Transient receptor potential melastatin 7 is involved in oestrogen receptor-negative metastatic breast cancer cells migration through its kinase domain.

Oestrogen receptor negative (ER(-)) invasive ductal carcinoma (IDC) represents a significant clinical challenge and therefore prompts the discovery of novel biomarkers. Transient receptor potential melastatin 7 (TRPM7), a channel protein that also contains a regulatory kinase domain, is overexpressed in IDC and regulates migration. However, the molecular mechanism remains poorly defined. Here, we examined whether TRPM7 regulates migration by its channel function or by its kinase domain. A Magnesium Inhibited Cation current was recorded in two ER(-) highly metastatic breast cancer cell lines. Down-regulation of TRPM7 neither affected Ca(2+)-, nor Mg(2+)-homoeostasis but significantly reduced cell migration via a Ca(2+)-independent pathway. Notably, the overexpression of the truncated kinase domain form of TRPM7 decreased cell migration, while the overexpression of the wild-type form strongly increased it. Concomitantly, TRPM7 silencing reduced the myosin IIA heavy chain phosphorylation. Furthermore, we found higher TRPM7 expression in ER(-) IDC tissues and lymph nodes than in the non-invasive tumoural samples. In conclusion, TRPM7 plays a critical role in breast cancer cell migration through its kinase domain, and our data support the consideration of using TRPM7 as a novel biomarker and a potential therapeutic target in the treatment of human ER(-) IDC.

Competition between PARP-1 and Ku70 control the decision between high-fidelity and mutagenic DNA repair.

Affinity maturation of antibodies requires a unique process of targeted mutation that allows changes to accumulate in the antibody genes while the rest of the genome is protected from off-target mutations that can be oncogenic. This targeting requires that the same deamination event be repaired either by a mutagenic or a high-fidelity pathway depending on the genomic location. We have previously shown that the BRCT domain of the DNA-damage sensor PARP-1 is required for mutagenic repair occurring in the context of IgH and IgL diversification in the chicken B cell line DT40. Here we show that immunoprecipitation of the BRCT domain of PARP-1 pulls down Ku70 and the DNA-PK complex although the BRCT domain of PARP-1 does not bind DNA, suggesting that this interaction is not DNA dependent. Through sequencing the IgL variable region in PARP-1(-/-) cells that also lack Ku70 or Lig4, we show that Ku70 or Lig4 deficiency restores GCV to PARP-1(-/-) cells and conclude that the mechanism by which PARP-1 is promoting mutagenic repair is by inhibiting high-fidelity repair which would otherwise be mediated by Ku70 and Lig4.

Efficacy and safety of a new immunoglobulin G product, Gammaplex(®), in primary immunodeficiency diseases.

This open-label multi-centre study evaluated a new intravenous immunoglobulin, Gammaplex®, in the treatment of 50 patients with primary immunodeficiency and significant hypogammglobulinaemia. Patients treated previously with other intravenous immunoglobulins received Gammaplex® on their same infusion schedule for 1 year; 22 were on a 21-day and 28 on a 28-day regimen (300-800 mg/kg/infusion). There were no serious, acute bacterial infections, whereas six subjects (12·0%) had at least one such infection in the 6 months before enrollment. Forty subjects (80·0%) had at least one non-serious infection; the median number of infective episodes per subject per year was 3·07. Antibiotics were taken by 38 subjects therapeutically and prophylactically by 16 at some time. Fewer than half (46·0%) missed any time off work or school because of infection or other illness. Trough immunoglobulin (Ig)G levels were above 6·00 g/l in all subjects at all assessments after 15 weeks with two exceptions. Overall, 21·2% of infusions were associated with an adverse event up to 72 h after infusion. The frequency of adverse events increased with infusion rate. Headache was the most common product-related adverse event (7·5% of 703 infusions). In conclusion, Gammaplex® is effective in primary immunodeficiency and is well tolerated.

LTRPC7 is a Mg.ATP-regulated divalent cation channel required for cell viability.

The molecular mechanisms that regulate basal or background entry of divalent cations into mammalian cells are poorly understood. Here we describe the cloning and functional characterization of a Ca2+- and Mg2+-permeable divalent cation channel, LTRPC7 (nomenclature compatible with that proposed in ref. 1), a new member of the LTRPC family of putative ion channels. Targeted deletion of LTRPC7 in DT-40 B cells was lethal, indicating that LTRPC7 has a fundamental and nonredundant role in cellular physiology. Electrophysiological analysis of HEK-293 cells overexpressing recombinant LTRPC7 showed large currents regulated by millimolar levels of intracellular Mg.ATP and Mg.GTP with the permeation properties of a voltage-independent divalent cation influx pathway. Analysis of several cultured cell types demonstrated small magnesium-nucleotide-regulated metal ion currents (MagNuM) with regulation and permeation properties essentially identical to the large currents observed in cells expressing recombinant LTRPC7. Our data indicate that LTRPC7, by virtue of its sensitivity to physiological Mg.ATP levels, may be involved in a fundamental process that adjusts plasma membrane divalent cation fluxes according to the metabolic state of the cell.

ADP-ribose gating of the calcium-permeable LTRPC2 channel revealed by Nudix motif homology.

Free ADP-ribose (ADPR), a product of NAD hydrolysis and a breakdown product of the calcium-release second messenger cyclic ADPR (cADPR), has no defined role as an intracellular signalling molecule in vertebrate systems. Here we show that a 350-amino-acid protein (designated NUDT9) and a homologous domain (NUDT9 homology domain) near the carboxy terminus of the LTRPC2/TrpC7 putative cation channel both function as specific ADPR pyrophosphatases. Whole-cell and single-channel analysis of HEK-293 cells expressing LTRPC2 show that LTRPC2 functions as a calcium-permeable cation channel that is specifically gated by free ADPR. The expression of native LTRPC2 transcripts is detectable in many tissues including the U937 monocyte cell line, in which ADPR induces large cation currents (designated IADPR) that closely match those mediated by recombinant LTRPC2. These results indicate that intracellular ADPR regulates calcium entry into cells that express LTRPC2.

Interaction between the Btk PH domain and phosphatidylinositol-3,4,5-trisphosphate directly regulates Btk.

Bruton's tyrosine kinase (Btk) binds to phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P(3)) through the Btk pleckstrin homology (PH) domain, an interaction thought to be required for Btk membrane translocation during B cell receptor signaling. Here, we report that interaction of PtdIns-3,4,5-P(3) with the PH domain of Btk directly induces Btk enzymatic activation in an in vitro kinase assay. A point mutation that reduces interaction of PtdIns-3,4,5-P(3) with the Btk PH domain blocks in vitro PtdIns-3,4,5-P(3)-dependent Btk activation, whereas the PH domain deletion enhances Btk basal activity but eliminates the PtdIns-3,4,5-P(3)-dependent stimulation. Btk kinase activity and the Btk activation loop phosphorylation site are both required for the PtdIns-3,4,5-P(3)-mediated stimulation of Btk kinase activity. Together, these results suggest that the Btk PH domain is positioned such that it normally suppresses both Btk kinase activity and access to substrates; when interacting with PtdIns-3,4,5-P(3), this suppression is relieved, producing apparent Btk activation. In addition, using Src family kinase inhibitors and Btk catalytically inactive mutants, we demonstrate that in vivo, the activation of Btk is due to both Lyn phosphorylation and PtdIns-3,4,5-P(3)-mediated direct activation. Thus, the Btk-PtdIns-3,4,5-P(3) interaction serves to translocate Btk to the membrane and directly regulate its signaling function.

SYK is upstream of phosphoinositide 3-kinase in B cell receptor signaling.

We have recently demonstrated that the D3-phosphoinositide phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P(3)) is critical for producing sustained calcium signals through its role in promoting the function of TEC family tyrosine kinases such as Bruton's tyrosine kinase. Although PtdIns-3,4,5-P(3) can potentially be synthesized by any of several types of phosphoinositide 3-kinases (PI3Ks), B cell receptor (BCR)-induced PtdIns-3,4,5-P(3) production is thought to occur primarily through the activation of the class Ia (p85/p110) PI3Ks. This process has been proposed to be mediated by an interaction between the Src family kinase LYN and the p85 subunit of PI3K and/or through p85 membrane recruitment mediated by CBL and/or CD19. However, calcium signaling and other PI3K-dependent signals are relatively preserved in a LYN kinase-deficient B lymphocyte cell line, suggesting that an alternative pathway for PI3K activation exists. As SYK/ZAP70 kinases are upstream from many BCR-initiated signaling events, we directly analyzed SYK-dependent accumulation of both PtdIns-3,4,5-P(3) and PtdIns-3,4-P(2) in B cell receptor signaling using both dominant negative and genetic knockout approaches. Both methods indicate that SYK is upstream of, and necessary for, a significant portion of BCR-induced PtdIns-3,4, 5-P(3) production. Whereas CD19 does not appear to be involved in this SYK-dependent pathway, the SYK substrate CBL is likely involved as the dominant negative SYK markedly attenuates CBL tyrosine phosphorylation and completely blocks the BCR-dependent association of CBL with p85 PI3K.

Paired immunoglobulin-like receptor B (PIR-B) inhibits BCR-induced activation of Syk and Btk by SHP-1.

Coligation of paired immunoglobulin-like receptor B (PIR-B) with B cell antigen receptor (BCR) blocks antigen-induced B cell activation. This inhibition is mediated in part by recruitment of SHP-1 and SHP-2 to the phosphorylated ITIMs in the cytoplasmic domain of PIR-B; however the molecular target(s) of these phosphatases remain elusive. Here we show that PIR-B ligation inhibits the BCR-induced tyrosine phosphorylation of Igalpha/Igbeta, Syk, Btk and phospholipase C (PLC)-gamma2. Overexpression of a catalytically inactive form of SHP-1 prevents the PIR-B-mediated inhibition of tyrosine phosphorylation of Syk, Btk, and PLC-gamma2. Dephosphorylation of Syk and Btk mediated by SHP-1 leads to a decrease of their kinase activity, which in turn inhibits tyrosine phosphorylation of PLC-gamma2. Furthermore, we define a requirement for Lyn in mediating tyrosine phosphorylation of PIR-B. Based on these results, we propose a model of PIR-B-mediated inhibitory signaling in which coligation of PIR-B and BCR results in phosphorylation of ITIMs by Lyn, subsequent recruitment of SHP-1, and a resulting inhibition of the BCR-induced inositol 1,4,5-trisphosphate generation by dephosphorylation of Syk and Btk.

The SH2 domain-containing inositol 5'-phosphatase (SHIP) recruits the p85 subunit of phosphoinositide 3-kinase during FcgammaRIIb1-mediated inhibition of B cell receptor signaling.

Coligation of FcgammaRIIb1 with the B cell receptor (BCR) or FcepsilonRI on mast cells inhibits B cell or mast cell activation. Activity of the inositol phosphatase SHIP is required for this negative signal. In vitro, SHIP catalyzes the conversion of the phosphoinositide 3-kinase (PI3K) product phosphatidylinositol 3,4, 5-trisphosphate (PIP3) into phosphatidylinositol 3,4-bisphosphate. Recent data demonstrate that coligation of FcgammaRIIb1 with BCR inhibits PIP3-dependent Btk (Bruton's tyrosine kinase) activation and the Btk-dependent generation of inositol trisphosphate that regulates sustained calcium influx. In this study, we provide evidence that coligation of FcgammaRIIb1 with BCR induces binding of PI3K to SHIP. This interaction is mediated by the binding of the SH2 domains of the p85 subunit of PI3K to a tyrosine-based motif in the C-terminal region of SHIP. Furthermore, the generation of phosphatidylinositol 3,4-bisphosphate was only partially reduced during coligation of BCR with FcgammaRIIb1 despite a drastic reduction in PIP3. In contrast to the complete inhibition of Tec kinase-dependent calcium signaling, activation of the serine/threonine kinase Akt was partially preserved during BCR and FcgammaRIIb1 coligation. The association of PI3K with SHIP may serve to activate PI3K and to regulate downstream events such as B cell activation-induced apoptosis.

The inhibitory receptor superfamily: potential relevance to atopy.

The role of inhibitory receptor superfamily (IRS) members in the pathophysiology of atopy is still largely unexplored but the past year or so has brought at least three important advances in the understanding of IRS member function: first, several targets of an inositol-phosphatase-dependent inhibitory signaling pathway utilized by some IRS members were identified; second, there is accumulating evidence from murine models supporting the importance of inhibitory Fc gamma receptors in modulating immune-complex-mediated inflammation; third, the inhibitory signaling capability of several previously identified candidate IRS members--including several expressed on cell types implicated in atopy and allergic reactions--has been demonstrated.

Classical conditioning increases membrane-bound protein kinase C in rabbit cerebellum.

We examined membrane-bound protein kinase C (PKC) in the cerebellum of rabbits given paired presentations of a tone conditioned stimulus (CS) that co-terminated with a periocular electrical stimulation unconditioned stimulus (US) or unpaired presentations of the CS and US or restraint in the experimental context. PKC activation was measured by quantitative film autoradiography of [3H]phorbol 12,13-dibutyrate ([3H]PBt2) binding in the molecular and granule cells layers of lobule HVI, anterior vermis and Crus I, and in the dentate/interpositus nuclei. There was a statistically significant increase in [3H]PBt2 binding within the molecular layer of lobule HVI in rabbits given paired training relative to controls. The results indicate PKC activation in lobule HVI may be important in acquisition of conditioned eyeblink responses.

Phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3)/Tec kinase-dependent calcium signaling pathway: a target for SHIP-mediated inhibitory signals.

Tec family non-receptor tyrosine kinases have been implicated in signal transduction events initiated by cell surface receptors from a broad range of cell types, including an essential role in B-cell development. A unique feature of several Tec members among known tyrosine kinases is the presence of an N-terminal pleckstrin homology (PH) domain. We directly demonstrate that phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3) interacting with the PH domain acts as an upstream activation signal for Tec kinases, resulting in Tec kinase-dependent phospholipase Cgamma (PLCgamma) tyrosine phosphorylation and inositol trisphosphate production. In addition, we show that this pathway is blocked when an SH2-containing inositol phosphatase (SHIP)-dependent inhibitory receptor is engaged. Together, our results suggest a general mechanism whereby PtdIns-3,4,5-P3 regulates receptor-dependent calcium signals through the function of Tec kinases.

Btk/Tec kinases regulate sustained increases in intracellular Ca2+ following B-cell receptor activation.

Bruton's tyrosine kinase (Btk) is essential for B-lineage development and represents an emerging family of non-receptor tyrosine kinases implicated in signal transduction events initiated by a range of cell surface receptors. Increased dosage of Btk in normal B cells resulted in a striking enhancement of extracellular calcium influx following B-cell antigen receptor (BCR) cross-linking. Ectopic expression of Btk, or related Btk/Tec family kinases, restored deficient extracellular Ca2+ influx in a series of novel Btk-deficient human B-cell lines. Btk and phospholipase Cgamma (PLCgamma) co-expression resulted in tyrosine phosphorylation of PLCgamma and required the same Btk domains as those for Btk-dependent calcium influx. Receptor-dependent Btk activation led to enhanced peak inositol trisphosphate (IP3) generation and depletion of thapsigargin (Tg)-sensitive intracellular calcium stores. These results suggest that Btk maintains increased intracellular calcium levels by controlling a Tg-sensitive, IP3-gated calcium store(s) that regulates store-operated calcium entry. Overexpression of dominant-negative Syk dramatically reduced the initial phase calcium response, demonstrating that Btk/Tec and Syk family kinases may exert distinct effects on calcium signaling. Finally, co-cross-linking of the BCR and the inhibitory receptor, FcgammaRIIb1, completely abrogated Btk-dependent IP3 production and calcium store depletion. Together, these data demonstrate that Btk functions at a critical crossroads in the events controlling calcium signaling by regulating peak IP3 levels and calcium store depletion.

Negative signaling pathways of the killer cell inhibitory receptor and Fc gamma RIIb1 require distinct phosphatases.

Inhibition of natural killer (NK) cells by the killer cell inhibitory receptor (KIR) involves recruitment of the tyrosine phosphatase SHP-1 by KIR and is prevented by expression of a dominant negative SHP-1 mutant. Another inhibitory receptor, the low affinity Fc receptor for immunoglobulin G (IgG) (Fc gamma RIIb1), has been shown to bind SHP-1 when cocross-linked with the antigen receptor on B cells (BCR). However, coligation of Fc gamma RIIb1 with BCR and with Fc epsilon RI on mast cells leads to recruitment of the inositol 5' phosphatase SHIP and to inhibition of mast cells from SHP-1-deficient mice. In this study, we evaluated the ability of these two inhibitory receptors to block target cell lysis by NK cells, and the contribution of SHP-1 and SHIP to inhibition. Recombinant vaccinia viruses encoding chimeric receptors and dominant negative mutants of SHP-1 and SHIP were used for expression in mouse and human NK cells. When the KIR cytoplasmic tail was replaced by that of Fc gamma RIIb1, recognition of HLA class I on target cells by the extracellular domain resulted in inhibition. A dominant negative mutant of SHP-1 reverted the inhibition mediated by the KIR cytoplasmic tail but not that mediated by Fc gamma RIIb1. In contrast, a dominant negative mutant of SHIP reverted only the inhibition mediated by the Fc gamma RIIb1 tail, providing functional evidence that SHIP plays a role in the Fc gamma RIIb1-mediated negative signal. These data demonstrate that inhibition of NK cells by KIR involves primarily the tyrosine phosphatase SHP-1, whereas inhibition mediated by Fc gamma RIIb1 requires the inositol phosphatase SHIP.

syk kinase activation by a src kinase-initiated activation loop phosphorylation chain reaction.

Activation of the syk tyrosine kinase occurs almost immediately following engagement of many types of antigen receptors, including Fc receptors, but the mechanism through which syk is activated is currently unclear. Here we demonstrate that Fc receptor-induced syk activation occurs as the result of phosphorylation of the syk activation loop by both src family kinases and other molecules of activated syk, suggesting that syk activation occurs as the result of a src kinase-initiated activation loop phosphorylation chain reaction. This type of activation mechanism predicts that syk activation would exhibit exponential kinetics, providing a potential explanation for its rapid and robust activation by even weak antigen receptor stimuli. We propose that a similar mechanism may be responsible for generating rapid activation of other cytoplasmic tyrosine kinases, such as those of the Bruton tyrosine kinase/tec family, as well.

Sequential involvement of Lck and SHP-1 with MHC-recognizing receptors on NK cells inhibits FcR-initiated tyrosine kinase activation.

Recognition of major histocompatibility (MHC) class I complexes on target cells by killer cell inhibitory receptors (KIR) blocks natural killer (NK) and T cell cytotoxic function. The inhibitory effect of KIR ligation requires the phosphotyrosine-dependent association of KIR with the cytoplasmic SH2-containing protein tyrosine phosphatase SHP-1. Using a somatic genetic model, we first define a requirement for the Src family protein tyrosine kinase (PTK) Lck in mediating KIR tyrosine phosphorylation. We then investigate how KIR ligation interrupts PTK-dependent NK cell activation signals. Specifically, we show that KIR ligation inhibits the Fc receptor (FcR)-induced tyrosine phosphorylation of the FcR-associated zeta signaling chain, the PTK ZAP-70, and phospholipase C gamma. Overexpression of catalytically inactive SHP-1 (acting as a dominant negative) restores the tyrosine phosphorylation of these signaling events and reverses KIR-mediated inhibition of NK cell cytotoxic function. These results suggest sequential roles for Lck and SHP-1 in the inhibition of PTK following MHC recognition by NK cells.

Characterization of Cbl tyrosine phosphorylation and a Cbl-Syk complex in RBL-2H3 cells.

Tyrosine phosphorylation of the Cbl protooncogene has been shown to occur after engagement of a number of different receptors on hematopoietic cells. However, the mechanisms by which these receptors induce Cbl tyrosine phosphorylation are poorly understood. Here we demonstrate that engagement of the high affinity IgE receptor (Fc epsilon R1) leads to the tyrosine phosphorylation of Cbl and analyze how this occurs. We show that at least part of Fc epsilon R1-induced Cbl tyrosine phosphorylation is mediated by the Syk tyrosine kinase, and that the Syk-dependent tyrosine phosphorylation of Cbl occurs mainly distal to the Cbl proline-rich region within the COOH-terminal 250 amino acids. Furthermore, we show by coprecipitation that Cbl is present in a complex with Syk before receptor engagement, that the proline-rich region of Cbl and a region of Syk comprised of the two SH2 domains and intradomain linker are required for formation of the complex, and that little or no tyrosine-phosphorylated Cbl is detected in complex with Syk. Overexpression of truncation mutants of Cbl capable of binding Syk has the effect of blocking tyrosine phosphorylation of endogenous Cbl. These results define a potentially important intramolecular interaction in mast cells and suggest a complex function for Cbl in intracellular signaling pathways.