Cytoplasmic Tyrosine Residues Research Articles

Ligand requirements for immunoreceptor triggering

Leukocytes interact with other cells using cell surface receptors. The largest group of such receptors are non-catalytic tyrosine phosphorylated receptors (NTRs), also called immunoreceptors. NTR signalling requires phosphorylation of cytoplasmic tyrosine residues by SRC-family tyrosine kinases. How ligand binding to NTRs induces this phosphorylation, also called NTR triggering, remains controversial, with roles suggested for size-based segregation, clustering, and mechanical force. Here we exploit a recently developed cell-surface generic ligand system to explore the ligand requirements for NTR triggering. We examine the effect of varying the ligand’s length, mobility and valency on the activation of representative members of four NTR families: SIRPβ1, Siglec 14, NKp44 and TREM-1. Increasing the ligand length impairs activation via NTRs, despite enhancing cell-cell conjugation, while varying ligand mobility has little effect on either conjugation or activation. Increasing the valency of the ligand, while enhancing cell-cell conjugation, does not enhance activation at equivalent levels of conjugation. These findings are more consistent with a role for size-based segregation, rather than mechanical force or clustering, in NTR triggering, suggesting a role for the kinetic-segregation model.

Differential in vivo roles of Mpl cytoplasmic tyrosine residues in murine hematopoiesis and myeloproliferative disease

Thrombopoietin (Tpo), which binds to its specific receptor, the Mpl protein, is the major cytokine regulator of megakaryopoiesis and circulating platelet number. Tpo binding to Mpl triggers activation of Janus kinase 2 (Jak2) and phosphorylation of the receptor, as well as activation of several intracellular signalling cascades that mediate cellular responses. Three tyrosine (Y) residues in the C-terminal region of the Mpl intracellular domain have been implicated as sites of phosphorylation required for regulation of major Tpo-stimulated signalling pathways: Mpl-Y565, Mpl-Y599 and Mpl-Y604. Here, we have introduced mutations in the mouse germline and report a consistent physiological requirement for Mpl-Y599, mutation of which resulted in thrombocytopenia, deficient megakaryopoiesis, low hematopoietic stem cell (HSC) number and function, and attenuated responses to myelosuppression. We further show that in models of myeloproliferative neoplasms (MPN), where Mpl is required for pathogenesis, thrombocytosis was dependent on intact Mpl-Y599. In contrast, Mpl-Y565 was required for negative regulation of Tpo responses; mutation of this residue resulted in excess megakaryopoiesis at steady-state and in response to myelosuppression, and exacerbated thrombocytosis associated with MPN.

Phagocytosis mediated by the human granulocyte receptor CEACAM3 is limited by the receptor-type protein tyrosine phosphatase PTPRJ

Carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) is a human granulocyte receptor mediating the efficient phagocytosis of a subset of human-restricted bacterial pathogens. Its function depends on phosphorylation of a tyrosine-based sequence motif, but the enzyme(s) responsible for reversing this modification are unclear. Here, we identify the receptor-type protein tyrosine phosphatase PTPRJ as a negative regulator of CEACAM3-mediated phagocytosis. We show depletion of PTPRJ results in a gain-of-function phenotype, while overexpression of a constitutively active PTPRJ phosphatase strongly reduces bacterial uptake via CEACAM3. We also determined that recombinant PTPRJ directly dephosphorylates the cytoplasmic tyrosine residues of purified full-length CEACAM3 and recognizes synthetic CEACAM3-derived phosphopeptides as substrates. Dephosphorylation of CEACAM3 by PTPRJ is also observed in intact cells, thereby limiting receptor-initiated cytoskeletal re-arrangements, lamellipodia formation, and bacterial uptake. Finally, we show that human phagocytes deficient for PTPRJ exhibit exaggerated lamellipodia formation and enhanced opsonin-independent phagocytosis of CEACAM3-binding bacteria. Taken together, our results highlight PTPRJ as a bona fide negative regulator of CEACAM3-initiated phagocyte functions, revealing a potential molecular target to limit CEACAM3-driven inflammatory responses.

Mechanism of Tim-3 regulation of CD8+ T cell function

Abstract Tim-3 or transmembrane immunoglobulin and mucin domain-3 is a type I membrane protein expressed by various immune cell types, and has been shown to have a co-stimulatory role in T cells through the PI3K pathway. Tim-3 comprises extracellular, transmembrane and intracellular domains, the latter of which contains five tyrosine residues. We and others have shown that these tyrosines participate in TCR-mediated signaling pathways. Using LCMV infection, we also found that Tim-3 expression influences the formation of short-lived effector and memory precursor CD8+ T cells. We hypothesize that Tim-3 signaling through the tyrosine residues in its cytoplasmic domain modulates CD8+ T cell activation and differentiation. To test this hypothesis, we have used LCMV-specific TCR transgenic (P14) mice crossed to truncated version of Tim-3 and CD8+ conditional deletion of Tim-3. CD8+ T cell activation and memory were analyzed by flow cytometry after infection with LCMV Armstrong to induce an acute infection. The effects of Tim-3 on CD8+ T cells were found to be most prominent at the effector stage of infection and RNA sequencing has shown that loss of Tim-3 signaling promotes acquisition of a memory-like phenotype. Transcription factors T-bet and Blimp-1 were found to be lower in Tim-3 KO CD8+ T cells, while Tcf-1 maintained at higher levels in the absence of Tim-3 signaling. Cytokine production by effector CD8+ T cells was also found to be lower when Tim-3 was absent. Thus, we conclude that Tim-3 signaling via its cytoplasmic tyrosine residues plays a role in CD8+ T cells in an acute infection. These results may lead to a better understanding of CD8+ T cell activation and memory development in different settings. Supported by R01 AI138504-01A1

Structural determinant of BST-2-mediated regulation of breast cancer cell motility: a role for cytoplasmic tail tyrosine residues.

There is now irrefutable evidence that overexpression of the innate immunity protein―BST-2, in breast cancer cells is implicated in tumor growth and progression. The cellular mechanisms that control BST-2-mediated effect in tumor progression involve enhancement of cancer cell motility―migration/invasion. However, the distinct structural elements of BST-2 that mediate breast cancer cell motility remain unknown. Here, we used various motility assays and different variants of BST-2 to examine the cellular and structural mechanisms controlling BST-2-mediated cell motility. We show that BST-2 silencing in various cancer cell lines inhibits cell motility. Restoration of BST-2 expression using construct expressing wild type BST-2 rescues cell motility. Mutational analysis identifies the cytoplasmic tail of BST-2 as a novel regulator of cancer cell motility, because cell motility was significantly abrogated by substitution of the BST-2 cytoplasmic tail tyrosine residues to alanine residues. Furthermore, in a spheroid invasion model, BST-2-expressing tumor spheroids are highly invasive inside 3D Matrigel matrices. In this model, the spreading distance of BST-2-expressing spheroids was significantly higher than that of BST-2-suppressed spheroids. Collectively, our data reveal that i) BST-2-expressing breast cancer cells in spheroids are more motile than their BST-2-supressed counterparts; ii) BST-2 cytoplasmic tail regulates non-proteolytic (migration) and proteolytic (invasion) mechanisms of breast cancer cell motility; and iii) replacement of the tyrosine residues at positions 6 and 8 in the cytoplasmic tail of BST-2 with alanine residues inhibits cell motility.

CD19 cytoplasmic tyrosine residues Y482 and Y513 are sufficient to promote normal B cell development and differentiation but induce a skewed humoral immune response.

Abstract CD19 amplifies BCR signal transduction by recruiting SH2 domain-containing proteins to its nine cytoplasmic phosphorylated tyrosine residues. Previous studies have shown that Y482 and Y513, which recruit PI3K are required for normal B cell development/differentiation and a productive T-dependent humoral response. However, it has not been determined if CD19 Y482 and Y513 are sufficient to promote normal B cell differentiation or antibody response to immunization. Furthermore, the effects of upstream CD19 tyrosine-mediated Vav/PLCγ2 and Grb2/Sos signaling are not well characterized. Using mice expressing CD19 mutated to only express tyrosines Y482 and Y513, we demonstrate that CD19 mediated Lyn/PI3K signaling is sufficient for B1 B cell development, marginal zone B cell maturation, and germinal center B cell formation. However, these mice exhibit an increased antibody response against T-independent antigens and are impaired in their ability to generate class-switched germinal center B cells during a T-dependent immune response, as well as long-lived, class-switched antibody secreting cells. These findings suggest that cross-talk between signal effectors recruited to Y482 and Y513 and those recruited to other CD19 tyrosine residues is important for determining the qualitative nature of the humoral response.

CD19 cytoplasmic tyrosines 482 and 513 mediating binding to PI3K are sufficient to reconstitute the T-dependent humoral immune response (IRM8P.713)

Abstract CD19 is a co-stimulatory receptor on B cells that has been shown to play a critical role in regulating B cell differentiation and the humoral immune response. In mice lacking CD19 expression, B1 and marginal zone (MZ) B cell subpopulations are absent and the T-dependent humoral response is severely impaired in association with defective GC B cell generation and GC responses resulting in loss of class switching, affinity maturation and ultimately the failure to generate Ag-specific memory B cells and Ab secreting cells (ASC) that produce class switched, high affinity Abs. Previous studies have demonstrated the selective mutation of tyrosines 482 and 513 (4/5F mice) in the cytoplasmic domain of CD19 is sufficient to recapitulate the phenotype associated with complete loss of CD19 expression. In the present study, mice were generated in which CD19 is expressed containing only Y482 and Y513 in the absence of the other seven cytoplasmic tyrosine residues (4/5Y mice). Analysis of 4/5Y mice revealed that these residues alone are sufficient to restore B1 and MZ B cell development as well as the T-dependent humoral immune response. Studies to examine mice expressing CD19 with a selective mutation to either tyrosine 482 or 513 revealed a preferential requirement for tyrosine 482 to promote a normal humoral response. These data support the conclusion that CD19 predominantly contributes to normal B cell development and the T-dependent humoral response through its recruitment of PI3K.

Phosphorylation Of The Cytoplasmic Tyrosine Residues Of c-Mpl Is Not Required To Preserve Intact Receptor Dependent Hematopoietic Stem Cell Survival Signaling

Thrombopoietin (TPO) and its cognate receptor c-Mpl are the primary regulators of megakaryopoiesis, and signaling through c-Mpl is necessary for maintaining normal platelet production in vivo. This signaling pathway is also critical for hematopoietic stem cell (HSC) survival and proliferation. Thrombopoietin-mediated Mpl dimerization results in phosphorylation of tyrosine residues in the c-Mpl cytoplasmic domain and activation of the tyrosine kinase Jak2 leading to activation of several additional downstream signaling pathways. To examine the role of phosphorylation of cytoplasmic c-Mpl tyrosine residues in megakaryopoiesis and HSC survival signaling in vivo, we generated transgenic mice expressing the WT c-Mpl cDNA or a variant c-Mpl cDNA, with all 5 intracellular tyrosine residues mutated to phenylalanines. Expression of the transgenes was directed by a 2-kb Mpl promotor and both transgenic mice were crossed with mpl-/- mice to eliminate Mpl production from the native gene. In mice expressing the WT Mpl transgene (Y-ALL), platelet counts were rescued to levels that were 3-5fold higher than WT mice. Surprisingly, mice expressing the tyrosine deficient Mpl (F-ALL) also demonstrated similarly elevated platelet counts. However, plasma TPO levels in F-ALL mice were markedly elevated, and in Y-ALL mice they were comparable to WT levels. Therefore, the unexpected thrombocytosis found in F-ALL mice was hypothesized to be caused by a disruption in normal TPO homeostasis secondary to impaired receptor endocytosis and TPO degradation caused by mutations of the cytoplasmic tyrosines Y78 and Y8. Signal transduction by the F-ALL and Y-ALL transgenes was further studied in 32D cells, a cytokine dependent murine myeloid cell line. Although appropriately expressed on the cell surface, the tyrosine deficient Mpl protein showed absent Mpl tyrosine phosphorylation and greatly diminished Jak2 phosphorylation when stimulated with TPO. Nevertheless, F-ALL expressing 32D cells proliferated in response to TPO, though these cells required about 10fold higher ligand levels than Y-ALL expressing 32Ds to achieve maximum proliferation rates. To study the relative degrees of restoration of HSC and megakaryocyte compartments by Y-ALL and F-ALL transgenes, under conditions of intact TPO homeostasis, we performed competitive repopulation assays, transplanting Y-ALL or F-ALL marrow cells plus GFP expressing WT marrow cells into lethally irradiated recipients. Peripheral blood platelets in chimeras generated with F-ALL marrow were almost all produced by WT competitor cells. In contrast, in chimeras made with Y-ALL marrow >80% of platelet production was by Y-ALL cells. The percentage of peripheral leukocytes produced by each of the competitor donor cells was used as a surrogate of HSC activity. In contrast to the widely disparate contributions to megakaryopoiesis by Y-ALL and F-ALL marrow cells in chimeras, the rescue of HSC activity by each of these transgenes was comparable. Together, our data suggests that phosphorylation of intracellular tyrosine residues is critical in megakaryopoiesis but dispensable for TPO induced survival and proliferation signaling in HSCs. Either, the markedly reduced levels of Jak2 phosphorylation induced by TPO stimulation of the F-ALL transgene is adequate to support the HSC compartment or alternate Mpl signal transduction pathways that do not involve Jak2 are also involved. Disclosures:No relevant conflicts of interest to declare.

Alpha1-adrenenoceptor stimulation inhibits cardiac excitation–contraction coupling through tyrosine phosphorylation of beta1-adrenoceptor

Adrenoceptor stimulation is a key determinant of cardiac excitation–contraction coupling mainly through the activation of serine/threonine kinases. However, little is known about the role of protein tyrosine kinases (PTKs) activated by adrenergic signaling on cardiac excitation–contraction coupling. A cytoplasmic tyrosine residue in β1-adrenoceptor is estimated to regulate Gs-protein binding affinity from crystal structure studies, but the signaling pathway leading to the phosphorylation of these residues is unknown. Here we show α1-adrenergic signaling inhibits β-adrenergically activated Ca2+ current, Ca2+ transients and contractile force through phosphorylation of tyrosine residues in β1-adrenoceptor by PTK. Our results indicate that inhibition of β-adrenoceptor-mediated Ca2+ elevation by α1-adrenoceptor-PTK signaling serves as an important regulatory feedback mechanism when the catecholamine level increases to protect cardiomyocytes from cytosolic Ca2+ overload.

The Cell Surface Glycoprotein CUB Domain-containing Protein 1 (CDCP1) Contributes to Epidermal Growth Factor Receptor-mediated Cell Migration

Epidermal growth factor (EGF) activation of the EGF receptor (EGFR) is an important mediator of cell migration, and aberrant signaling via this system promotes a number of malignancies including ovarian cancer. We have identified the cell surface glycoprotein CDCP1 as a key regulator of EGF/EGFR-induced cell migration. We show that signaling via EGF/EGFR induces migration of ovarian cancer Caov3 and OVCA420 cells with concomitant up-regulation of CDCP1 mRNA and protein. Consistent with a role in cell migration CDCP1 relocates from cell-cell junctions to punctate structures on filopodia after activation of EGFR. Significantly, disruption of CDCP1 either by silencing or the use of a function blocking antibody efficiently reduces EGF/EGFR-induced cell migration of Caov3 and OVCA420 cells. We also show that up-regulation of CDCP1 is inhibited by pharmacological agents blocking ERK but not Src signaling, indicating that the RAS/RAF/MEK/ERK pathway is required downstream of EGF/EGFR to induce increased expression of CDCP1. Our immunohistochemical analysis of benign, primary, and metastatic serous epithelial ovarian tumors demonstrates that CDCP1 is expressed during progression of this cancer. These data highlight a novel role for CDCP1 in EGF/EGFR-induced cell migration and indicate that targeting of CDCP1 may be a rational approach to inhibit progression of cancers driven by EGFR signaling including those resistant to anti-EGFR drugs because of activating mutations in the RAS/RAF/MEK/ERK pathway.

BIT/SHPS-1 promotes antiapoptotic effect of BDNF on low potassium-induced cell death of cultured cerebellar granule neurons.

Brain immunoglobulin-like molecule with tyrosine-based activation motifs/SHP substrate 1 (BIT/SHPS-1) is a neuronal adhesion molecule that is highly expressed in cerebellar granule neurons (CGNs); however its function in CGNs remains unclear. Our previous studies indicated that BIT/SHPS-1 is able to modulate the antiapoptotic effect of brain-derived neurotrophic factor (BDNF) on CNS neurons by cell type-specific mechanisms. In this article, we have studied the role of BIT/SHPS-1 in the antiapoptotic function of BDNF on low potassium (LK)-induced cell death of cultured CGNs which is an in vitro model system of neuronal apoptosis during brain development. Cultured rat CGNs were transduced with wild-type rat BIT/SHPS-1 (BIT/SHPS-1(WT)), its 4F-mutant (BIT/SHPS-1(4F), in which all cytoplasmic tyrosine residues were substituted with phenylalanine), or nuclear localization signal-attached beta-galactosidase (NLS-LacZ, as control)-expressing adenoviruses. Expression of BIT/SHPS-1(WT) and BIT/SHPS-1(4F) alone did not affect steady-state cell viability. Tyrosine phosphorylation of BIT/SHPS-1 was only detected in BIT/SHPS-1(WT)-expressing cultures in the presence and the absence of BDNF. When subjected to LK in the presence of BDNF, BIT/SHPS-1(WT)- and BIT/SHPS-1(4F)-expressing cultures showed a significant resistance to cell death, while the control virus-transfected culture did not. In addition, a phosphatidylinositol 3-kinase (PI3-K) inhibitor, LY294002, attenuated the antiapoptotic effect of BDNF on BIT/SHPS-1(WT)-, and BIT/SHPS-1(4F)-expressing cultures. These results demonstrated that in both tyrosine phosphorylation-independent and PI3-K-dependent manners, BIT/SHPS-1 promotes the antiapoptotic effect of BDNF on the LK-induced cell death of CGNs.

Intronic CD22 Gene Mutations as A Pathogenic Mechanism of Human B-Precursor Leukemia

AbstractAbstract 149CD22 is an inhibitory co-receptor of B-cells and B-cell precursors that acts as a negative regulator of multiple signal transduction pathways critical for B-cell homeostasis. The inhibitory and apoptosis-promoting signaling function of CD22 is dependent on recruitment of the Src homology 2 domain–containing tyrosine phosphatase (SHP)-1 to the immunoreceptor tyrosine-based inhibitory motifs (ITIMs) of its cytoplasmic domain. Collective genetic evidence from CD22-deficient or SHP-1-deficient mice shows that disruption of the CD22-SHP1 signaling network can result in development of a B-cell lymphoproliferative state associated with defective apoptosis and maturation. However, direct genetic evidence for the functional significance of CD22 in human B-cell ontogeny or its implied tumor suppressor role has been lacking. Here, we report that primary leukemic cells from infants with newly diagnosed B-precursor leukemia (BPL) express a truncated and functionally defective CD22 co-receptor protein that is unable to transmit apoptotic signals. Expression of this structurally and functionally abnormal CD22 protein is associated with a very aggressive in vivo growth of patients' primary leukemia cells causing disseminated overt leukemia in SCID mice. The abnormal CD22 co-receptor is encoded by a profoundly aberrant mRNA arising from a splicing defect that causes the deletion of exon 12 (c.2208-c.2327) (CD22DE12). CD22DE12 involves an exact splice with no other mutation at the splice junction. The deletion of exon 12 in CD22 mRNA results in a truncating frameshift mutation starting at K736 with an insertion of 15 amino acids not seen in wild-type CD22 sequence followed by a TGA termination codon. Mutant CD22DE12 protein lacks the conserved tyrosines and ITIMs that provide docking sites for the SH2 domains of the tyrosine phosphatase SHP1. It also lacks regions homologous to ITAMs, tyrosine-based activation motifs, which are docking sites for SH2 containing proteins. Thus, CD22DE12 mRNA encodes a truncated CD22 protein lacking most of the intracellular domain including the key regulatory signal transduction elements and all of the cytoplasmic tyrosine residues. The splicing defect is associated with multiple homozygous mutations within a 132-bp segment of the intronic sequence between exons 12 and 13. These mutations cause marked changes in the predicted secondary structures of the mutant CD22 pre-mRNA sequences that affect the target motifs for the splicing factors hnRNP-L, PTB, and PCBP that are up-regulated in infant leukemia cells. Forced expression of the mutant CD22DE12 protein in transgenic mice, under control of the immunoglobulin enhancer Eμ that is activated in early B-cell ontogeny prior to Ig gene rearrangements, perturbs B-cell development, as evidenced by B-precursor/B-cell hyperplasia at 6 weeks of age and corrupts the regulation of gene expression, causing reduced expression levels of several genes with a tumor suppressor function. This CD22DE12-associated unique 12-gene signature transcriptome included (i) tumor suppressor genes TP53 (as well as TP53 regulator MDM2), neurofibromatosis 2 (NF2) (as well as NF2 regulator RAC1), and the adenomatous polyposis coli (APC) gene, a tumor suppressor known to regulate the Wnt/beta-catenin signaling, (ii) genes for chromatin remodeling/global gene expression regulators with a tumor suppressor function IKZF1/IKAROS and SATB1, as well as (iii) cell cycle regulatory genes CDKN1C, CCNG1, and NOTCH4. By comparing gene expression profiles of primary leukemia cells from 31 infants vs. 30 non-infant children with ALL we found that reduced expression levels of 6 of the 9 CD22DE12 signature genes that were represented on the human cDNA arrays, including TP53 and APC as well as MDM2, SATB1, CCNG1 and GNB2 discriminated infant BPL from non-infant BPL. The documented molecular and functional abnormalities involving CD22 in primary leukemic cells from patients with newly diagnosed infant BPL uniquely implicate deficiency of this B-lineage restricted co-receptor protein in the pathogenesis of infant leukemia. This collection of experimental data is unprecedented in that it links a genetic defect involving a B-lineage specific regulatory gene to the most aggressive human B-lineage lymphoid malignancy that arises from an uncoupling of proliferation and differentiation of B-precursors during the earliest stages of B-cell ontogeny. Disclosures:No relevant conflicts of interest to declare.

The role of Ig-α/β in B cell antigen receptor internalization

The B cell antigen receptor (BCR) is expressed on the surface of B lymphocytes where it can bind antigen then transmit signals which regulate activation, growth, and differentiation. These signals can induce a number of cytoskeletal rearrangements leading to dynamic cellular processes including internalization of the bound antigen which is then processed and presented to T cells on MHC II. The relative importance of regions within the Igα and Igβ cytoplasmic domains has been well studied in terms of signaling but their roles in BCR internalization and trafficking are less clear. We hypothesize that the Igα and Igβ cytoplasmic domains is important for normal internalization and trafficking of the 4 chain BCR. An Igα and Igβ deficient murine lymphoid cell line was used to express mIgM along with a panel of Igα and Igβ mutants in order to compare their internalization and subcellular localization. Here we show that the Igα and Igβ cytoplasmic domains are each sufficient for internalization, though Igα is dominant in this process. We also show that the internalization signal is contained in a region past the first cytoplasmic tyrosine residue of Igα and Igβ, Y176 and Y195 respectively. We also show that a 4 amino acid motif normally contained within the Igα ITAM is sufficient to rescue aberrant internalization. In terms of receptor trafficking, each cytoplasmic domain is sufficient for trafficking to lysosomal compartments but that a normal rate of trafficking likely requires the tandem effects of both Igα and Igβ.

CD22 EXON 12 deletion as a pathogenic mechanism of human B-precursor leukemia

Here, we report that primary leukemic cells from infants with newly diagnosed B-precursor leukemia express a truncated and functionally defective CD22 coreceptor protein that is unable to transmit apoptotic signals because it lacks most of the intracellular domain, including the key regulatory signal transduction elements and all of the cytoplasmic tyrosine residues. Expression of this structurally and functionally abnormal CD22 protein is associated with a very aggressive in vivo growth of patients' primary leukemia cells causing disseminated overt leukemia in SCID mice. The abnormal CD22 coreceptor is encoded by a profoundly aberrant mRNA arising from a splicing defect that causes the deletion of exon 12 (c.2208-c.2327) (CD22ΔE12) and results in a truncating frameshift mutation. The splicing defect is associated with multiple homozygous mutations within a 132-bp segment of the intronic sequence between exons 12 and 13. These mutations cause marked changes in the predicted secondary structures of the mutant CD22 pre-mRNA sequences that affect the target motifs for the splicing factors hnRNP-L, PTB, and PCBP that are up-regulated in infant leukemia cells. Forced expression of the mutant CD22ΔE12 protein in transgenic mice perturbs B-cell development, as evidenced by B-precursor/B-cell hyperplasia, and corrupts the regulation of gene expression, causing reduced expression levels of several genes with a tumor suppressor function. We further show that CD22ΔE12-associated unique gene expression signature is a discriminating feature of newly diagnosed infant leukemia patients. These striking findings implicate CD22ΔE12 as a previously undescribed pathogenic mechanism in human B-precursor leukemia.

Adaptor protein is essential for insect cytokine signaling in hemocytes

Growth-blocking peptide (GBP) is an insect cytokine that stimulates a class of immune cells called plasmatocytes to adhere to one another and to foreign surfaces. Although extensive structure-activity studies have been performed on the GBP and its mutants in Lepidoptera Pseudaletia separata, the signaling pathway of GBP-dependent activation of plasmatocytes remains unknown. We identified an adaptor protein (P77) with a molecular mass of 77 kDa containing SH2/SH3 domain binding motifs and an immunoreceptor tyrosine-based activation motif (ITAM)-like domain in the cytoplasmic region of the C terminus. Although P77 showed no capacity for direct binding with GBP, its cytoplasmic tyrosine residues were specifically phosphorylated within seconds after GBP was added to a plasmatocyte suspension. Tyrosine phosphorylation of P77 also was observed when hemocytes were incubated with Enterobactor cloacae or Micrococcus luteus, but this phosphorylation was found to be induced by GBP released from hemocytes stimulated by the pathogens. Tyrosine phosphorylation of the integrin beta subunit also was detected in plasmatocytes stimulated by GBP. Double-stranded RNAs targeting P77 not only decreased GBP-dependent tyrosine phosphorylation of the integrin beta subunit, but also abolished GBP-induced spreading of plasmatocytes on foreign surfaces. P77 RNAi larvae also showed significantly higher mortality than control larvae after infection with Serratia marcescens, indicating that P77 is essential for GBP to mediate a normal innate cellular immunity in insects. These results demonstrate that GBP signaling in plasmatocytes requires the adaptor protein P77, and that active P77-assisted tyrosine phosphorylation of integrins is critical for the activation of plasmatocytes.

Site-directed mutagenesis reveals a unique requirement for tyrosine residues in IL-7Rα and TSLPR cytoplasmic domains in TSLP-dependent cell proliferation

BackgroundThymic stromal lymphopoietin (TSLP) is an interleukin-7 (IL-7) like cytokine, which plays an important role in the regulation of immune responses to allergens. TSLP binds to a heterodimeric receptor complex composed of the IL-7 receptor α chain (IL-7Rα) and the TSLP receptor (TSLPR, also known as CRLF2). It has previously been suggested that the lone tyrosine residue in the mouse TSLPR cytoplasmic domain is required for cell proliferation using chimeric receptor systems. Also the role of tyrosine residues in the IL-7Rα cytoplasmic domain in TSLP signaling has not yet been investigated. We undertook a systematic analysis to test the role of tyrosine residues of both the IL-7Rα and the TSLPR in inducing cell proliferation in a growth factor dependent cell line, Ba/F3.ResultsA multiple sequence alignment of the IL-7Rα and TSLPR cytoplasmic domains revealed conservation of most, but not all, cytoplasmic tyrosine residues across several species. Our site-directed mutagenesis experiments revealed that the single tyrosine residue in human TSLPR was not required for TSLP-dependent cell proliferation. It has previously been reported that Y449 of human IL-7Rα is required for IL-7 dependent proliferation. Interestingly, in contrast to IL-7 signaling, none of tyrosine residues in the human IL-7Rα cytoplasmic domain were required for TSLP-dependent cell proliferation in the presence of a wild type TSLPR. However, the mutation of all cytoplasmic four tyrosine residues of human IL-7Rα and human TSLPR to phenylalanine residues abolished the proliferative ability of the TSLP receptor complex in response to TSLP.ConclusionThese results suggest that TSLP requires at least one cytoplasmic tyrosine residue to transmit proliferative signals. Unlike other members of IL-2 cytokine family, tyrosine residues in IL-7Rα and TSLPR cytoplasmic domains play a redundant role in TSLP-mediated cell growth.

Anthrax toxin triggers the activation of src -like kinases to mediate its own uptake

AB-type toxins, like other bacterial toxins, are notably opportunistic molecules. They rely on target cell receptors to reach the appropriate location within the target cell where translocation of their enzymatic subunits occurs. The anthrax toxin, however, times its own uptake, suggesting that toxin binding triggers specific signaling events. Here we show that the anthrax toxin triggers tyrosine phosphorylation of its own receptors, capillary morphogenesis gene 2 and tumor endothelial marker 8, which are not endowed with intrinsic kinase activity. This is required for efficient toxin uptake because endocytosis of the mutant receptor lacking the cytoplasmic tyrosine residues is strongly delayed. Phosphorylation of the receptors was dependent on src-like kinases, which where activated upon toxin binding. Importantly, src-dependent phosphorylation of the receptor was required for its subsequent ubiquitination, which in turn was required for clathrin-mediated endocytosis. Consistently, we found that uptake of the anthrax toxin and processing of the lethal factor substrate MEK1 are inhibited by silencing of src and fyn, as well as in src and fyn knockout cells.

Mechanism for Amyloid Precursor-like Protein 2 Enhancement of Major Histocompatibility Complex Class I Molecule Degradation

Earlier studies have demonstrated interaction of the murine major histocompatibility complex (MHC) class I molecule K(d) with amyloid precursor-like protein 2 (APLP2), a ubiquitously expressed member of the amyloid precursor protein family. Our current findings indicate that APLP2 is internalized in a clathrin-dependent manner, as shown by utilization of inhibitors of the clathrin pathway. Furthermore, we demonstrated that APLP2 and K(d) bind at the cell surface and are internalized together. The APLP2 cytoplasmic tail contains two overlapping consensus motifs for binding to the adaptor protein-2 complex, and mutation of a tyrosine shared by both motifs severely impaired APLP2 internalization and ability to promote K(d) endocytosis. Upon increased expression of wild type APLP2, K(d) molecules were predominantly directed to the lysosomes rather than recycled to the plasma membrane. These findings suggest a model in which APLP2 binds K(d) at the plasma membrane, facilitates uptake of K(d) in a clathrin-dependent manner, and routes the endocytosed K(d) to the lysosomal degradation pathway. Thus, APLP2 has a multistep trafficking function that influences the expression of major histocompatibility complex class I molecules at the plasma membrane.

Colony-stimulating factor-1–induced oscillations in phosphatidylinositol-3 kinase/AKT are required for caspase activation in monocytes undergoing differentiation into macrophages

AbstractThe differentiation of human peripheral blood monocytes into resident macrophages is driven by colony-stimulating factor-1 (CSF-1), which upon interaction with CSF-1 receptor (CSF-1R) induces within minutes the phosphorylation of its cytoplasmic tyrosine residues and the activation of multiple signaling complexes. Caspase-8 and -3 are activated at day 2 to 3 and contribute to macrophage differentiation, for example, through cleavage of nucleophosmin. Here, we show that the phosphatidylinositol-3 kinase and the downstream serine/threonine kinase AKT connect CSF-1R activation to caspase-8 cleavage. Most importantly, we demonstrate that successive waves of AKT activation with increasing amplitude and duration are required to provoke the formation of the caspase-8–activating molecular platform. CSF-1 and its receptor are both required for oscillations in AKT activation to occur, and expression of a constitutively active AKT mutant prevents the macrophage differentiation process. The extracellular receptor kinase 1/2 pathway is activated with a coordinated oscillatory kinetics in a CSF-1R–dependent manner but plays an accessory role in caspase activation and nucleophosmin cleavage. Altogether, CSF-1 stimulation activates a molecular clock that involves phosphatidylinositol-3 kinase and AKT to promote caspase activation. This oscillatory signaling pathway, which is coordinated with extracellular receptor kinase 1/2 oscillatory activation, involves CSF-1 and CSF-1R and controls the terminal differentiation of macrophages.

An Activating and Inhibitory Signal from an Inhibitory Receptor LMIR3/CLM-1: LMIR3 Augments Lipopolysaccharide Response through Association with FcRγ in Mast Cells

Leukocyte mono-Ig-like receptor 3 (LMIR3) is an inhibitory receptor mainly expressed in myeloid cells. Coengagement of Fc epsilonRI and LMIR3 impaired cytokine production in bone marrow-derived mast cells (BMMCs) induced by Fc epsilonRI crosslinking alone. Mouse LMIR3 possesses five cytoplasmic tyrosine residues (Y241, Y276, Y289, Y303, Y325), among which Y241 and Y289 (Y241/289) or Y325 fit the consensus sequence of ITIM or immunotyrosine-based switch motif (ITSM), respectively. The inhibitory effect was abolished by the replacement of Y325 in addition to Y241/289 with phenylalanine (Y241/189/325/F) in accordance with the potential of Y241/289/325 to cooperatively recruit Src homology region 2 domain-containing phosphatase 1 (SHP)-1 or SHP-2. Intriguingly, LMIR3 crosslinking alone induced cytokine production in BMMCs expressing LMIR3 (Y241/276/289/303/325F) mutant as well as LMIR3 (Y241/289/325F). Moreover, coimmunoprecipitation experiments revealed that LMIR3 associated with ITAM-containing FcRgamma. Analysis of FcRgamma-deficient BMMCs demonstrated that both Y276/303 and FcRgamma played a critical role in the activating function of this inhibitory receptor. Importantly, LMIR3 crosslinking enhanced cytokine production of BMMCs stimulated by LPS, while suppressing production stimulated by other TLR agonists or stem cell factor. Thus, an inhibitory receptor LMIR3 has a unique property to associate with FcRgamma and thereby functions as an activating receptor in concert with TLR4 stimulation.