P56lck Kinase Activity Research Articles

ChemInform Abstract: Synthesis and Protein-Tyrosine Kinase Inhibitory Activities of Flavonoid Analogues.

Treatment of o-hydroxyacetophenones 2a-e with excess lithium bis(trimethylsilyl)amide followed by dialkyl carbonates gave alkyl 3-(2-hydroxyaryl)-3-oxopropanoates 3a-e. The latter substances were transformed through the reaction of their magnesium chelates with benzoyl chlorides into a series of 3-(alkoxycarbonyl)-2-arylflavones, which were subsequently elaborated into a variety of flavonoids. These compounds were tested for their abilities to inhibit the in vitro protein-tyrosine kinase activity of p56lck, an enzyme which is thought to play a key role in mediating signal transduction from the CD4 receptor during lymphocyte activation. All of the active compounds had either an amino or a hydroxyl substituent at the 4'-position of the 2-aryl ring. The most active substance prepared in this study is compound 17c, which is approximately 1 order of magnitude more potent than the natural product quercetin (1). Compound 17c was a competitive inhibitor of p56lck with respect to ATP and was highly selective for the inhibition of protein-tyrosine over protein-serine/threonine kinases.

Protein tyrosine kinase p56 lck-deficiency confers hypersusceptibility to ρ-fluorophenylalanine (pFPhe)-induced apoptosis by augmenting mitochondrial apoptotic pathway in human Jurkat T cells

Phenylalanine analog, ρ-fluorophenylalanine (pFPhe)-mediated cytotoxicity and several apoptotic events including mitochondrial cytochrome c release, activation of caspase-9, -3, and -8, Bid cleavage, degradation of PARP and PLCγ-1, and DNA fragmentation were more significant in p56 lck-deficient Jurkat T cells (JCaM1.6) than in wild-type Jurkat T cells (E6.1). The susceptibility of JCaM1.6 toward apoptogenic activity of pFPhe decreased after acquisition of p56 lck by transfection. The p56 lck kinase activity increased 1.6-fold at 15–30 min after pFPhe treatment. The pan-caspase inhibitor (z-VAD-fmk) completely blocked the pFPhe-mediated apoptotic changes except caspase-9 activation. The caspase-8 inhibitor (z-IETD-fmk), which failed to influence pFPhe-induced caspase-9 activation, completely blocked caspase-8 activation and PLCγ-1 degradation with a marked reduction in caspase-3 activation and PARP degradation, indicating pFPhe-induced caspase-8 activation as a downstream event of mitochondria-dependent activation of caspase-9. These results indicate that the deficiency of p56 lck augments pFPhe-induced mitochondrial cytochrome c release and resultant apoptotic cell death in Jurkat T cells.

Chronic Disseminated Mycobacterium xenopi Infection in a Cat with Idiopathic CD4+ T Lymphocytopenia

A 3-year-old castrated male domestic shorthair cat was referred for examination in August 2000 with a 3-week history of poor appetite, vomiting, and weight loss. One week before referral, evaluation by the referring veterinarian identified anemia (PCV 21% [reference range 29–48%]) and moderate renal insufficiency (BUN 55mg/dL [reference range 14–36mg/dL], serum creatinine 4.2mg/dL [reference range 0.6–2.4mg/dL], urine specific gravity 1.015) with an inflammatory urine sediment (proteinuria, white blood cells, bacteria) on a voided sample. The results of enzyme-linked immunosorbent assays for feline leukemia virus (FeLV) antigen and feline immunodeficiency virus (FIV) antibodies were negative. Radiographs disclosed bilateral renomegaly and microcardia. Abdominal ultrasound examination identified a thin cortex in the left kidney and both kidneys were hyperechoic. The cat was treated with lactated Ringer’s solution IV and enrofloxacin 6mg/kg/d IV for 3 days. Upon examination at the referral hospital, the cat was quiet and responsive. Physical examination abnormalities were limited to bilaterally enlarged kidneys that appeared to be painful on palpation and a body condition score of 3/9. Body weight was 3.6 kg. On CBC there was a severe nonregenerative macrocytic normochromic anemia (PCV 17% [reference range 30–47%], MCV 55 fL [reference range 41–51 fL], MCHC 31 g/dL [reference range 31–35 g/dL], aggregate reticulocytes 20,880/mL) in the presence of normal serum erythropoietin concentration (17mU/mL [reference range 10–30mU/mL]). The WBC count (10,200/mL [reference range 5,500–19,500/ mL]), differential cell count (segmented neutrophils 7,040/mL [reference range 2,500–12,500/mL], lymphocytes 2,860/mL [reference range 1,500–7,000/mL], monocytes 260/mL [reference range 0–800/mL], eosinophils 50/mL [reference range 0–1,500/mL]), and platelet count (485,000/mL [reference range 300,000–800,000/ mL]) were normal. Serum chemistry results indicated moderate azotemia (BUN 36mg/dL [reference range 16–34mg/dL], serum creatinine 3.5mg/dL [reference range 0.7–2.3mg/dL]), increased AST activity (173U/L [reference range 5–30U/L]), and hypoalbuminemia (1.8 g/dL [reference range 2.0–3.1 g/dL]). Results of urinalysis collected by cystocentesis indicated isosthenuria (USG 1.011), proteinuria, and bacteruria. Aerobic urine culture was negative, and plasma prothrombin and partial thromboplastin times were within normal limits. Ultrasonographic examination of the abdomen identified moderate bilateral renomegaly with pyelectasia, mesenteric lymphadenopathy, and an enlarged hypoechoic pancreas. By means of ultrasound guidance, fineneedle tissue aspirates were collected from kidney, mesenteric lymph node, and pancreas, and slides were prepared for cytologic evaluation using Wright-Giemsa stain. Preparations obtained from the kidney were very cellular, consisting primarily of large, epitheliod macrophages arranged in sheets and individually. The cytoplasm of many of the macrophages contained long, thin, negatively staining, rod-shaped inclusions consistent withMycobacteria spp. (Fig 1). Renal epithelial cells were also observed in the preparation.Macrophages containing similar negatively staining rods were observed in tissue aspirate preparations from the pancreas and a mesenteric lymph node, along with pancreatic epithelium and lym-

An octapeptide analogue of HIV gp120 modulates protein tyrosine kinase activity in activated peripheral blood T lymphocytes.

Following infection with HIV, patients exhibit lymphocyte dysfunction before the loss of CD4+ T cells. The major HIV surface glycoprotein, gp120, can modulate lymphocyte function in vitro; however, the mechanism by which gp120 affects T lymphocyte signal transduction is controversial. We have used Peptide T, a synthetic octapeptide derived from a conserved, CD4 binding region of gp120, to examine gp120-related modulation of lymphocyte signal transduction. Activation of lymphocytes through the T cell receptor (TCR) in collaboration with cell surface accessory molecules results in rapid increases in tyrosine phosphorylation, probably through the recruitment and activation of src-family protein tyrosine kinases (PTK) such as lck and fyn which have been implicated in mediating the proximal signalling events mediated through the TCR. To identify potential mechanisms by which gp120 could modulate the function of T lymphocytes, we determined the effect of Peptide T on normal, activated peripheral blood lymphoblasts. Treatment of normal, activated peripheral blood lymphoblasts with Peptide T (10(-9) M) for 60 min transiently reduced levels of protein tyrosine phosphorylation (ptyr). Reduction in levels of cellular ptyr was associated with transient inhibition of the activity of total cellular and CD4-associated p56lck kinase activity (80%). Peptide T also induced a small delayed reduction in the p59fyn activity (up to 42%). Despite the decrease in total cellular ptyr levels, pp60c-src kinase activity was increased 11-fold following treatment with Peptide T. Peptide T pretreatment also induced tyrosine phosphorylation of a 48-kD CD4-associated protein, indicating that Peptide T may have multiple effects. Peptide T did not alter the levels of total cellular p56lck enzyme, nor did it directly inhibit the activity of purified p56lck. These results are consistent with a Peptide T-dependent modulation of PTK regulation, and support the potential of gp120 to interfere with T lymphocyte signal transduction in activated T lymphocytes.

The Siva protein is a novel intracellular ligand of the CD4 receptor that promotes HIV-1 envelope-induced apoptosis in T-lymphoid cells

In addition to its positive signaling function in the antigen presentation process, CD4 acts as the primary receptor for HIV-1. Contact between CD4 and the viral envelope leads to virus entry, but can also trigger apoptosis of uninfected CD4+ T-cells through a mechanism that is poorly understood. We show that Siva-1, a death domain-containing proapoptotic protein, associates with the cytoplasmic domain of CD4. This interaction is mediated by the cysteine-rich region found in the C-terminal part of the Siva-1 protein. Expression of Siva-1 specifically increases the susceptibility of both T-cell lines and unstimulated human primary CD4+ T-lymphocytes to CD4-mediated apoptosis triggered by the HIV-1 envelope, and results in activation of a caspase-dependent mitochondrial pathway. The same susceptibility is observed in T-cells expressing a truncated form of CD4 that is able to recruit Siva-1 but fails to associate with p56Lck, indicating that Siva-1 participates in a pathway independent of the p56Lck kinase activity. Altogether, these results suggest that Siva-1 might participate in the CD4-initiated signaling apoptotic pathway induced by the HIV-1 envelope in T-lymphoid cells.

Epitope‐specific crosslinking of CD45 down‐regulates membrane‐associated tyrosine phosphatase activity and triggers early signalling events in human activated T cells

CD45 engagement by monoclonal antibodies on human activated T cells triggers tumour necrosis factor-alpha (TNF-alpha) gene transcription in an epitope-specific manner. To dissect the early signalling events leading to TNF-alpha gene expression, we established that CD45 crosslinking resulted in tyrosine phosphorylation of p56lck, ZAP-70, CD3-zeta, LAT and Vav. This was accompanied by down-regulation of membrane-associated protein tyrosine phosphatase activity in the absence of demonstration of enhanced p56lck, p72syk and ZAP-70 kinase activity, which remained constitutive. These early events eventually triggered an intracellular Ca(2+) rise and phosphoinositide turnover. We conclude that down-regulation of membrane-associated tyrosine phosphatase activity by CD45 extracytoplasmic domain multimerization led, in an epitope-specific fashion, to unopposed tyrosine kinase activity and to the activation of the T-cell receptor/CD3 complex signalling cascade, resulting in TNF-alpha gene expression. This model strongly suggests that CD45 extracytoplasmic tail multimerization may contribute to the modulation T-cell functions.

A regulatory role for CD37 in T cell proliferation.

CD37 is a leukocyte-specific protein belonging to the tetraspanin superfamily. Previously thought to be predominantly a B cell molecule, CD37 is shown in this study to regulate T cell proliferation. CD37-deficient (CD37(-/-)) T cells were notably hyperproliferative in MLR, in response to Con A, or CD3-TCR engagement particularly in the absence of CD28 costimulation. Hyperproliferation was not due to differences in memory to naive T cell ratios in CD37(-/-) mice, apoptosis, or TCR down-modulation. Division cycle analyses revealed CD37(-/-) T cells to enter first division earlier than wild-type T cells. Importantly, proliferation of CD37(-/-) T cells was preceded by enhanced early IL-2 production. We hypothesized CD37 to be involved in TCR signaling and this was supported by the observation that CD4/CD8-associated p56(Lck) kinase activity was increased in CD37(-/-) T cells. Remarkably, CD37 cross-linking on human T cells transduced signals that led to complete inhibition of CD3-induced proliferation. In the presence of CD28 costimulation, CD37 engagement still significantly reduced proliferation. Taken together, these results demonstrate a regulatory role for CD37 in T cell proliferation by influencing early events of TCR signaling.

An oncogenic tyrosine kinase inhibits DNA repair and DNA-damage-induced Bcl-x L deamidation in T cell transformation

A transgenic mouse model of T cell lymphoma was used to investigate the transforming events mediated by an oncogenic tyrosine kinase in pretumorigenic CD4 −CD8 − (DN) thymocytes. Parental CD45 −/− and p56 lck-F505Y mice do not develop tumors, whereas their CD45 −/−p56 lck-F505Y progeny develop T lymphomas. Increased but nononcogenic p56 lck kinase activity in p56 lck-F505Y mice DN thymocytes causes cell-cycle progression, survival, and Bcl-X L upregulation. Additional unique oncogenic signals occur in pretumorigenic CD45 −/−p56 lck-F505Y thymocytes in which p56 lck kinase activity is 2- to 3-fold higher relative to p56 lck-F505Y: inhibition of DNA repair, inhibition of DNA-damage-induced Bcl-X L deamidation, Bax conformational change and mitochondrial translocation, cytochrome c release, and the apoptotic caspase execution cascade. Inhibition of Bcl-X L deamidation may be a critical switch in oncogenic kinase-induced T cell transformation.

Lack of evidence for aggregation‐dependent enhancement of p56lck in the signal transduction upon major histocompatibility complex recognition by mature T cells

The kinase activity of lymphocyte-specific tyrosine kinase p56lck (Lck) upon physiological major histocompatibility complex (MHC) recognition by normal mature T cells was examined. Recognition of the target MHC molecules by T cells induced phosphorylation of the zeta-chain without obvious enhancement of the background Lck activity. There was no sign of enhancement of Lck through putative T-cell receptor (TCR)-independent class II MHC/CD4 interactions either. As has been reported, cross-linking of CD4 molecules by antibodies induced a marked enhancement of Lck activity. However, it did not have an immediate relevance to TCR-mediated signal transduction, as judged from the lack of detectable de novo phosphorylation of zeta-chain and the absence of functional responses of T cells. These results strongly favour the model in which TCR-mediated signal transduction does not involve aggregation-dependent enhancement of Lck, suggesting that the signal can be triggered simply by the recruitment of already active Lck with basal kinase activity through the formation of a TCR/MHC/CD4 ternary complex.

CTL activation is induced by cross-linking of TCR/MHC-peptide-CD8/p56lck adducts in rafts.

To investigate the role of the coreceptor CD8 and lipid rafts in cytotoxic T lymphocyte (CTL) activation, we used soluble mono-and multimeric H-2Kd-peptide complexes and cloned S14 CTL specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite (PbCS) peptide 252-260 [PbCS(ABA)]. We report that activation of CTL in suspension requires multimeric Kd-PbCS(ABA) complexes co-engaging TCR and CD8. Using TCR ligand photo-cross-linking, we find that monomeric Kd-PbCS(ABA) complexes promote association of TCR/CD3 with CD8/p56lck. Dimerization of these adducts results in activation of p56lck in lipid rafts, where phosphatases are excluded. Additional cross-linking further increases p56lck kinase activity, induces translocation of TCR/CD3 and other signaling molecules to lipid rafts and intracellular calcium mobilization. These events are prevented by blocking Src kinases or CD8 binding to TCR-associated Kd molecules, indicating that CTL activation is initiated by cross-linking of CD8-associated p56lck. They are also inhibited by methyl-beta-cyclodextrin, which disrupts rafts and by dipalmitoyl phosphatidylethanolamine, which interferes with TCR signaling. Because efficient association of CD8 and p56lck takes place in rafts, both reagents, though in different ways, impair coupling of p56lck to TCR, thereby inhibiting the initial and essential activation of p56lck induced by cross-linking of engaged TCR.

The Src Homology-2 Domains (SH2 Domains) of the Protein Tyrosine Kinase p56 lck Structure, Mechanism and Drug Design

Src homology 2 (SH2) domains are found in many intercellular signal-transduction proteins which bind phosphotyrosine containing polypeptide sequences with high affinity and specificity and are considered potential targets for drug discovery. The protein p56lck is a member of the family of Src tyrosine kinase. The SH2 domain is thought to be responsible for the recruitment and regulation of p56lck kinase activity. There have been enormous efforts in the development of SH2 domain inhibitors for diseases such as cancer, osteoporosis and other diseases. This review focuses on current understanding of SH2 domain structure, mechanism and drug discovery with an emphasis on p56lck SH2 domain. A potential impact of the accumulated crystallographic effort on the development of methods for structure-based drug design is briefly addressed.

CD45-associated protein is not essential for the regulation of antigen receptor-mediated signal transduction.

CD45 is a transmembrane protein tyrosine phosphatase required for signaling through the T-and B-cell antigen receptors. In lymphocytes, CD45 interacts with CD45-associated protein (CD45AP), a 32 000 Mr phosphoprotein, through their respective transmembrane domains. To determine whether CD45AP affects the ability of CD45 to regulate antigen receptor signaling, CD45AP-deficient mice were generated. Thymocyte development was grossly normal. Moreover, the cellularity of the thymus and spleens were normal. CD45 expression on thymocytes and splenocytes, ascertained by flow cytometry, was comparable between CD45AP-deficient mice and littermate controls. In contrast to a previous report (Matsuda et al., J. Exp. Med. 1998 187: 1863 - 1870). CD45AP-deficient and normal thymocytes and splenocytes proliferated similarly in response to various mitogens or antigen receptor cross-linking. Furthermore, thymocyte CD45-associated p56(lck) kinase activity was similar between CD45AP-deficient and normal cells. We conclude that CD45AP is not essential for the regulation of Src-family kinase activity by CD45.

Inhibition of p56lckTyrosine Kinase by Isothiazolones

Lck encodes a 56-kDa protein–tyrosine kinase, predominantly expressed in T lymphocytes, crucial for initiating T cell antigen receptor (TCR) signal transduction pathways, culminating in T cell cytokine gene expression and effector functions. As a consequence of a high-throughput screen for selective, novel inhibitors of p56lck, an isothiazolone compound was identified, methyl-3-(N-isothiazolone)-2-thiophenecarboxylate(A-125800), which inhibits p56lckkinase activity with IC50= 1–7 μM. Under similar assay conditions, the isothiazolone compound was equipotent in blocking the ZAP-70 tyrosine kinase activity but was 50 to 100 times less potent against the catalytic activities of p38 MAP kinase and c-JunN-terminal kinase 2α. A-125800 blocked activation-dependent TCR tyrosine phosphorylation and intracellular calcium mobilization in Jurkat T cells (IC50= 35 μM) and blocked T cell proliferation in response to alloantigen (IC50= 14 μM) and CD3/CD28-induced IL-2 secretion (IC50= 2.2 μM) in primary T cell cultures. Inhibition of p56lckby A-125800 was dose- and time-dependent and was irreversible. A substitution of methylene for the sulfur atom in the isothiazolone ring of the compound completely abrogated the ability to inhibit p56lckkinase activity and TCR-dependent signal transduction. Incubation with thiols such as β-ME or DTT also blocked the ability of the isothiazolone to inhibit p56lckkinase activity. LC/MS analysis established the covalent modification of p56lckat cysteine residues 378, 465, and 476. Together these data support an inhibitory mechanism, whereby cysteine -SH groups within the p56lckcatalytic domain react with the isothiazolone ring, leading to ring opening and disulfide bond formation with the p56lckenzyme. Loss of p56lckactivity due to -SH oxidation has been suggested to play a role in the pathology of AIDS. Consequently, a similar mechanism of sulfhydryl oxidation leading to p56lckinhibition, described in this report, may occur in the intact T cell and may underlie certain T cell pathologies.

Ex vivo activation of tumor-draining lymph node T cells reverses defects in signal transduction molecules.

The adoptive transfer of tumor-draining lymph node (LN) T cells activated ex vivo with anti-CD3 and interleukin 2 (IL-2) mediates the regression of the poorly immunogenic murine melanoma D5. The efficacy of the activated LN cells is augmented when the sensitizing tumor is a genetically modified variant (designated D5G6) that secretes granulocyte/macrophage-colony-stimulating factor. In contrast to anti-CD3/IL-2-activated LN cells, adoptive transfer of freshly isolated tumor-draining LN T cells has no therapeutic activity. To determine whether the acquisition of antitumor function during ex vivo activation is associated with modifications in signal transduction capacity, the protein tyrosine kinases p56lck and p59fyn and proteins of the NF-kappaB family were analyzed in tumor-draining LN T cells. The levels of p56lck and p59fyn were lower in tumor-draining than in normal LN T cells and production of tyrosine-phosphorylated substrates was markedly depressed following anti-CD3 stimulation. After 5-day anti-CD3/IL-2 activation, levels of p56lck and p59fyn and protein tyrosine kinase activity increased. Interestingly, the levels of p56lck, p59fyn, and tyrosine kinase activity were higher in activated T cells derived from LN that drained D5G6 than they were in those from D5 tumors. In contrast, the cytoplasmic levels of c-Rel and Rel A were normal in freshly isolated tumor-draining LN, as was nuclear kappaB DNA-binding activity induced by anti-CD3 mAb or phorbol myristate acetate. Stimulation of activated LN cells with D5 tumor cells induced the nuclear translocation of NF-kappaB. These findings indicate that the recovery of proteins mediating signal transduction through the T cell receptor/CD3 complex in LN T cells activated ex vivo was associated with the acquisition of antitumor function.

T-Cell Receptor Signaling Pathway Exerts a Negative Control on Thrombin-Mediated Increase in [Ca2+]i and p38 MAPK Activation in Jurkat T Cells: Implication of the Tyrosine Kinase p56Lck

Activation of the mitogen-activated protein kinase (Erk) and c-Jun terminal kinase is a well-documented mechanism for the seven transmembrane spanning receptors. We have previously shown that thrombin stimulation of the T-leukemic cell line Jurkat induced a transient increase in [Ca2+]i and tyrosine phosphorylation of several cellular proteins. Here, we have analyzed p42-44 MAPK, JNK and p38 MAPK activation using Jurkat T-cell lines deficient in either the tyrosine kinase p56Lck (JCaM1) or the tyrosine phosphatase CD45 (J45.01). Our results demonstrate that p56Lck and CD45 exert a negative control on thrombin-induced p38 MAPK activation and [Ca2+]i release in Jurkat cells. Thrombin receptor expression was identical on the different cell lines as assessed by FACS analysis. Tyrosine phosphorylation of p38 MAPK was drastically increased after thrombin stimulation of JCaM1 or J45.01 cells, as compared with parental cells (JE6.1). P42-44 MAPK and JNK activity also enhanced after thrombin treatment of JE6.1 and JCaM1 cell lines, whereas basal kinase activity was higher in J45.01 cells and was not further stimulated by thrombin. Thrombin and thrombin receptor agonist peptide-induced [Ca2+]imobilization paralleled p38 MAPK activation in JCaM1 and J45.01 cells. Moreover, reconstitution of J45.01 and JCaM1 cell lines with either CD45 or Lck is accompanied by restoration of a normal thrombin-induced [Ca2+]i response and p38MAPK phosphorylation. These data show that a component of the T-cell receptor signaling pathway exerts a negative control on thrombin-induced responses in Jurkat T cells. Accordingly, we found that thrombin enhanced tyrosine phosphorylation of p56Lck and decreased p56Lck kinase activity in J45.01 cells. Our results are consistent with a negative role for p56Lck on thrombin-induced [Ca2+]i release and p38 MAPK activation in Jurkat T-cell lines.

T-Cell Receptor Signaling Pathway Exerts a Negative Control on Thrombin-Mediated Increase in [Ca2+]i and p38 MAPK Activation in Jurkat T Cells: Implication of the Tyrosine Kinase p56Lck

AbstractActivation of the mitogen-activated protein kinase (Erk) and c-Jun terminal kinase is a well-documented mechanism for the seven transmembrane spanning receptors. We have previously shown that thrombin stimulation of the T-leukemic cell line Jurkat induced a transient increase in [Ca2+]i and tyrosine phosphorylation of several cellular proteins. Here, we have analyzed p42-44 MAPK, JNK and p38 MAPK activation using Jurkat T-cell lines deficient in either the tyrosine kinase p56Lck (JCaM1) or the tyrosine phosphatase CD45 (J45.01). Our results demonstrate that p56Lck and CD45 exert a negative control on thrombin-induced p38 MAPK activation and [Ca2+]i release in Jurkat cells. Thrombin receptor expression was identical on the different cell lines as assessed by FACS analysis. Tyrosine phosphorylation of p38 MAPK was drastically increased after thrombin stimulation of JCaM1 or J45.01 cells, as compared with parental cells (JE6.1). P42-44 MAPK and JNK activity also enhanced after thrombin treatment of JE6.1 and JCaM1 cell lines, whereas basal kinase activity was higher in J45.01 cells and was not further stimulated by thrombin. Thrombin and thrombin receptor agonist peptide-induced [Ca2+]imobilization paralleled p38 MAPK activation in JCaM1 and J45.01 cells. Moreover, reconstitution of J45.01 and JCaM1 cell lines with either CD45 or Lck is accompanied by restoration of a normal thrombin-induced [Ca2+]i response and p38MAPK phosphorylation. These data show that a component of the T-cell receptor signaling pathway exerts a negative control on thrombin-induced responses in Jurkat T cells. Accordingly, we found that thrombin enhanced tyrosine phosphorylation of p56Lck and decreased p56Lck kinase activity in J45.01 cells. Our results are consistent with a negative role for p56Lck on thrombin-induced [Ca2+]i release and p38 MAPK activation in Jurkat T-cell lines.

Enhancement of HIV-1-Induced Syncytium Formation in T Cells by the Tyrosyl Kinase p56lck

The CD4 glycoprotein is the primary cellular receptor for human immunodeficiency virus type 1 (HIV-1) and has also been reported to be physically associated with p56lck, a tyrosyl protein kinase. p56lckis a member of thesrcfamily of nonreceptor protein-tyrosine kinases and is expressed predominantly in T lymphocytes. Our objective was to study the effect of p56lckon the biology of HIV-1. For this purpose, we have stably transfected two human p56lck-negative T cell lines (C8166-45 and MT-2) with plasmids encoding for this cellular protein. Following coculture with HIV-1-infected cells or infection with cell-free virus, p56lck-expressing cell lines showed a greater propensity for virus-mediated syncytium formation than parental p56lck-negative cells. The enhancement of HIV-1-induced syncytium formation was not associated with the kinase activity of p56lck, as demonstrated by experiments using a kinase-deficient mutant. However, the physical interaction between CD4 and p56lckwas shown to be necessary to obtain the enhancement of syncytium formation since a mutated version of p56lck, which is deficient in its capacity to associate with CD4, did not lead to an increase in virus-mediated cell-to-cell fusion events. Finally, we determined that cells transfected with wild-type and kinase-negative mutant p56lckshowed a reduced rate of CD4 endocytosis compared to parental p56lck-negative cells. Together, these results suggest that p56lckcan be seen as an accessory molecule facilitating HIV-1-mediated syncytium formation in T cells by a mechanism involving the stabilization of the CD4 molecule at the cell surface.

Gene activating and proapoptotic potential are independent properties of different CD4 epitopes

CD4 engagement triggers an early signaling cascade which initiates late events such as transcription factor activation. The outcome of CD4 engagement is T-cell commitment to alternative, dramatically different fates, such as activation and apoptosis. We have tested a panel of anti-CD4 mAbs specific for different CD4 epitopes, as well as HIV-1 gp120, for the capacity to activate crucial early events such as enhancement of p56lck kinase activity and Shc phosphoryation. The same CD4 epitopes were characterized for their capacity both to deliver a gene activating signal and to program T-cells to activation dependent death. No correlation could be found between capacity of specific CD4 epitopes to deliver a gene activating signal and capacity to prime T-cells to apoptotis, suggesting that gene activating and proapoptotic potential are independent functions of CD4 epitopes. Furthermore, while triggering of the calcium pathway appears critical in NF-AT activation, optimal p56lck activation and Shc phosphorylation might be required for initiation of the apoptotic pathway.

The preparation and sar of 4-(anilino), 4-(phenoxy), and 4-(thiophenoxy)-quinazolines: Inhibitors of p56 lck and EGF-R tyrosine kinase activity

We report herein our preliminary results of a SAR study of quinazoline-based inhibitors of p56 lck and EGF-R tyrosine kinase activity. 1 The most potent inhibitor of p56 lck identified, RPR-108518A ( 10), has an IC 50 of 0.50 μM. The 3-chlorophenoxy- and 3-chlorothiophenoxy- derivatives 5 and 6 were also shown to be extremely potent EGF-R inhibitors.

Herpesvirus saimiri Tip-484 membrane protein markedly increases p56lck activity in T cells.

Herpesvirus saimiri (HVS) is a T-cell-specific transforming and oncogenic virus. A protein encoded by HVS known as Tip-484 (for tyrosine kinase interacting protein from HVS strain 484) is required for this transformation. Tip-484 binds specifically to the nonreceptor protein tyrosine kinase p56lck. By transfecting Tip-484 into T cells, we now show that this interaction leads to a several hundred-fold increase in the kinase activity of p56lck. Tip-484 is part of a protein complex which is dependent on the presence of p56lck and is phosphorylated. We also show that two of the complexed proteins represent two phosphorylated forms of Tip-484. Furthermore, the p56lck kinase activity in HVS-infected human peripheral blood T lymphocytes was at least ninefold higher than that in noninfected control cells and significantly decreased in cells infected with a Tip-484 deletion mutant virus. Finally, we report that Tip-484 is required for oncogenesis in rabbits by the survival of rabbits inoculated with Tip-484 deletion mutant HVS. The data demonstrate dramatic stimulation of the signaling pathway of p56lck. This effect can contribute to the molecular mechanisms that lead to sustained autocrine secretion of growth factors, permanent T-cell growth, and ultimately lymphocytic tumor formation.