Dynamic actin polymerization drives T cell receptor-induced spreading: a role for the signal transduction adaptor LAT.

T cell activation induces functional changes in cell shape and cytoskeletal architecture. To facilitate the collection of dynamic, high-resolution images of activated T cells, we plated T cells on coverslips coated with antibodies to the T cell receptor (TCR). Using these images, we were able to quantitate the morphological responses of individual cells over time. Here, we show that TCR engagement triggers the formation and expansion of contacts bounded by continuously remodeled actin-rich rings. These processes are associated with the extension of lamellipodia and require actin polymerization, tyrosine kinase activation, cytoplasmic calcium increases, and LAT, an important hematopoietic adaptor. In addition, the maintenance of the resulting contact requires sustained calcium influxes, an intact microtubule cytoskeleton, and functional LAT.

Deficiency of PTEN in Jurkat T cells causes constitutive localization of Itk to the plasma membrane and hyperresponsiveness to CD3 stimulation.

Pleckstrin homology (PH) domain binding to D3-phosphorylated phosphatidylinositides (PI) provides a reversible means of recruiting proteins to the plasma membrane, with the resultant change in subcellular localization playing a key role in the activation of multiple intracellular signaling pathways. Previously we found that the T-cell-specific PH domain-containing kinase Itk is constitutively membrane associated in Jurkat T cells. This distribution was unexpected given that the closely related B-cell kinase, Btk, is almost exclusively cytosolic. In addition to constitutive membrane association of Itk, unstimulated JTAg T cells also exhibited constitutive phosphorylation of Akt on Ser-473, an indication of elevated basal levels of the phosphatidylinositol 3-kinase (PI3K) products PI-3,4-P(2) and PI-3,4,5-P(3) in the plasma membrane. Here we describe a defect in expression of the D3 phosphoinositide phosphatase, PTEN, in Jurkat and JTAg T cells that leads to unregulated PH domain interactions with the plasma membrane. Inhibition of D3 phosphorylation by PI3K inhibitors, or by expression of PTEN, blocked constitutive phosphorylation of Akt on Ser-473 and caused Itk to redistribute to the cytosol. The PTEN-deficient cells were also hyperresponsive to T-cell receptor (TCR) stimulation, as measured by Itk kinase activity, tyrosine phosphorylation of phospholipase C-gamma1, and activation of Erk compared to those in PTEN-replete cells. These data support the idea that PH domain-mediated association with the plasma membrane is required for Itk activation, provide evidence for a negative regulatory role of PTEN in TCR stimulation, and suggest that signaling models based on results from Jurkat T-cell lines may underestimate the role of PI3K in TCR signaling.

The conserved carboxyl-terminal half of herpes simplex virus type 1 regulatory protein ICP27 is dispensable for viral growth in the presence of compensatory mutations.

ICP27 is an essential herpes simplex virus type 1 (HSV-1) immediate-early protein that regulates viral gene expression by poorly characterized mechanisms. Previous data suggest that its carboxyl (C)-terminal portion is absolutely required for productive viral infection. In this study, we isolated M16R, a second-site revertant of a viral ICP27 C-terminal mutant. M16R harbors an intragenic reversion, as demonstrated by the fact that its cloned ICP27 allele can complement the growth of an HSV-1 ICP27 deletion mutant. DNA sequencing demonstrated that the intragenic reversion is a frameshift alteration in a homopolymeric run of C residues at codons 215 to 217. This results in the predicted expression of a truncated, 289-residue molecule bearing 72 novel C-terminal residues derived from the +1 reading frame. Consistent with this, M16R expresses an ICP27-related molecule of the predicted size in the nuclei of infected cells. Transfection-based viral complementation assays confirmed that the truncated, frameshifted protein can partially substitute for ICP27 in the context of viral infection. Surprisingly, its novel C-terminal residues are required for this activity. To see if the frameshift mutation is all that is required for M16R's viability, we re-engineered the M16R ICP27 allele and inserted it into a new viral background, creating the HSV-1 mutant M16exC. An additional mutant, exCd305, was constructed which possesses the frameshift in the context of an ICP27 gene with the C terminus deleted. We found that both M16exC and exCd305 are nonviable in Vero cells, suggesting that one or more extragenic mutations are also required for the viability of M16R. Consistent with this interpretation, we isolated two viable derivatives of exCd305 which grow productively in Vero cells despite being incapable of encoding the C-terminal portion of ICP27. Studies of viral DNA synthesis in mutant-infected cells indicated that the truncated, frameshifted ICP27 protein can enhance viral DNA replication. In summary, our results demonstrate that the C-terminal portion of ICP27, conserved widely in herpesviruses and previously believed to be absolutely essential, is dispensable for HSV-1 lytic replication in the presence of compensatory genomic mutations.

Mortality impact of an integrated community cardiovascular health program.

BACKGROUND: Preventing cardiovascular disease through community interventions makes theoretical sense but has been difficult to demonstrate. We set out to determine whether a community cardiovascular health program had an impact on mortality. DESIGN: Program evaluation plus ecologic observational analysis of program encounters and mortality rates with external comparisons. SETTING: Franklin County and two comparison counties in rural Maine. PARTICIPANTS: Program encountered >50% of regional adults, broadly distributed by site, gender, and age. INTERVENTIONS: From 1974 to 1994, a community program, integrated with primary medical care and staffed by professional nurses, provided education, screening, counseling, referral, tracking, and follow-up for cardiovascular risk factors. MAIN OUTCOME MEASURES: Age-adjusted mortality rates (total, heart, coronary, cerebrovascular, cancer) for three counties and Maine, plus annual program encounters. RESULTS: Relative to Maine, the Franklin heart disease death rate was 0.97 at baseline (1960-1969; 95% confidence interval, 0.91 to 1.03), 0.91 during the program (0.85 to 0.97), 0.83 during the 11 years of program growth (0.78 to 0.88), but 1.0 during the 10 years of decreasing encounters. Franklin's total death rate was 1.01 at baseline, 0.95 during the program (0.92 to 0.98), and 0.90 during program growth (0.86 to 0. 94). Results were similar for coronary disease, stroke, and cancer. Relative death rates did not fall in either comparison county. Nurse-client encounters totaled 120,280 over 21 years. Relative to Maine, heart disease death rates correlated inversely with program encounters (r = -0.53) but not with unemployment or physician supply. CONCLUSIONS: Integrated with primary medical care, a comprehensive, nurse-mediated community cardiovascular health program in rural Maine has been associated with significant time-dependent and dose-dependent reductions in cardiovascular and total mortality.

Biochemical interactions integrating Itk with the T cell receptor-initiated signaling cascade.

Itk, a Tec family tyrosine kinase, acts downstream of Lck and phosphatidylinositol 3'-kinase to facilitate T cell receptor (TCR)-dependent calcium influxes and increases in extracellular-regulated kinase activity. Here we demonstrate interactions between Itk and crucial components of TCR-dependent signaling pathways. First, the inositide-binding pocket of the Itk pleckstrin homology domain directs the constitutive association of Itk with buoyant membranes that are the primary site of TCR activation and are enriched in both Lck and LAT. This association is required for the transphosphorylation of Itk. Second, the Itk proline-rich region binds to Grb2 and LAT. Third, the Itk Src homology (SH3) 3 and SH2 domains interact cooperatively with Syk-phosphorylated SLP-76. Notably, SLP-76 contains a predicted binding motif for the Itk SH2 domain and binds to full-length Itk in vitro. Finally, we show that kinase-inactive Itk can antagonize the SLP-76-dependent activation of NF-AT. The inhibition of NF-AT activation depends on the Itk pleckstrin homology domain, proline-rich region, and SH2 domain. Together, these observations suggest that multivalent interactions recruit Itk to LAT-nucleated signaling complexes and facilitate the activation of LAT-associated phospholipase Cgamma1 by Itk.

T cell receptor-initiated calcium release is uncoupled from capacitative calcium entry in Itk-deficient T cells.

Itk, a Tec family tyrosine kinase, plays an important but as yet undefined role in T cell receptor (TCR) signaling. Here we show that T cells from Itk-deficient mice have a TCR-proximal signaling defect, resulting in defective interleukin 2 secretion. Upon TCR stimulation, Itk-/- T cells release normal amounts of calcium from intracellular stores, but fail to open plasma membrane calcium channels. Since thapsigargin-induced store depletion triggers normal calcium entry in Itk-/- T cells, an impaired biochemical link between store depletion and channel opening is unlikely to be responsible for this defect. Biochemical studies indicate that TCR-induced inositol 1,4,5 tris-phosphate (IP3) generation and phospholipase C gamma1 tyrosine phosphorylation are substantially reduced in Itk-/- T cells. In contrast, TCR-zeta and ZAP-70 are phosphorylated normally, suggesting that Itk functions downstream of, or in parallel to, ZAP-70 to facilitate TCR-induced IP3 production. These findings support a model in which quantitative differences in cytosolic IP3 trigger distinct responses, and in which only high concentrations of IP3 trigger the influx of extracellular calcium.

Lck phosphorylates the activation loop tyrosine of the Itk kinase domain and activates Itk kinase activity.

The Tec family tyrosine kinase Itk has been implicated in T cell receptor (TCR) signaling, yet its precise role and mechanism of activation remain undefined. To investigate these issues, we examined the biochemical response of Itk to TCR stimulation. We found that Itk is tyrosine-phosphorylated after TCR cross-linking and that this phosphorylation depends on the presence of functional Lck. To determine if this Lck dependence results from direct phosphorylation of Itk by Lck, we generated recombinant Itk and Lck using a baculovirus expression system and used these proteins in subsequent biochemical analyses. We found that Lck phosphorylates Itk upon co-expression in insect cells and, further, that this phosphorylation of Itk results in increased Itk in vitro kinase activity. The major site of Lck phosphorylation on Itk was mapped to the conserved tyrosine (Tyr511) in the activation loop of the Itk kinase domain. Substitution of this tyrosine with phenylalanine abolishes Itk kinase activity in insect cells, indicating that phosphorylation at this site plays a critical role in regulating Itk function.

Regulatory intramolecular association in a tyrosine kinase of the Tec family.

The T-cell-specific tyrosine kinase Itk is a member of the Tec family of non-receptor tyrosine kinases, and is required for signalling through the T-cell antigen receptor (TCR). The role of Itk in TCR signalling and the manner in which Itk activity is regulated are not well understood. Substrate binding and enzymatic activity of the structurally related Src kinases are regulated by an intramolecular interaction between the Src-homology-2 (SH2) domain and a phosphotyrosine. Although Itk also contains SH3, SH2 and tyrosine kinase domains, it lacks the corresponding regulatory phosphorylation site, and therefore must be regulated by an alternative mechanism. The proline-rich sequence adjacent to the SH3 domain of Tec family kinases contains an SH3 ligand, potentially allowing a different intramolecular interaction. By using multidimensional nuclear magnetic resonance we have determined the structure of a fragment of Itk, confirming that these domains interact intramolecularly. Formation of this intramolecular SH3-ligand complex prevents the Itk SH3 domain and proline-rich region from interacting with their respective protein ligands, Sam68 and Grb-2. We believe that this structure represents the first example of an intramolecular interaction between an SH3 domain and a proline-rich ligand, and has implications for the regulation of Tec family kinases.

Identification of Itk/Tsk Src homology 3 domain ligands.

The tyrosine kinase Itk/Tsk is a T cell specific analog of Btk, the tyrosine kinase defective in the human immunodeficiency X-linked agammaglobulinemia and in xid mice. T lymphocytes from Itk-deficient mice are refractory to mitogenic stimuli delivered through the T cell receptor (TCR). To gain insights into the biochemical role of Itk, the binding properties of the Itk SH3 domain were examined. An optimal Itk SH3 binding motif was derived by screening biased phage display libraries; peptides based on this motif bound with high affinity and selectivity to the Itk SH3 domain. Initial studies with T cell lysates indicated that the Itk SH3 domain bound Cbl, Fyn, and other tyrosine phosphoproteins from TCR-stimulated Jurkat cells. Under conditions of increased detergent stringency Sam 68, Wiskott-Aldrich Syndrome protein, and hnRNP-K, but not Cbl and Fyn, were bound to the Itk SH3 domain. By examining the ability of different SH3 domains to interact with deletion variants of Sam 68 and WASP, we demonstrated that the Itk-SH3 domain and the SH3 domains of Src family kinases bind to overlapping but distinct sets of proline-rich regions in Sam 68 and WASP.

p56Lck and p59Fyn regulate CD28 binding to phosphatidylinositol 3-kinase, growth factor receptor-bound protein GRB-2, and T cell-specific protein-tyrosine kinase ITK: implications for T-cell costimulation.

T-cell activation requires cooperative signals generated by the T-cell antigen receptor zeta-chain complex (TCR zeta-CD3) and the costimulatory antigen CD28. CD28 interacts with three intracellular proteins-phosphatidylinositol 3-kinase (PI 3-kinase), T cell-specific protein-tyrosine kinase ITK (formerly TSK or EMT), and the complex between growth factor receptor-bound protein 2 and son of sevenless guanine nucleotide exchange protein (GRB-2-SOS). PI 3-kinase and GRB-2 bind to the CD28 phosphotyrosine-based Tyr-Met-Asn-Met motif by means of intrinsic Src-homology 2 (SH2) domains. The requirement for tyrosine phosphorylation of the Tyr-Met-Asn-Met motif for SH2 domain binding implicates an intervening protein-tyrosine kinase in the recruitment of PI 3-kinase and GRB-2 by CD28. Candidate kinases include p56Lck, p59Fyn, zeta-chain-associated 70-kDa protein (ZAP-70), and ITK. In this study, we demonstrate in coexpression studies that p56Lck and p59Fyn phosphorylate CD28 primarily at Tyr-191 of the Tyr-Met-Asn-Met motif, inducing a 3- to 8-fold increase in p85 (subunit of PI 3-kinase) and GRB-2 SH2 binding to CD28. Phosphatase digestion of CD28 eliminated binding. In contrast to Src kinases, ZAP-70 and ITK failed to induce these events. Further, ITK binding to CD28 was dependent on the presence of p56Lck and is thus likely to act downstream of p56Lck/p59Fyn in a signaling cascade. p56Lck is therefore likely to be a central switch in T-cell activation, with the dual function of regulating CD28-mediated costimulation as well as TCR-CD3-CD4 signaling.

Pharmacokinetics of clarithromycin, a new macrolide, after single ascending oral doses.

The pharmacokinetics and safety of single ascending doses of clarithromycin (6-0-methylerythromycin A) were assessed in a placebo-controlled, double-blind, randomized trial with 39 healthy male volunteers. Subjects were randomized to receive single doses of either placebo or 100, 200, 400, 600, 800, or 1,200 mg of clarithromycin. Blood and urine collections were performed over the 24 h following administration of the test preparation. Biological specimens were analyzed for clarithromycin and 14(R)-hydroxyclarithromycin content by a high-performance liquid chromatographic technique. The pharmacokinetics of clarithromycin appeared to be dose dependent, with terminal disposition half-life ranging from 2.3 to 6.0 h and mean +/- standard deviation area under the concentration-versus-time curve from time 0 to infinity for plasma ranging from 1.67 +/- 0.48 to 3.72 +/- 1.26 mg/liter.h per 100-mg dose over the 100- to 1,200-mg dose range. Similar dose dependency was noted in the pharmacokinetics of the 14(R)-hydroxy metabolite. Mean urinary excretion of clarithromycin and its 14(R)-hydroxy metabolite ranged from 11.5 to 17.5% and 5.3 to 8.8% of the administered dose, respectively. Urinary excretion data and plasma metabolite/parent compound concentration ratio data suggested that capacity-limited formation of the active metabolite may account, at least in part, for the nonlinear pharmacokinetics of clarithromycin. No substantive dose-related trend was observed for the renal clearance of either compound. There were no clinically significant drug-related alterations in laboratory and nonlaboratory safety parameters. In addition, there was no significant difference between placebo and clarithromycin recipients in the incidence or severity of adverse events. Clarithromycin appears to be safe and well tolerated.

The efficacy of personalized audiovisual patient-education materials.

Patient education is considered an important component of primary care medicine. The traditional methods of patient education have been physician-patient dialogue and printed handouts. This study compares the relative efficacy of pamphlets, one-to-one dialogue, and audiovisual presentations. The results indicate that the slide and sound presentation was most effective in conveying patient information.

PIP: 2 primary care sites, a clinic and a private physician's office, were the sites of a study designed to compare the relative efficacy of pamphlets, 1-to-1 dialogue, and audiovisual presentations used in patient education. Respondents were female patients who presented at either site requesting contraception. The final sample of 100 patients consisted of 70 from the clinic setting and 30 from the private setting. The sample was predominantly white (63%), of low income (54% had family incomes of less than $8000), and Protestant (85%). The mean age of the subjects was 21.5 years. All 100 patients received a presentation of the same information on contraceptive methods extracted from a pamphlet distributed by a leading manufacturer of contraceptives. Before data collection, the 100 participants were randomly divided into 5 groups of 20 patients with each group receiving the educational material in 1 of 5 ways. The group that received a combination of presentations evidenced the most knowledgeable gain. Subjects who received the oral presentation alone showed the least gain. Although the combination group made the most improvement, this change was not significantly different from the increases made by the patients receiving information through the slide and sound presentations alone. The changes made by all 3 audiovisual groups were significantly better than the pamphlet or the oral group. The difference in improvement scores across various demographic groups was not statistically significant. Patients who received the combination of presentations were most satisfied with the information, while those receiving the pamphlet were least satisfied. Patients receiving the information through the combination of presentations felt that they learned the most. The amount of time physicians spent discussing contraceptives varied across groups. In particular, the time spent when the physicians were told to provide oral instructions (oral and combination groups) was significantly different from the amount of time spent when the physicians presented the contraceptive information in another manner.