Inhibitory Adaptor Protein Research Articles

TOLLIP inhibits lipid accumulation and the integrated stress response in alveolar macrophages to control Mycobacterium tuberculosis infection.

A polymorphism causing deficiencies in Toll-interacting protein (TOLLIP), an inhibitory adaptor protein affecting endosomal trafficking, is associated with increased tuberculosis (TB) risk. It is, however, unclear how TOLLIP affects TB pathogenesis. Here we show that TB severity is increased in Tollip-/- mice, characterized by macrophage- and T cell-driven inflammation, foam cell formation and lipid accumulation. Tollip-/- alveolar macrophages (AM) specifically accumulated lipid and underwent necrosis. Transcriptional and protein analyses of Mycobacterium tuberculosis (Mtb)-infected, Tollip-/- AM revealed increased EIF2 signalling and downstream upregulation of the integrated stress response (ISR). These phenotypes were linked, as incubation of the Mtb lipid mycolic acid with Mtb-infected Tollip-/- AM activated the ISR and increased Mtb replication. Correspondingly, the ISR inhibitor, ISRIB, reduced Mtb numbers in AM and improved Mtb control, overcoming the inflammatory phenotype. In conclusion, targeting the ISR offers a promising target for host-directed anti-TB therapy towards improved Mtb control and reduced immunopathology.

The role of LNK/SH2B3 genetic alterations in myeloproliferative neoplasms and other hematological disorders.

Malignant hematological diseases are mainly because of the occurrence of molecular abnormalities leading to the deregulation of signaling pathways essential for precise cell behavior. High-resolution genome analysis using microarray and large-scale sequencing have helped identify several important acquired gene mutations that are responsible for such signaling deregulations across different hematological malignancies. In particular, the genetic landscape of classical myeloproliferative neoplasms (MPNs) has been in large part completed with the identification of driver mutations (targeting the cytokine receptor/Janus-activated kinase 2 (JAK2) pathway) that determine MPN phenotype, as well as additional mutations mainly affecting the regulation of gene expression (epigenetics or splicing regulators) and signaling. At present, most efforts concentrate in understanding how all these genetic alterations intertwine together to influence disease evolution and/or dictate clinical phenotype in order to use them to personalize diagnostic and clinical care. However, it is now evident that factors other than somatic mutations also play an important role in MPN disease initiation and progression, among which germline predisposition (single-nucleotide polymorphisms and haplotypes) may strongly influence the occurrence of MPNs. In this context, the LNK inhibitory adaptor protein encoded by the LNK/SH2B adaptor protein 3 (SH2B3) gene is the target of several genetic variations, acquired or inherited in MPNs, lymphoid leukemia and nonmalignant hematological diseases, underlying its importance in these pathological processes. As LNK adaptor is a key regulator of normal hematopoiesis, understanding the consequences of LNK variants on its protein functions and on driver or other mutations could be helpful to correlate genotype and phenotype of patients and to develop therapeutic strategies to target this molecule. In this review we summarize the current knowledge of LNK function in normal hematopoiesis, the different SH2B3 mutations reported to date and discuss how these genetic variations may influence the development of hematological malignancies.

Role of Toll-Interacting Protein Gene Polymorphisms in Leprosy Mexican Patients

Background. Leprosy is a debilitating infectious disease of human skin and nerves. Genetics factors of the host play an important role in the disease susceptibility. Toll-interacting protein (TOLLIP) is an inhibitory adaptor protein within the toll-like receptor (TLR) pathway, which recognizes structurally conserved molecular patterns of microbial pathogens, initiating immune responses. The objective of this study was to investigate the association of variants in the TOLLIP gene with susceptibility to leprosy in Mexican patients. Methods. TOLLIP polymorphisms were studied using a case-control design of Mexican patients with lepromatous leprosy (LL). The polymorphisms of TOLLIP at loci −526 C>G (rs5743854), 1309956C>T (rs3750920), 1298430C>A (rs5744015), and 1292831 G>A (rs3750919) were analyzed by PCR, with sequence-specific primers in LL patients and healthy subjects (HS) as controls. Results. Genotype distributions were in Hardy Weinberg equilibrium for all sites except for rs3750920. Neither genotype nor allele frequencies were statistically different between LL patients and controls (P > 0.05). The maximum pairwise D' coefficient reached was 0.44 of linkage (P = 0.01) for all the polymorphisms except for rs5743854. The three loci haplotype comparison yielded no significant differences between groups. Conclusions. Just the individuals with genotype C/C of rs3750920 have a trend of protective effect to developing LL.

Homeostasis of hematopoietic stem cells regulated by the myeloproliferative disease associated-gene product Lnk/Sh2b3 via Bcl-xL

Hematopoietic stem cells (HSCs) are maintained at a very low frequency in adult bone marrow under steady-state conditions. However, it is not fully understood how homeostasis of bone marrow HSCs is maintained. We attempted to identify a key molecule involved in the regulation of HSC numbers, a factor that, in the absence of Lnk, leads to HSC expansion. Here, we demonstrate that upon stimulation with thrombopoietin, expression of Bcl-xL, an antiapoptotic protein, was highly enhanced in Lnk-deficient HSCs compared to normal HSCs. As a result, Lnk-deficient HSCs underwent reduced apoptosis following exposure to lethal radiation. Downregulation of Bcl-xL expression in Lnk-deficient HSCs by short-hairpin RNA resulted in a great reduction of their capacity for reconstitution. These findings suggest that Lnk/Sh2b3 constrains the expression of Bcl-xL and that the loss of Lnk/Sh2b3 function enhances survival of HSCs by inhibiting apoptosis. Furthermore, our observations indicate that HSCs in patients with an Lnk/Sh2b3 mutation might become resistant to apoptosis due to thrombopoietin-mediated enhanced expression of Bcl-xL. Consequently, reduced apoptosis could facilitate accumulation of HSCs with oncogenic mutations leading to development of myeloproliferative disorders.

Genetics of the myeloproliferative neoplasms

Since the discovery of the JAK2V617F mutation in 2005, an increasing number of somatic and germline genetic events responsible for myeloproliferative neoplasm (MPN) pathogenesis have been uncovered. The purpose of this review is to outline the most recent discoveries of the genetic alterations found in patients with MPNs. In addition to the JAK2V617F mutation, additional mutations in the JAK–STAT pathway have been discovered including a series of mutations in exon 12 of JAK2, the thrombopoietic receptor gene MPL, and in the gene encoding the JAK–STAT inhibitory adaptor protein LNK. Additionally, mutations in genes which appear to affect the epigenome of MPN patients have been discovered including mutations in TET2, IDH1/ 2, EZH2, and ASXL1. Lastly, some insights into the genetic events which contribute to transformation of a chronic MPN phenotype to acute myeloid leukemia have been elucidated, including deletion of the transcription factor Ikaros. The spectrum of genetic abnormalities found in the classic MPNs has increased over the last 6 years and somatic mutations in JAK2, MPL, LNK, TET2, EZH2, ASXL1, and IDH1/2 have all been described. Despite this, the initiating genetic events responsible for the development of MPNs is still not totally understood.

Expression level and differential JAK2-V617F–binding of the adaptor protein Lnk regulates JAK2-mediated signals in myeloproliferative neoplasms

AbstractActivating mutations in signaling molecules, such as JAK2-V617F, have been associated with myeloproliferative neoplasms (MPNs). Mice lacking the inhibitory adaptor protein Lnk display deregulation of thrombopoietin/thrombopoietin receptor signaling pathways and exhibit similar myeloproliferative characteristics to those found in MPN patients, suggesting a role for Lnk in the molecular pathogenesis of these diseases. Here, we showed that LNK levels are up-regulated and correlate with an increase in the JAK2-V617F mutant allele burden in MPN patients. Using megakaryocytic cells, we demonstrated that Lnk expression is regulated by the TPO-signaling pathway, thus indicating an important negative control loop in these cells. Analysis of platelets derived from MPN patients and megakaryocytic cell lines showed that Lnk can interact with JAK2-WT and V617F through its SH2 domain, but also through an unrevealed JAK2-binding site within its N-terminal region. In addition, the presence of the V617F mutation causes a tighter association with Lnk. Finally, we found that the expression level of the Lnk protein can modulate JAK2-V617F–dependent cell proliferation and that its different domains contribute to the inhibition of multilineage and megakaryocytic progenitor cell growth in vitro. Together, our results indicate that changes in Lnk expression and JAK2-V617F–binding regulate JAK2-mediated signals in MPNs.

Novel mutations in the inhibitory adaptor protein LNK drive JAK-STAT signaling in patients with myeloproliferative neoplasms

AbstractDysregulated Janus kinase–signal transducer and activator of transcription (JAK-STAT) signaling due to activation of tyrosine kinases is a common feature of myeloid malignancies. Here we report the first human disease-related mutations in the adaptor protein LNK, a negative regulator of JAK-STAT signaling, in 2 patients with JAK2 V617F–negative myeloproliferative neoplasms (MPNs). One patient exhibited a 5 base-pair deletion and missense mutation leading to a premature stop codon and loss of the pleckstrin homology (PH) and Src homology 2 (SH2) domains. A second patient had a missense mutation (E208Q) in the PH domain. BaF3-MPL cells transduced with these LNK mutants displayed augmented and sustained thrombopoietin-dependent growth and signaling. Primary samples from MPN patients bearing LNK mutations exhibited aberrant JAK-STAT activation, and cytokine-responsive CD34+ early progenitors were abnormally abundant in both patients. These findings indicate that JAK-STAT activation due to loss of LNK negative feedback regulation is a novel mechanism of MPN pathogenesis.

Abstract B6: Identification of novel mutations in the inhibitory adaptor protein LNK in patients with JAK2 V617F-negative and -positive chronic myeloproliferative neoplasms

Abstract Dysregulated JAK-STAT signaling is a hallmark of myeloproliferative neoplasms (MPNs), as evidenced by the identification of activating mutations in JAK2, and the thrombopoietin (TPO) receptor MPL in a subset of MPN patients. Clinical trials with highly specific inhibitors of JAK2 are currently ongoing, and clinical responses have been observed in the majority of MPN patients, validating JAK2 as an important therapeutic target in these patients. In addition, responses have been observed in patients lacking known mutations in JAK2 or MPL, suggesting that other regulatory elements in this pathway are altered. However, the molecular basis for this observation is not well understood. One regulator of JAK-STAT signaling is LNK (SH2B3), a member of a family of adaptor proteins that share several structural motifs, including a proline-rich N-terminal dimerization domain (Pro/DD), a pleckstrin homology (PH) domain, an SH2 domain, and a conserved C-terminal tyrosine residue. LNK binds to MPL via its SH2 domain and co-localizes to the plasma membrane via its PH domain. Upon cytokine stimulation with TPO, LNK binds strongly to JAK2 and inhibits downstream STAT activation, thereby providing critical negative feedback regulation. LNK-/- mice exhibit an MPN phenotype, including an expanded hematopoietic stem cell compartment, megakaryocyte hyperplasia, splenomegaly, leukocytosis, and thrombocytosis. We sequenced LNK in a cohort of MPN patients, leading to the identification of novel mutations in 7/159 (4.4%) patients. One patient with JAK2 V617F-negative primary myelofibrosis (PMF) exhibited a 5 base-pair deletion and missense mutation (DEL) leading to a premature stop codon and loss of the PH and SH2 domains. Six additional patients were found to have point mutations affecting conserved residues in the PH domain. Interestingly, a point mutation leading to an E208Q substitution was found in one JAK2 V617F- negative patient with essential thrombocythemia (ET), as well as one JAK2 V617F-positive ET patient. Similarly, a P242S substitution was also found in both a JAK2 V617F-negative ET patient, as well as a JAK2 V617F-positive patient with post-polycythemic myelofibrosis. These latter findings suggest that even in the presence of the JAK2 V617F mutation, abrogation of LNK function may be a cooperating pathogenetic mutation. TPO-dependent BaF3-MPL cells transduced with the LNK DEL mutant exhibited augmented and sustained TPO-dependent growth and activation of JAK2-STAT3/5. The E208Q mutation resulted in partial loss of LNK function, suggesting that LNK mutations may confer a spectrum of phenotypes. Primary patient samples from MPN patients bearing the LNK DEL and E208Q mutations exhibited aberrant JAK-STAT activation, and cytokine-responsive CD34+ early progenitors were abnormally abundant. The STAT3/5 activation response was abrogated by JAK inhibition, suggesting that JAK2 inhibitors may be a feasible option for MPN patients bearing LNK mutations. Our identification of mutations in LNK, the first reported in human disease, demonstrates that loss of JAK-STAT negative feedback control is a novel mechanism of MPN pathogenesis. As each of these LNK mutations localizes to the PH domain and appears to be heterozygous, mislocalized mutant LNK may exert a dominant negative effect by binding and sequestering wild-type LNK. These findings may also partly explain why some MPN patients lacking JAK2 or MPL mutations respond to treatment with JAK2 inhibitors, and highlight the importance of a more complete understanding of the role of inhibitory pathways in MPN pathogenesis. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B6.

Abstract 239: Mutation of the inhibitory adaptor protein LNK drives potentiated JAK-STAT signaling in patients with JAK2 V617F-negative myeloproliferative neoplasms

Abstract Dysregulated JAK-STAT signaling via activating mutations in tyrosine kinases (e.g. JAK2 and MPL) is a hallmark of chronic myeloproliferative neoplasms (MPNs). Even in the absence of mutations in JAK2 or MPL, JAK-STAT activation can be demonstrated, suggesting that alterations of other regulatory elements in this pathway may contribute to MPN pathobiology. One regulator of JAK-STAT signaling is LNK (SH2B3), an adaptor protein that binds to MPL via it SH2 domain and co-localizes to the plasma membrane via its pleckstrin homology (PH) domain. Upon cytokine stimulation, LNK binds strongly to JAK2 and dampens or terminates downstream STAT activation. LNK−/- mice exhibit features consistent with an MPN phenotype, including splenomegaly, leukocytosis, and thrombocytosis. We therefore sequenced LNK in 34 JAK2 V617F-negative MPN patients, and report the identification of novel mutations in exon 2 of LNK in two patients. In a patient with primary myelofibrosis, a 5 base-pair deletion and missense mutation (DEL) leading to a premature stop codon and loss of the PH and SH2 domains was identified. A second patient with essential thrombocythemia exhibited a missense mutation leading to an E208Q substitution in the PH domain. DNA isolated from cultured skin fibroblasts revealed wild-type (WT) sequence, confirming that these mutations were somatic. TPO-dependent BaF3-MPL cells were transduced with WT and mutant LNK. While WT LNK inhibited TPO-dependent growth and activation of JAK2-STAT3/5, the DEL mutation led to loss of these negative feedback properties, thereby permitting augmented and sustained JAK-STAT activation in response to TPO stimulation. The E208Q mutation resulted in partial loss of LNK function, suggesting that LNK mutations may confer a spectrum of phenotypes. In peripheral blood samples obtained from MPN patients, stimulation with TPO or G-CSF revealed a unique phosphorylated STAT3/5 (pSTAT3+/5+) subpopulation that was increased in DEL compared with normal donor samples. A similar pSTAT3+/5+ subpopulation was seen with JAK2 V617F and MPL W515L-positive samples, suggesting that this may be a shared feature of MPNs. E208Q cells exhibited STAT3/5 phosphorylation in response to TPO, but not G-CSF, indicating that a partial loss of LNK function may generate differential STAT activation profiles in response to specific cytokines. The cytokine-responsive pSTAT3+/5+ cells from DEL were primarily CD34+, and the DEL mutation was detected in this subset, suggesting that LNK mutations arise in a hematopoietic stem or progenitor cell. Finally, the pSTAT3+/5+ response was abrogated by JAK inhibition, suggesting that JAK2 inhibitors may be a feasible option for MPN patients bearing LNK mutations. Thus, mutations in LNK, the first reported in human disease, lead to loss of LNK negative feedback function and represent a novel mechanism of MPN pathogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 239.

Lnk Constrains Oncogenic JAK2-Induced Myeloproliferative Disease in Mice.

Abstract 1437Poster Board I-460Hematopoietic stem cell (HSC) homeostasis and self-renewal are regulated by intrinsic cytokine signaling pathways. One important signaling axis for HSC is the cell surface receptor, Mpl, and its ligand, thrombopoietin (Tpo). Upon Tpo stimulation, Mpl activates Janus Kinase (JAK2), which in turn triggers a cascade of downstream signal transduction pathways that regulate key aspects of cell development. Mice that lack the inhibitory adaptor protein Lnk harbor a vastly expanded HSC pool with enhanced self-renewal. We previously demonstrated that Lnk controls HSC self-renewal predominantly through the Mpl/JAK2 pathway. Lnk binds directly to phosphorylated tyrosine 813 in JAK2 upon Tpo stimulation. Moreover, Lnk-deficient HSCs display potentiated JAK2 activation.Dysregulation of cytokine receptor signaling pathways frequently lead to hematological malignancies. Abnormal activation of JAK2 by a chromosomal translocation between the transcription factor Tel and JAK2 (Tel/JAK2) was shown to cause atypical Chronic Myelogenous Leukemia (aCML) in human patients. Moreover, the JAK2 V617F mutation has been observed at high frequency in several myeloproliferative diseases (MPDs). The JAK2V617F retains Lnk binding, suggesting that alterations in Lnk could influence MPD development. Indeed, we found that loss of Lnk accelerates and exacerbates oncogenic JAK2-induced MPD in mouse transplant models. Specifically, Lnk deficiency enhanced cytokine signaling, thereby augmenting the ability of oncogenic JAK2 to expand myeloid progenitors. To test whether the interaction between Lnk and JAK2V617F directly constrains MPD development in mice, we transplanted wild-type bone marrow cells expressing the JAK2V617F/Y813F double mutant that does not interact with Lnk (WT;JAK2VF/YF). WT;JAK2VF/YF engrafted mice exhibited increased myeloid expansion when compared to WT;JAK2VF mice, and conferred accelerated polycythemia vera development in secondary transplants. In summary, we identified Lnk as a physiological negative regulator of JAK2 in stem cells that may constrain leukemic transformation conferred by oncogenic JAK2. DisclosuresNo relevant conflicts of interest to declare.

Lnk Controls Hematopoietic Stem Cell Self-Renewal through Direct Interactions with JAK2 and Contributes to Oncogenic JAK2-Induced Myeloproliferative Diseases in Mice

Hematopoietic stem cell (HSC) homeostasis and self-renewal are regulated by intrinsic cytokine signaling pathways. One important signaling axis for HSC is the cell surface receptor, Mpl, and its ligand, thrombopoietin (Tpo). Upon Tpo stimulation, Mpl activates Janus Kinase (JAK2) that triggers a cascade of downstream signal transduction pathways that regulates many aspects of cell development. Under steady-state conditions, mice lacking the inhibitory adaptor protein Lnk harbor an expanded HSC pool with enhanced self-renewal. Surprisingly, we found that Lnk−/− HSCs have an increased quiescent fraction, decelerated cell cycle kinetics, and enhanced resistance to repeated 5-Florouracil (5-FU) treatments in vivo compared to wild type HSCs. We further provided genetic evidence demonstrating that Lnk controls HSC quiescence and self-renewal predominantly through Mpl. Consistent with this observation, Lnk deficiency in HSCs potentiates JAK2 activation in response to TPO. Biochemical experiments reveal that Lnk directly binds to phosphorylated tyrosine residues in JAK2 following TPO stimulation. Dysregulation of cytokine receptor signaling pathways leads to hematological malignancies. Abnormal activation of JAK2 by a chromosomal translocation between the transcription factor Tel and JAK2 (Tel/JAK2) was shown to cause atypical Chronic Myelogenous Leukemia (aCML). Recently, the JAK2 V617F mutation has been observed at high frequencies in several myeloproliferative diseases (MPDs). The JAK2V617F mutant retains Lnk binding ability, suggesting Lnk status could modify MPD development. Indeed, we found that loss of Lnk accelerates oncogenic JAK2- induced CML/MPD in the mouse transplant models. Therefore, we identified Lnk as a physiological negative regulator of JAK2 in stem cells that may contribute to leukemic transformation conferred by oncogenic JAK2.

Lnk controls mouse hematopoietic stem cell self-renewal and quiescence through direct interactions with JAK2

In addition to its role in megakaryocyte production, signaling initiated by thrombopoietin (TPO) activation of its receptor, myeloproliferative leukemia virus protooncogene (c-Mpl, or Mpl), controls HSC homeostasis and self-renewal. Under steady-state conditions, mice lacking the inhibitory adaptor protein Lnk harbor an expanded HSC pool with enhanced self-renewal. We found that HSCs from Lnk-/- mice have an increased quiescent fraction, decelerated cell cycle kinetics, and enhanced resistance to repeat treatments with cytoablative 5-fluorouracil in vivo compared with WT HSCs. We further provide genetic evidence demonstrating that Lnk controls HSC quiescence and self-renewal, predominantly through Mpl. Consistent with this observation, Lnk-/- HSCs displayed potentiated activation of JAK2 specifically in response to TPO. Biochemical experiments revealed that Lnk directly binds to phosphorylated tyrosine residues in JAK2 following TPO stimulation. Of note, the JAK2 V617F mutant, found at high frequencies in myeloproliferative diseases, retains the ability to bind Lnk. Therefore, we identified Lnk as a physiological negative regulator of JAK2 in stem cells and TPO/Mpl/JAK2/Lnk as a major regulatory pathway in controlling stem cell self-renewal and quiescence.

Association of toll-interacting protein gene polymorphisms with atopic dermatitis.

BackgroundAtopic dermatitis (AD) is a common inflammatory skin disorder, affecting up to 15% of children in industrialized countries. Toll-interacting protein (TOLLIP) is an inhibitory adaptor protein within the toll-like receptor (TLR) pathway, a part of the innate immune system that recognizes structurally conserved molecular patterns of microbial pathogens, leading to an inflammatory immune response.MethodsIn order to detect a possible role of TOLLIP variation in the pathogenesis of AD, we screened the entire coding sequence of the TOLLIP gene by SSCP in 50 AD patients. We identified an amino acid exchange in exon 6 (Ala222Ser) and a synonymous variation in exon 4 (Pro139Pro). Subsequently, these two variations and four additional non-coding polymorphisms (-526 C/G, two polymorphisms in intron 1 and one in the 3'UTR) were genotyped in 317 AD patients and 224 healthy controls.ResultsThe -526G allele showed borderline association with AD in our cohort (p = 0.012; significance level after correction for multiple testing 0.0102). Haplotype analysis did not yield additional information. Evaluation of mRNA expression by quantitative real-time polymerase chain reaction in six probands with the CC and six with the GG genotype at the -526 C/G locus did not reveal significant differences between genotypes.ConclusionVariation in the TOLLIP gene may play a role in the pathogenesis of AD. Yet, replication studies in other cohorts and populations are warranted to confirm these association results.

Dexamethasone Up-Regulates the Inhibitory Adaptor Protein Dok-1 and Suppresses Downstream Activation of the Mitogen-Activated Protein Kinase Pathway in Antigen-Stimulated RBL-2H3 Mast Cells

The glucocorticoid dexamethasone suppresses antigen-induced degranulation, cytokine production, and intermediate signaling events in RBL-2H3 mast cells, although the exact mechanisms are uncertain. By microarray analysis, we discovered that expression of the inhibitory adaptor protein, downstream of tyrosine kinase (Dok)-1, was up-regulated 4-fold in dexamethasone-treated RBL-2H3 cells. The up-regulation was apparent with as little as 1 to 10 nM dexamethasone. Treatment with dexamethasone also enhanced tyrosine phosphorylation of Dok-1, augmented recruitment of Ras GTPase-activating protein (RasGAP) by Dok-1, and inhibited activation of the mitogen-activated protein (MAP) kinase pathway in antigen-stimulated cells. The same effects were obtained by transient overexpression of Dok-1 but not by overexpression of Dok-1 that was mutated in RasGAP-binding domain. The negative regulatory role of Dok-1 was further validated by the expression of small interfering RNA directed against Dok-1, which enhanced activation of MAP kinase and subsequent release of arachidonic acid and tumor necrosis factor-alpha. These findings identify Dok-1 as mediator of the antiallergic actions of dexamethasone and as a negative regulator of the MAP kinase pathway and downstream release of inflammatory mediators.