Alterations in inflammatory markers after a 12-week exercise program in individuals with schizophrenia-a randomized controlled trial.

Background: In individuals with schizophrenia, inflammation is associated with depression, somatic comorbidity and reduced quality of life. Physical exercise is known to reduce inflammation in other populations, but we have only limited knowledge in the field of schizophrenia. We assessed inflammatory markers in plasma samples from individuals with schizophrenia participating in an exercise intervention randomized controlled trial. We hypothesized that (i) physical exercise would reduce levels of inflammatory markers and (ii) elevated inflammatory status at baseline would be associated with improvement in cardiorespiratory fitness (CRF) following intervention. Method: Eighty-two individuals with schizophrenia were randomized to a 12-week intervention of either high-intensity interval training (HIIT, n = 43) or active video gaming (AVG, n = 39). Participants were assessed at baseline, post intervention and four months later. The associations between exercise and the inflammatory markers soluble urokinase plasminogen activator receptor, c-reactive protein, tumor necrosis factor (TNF), soluble TNF receptor 1 and interleukin 6 (IL-6) were estimated using linear mixed effect models for repeated measures. For estimating associations between baseline inflammation and change in CRF, we used linear regression models. Results: Our main findings were (i) TNF and IL-6 increased during the intervention period for both groups. Other inflammatory markers did not change during the exercise intervention period; (ii) baseline inflammatory status did not influence change in CRF during intervention, except for a positive association between baseline IL-6 levels and improvements of CRF to post intervention for both groups. Conclusion: In our study, HIIT and AVG for 12-weeks had no reducing effect on inflammatory markers. Patients with high baseline IL-6 levels had a positive change in CRF during intervention. In order to increase our knowledge regarding association between inflammatory markers and exercise in individuals with schizophrenia, larger studies with more frequent and longer exercise bout duration are warranted.

Exploring low grade inflammation by soluble urokinase plasminogen activator receptor levels in schizophrenia: a sex-dependent association with depressive symptoms.

BACKGROUND: There is evidence of increased low grade inflammation (LGI) in schizophrenia patients. However, the inter-individual variation is large and the association with demographic, somatic and psychiatric factors remains unclear. Our aim was to explore whether levels of the novel LGI marker soluble urokinase plasminogen activator receptor (suPAR) were associated with clinical factors in schizophrenia and if such associations were sex-dependent. METHOD: In this observational study a total of 187 participants with schizophrenia (108 males, 79 females) underwent physical examination and assessment with clinical interviews (Positive and Negative Syndrome Scale (PANSS), Calgary Depression Scale for Schizophrenia (CDSS), Alcohol Use Disorder Identification Test (AUDIT), and Drug Use Disorder Identification Test (DUDIT)). Blood levels of suPAR, glucose, lipids, and high sensitivity C-reactive protein (hsCRP) were determined and body mass index (BMI) calculated. Multivariable linear regression analyses were used adjusting for confounders, and sex interaction tested in significant variables. RESULTS: Adjusting for sex, age, current tobacco smoking and BMI, we found that levels of hsCRP and depressive symptoms (CDSS) were positively associated with levels of suPAR (p < 0.001). The association between suPAR and CDSS score was significant in females (p < 0.001) but not in males. Immune activation measured by hsCRP was not associated with depressive symptoms after adjusting for BMI. CONCLUSION: Our findings indicate that increased suPAR levels are associated with depressive symptoms in females with schizophrenia, suggesting aberrant immune activation in this subgroup. Our results warrant further studies, including longitudinal follow-up of suPAR levels in schizophrenia and experimental studies of mechanisms.

A switch-variant model integrates the functions of an autoimmune variant of the phosphatase PTPN22.

The R620W polymorphism in protein tyrosine phosphatase nonreceptor type 22 (PTPN22) predisposes carriers to several autoimmune diseases. Two papers in Science Immunology and Science Signaling on this human disease-associated variant lead us to propose a new "switch-of-function" model.

Effect of a physical activity intervention on suPAR levels: A randomized controlled trial.

OBJECTIVES: Soluble urokinase-type plasminogen activator receptor (suPAR) is a novel inflammatory marker, associated with lifestyle diseases and mortality risk. No studies have investigated whether physical activity may reduce suPAR levels using a randomized controlled design. DESIGN AND METHODS: suPAR and C-reactive protein (CRP) levels were determined in blood samples from a previous randomized controlled trial with Pakistani immigrants in Norway, 2008. The study included physically inactive men that were randomized to an intervention group (supervised group exercises) or a control group and followed for 5 months. A linear regression model was used and adjusted for age, inactivity level at baseline, and mean difference in CRP levels. RESULTS: Overall, 80 and 53 participants were included in the intervention and control group, respectively. Obesity and smoking were associated with higher suPAR levels at baseline. The intervention group had a mean suPAR level of 2.65 (95% CI=2.48-2.78)ng/mL at baseline compared to 2.80 (95% CI=2.65-2.95)ng/mL at post-test, and thereby significantly increased suPAR levels after intervention (p=0.02). In the control group, mean suPAR level significantly increased from 2.93 (95% CI=2.68-3.16)ng/mL at baseline to 3.09 (95% CI=2.81-3.38)ng/mL at post-test (p=0.04). When comparing change from baseline to post-test in suPAR levels for the intervention group versus the control group, no significant change in the unadjusted model was found (ß=-0.002, 95% CI=-0.219-0.215). Similar results were found for CRP levels. CONCLUSION: There was no change in suPAR levels after regular exercise compared to a control group implying that suPAR rather reflects underlying harmful inflammatory responses associated with disease development.

Antipsychotic-associated psoriatic rash - a case report.

BACKGROUND: Antipsychotics are a heterogeneous group of drugs. Although, antipsychotics have been used for years, unexpected side effects may still occur. With this case report we focus on a possible association between psoriasis and antipsychotics. Data on the patient's course of psychiatric disease, onset of psoriasis and its evolution were extracted from the patient's medical files. CASE PRESENTATION: We present a case of a 21-year-old female diagnosed with schizophrenia. She was initially treated with quetiapine, and later switched to aripiprazole due to weight gain. After initiation of antipsychotic treatment, the patient suffered from severe psoriasis lesions. CONCLUSIONS: Antipsychotics may possess immunological properties that may be involved in immune-mediated conditions, such as psoriatic rash. Further studies are warranted to determine causality and mechanism.

Diabetic ketoacidosis and diabetes associated with antipsychotic exposure among a previously diabetes-naive population with schizophrenia: a nationwide nested case-control study.

AIMS/HYPOTHESIS: Diabetic ketoacidosis (DKA) is a potentially fatal metabolic emergency of both type 1 and type 2 diabetes. Although there is a reduced risk of type 1 diabetes in schizophrenia, the incidence of DKA is tenfold higher than that of the general population. Thus, we aimed to investigate associations between exposure to antipsychotic medication (within 3 months prior to event) and DKA, type 1 diabetes and type 2 diabetes. We also reported related, clinically relevant outcomes. METHODS: Using a nested case-control study design, we identified cases of DKA, type 1 diabetes and type 2 diabetes in a previously diabetes-naive population with schizophrenia in Denmark from 1995 to 2014. Cases were matched (by age, sex and year of schizophrenia onset) 1:5 to schizophrenic control individuals who were alive and had not emigrated prior to event. Conditional logistic regression was used to compute ORs with 95% CIs. Other outcomes included diabetes aetiology of DKA, in-hospital mortality, DKA readmissions and temporal trends of use of insulin and oral glucose-lowering agents. RESULTS: Of 29,955 individuals with schizophrenia, we identified 28 individuals with DKA, 90 with type 1 diabetes and 2140 with type 2 diabetes. These were matched to 137, 410 and 9861 individuals in the control group, respectively. Antipsychotic exposure was associated with DKA (OR 2.60; 95% CI 1.06, 6.38) and type 2 diabetes (OR 1.64; 95% CI 1.48, 1.83). A trend towards increased risk of type 1 diabetes was found but remained insignificant (OR 1.38; 95% CI 0.84, 2.29). Diabetes aetiology of DKA was type 1 in eight cases and type 2 in 14 cases. Of the remaining six cases of DKA, aetiology could not be determined, as four were fatal within 8 days and for two, no prescriptions for insulin and oral glucose-lowering agents were redeemed. Of all DKA cases, six had more than one episode of DKA, and of all type 1 diabetes and type 2 diabetes cases, four and 11, respectively, had at least one episode. Use of insulin and oral glucose-lowering agents was higher among individuals with DKA relative to those with type 1 diabetes and type 2 diabetes. CONCLUSIONS/INTERPRETATION: Antipsychotic exposure was associated with DKA and type 2 diabetes in a previously diabetes-naive schizophrenia population. Antipsychotic-associated DKA is relevant not only for psychiatrists but also for other physicians who may manage and admit such patients.

Diabetic ketoacidosis in patients exposed to antipsychotics: a systematic literature review and analysis of Danish adverse drug event reports.

RATIONALE: Patients exposed to second-generation antipsychotics (SGAs) have approximately 10 times increased risk of diabetic ketoacidosis (DKA) compared with the general population. However, as DKA is a rare complication of type 2 diabetes mellitus, and susceptible patients exposed to antipsychotics may rapidly develop DKA independently of treatment duration and weight gain, this is rather suggestive of type 1 diabetes mellitus (T1DM) or latent autoimmune diabetes in adults. OBJECTIVES: We performed a systematic review of current studies regarding antipsychotic-associated DKA with type 1 etiology and analyzed Danish adverse drug event (ADE) reports (previously unpublished cases). METHODS: PubMed, Embase, and the Cochrane Library were searched for all relevant studies, and the Danish Medicines Agency retrieved ADE reports using the Danish ADE database (up to date as of June 28, 2016). Diagnosis of antipsychotic-associated DKA with type 1 etiology was either considered confirmed or possible depending on authors' conclusions in the studies and/or clinical aspects. In addition, clinico-demographic risk factors were extracted. RESULTS: A total of 655 records and 11 ADE reports were identified, and after screening for eligibility, we included 21 case reports/series and two ADE reports (n = 24). No relevant clinical studies were included. Although fatal cases were identified, these were excluded because of diagnostic uncertainties (n = 15). DKA occurred in 15 males (62.5 %) and nine females (37.5 %), with a mean age ± standard deviation of 34.8 ± 12.4 years. Median time to DKA was 5 months (interquartile range: 1.4-11 months). Associated antipsychotics were olanzapine (n = 9, 36 %), aripiprazole (n = 6, 24 %), risperidone (n = 6, 24 %), clozapine (n = 3, 12 %), and quetiapine (n = 1, 4 %). Nine patients (37.5 %) were confirmedly diagnosed with T1DM following DKA resolution, whereas 15 patients (62.5 %) had possible T1DM. In 22 patients (91.7 %), ongoing insulin treatment was required for glycemic control. CONCLUSIONS: Increased awareness of the potential risk of antipsychotic-associated DKA and subsequent T1DM diagnosis, with insulin requirements for glycemic control, is warranted. The underlying mechanisms are poorly understood but most probably multifactorial. Certainly, further studies are warranted. Clinicians must utilize appropriate monitoring in susceptible patients and consider the possibility of continuing antipsychotic treatment with appropriate diabetic care.

Eosinophilic myocarditis during treatment with olanzapine - report of two possible cases.

BACKGROUND: Drug-induced eosinophilic myocarditis is a life-threatening and frequently overlooked condition. The prevalence of myocarditis in clozapine-treated patients may be as high as 3 %. An association between olanzapine and myocarditis has not previously been described, but given the chemical similarity between olanzapine and clozapine, we hypothesized the existence of such an association. We searched the spontaneous adverse drug reports database of the Danish Health and Medicines Authority for olanzapine and myocarditis in the period from October 21, 1996 to - June 03, 2015. We identified two fatal cases of eosinophilic myocarditis associated with the use of olanzapine. CASE PRESENTATION: Case 1 was a 39-year-old Caucasian man with known substance abuse and schizophrenia. He was found dead in his home. Olanzapine was prescribed at day -54, and dose at time of death was 40 mg/day. Post-mortem toxicological examination demonstrated presence of olanzapine, morphine, venlafaxine and oxazepam. Syringes indicating substance abuse were found in his home. Case 2 was a 36-year-old Caucasian man diagnosed with schizophrenia was found dead unexpectedly. There was no history of substance abuse. Current treatment was olanzapine 20 mg/day +5 mg as PRN (prescribed for almost 4 years), aripiprazole 30 mg/day (prescribed for 6 months) and mirtazapine 30 mg/day (prescribed for 6 months). Both cases of eosinophilic myocarditis were confirmed by autopsy findings and both patients received olanzapine in doses exceeding the recommendations. CONCLUSION: Olanzapine may have contributed to and/or worsened the two reported fatal cases of myocarditis. Additional studies are required to establish a causal link between olanzapine and eosinophilic myocarditis.

Soluble urokinase-type plasminogen activator receptor levels in patients with schizophrenia.

BACKGROUND: The etiology of schizophrenia remains largely unknown but alterations in the immune system may be involved. In addition to the psychiatric symptoms, schizophrenia is also associated with up to 20 years reduction in life span. Soluble urokinase-type plasminogen activator receptor (suPAR) is a protein that can be measured in blood samples and reflects the levels of inflammatory activity. It has been associated with mortality and the development of type 2 diabetes and cardiovascular disease. METHODS: suPAR levels in patients with schizophrenia were compared to healthy controls from the Danish Blood Donor Study. SuPAR levels were dichotomized at >4.0 ng/ml, which is considered the threshold for low grade inflammation. A multiple logistic regression model was used and adjusted for age, sex, and current smoking. RESULTS: In total we included 1009 subjects, 105 cases with schizophrenia (10.4%) and 904 controls (89.6%). The mean suPAR values were 4.01 ng/ml (SD = 1.43) for the cases vs 1.91 ng/ml (SD = 1.35) for the controls (P < .001). Multiple logistic regression with odds ratio (OR) for suPAR levels >4.0 ng/ml yielded: schizophrenia, OR: 46.15 95% CI 22.69-93.87, P < .001; age, OR: 1.02 95% CI 0.99-1.02, P = .15; male sex, OR: 0.70 95% CI 0.35-1.36, P = .29; and current smoking, OR: 3.51 95% CI 1.78-6.94, P < .001. CONCLUSIONS: Patients with schizophrenia had significantly higher suPAR levels than healthy controls. Further studies are warranted to clarify if elevated suPAR levels are involved in the pathophysiology of schizophrenia and/or the increased mortality found in patients with schizophrenia.

The autoimmune-predisposing variant of lymphoid tyrosine phosphatase favors T helper 1 responses.

The C1858T single nucleotide polymorphism in PTPN22, which is the gene encoding lymphoid tyrosine phosphatase (LYP), confers increased risk for various autoimmune disorders in Caucasians. Although the disease-associated LYP allele (LYP*W620) is a gain-of-function variant that has higher catalytic activity than the major allele (LYP*R620), it is still unclear how LYP*W620 predisposes for autoimmunity. Here, we compared both T cell signaling and T cell function in healthy human donors homozygous for either LYP*R620 or LYP*W620. Generally, the presence of LYP*W620 caused reduced proximal T cell antigen receptor-mediated signaling (e.g. ζ chain phosphorylation) but augmented CD28-associated signaling (e.g. AKT activation). Altered ligand binding properties of the two LYP variants could explain these findings since LYP*R620 interacted more strongly with the p85 subunit of PI3K. Variation in signaling between cells expressing either LYP*R620 or LYP*W620 also affected the differentiation of conventional CD4(+) T cells. For example, LYP*W620 homozygous donors displayed exaggerated Th1 responses (e.g. IFNγ production) and reduced Th17 responses (e.g. IL-17 production). Importantly, while regulatory T cells normally suppressed Th1-mediated IFNγ production in LYP*R620 homozygous individuals, such suppression was lost in LYP*W620 homozygous individuals. Altogether, these findings provide a molecular and cellular explanation for the autoimmune phenotype associated with LYP*W620.

LYP inhibits T-cell activation when dissociated from CSK.

Lymphoid tyrosine phosphatase (LYP) and C-terminal Src kinase (CSK) are negative regulators of signaling mediated through the T-cell antigen receptor (TCR) and are thought to act in a cooperative manner when forming a complex. Here we studied the spatiotemporal dynamics of the LYP-CSK complex in T cells. We demonstrate that dissociation of this complex is necessary for recruitment of LYP to the plasma membrane, where it downmodulates TCR signaling. Development of a potent and selective chemical probe of LYP confirmed that LYP inhibits T-cell activation when removed from CSK. Our findings may explain the reduced TCR-mediated signaling associated with a single-nucleotide polymorphism that confers increased risk for certain autoimmune diseases, including type 1 diabetes and rheumatoid arthritis, and results in expression of a mutant LYP that is unable to bind CSK. Our compound also represents a starting point for the development of a LYP-based treatment of autoimmunity.

T cell-signaling network analysis reveals distinct differences between CD28 and CD2 costimulation responses in various subsets and in the MAPK pathway between resting and activated regulatory T cells.

To uncover signaling system differences between T cell stimuli and T cell subsets, phosphorylation status of 18 signaling proteins at six different time points following TCR triggering and CD28/CD2 costimulation was examined in human T cell subsets by phospho-epitope-specific flow cytometry of fluorescent cell barcoded samples, thereby providing a high-resolution signaling map. Compared with effector/memory T cells, naive T cells displayed stronger activation of proximal signaling molecules after TCR triggering alone. Conversely, distal phosphorylation events, like pErk and pS6-ribosomal protein, were stronger in effector/memory subsets. CD28 costimulation specifically induced signaling necessary for proper NF-κB activation, whereas CD2 signaled more strongly to S6-ribosomal protein. Analysis of resting regulatory T cells (rTregs; CD4(+)CD45RA(+)FOXP3(+)) and activated regulatory T cells (actTregs; CD4(+)CD45RA(-)FOXP3(++)) revealed that, although rTregs had low basal, but inducible, Erk activity, actTregs displayed high basal Erk phosphorylation and little or no Akt activation. Interestingly, the use of Mek inhibitors to block Erk activation inhibited activation-dependent FOXP3 upregulation in rTregs, their transition to actTregs, and the resulting increase in suppressive capacity. In summary, our systems approach unraveled distinct differences in signaling elicited by CD28 and CD2 costimulation and between rTregs and actTregs. Blocking rTreg transition to highly suppressive actTregs by Mek inhibitors might have future therapeutic applications.

Inhibition of lymphoid tyrosine phosphatase by benzofuran salicylic acids.

The lymphoid tyrosine phosphatase (Lyp, PTPN22) is a critical negative regulator of T cell antigen receptor (TCR) signaling. A single-nucleotide polymorphism (SNP) in the ptpn22 gene correlates with the incidence of various autoimmune diseases, including type 1 diabetes, rheumatoid arthritis, and systemic lupus erythematosus. Since the disease-associated allele is a more potent inhibitor of TCR signaling, specific Lyp inhibitors may become valuable in treating autoimmunity. Using a structure-based approach, we synthesized a library of 34 compounds that inhibited Lyp with IC(50) values between 0.27 and 6.2 µM. A reporter assay was employed to screen for compounds that enhanced TCR signaling in cells, and several inhibitors displayed a dose-dependent, activating effect. Subsequent probing for Lyp's direct physiological targets by immunoblot analysis confirmed the ability of the compounds to inhibit Lyp in T cells. Selectivity profiling against closely related tyrosine phosphatases and in silico docking studies with the crystal structure of Lyp yielded valuable information for the design of Lyp-specific compounds.

Multidentate small-molecule inhibitors of vaccinia H1-related (VHR) phosphatase decrease proliferation of cervix cancer cells.

Loss of VHR phosphatase causes cell cycle arrest in HeLa carcinoma cells, suggesting that VHR inhibition may be a useful approach to halt the growth of cancer cells. We recently reported that VHR is upregulated in several cervix cancer cell lines as well as in carcinomas of the uterine cervix. Here we report the development of multidentate small-molecule inhibitors of VHR that inhibit its enzymatic activity at nanomolar concentrations and exhibit antiproliferative effects on cervix cancer cells. Chemical library screening was used to identify hit compounds, which were further prioritized in profiling and kinetic experiments. SAR analysis was applied in the search for analogs with improved potency and selectivity, resulting in the discovery of novel inhibitors that are able to interact with both the phosphate-binding pocket and several distinct hydrophobic regions within VHR's active site. This multidentate binding mode was confirmed by X-ray crystallography. The inhibitors decreased the proliferation of cervix cancer cells, while growth of primary normal keratinocytes was not affected. These compounds may be a starting point to develop drugs for the treatment of cervical cancer.

The heterotrimeric G-protein alpha-subunit Galphaq regulates TCR-mediated immune responses through an Lck-dependent pathway.

Here, we examined the functional involvement of heterotrimeric G-proteins in TCR-induced immune responses. TCR/CD3 crosslinking resulted in activation of both Galphaq and Galphas, but not Galphai-2. Targeting of Galphas, Galphai-2 and Galphaq using siRNA demonstrated a specific role of Galphaq in TCR signaling. Jurkat TAg T cells with Galphaq knockdown displayed reduced activation of Lck and LAT phosphorylation, but paradoxically showed sustained ERK1/2 phosphorylation and increased NFAT-AP-1-reporter activity implicating Galphaq in the negative control of downstream signaling and IL-2-promoter activity. Primary T cells isolated from Galphaq-deficient mice had a similar TCR signaling response with reduced proximal LAT phosphorylation, sustained ERK1/2 phosphorylation and augmented immune responses including increased secretion of IL-2, IL-5, IL-12 and TNF-alpha. The effects on NFAT-AP-1-reporter activity were sensitive to the Src family kinase inhibitor PP2 and were reversed by transient expression of constitutively active Lck. Furthermore, expression of constitutively active Galphaq Q209L elevated Lck activity and Zap-70 phosphorylation. Together these data argue for a role of Galphaq in the fine-tuning of proximal TCR signals at the level of Lck and a negative regulatory role of Galphaq in transcriptional activation of cytokine responses.

TCR-induced downregulation of protein tyrosine phosphatase PEST augments secondary T cell responses.

We report that the protein tyrosine phosphatase PTP-PEST is expressed in resting human and mouse CD4(+) and CD8(+) T cells, but not in Jurkat T leukemia cells, and that PTP-PEST protein, but not mRNA, was dramatically downregulated in CD4(+) and CD8(+) primary human T cells upon T cell activation. This was also true in mouse CD4(+) T cells, but less striking in mouse CD8(+) T cells. PTP-PEST reintroduced into Jurkat at levels similar to those in primary human T cells, was a potent inhibitor of TCR-induced transactivation of reporter genes driven by NFAT/AP-1 and NF-kappaB elements and by the entire IL-2 gene promoter. Introduction of PTP-PEST into previously activated primary human T cells also reduced subsequent IL-2 production by these cells in response to TCR and CD28 stimulation. The inhibitory effect of PTP-PEST was associated with dephosphorylation the Lck kinase at its activation loop site (Y394), reduced early TCR-induced tyrosine phosphorylation, reduced ZAP-70 phosphorylation and inhibition of MAP kinase activation. We propose that PTP-PEST tempers T cell activation by dephosphorylating TCR-proximal signaling molecules, such as Lck, and that down-regulation of PTP-PEST may be a reason for the increased response to TCR triggering of previously activated T cells.

Protein tyrosine phosphatases in autoimmunity.

Protein tyrosine phosphatases (PTPs) are important regulators of many cellular functions and a growing number of PTPs have been implicated in human disease conditions, such as developmental defects, neoplastic disorders, and immunodeficiency. Here, we review the involvement of PTPs in human autoimmunity. The leading examples include the allelic variant of the lymphoid tyrosine phosphatase (PTPN22), which is associated with multiple autoimmune diseases, and mutations that affect the exon-intron splicing of CD45 (PTPRC). We also find it likely that additional PTPs are involved in susceptibility to autoimmune and inflammatory diseases. Finally, we discuss the possibility that PTPs regulating the immune system may serve as therapeutic targets.

A weak Lck tail bite is necessary for Lck function in T cell antigen receptor signaling.

Src family kinases are suppressed by a "tail bite" mechanism, in which the binding of a phosphorylated tyrosine in the C terminus of the protein to the Src homology (SH) 2 domain in the N-terminal half of the protein forces the catalytic domain into an inactive conformation stabilized by an additional SH3 interaction. In addition to this intramolecular suppressive function, the SH2 domain also mediates intermolecular interactions, which are crucial for T cell antigen receptor (TCR) signaling. To better understand the relative importance of these two opposite functions of the SH2 domain of the Src family kinase Lck in TCR signaling, we created three mutants of Lck in which the intramolecular binding of the C terminus to the SH2 domain was strengthened. The mutants differed from wild-type Lck only in one to three amino acid residues following the negative regulatory tyrosine 505, which was normally phosphorylated by Csk and dephosphorylated by CD45 in the mutants. In the Lck-negative JCaM1 cell line, the Lck mutants had a much reduced ability to transduce signals from the TCR in a manner that directly correlated with SH2-Tyr(P)(505) affinity. The mutant with the strongest tail bite was completely unable to support any ZAP-70 phosphorylation, mitogen-activated protein kinase activation, or downstream gene activation in response to TCR ligation, whereas other mutants had intermediate abilities. Lipid raft targeting was not affected. We conclude that Lck is regulated by a weak tail bite to allow for its activation and service in TCR signaling, perhaps through a competitive SH2 engagement mechanism.

Hypophosphorylated TCR/CD3zeta signals through a Grb2-SOS1-Ras pathway in Lck knockdown cells.

Despite the loss of proximal TCR-dependent signaling events, downstream T cell responses are paradoxically augmented in T cells with siRNA-mediated Lck knockdown (Methi et al., J. Immunol. 2005. 175: 7398-7406). This indicates that alternative Lck-independent pathways of T cell activation exist or that low levels of Lck elicit other signals than normal T cell activation. Here we report the recruitment of Grb2-SOS1 to CD3zeta of the TCR complex after prolonged anti-CD3 (OKT3) stimulation in T cells with Lck knockdown. Grb2 bound to incompletely phosphorylated ITAM1 with the pY-Y configuration in a solid-phase assay, but was excluded by ZAP-70 in the doubly phosphorylated pY-pY conformation. Ras and ERK1/2 activation was augmented after prolonged stimulation in T cells with Lck knockdown compared to control, leading to increased activation of the proximal IL-2 promoter (NFAT-AP-1). Finally, the phosphorylation of Ras-GAP was strongly suppressed in Lck knockdown cells, indicating that a Ras negative feedback mechanism is dependent on Lck.