2+]i and inositol phosphate production, after activation of the B cell antigen receptor, indicates that a tyrosine kinase acts as an essential link between the B cell antigen receptor and PLC.

Transmission of Signals from the T Lymphocyte Antigen Receptor to the Genes Responsible for Cell Proliferation and Immune Function: The Missing Link

Article de synthese sur la lyse des cellules cibles par les lymphocytes T. L'interet est porte ― sur la cytolyse mediee par les granules cytoplasmiques qui contiennent plusieurs esterases a serine qui sont liberees par exocytose ― sur la perforine et son mode d'action

B lymphocyte antigen receptors (mIg) are non-covalently associated with a disulfide linked, inducibly phosphorylated glycoprotein complex.

T and B lymphocyte antigen receptors exhibit single transmembrane spanning regions and very short, three to five amino acid, C-terminal cytoplasmic tails. Ligation of these receptors leads, apparently through GTP binding protein activation, to rapid stimulation of a polyphosphoinositide specific phosphodiesterase (PPI-PDE). T lymphocyte antigen receptors (alpha beta) are coupled to PPI-PDE via a receptor associated complex of membrane proteins, designated CD3. Although an analogous transducer complex is presumed to exist in B cells, no such structure has been defined. We utilized in vitro [32P]phosphorylation to identify and characterize a membrane immunoglobulin (mIg) associated phosphoprotein complex which appears to represent a B cell analog of CD3. The phosphoprotein complex consists of three N-glycosylated polypeptides which occur as disulfide linked dimers, non-covalently associated with mIg. The complex associated with mIgM (pp32, pp34 and pp37 subunits) differs from that associated with mIgD (pp33, pp34 and pp37 subunits), and the isotype specific phosphoprotein (pp32 or pp33) appears to exist as a disulfide linked heterodimer with either pp34 or pp37. Aluminum fluoride stimulates phosphorylation of all of the subunits, and at least one of the proteins is phosphorylated on a tyrosine residue(s).