Localization of a lymphocyte-specific protein tyrosine kinase gene (lck) at a site of frequent chromosomal abnormalities in human lymphomas.

The murine lck gene is closely related to a family of cellular protooncogenes and encodes a lymphocyte-specific, membrane-associated protein tyrosine kinase. We and others have demonstrated that the lck gene is rearranged and overexpressed in the murine lymphoma LSTRA, most likely as a result of the insertion of Moloney murine leukemia virus DNA immediately adjacent to the gene. We now report that the lck gene is located at the distal end of murine chromosome 4 and on human chromosome 1 at position 1p32-35 near a site of frequent structural abnormalities in human lymphomas and neuroblastomas. These results raise the possibility that structural alteration of the lck gene through chromosomal rearrangement may contribute to transformation in human malignant disease.     

Ras GTPase-activating protein: a substrate and a potential binding protein of the protein-tyrosine kinase p56lck.

Ras GTPase-activating protein (GAP) is a cytoplasmic factor that regulates the GTPase activity of p21ras. Phosphorylation of GAP on tyrosine has recently been reported by several groups and may be an important step in linking signaling pathways involving p21ras and protein-tyrosine kinases. p56lck, a src-like protein-tyrosine kinase, seems to play a crucial role in T-cell development and T-cell activation. However, the molecular mechanisms of T-cell signaling involving p56lck and the substrates of p56lck have not yet been identified. To test whether GAP is a substrate of p56lck, in vitro kinase reactions were performed with purified, recombinant GAP and p56lck. We found that GAP became specifically phosphorylated on tyrosine within one tryptic peptide. Furthermore, coimmunoprecipitation studies provided evidence that the tyrosine-phosphorylated form of GAP is bound to p56lck. These results suggest that in T cells the function of GAP might be regulated through its phosphorylation on tyrosine and binding to the protein-tyrosine kinase p56lck.     

T-lymphocyte interleukin 2-dependent tyrosine protein kinase signal transduction involves the activation of p56lck.

Addition of interleukin 2 (IL-2) to IL-2-dependent T cells results in tyrosine protein kinase signal transduction events even though the IL-2 receptor alpha and beta chains lack intrinsic enzymatic activity. Here we report that addition of IL-2 to IL-2-dependent human T cells transiently stimulates the specific activity of p56lck, a member of the src family of nonreceptor tyrosine protein kinases expressed at high levels in T lymphocytes. The ability of IL-2 to induce p56lck activation was found to be independent of the capacity of p56lck to associate with either CD4 or CD8. Following IL-2 treatment, p56lck was found to undergo serine/threonine phosphorylation modifications that resulted in altered mobility of the lck gene product on polyacrylamide gels. These observations raise the possibility that p56lck participates in IL-2-mediated signal transduction events in T cells.     

Murine T-lymphocyte proliferation induced by interleukin 2 correlates with a transient increase in p56lck kinase activity and the tyrosine phosphorylation of a 97-kDa protein.

The addition of recombinant interleukin 2 (rIL-2) to anti-CD3-activated murine G0 phase T cells results in an increased level of tyrosine phosphorylation of a single 97-kDa protein. The degree of tyrosine phosphorylation paralleled the amount of rIL-2 added and correlated with the extent of DNA synthesis. IL-2 treatment resulted in a transient increase in p56lck kinase activity without detectable modification of its level of tyrosine phosphorylation and gel mobility. When G0 T cells were activated by phorbol dibutyrate in the absence of IL-2, the high-affinity IL-2 receptor (IL-2R) expressed failed to induce a proliferative signal, and neither the tyrosine phosphorylation of the 97-kDa protein nor the transient increase in p56lck kinase activity was detected. Northern analysis of the total RNA extracted from these cells showed the accumulation of IL-2R alpha chain-specific mRNA but neither c-myc nor cdc2 mRNA was expressed. The addition of 100 nM rIL-2 to T cells activated by phorbol dibutyrate was able to induce a proliferative response, and under these conditions tyrosine phosphorylation of the 97-kDa protein, the transient increase in p56lck kinase activity, and specific mRNA for IL-2R alpha chain, c-myc, and cdc2 were detected. Unstimulated G0 T cells responded to 100 nM rIL-2 in the same manner as phorbol dibutyrate-activated cells. Irrespective of the signal-transducing structures involved, the IL-2-induced proliferative response closely correlates with an increase in p56lck kinase activity along with the tyrosine phosphorylation of a 97-kDa protein.     

Phosphorylation of Ser-42 and Ser-59 in the N-terminal region of the tyrosine kinase p56lck.

Ser-42 and Ser-59 in the N-terminal region have been identified as the major phorbol ester-induced phosphorylation sites of p56lck. Phosphorylation of Ser-59 results in a gel shift from 56 kDa to 61 kDa. Simultaneous phosphorylation of Ser-42 and Ser-59 results in a further gel shift to 63 kDa. In vitro kinase assays show that Ser-59 can be uniquely phosphorylated by mitogen-activated protein kinase and that Ser-42 can be phosphorylated by either protein kinase A or protein kinase C.     

Rapid activation of the T-cell tyrosine protein kinase pp56lck by the CD45 phosphotyrosine phosphatase.

T lymphocytes express a tyrosine protein kinase (TPK; protein-tyrosine kinase; ATP:protein-tyrosine O-phosphotransferase, EC 2.7.1.112), pp56lck that is encoded by the lck protooncogene. This TPK was recently found to be associated with the intracellular domain of the T-cell surface glycoproteins, CD4 and CD8, suggesting that it plays an important role in T-cell development and activation. We have studied the regulation of pp56lck and found that this kinase can be rapidly activated by an endogenous mechanism present in T-lymphocyte membranes. This activation was sensitive to sodium orthovanadate and O-phosphotyrosine, consistent with the involvement of a phosphotyrosine phosphatase (PTPase; protein-tyrosine-phosphatase; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) in pp56lck activation. Based on a recent report demonstrating that CD45, the leukocyte common antigen, is a membrane-bound PTPase, we analyzed its role in pp56lck activation. CD45 was found to be the major (greater than 90%) PTPase in membranes of the murine T-lymphoma line BW5147. Moreover, activation of pp56lck was undetectable in a mutant BW5147 line lacking CD45 expression (and the associated PTPase activity). In contrast, activation of pp56lck was readily detected in the wild-type lymphoma line. More important, when immunoprecipitated CD45 was added to pp56lck, the TPK activity of the latter increased greater than 2-fold within minutes. This effect of CD45 was completely blocked by sodium orthovanadate. These findings indicate an important role for the CD45 PTPase in pp56lck activation. This role could be mediated by direct dephosphorylation of a regulatory tyrosine residue in pp56lck.     

Modification of Ser59 in the unique N-terminal region of tyrosine kinase p56lck regulates specificity of its Src homology 2 domain.

During T-cell activation, Ser59 in the unique N-terminal region of p56lck is phosphorylated. Mutation of Ser59 to Glu59 mimics Ser59 phosphorylation, and upon CD4 crosslinking, this mutant p56lck induces tyrosine phosphorylation of intracellular proteins distinct from those induced by wild-type p56lck. Mutant and wild-type p56lck have similar affinities for CD4 and similar kinase activities. In glutathione S-transferase fusion proteins, the p56lck Src homology 2 (SH2) domain with the SH3 domain and the unique N-terminal region (including Ser59) has a different binding specificity for phosphotyrosyl proteins than the SH2 domain alone. Either deletion of the unique N-terminal region or mutation of Ser59 to Glu59 in the fusion protein reverts the phosphotyrosyl protein binding specificity back to that of the SH2 domain alone. These results suggest that phosphorylation of Ser59 regulates the function of p56lck by controlling binding specificity of its SH2 domain.     

The coreceptor CD4 is expressed in distinct nanoclusters and does not colocalize with T-cell receptor and active protein tyrosine kinase p56lck

CD4 molecules on the surface of T lymphocytes greatly augment the sensitivity and activation process of these cells, but how it functions is not fully understood. Here we studied the spatial organization of CD4, and its relationship to T-cell antigen receptor (TCR) and the active form of Src kinase p56lck (Lck) using single and dual-color photoactivated localization microscopy (PALM) and direct stochastic optical reconstruction microscopy (dSTORM). In nonactivated T cells, CD4 molecules are clustered in small protein islands, as are TCR and Lck. By dual-color imaging, we find that CD4, TCR, and Lck are localized in their separate clusters with limited interactions in the interfaces between them. Upon T-cell activation, the TCR and CD4 begin clustering together, developing into microclusters, and undergo a larger scale redistribution to form supramolecluar activation clusters (SMACs). CD4 and Lck localize in the inner TCR region of the SMAC, but this redistribution of disparate cluster structures results in enhanced segregation from each other. In nonactivated cells these preclustered structures and the limited interactions between them may serve to limit spontaneous and random activation events. However, the small sizes of these island structures also ensure large interfacial surfaces for potential interactions and signal amplification when activation is initiated. In the later activation stages, the increasingly larger clusters and their segregation from each other reduce the interfacial surfaces and could have a dampening effect. These highly differentiated spatial distributions of TCR, CD4, and Lck and their changes during activation suggest that there is a more complex hierarchy than previously thought.For helper T cells, CD4 has been termed a coreceptor based on its important role in antigen recognition class II major histocompatibility complex (MHC)–peptide complexes by the αβ T-cell receptor (TCR) as well as in signal transduction. Indeed, CD4 significantly increases T-cell sensitivity to antigen upon activation (1–4). This ability of CD4 to enhance antigen recognition has often been connected to the fact that the N-terminal Ig domain of CD4 has specific affinity to invariant sites on MHC class II molecules (5, 6). It has been suggested that CD4 stabilizes the molecular complex of TCR and peptide–MHC (pMHC) by binding to the same MHC either simultaneously with the TCR (7) or shortly after TCR–pMHC engagement (2, 3). However, from more recent 2D measurements, CD4 blockades showed no effect on the stability of TCR binding to agonist peptide–MHC complexes in a synapse (8). In terms of signal transduction, the role of CD4 has been studied based on the binding ability of a cysteine motif in the cytoplasmic tail of CD4 to Src kinase p56lck (Lck) (9), which is responsible for the phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) sequences in TCR–CD3 complex as the earliest observable biochemical event during T-cell activation (10). It has been proposed that CD4 mainly contributes to the sensitivity of T cells by facilitating the recruitment of Lck to TCR–CD3s that are actively engaged in ligand recognition (11, 12). Nevertheless, the absence of CD4 does not preclude T cells from being generated at the thymus or being activated by TCR–pMHC engagement (13, 14).It is now well appreciated that spatial reorganization and distribution of some of the membrane receptors and signaling molecules is one of the critical regulating mechanisms in T-cell activation. The molecular assembly and clusters such as supramolecular activation clusters (SMACs) (15) of immunological synapse (IS) (14), microclusters (16–20), and their roles in T-cell signaling have been widely studied. More recently, the presence and unique roles of smaller-sized protein clusters, termed “nanoclusters” or “protein islands,” of TCR–CD3 complex (21–24), linker for activation of T cell (LAT) (21, 22, 24, 25), Lck (26), and other signaling molecules (24) were revealed by electron microscopy and the newly available superresolution fluorescence microscopy.Considering that the TCR–CD3 complex, CD4, and Lck are constitutively expressed in nonactivated T cells, it is highly likely that the interaction dynamics between these components would also be controlled spatially during the T-cell activation process. Here, we studied the relative molecular distribution of these molecules using single- and dual-color photoactivated localization microscopy (PALM) (27) and direct stochastic optical reconstruction microscopy (dSTORM) (28, 29) in live and fixed T cells for both nonactivated and activating conditions. The corresponding spatial analyses were also used to quantitatively determine the sizes, degree of clustering, and degree of interactions of these clusters. We found that CD4 is also expressed in preclustered structures, separate from TCR–CD3 and LAT, and composed of three to six molecules per cluster. The interactions between these molecules occurred only in the interfaces between the clusters. Upon T-cell activation, the TCR–CD3 and CD4 molecules increased the size of their own clusters without appreciable mixing. Instead, their molecular segregation increased, whereas the T cell develops a synapse structure, often in the SMAC or “bull’s eye” pattern, with the TCR–CD3 in the central supramolecular activation cluster (cSMAC) with the CD4 and Lck clusters localizing around it. These observed clustering behaviors accompanying reorganization of spatial distributions of CD4, Lck, and TCR might be a general and effective mechanism to activate and regulate the T-cell signaling by controlling the magnitude of interfacial interactions between signaling components in each cluster.     

Purification and initial characterization of the lymphoid-cell protein-tyrosine kinase p56lck from a baculovirus expression system.

The lymphocyte-specific protein-tyrosine kinase p56lck has been purified 90-fold to approximately 30% purity in 30% yield from a baculovirus expression system by a two-column purification procedure. At least two forms of p56lck were isolated, differing in the extent of phosphorylation and migrating as 56- and 59-kDa species on SDS/PAGE but as a single 56-kDa band after treatment with potato acid phosphatase. Autophosphorylation of purified p56lck occurred at a rate of 25 fmol/min to a maximum incorporation of approximately 2 mol of phosphate per mol of p56lck with tyrosine-394 (but not tyrosine-505) and other, unidentified tyrosine residue(s) being the major sites of phosphorylation in vitro. Phosphorylation of tyrosine-containing peptides was monitored using an automated HPLC system. Although peptide substrate Km values were in the 1-5 mM range, the Vmax for the 13-amino acid peptide RRLIEDAEYAARG (modified p60src autophosphorylation site) was 120 min-1 (350 min-1 when adjusted for p56lck purity), suggesting that the enzyme purified from recombinant baculovirus-infected Sf9 cells has a high catalytic turnover compared with other tyrosine kinases.     

Phosphotyrosine-independent binding of a 62-kDa protein to the src homology 2 (SH2) domain of p56lck and its regulation by phosphorylation of Ser-59 in the lck unique N-terminal region.

A previously undescribed 62-kDa protein (p62) that does not contain phosphotyrosine but, nevertheless, binds specifically to the isolated src homology 2 (SH2) domain of p56lck has been identified. The additional presence of the unique N-terminal region of p56lck prevents p62 binding to the SH2 domain. However, phosphorylation at Ser-59 (or alternatively, its mutation to Glu) reverses the inhibition and allows interaction of the p56lck SH2 domain with p62. Moreover, p62 is associated with a serine/threonine kinase activity and also binds to ras GTPase-activating protein, a negative regulator of the ras signaling pathway. Thus, phosphotyrosine-independent binding of p62 to the p56lck SH2 domain appears to provide an alternative pathway for p56lck signaling that is regulated by Ser-59 phosphorylation.     

CD5 acts as a tyrosine kinase substrate within a receptor complex comprising T-cell receptor zeta chain/CD3 and protein-tyrosine kinases p56lck and p59fyn.

T-cell antigens including CD2, CD4, CD6, CD8, and CD28 serve as coreceptors with the T-cell receptor (TCR)/CD3 complex in control of T-cell growth. The molecular basis by which these antigens fulfill this role has remained a major issue. An initial clue to this question came with our finding that the sensitivity of in vitro kinase labeling (specifically using protein-tyrosine kinase p56lck) allowed detection of a physical association between CD4-p56lck and the TCR/CD3 complexes. Another T-cell antigen, CD5, is structurally related to the macrophage scavenger receptor family and, as such, can directly stimulate and/or potentiate T-cell proliferation. In this study, we reveal that in Brij 96-based cell lysates, anti-CD5 antibodies coprecipitated TCR zeta chain (TCR zeta)/CD3 subunits as well as the protein-tyrosine kinases p56lck and p59fyn. Conversely, anti-CD3 antibody coprecipitated CD5, p56lck, and p59fyn. Indeed, anti-CD5 and anti-CD3 gel patterns were virtually identical, except for a difference in relative intensity of polypeptides. Anti-CD4 coprecipitated p56lck, p32, and CD3/TCR zeta subunits but precipitated less CD5, suggesting the existence of CD4-TCR zeta/CD3 complexes distinct from the CD5-TCR zeta/CD3 complexes. Consistent with the formation of a multimeric CD5-TCR zeta/CD3 complex, anti-CD5 crosslinking induced tyrosine phosphorylation of numerous T-cell substrates, similar to those phosphorylated by TCR zeta/CD3 ligation. Significantly, as for TCR zeta, CD5 was found to act as a tyrosine kinase substrate induced by TCR/CD3 ligation. The kinetics of phosphorylation of CD5 (t1/2 = 20 sec) was among the earliest of activation events, more rapid than seen for TCR zeta (t1/2 = 1 min). CD5 represents a likely TCR/CD3-associated substrate for protein-tyrosine kinases (p56lck or p59fyn) and an alternative signaling pathway within a multimeric TCR complex.     

Molecular cloning of a phosphotyrosine-independent ligand of the p56lck SH2 domain.

A novel human cDNA encoding a cytosolic 62-kDa protein (p62) that binds to the Src homology 2 (SH2) domain of p56lck in a phosphotyrosine-independent manner has been cloned. The cDNA is composed of 2074 nucleotides with an open reading frame encoding 440 amino acids. Northern analysis suggests that p62 is expressed ubiquitously in all tissues examined. p62 is not homologous to any known protein in the data base. However, it contains a cysteine-rich region resembling a zinc finger motif, a potential G-protein-binding region, a PEST motif, and several potential phosphorylation sites. Using T7-epitope tagged p62 expression in HeLa cells, the expressed protein was shown to bind to the lck SH2 domain. Deletion of the N-terminal 50 amino acids abolished binding, but mutagenesis of the single tyrosine residue in this region had no effect on binding. Thus, the cloned cDNA indeed encodes the p62 protein, which is a phosphotyrosine-independent ligand for the lck SH2 domain. Its binding mechanism is unique with respect to binding modes of other known ligands for SH2 domains.     

p21ras activation via hemopoietin receptors and c-kit requires tyrosine kinase activity but not tyrosine phosphorylation of p21ras GTPase-activating protein.

Products of the ras gene family, termed p21ras, are GTP-binding proteins that have been implicated in signal transduction via receptors encoding tyrosine kinase domains. Recent findings have defined a superfamily of hemopoietin receptors that includes receptors for a number of interleukins and colony-stimulating factors. The intracellular portions of these receptors show only restricted homologies, have no tyrosine kinase domain, and provide no clues to the mode of signal transduction. However, in most cases the factors stimulate tyrosine phosphorylation. We demonstrate here that ligand-induced activation of the interleukin (IL)-2, IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor receptors resulted in activation of p21ras in various hemopoietic cell lines. The only cytokine tested that binds to a hemopoietin receptor and that did not activate p21ras was IL-4. Activation of p21ras was also observed in response to Steel factor, which stimulates the endogenous tyrosine kinase activity of the c-kit receptor, as well as with phorbol esters, which activate protein kinase C. Experiments with protein kinase inhibitors implicated tyrosine kinase activity, but not protein kinase C activity, as the upstream signal in p21ras activation via these growth factor receptors. Attempts to demonstrate tyrosine phosphorylation of the p21ras GTPase-activating protein (GAP) were negative, suggesting that phosphorylation of GAP may not be the major mechanism for regulation of p21ras activity by tyrosine kinases.     

Granulocyte-macrophage colony-stimulating factor-induced protein tyrosine phosphorylation of microtubule-associated protein kinase in human neutrophils.

Granulocyte-macrophage colony-stimulating factor (GM-CSF), formylmethionylleucylphenylalanine, tumor necrosis factor alpha, platelet-activating factor, phorbol ester (phorbol 12-myristate 13-acetate), and calcium ionophore A23187 are able to increase the level of tyrosine phosphorylation of different protein substrates, as demonstrated by Western blotting with anti-phosphotyrosine antibody (anti-PY). A protein of 41 kDa (p41) consistently showed more intense reactivity to anti-PY than controls. Blots treated with anti-PY, stripped of the antibody, and reblotted with microtubule-associated protein kinase (MAPK, p42MAPK) antibody show only one band. The molecular mass of that band exactly matches that of p41. MAPK-reactive protein is present in control and stimulated cells, although the intensity of the band is greater in the latter. GM-CSF-stimulated phosphorylation of p41 is time- and dose-dependent. Anti-MAPK antibody detects a single band of 41 kDa, whose intensity increases with time of incubation and concentration of the agonist. Thus, the anti-MAPK antibody appears to react better to the phosphorylated form of p41 from GM-CSF-stimulated cells than to the dephosphorylated form. The p41 and MAPK proteins are localized in the cytosol. Finally, MAPK immunoprecipitates were probed with anti-PY in Western blots and a band of 41 kDa was found. In summary, these results suggest that this 41-kDa protein in neutrophils that is tyrosine phosphorylated in response to GM-CSF and other stimuli is MAPK. Its phosphorylation may represent an early and crucial signal associated with the GM-CSF neutrophil stimulation cascade.     

Human spleen tyrosine kinase p72Syk associates with the Src-family kinase p53/56Lyn and a 120-kDa phosphoprotein.

The 72-kDa spleen tyrosine kinase (Syk) and Src-family kinase p53/56Lyn (Lyn) contribute to signaling via the B-cell antigen receptor complex. Here we show that Syk and Lyn from human B lymphocytes can interact directly. Syk and Lyn coimmunoprecipitated from mature and activated B-cell lines, and gel-purified Syk and Lyn reassociated in vitro, demonstrating their direct interaction. This Syk-Lyn interaction may be dependent on the stage of B-cell differentiation, since Syk-Lyn associations were not detected in pre-B and myeloma cell lines and Syk from an immature B-cell line did not reassociate with Lyn in vitro. Serine/threonine kinase activity was also associated with Syk. Crosslinking of cell surface IgM led to rapid activation of both tyrosine and serine/threonine protein kinase activities that resulted in phosphorylation in vitro of proteins coprecipitating with Syk--in particular, a serine/threonine phosphorylated protein 120 kDa in size (pp120). Several phosphoproteins, including one of 72 kDa and one of 120 kDa, coprecipitated with phospholipase C-gamma 1 (PLC gamma 1). Sequential immunoprecipitation identified the 72-kDa protein associated with PLC gamma 1 as Syk. The 120-kDa serine/threonine phosphorylated protein that coprecipitated with PLC gamma 1 resembled the Syk-associated pp120 by several criteria. Thus, pp120 may serve as a link between Syk and PLC gamma 1, coupling the B-cell antigen receptor to the phosphatidylinositol pathway.     

The Src homology 2 domain of the protein-tyrosine kinase p56lck mediates both intermolecular and intramolecular interactions.

A key event in signaling by many cell surface receptors is the activation of Src-like protein-tyrosine kinases and the assembly of protein complexes at the plasma membrane mediated by Src homology 2 and 3 (SH2 and SH3) domains. p56lck is a Src-related protein-tyrosine kinase which has SH2 and SH3 domains and is involved in T-cell signaling and oncogenic transformation. Here we demonstrate that purified recombinant SH2 and HSH3/SH2 domains of p56lck can mediate intermolecular interactions with a number of tyrosine-phosphorylated proteins present in lysates of NIH 3T3 cells transformed by a constitutively activated form of p56lck (p56lckF505). Two of the interacting tyrosine-phosphorylated proteins were identified as the p85 subunit of phosphatidylinositol 3-kinase and the GTPase-activating protein of p21ras. Using a synthetic phosphopeptide corresponding to the tyrosine-phosphorylated carboxyl terminus of p56lck (amino acids 494-509), purified recombinant Lck SH2 domain, and differentially phosphorylated forms of p56lck we provide evidence that the SH2 domain of p56lck can also mediate intramolecular interactions with the phosphorylated carboxyl terminus. Together these results suggest that the SH2 domain of p56lck has a dual function: (i) it can mediate intermolecular interactions with cellular proteins phosphorylated on tyrosine and thus might be involved in building up signaling complexes at the plasma membrane and (ii) it can bind to the tyrosine-phosphorylated carboxyl terminus of p56lck in an intramolecular fashion and thereby might be involved in the regulation of its intrinsic protein-tyrosine kinase activity. Phosphorylation/dephosphorylation of the regulatory tyrosine residue 505 might serve as a switch between these two functions.     

Determination of the substrate-docking site of protein tyrosine kinase C-terminal Src kinase

Protein tyrosine kinases (PTK) are key enzymes of mammalian signal transduction. For the fidelity of signal transduction, each PTK phosphorylates only one or a few proteins on specific Tyr residues. Substrate specificity is thought to be mediated by PTK-substrate docking interactions and recognition of the phosphorylation site sequence by the kinase active site. However, a substrate-docking site has not been determined on any PTK. C-terminal Src kinase (Csk) is a PTK that specifically phosphorylates Src family kinases on a C-terminal Tyr. In this study, by sequence alignment and site-specific mutagenesis, we located a substrate-docking site on Csk. Mutations in the docking site disabled Csk to phosphorylate, regulate, and complex with Src but only moderately affected its general kinase activity. A peptide mimicking the docking site potently inhibited (IC50 = 21 microM) Csk phosphorylation of Src but only moderately inhibited (IC50 = 422 microM) its general kinase activity. Determination of the substrate-docking site provides the structural basis of substrate specificity in Csk and a model for understanding substrate specificity in other PTKs.     

Identification of Tyr-185 as the site of tyrosine autophosphorylation of recombinant mitogen-activated protein kinase p42mapk.

Tyrosine phosphorylation of 42-kDa mitogen-activated protein kinase (p42mapk) occurs during expression of the recombinant protein in Escherichia coli, as well as during in vitro phosphorylation of the protein purified from this source. Structural analyses were performed to identify the site(s) of tyrosine phosphorylation of recombinant p42mapk, both during expression of the protein in E. coli and during in vitro incubations with ATP/Mg2+/Mn2+. Mass spectrometry and phosphopeptide mapping showed that tyrosine phosphorylation of recombinant p42mapk occurs on Tyr-185, the site of regulatory tyrosine phosphorylation that occurs in mitogen-stimulated mammalian cells.     

Reactive oxygen intermediates activate NF-kappa B in a tyrosine kinase- dependent mechanism and in combination with vanadate activate the p56lck and p59fyn tyrosine kinases in human lymphocytes

We have previously observed that ionizing radiation induces tyrosine phosphorylation in human B-lymphocyte precursors by stimulation of unidentified tyrosine kinases and this phosphorylation is substantially augmented by vanadate. Ionizing radiation generates reactive oxygen intermediates (ROI). Because H2O2 is a potent ROI generator that readily crosses the plasma membrane, we used H2O2 to examine the effects of ROI on signal transduction. We now provide evidence that the tyrosine kinase inhibitor herbimycin A and the free radical scavenger N- acetyl-cysteine inhibit both radiation-induced and H2O2-induced activation of NF-kappa B, indicating that activation triggered by ROI is dependent on tyrosine kinase activity. H2O2 was found to stimulate Ins-1,4,5-P3 production in a tyrosine kinase-dependent manner and to induce calcium signals that were greatly augmented by vanadate. The synergistic induction of tyrosine phosphorylation by H2O2 plus vanadate included physiologically relevant proteins such as PLC gamma 1. Although treatment of cells with H2O2 alone did not affect the activity of src family kinases, treatment with H2O2 plus vanadate led to activation of the p56lck and p59fyn tyrosine kinases. The combined inhibition of phosphatases and activation of kinases provides a potent mechanism for the synergistic effects of H2O2 plus vanadate. Induction of tyrosine phosphorylation by ROI may thus lead to many of the pleiotropic effects of ROI in lymphoid cells, including downstream activation of PLC gamma 1 and NF-kappa B.