Functional complementation of BLNK by SLP-76 and LAT linker proteins

Recent studies have demonstrated a requirement for the SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa) and LAT (linker for activation of T cells) adaptor/linker proteins in T cell antigen receptor activation and T cell development as well as the BLNK (B cell linker) linker protein in B cell antigen receptor (BCR) signal transduction and B cell development. Whereas the SLP-76 and LAT adaptor proteins are expressed in T, natural killer, and myeloid cells and platelets, BLNK is preferentially expressed in B cells and monocytes. Although BLNK is structurally homologous to SLP-76, BLNK interacts with a variety of downstream signaling proteins that interact directly with both SLP-76 and LAT. Here, we demonstrate that neither SLP-76 nor LAT alone is sufficient to restore the signaling deficits observed in BLNK-deficient B cells. Conversely, the coexpression of SLP-76 and LAT together restored BCR-inducible calcium responses as well as activation of all three families of mitogen-activated protein kinases. Together, these data suggest functional complementation of SLP-76 and LAT in T cell antigen receptor function with BLNK in BCR function.     

Hematopoietic progenitor kinase 1 associates physically and functionally with the adaptor proteins B cell linker protein and SLP-76 in lymphocytes

B cell linker protein (BLNK) is a SLP-76-related adaptor protein essential for signal transduction from the BCR. To identify components of BLNK-associated signaling pathways, we performed a phosphorylation-dependent yeast two-hybrid analysis using BLNK probes. Here we report that the serine/threonine kinase hematopoietic progenitor kinase 1 (HPK1), which is activated upon antigen-receptor stimulation and which has been implicated in the regulation of MAP kinase pathways, interacts physically and functionally with BLNK in B cells and with SLP-76 in T cells. This interaction requires Tyr(379) of HPK1 and the Src homology 2 (SH2) domain of BLNK/SLP-76. Via homology modeling, we defined a consensus binding site within ligands for SLP family SH2 domains. We further demonstrate that the SH2 domain of SLP-76 participates in the regulation of AP-1 and NFAT activation in response to T cell receptor (TCR) stimulation and that HPK1 inhibits AP-1 activation in a manner partially dependent on its interaction with SLP-76. Our data are consistent with a model in which full activation of HPK1 requires its own phosphorylation on tyrosine and subsequent interaction with adaptors of the SLP family, providing a mechanistic basis for the integration of this kinase into antigen receptor signaling cascades.     

Hematopoietic growth factors activate the tyrosine phosphorylation of distinct sets of proteins in interleukin-3-dependent murine cell lines.

By immunoblotting with antibodies for phosphotyrosine, we have demonstrated that the hematopoietic growth factors interleukin-2, interleukin-3, interleukin-4, and granulocyte-macrophage colony-stimulating factor stimulate the tyrosine phosphorylation of specific sets of proteins in murine hematopoietic progenitor cell lines. The stimulation of tyrosine phosphorylation is a receptor-dependent transient event. The effect of these hematopoietic growth factors on protein tyrosine phosphorylation was not mediated through protein kinase C.     

Regulation of highly cytokinergic IgE-induced mast cell adhesion by Src, Syk, Tec, and protein kinase C family kinases

Mast cells play a critical role in IgE-dependent immediate hypersensitivity. Recent studies have shown that, contrary to the traditional view, binding of monomeric IgE to Fc epsilon RI results in a number of biological outcomes in mast cells, including survival. However, IgE molecules display heterogeneity in inducing cytokine production; highly cytokinergic (HC) IgEs cause extensive Fc epsilon RI aggregation, which leads to potent enhancement of survival and other activation events, whereas poorly cytokinergic (PC) IgEs can do so inefficiently. The present study demonstrates that HC, but not PC, IgEs can efficiently induce adhesion and spreading of mouse mast cells on fibronectin-coated plates in slow and sustained kinetics. HC IgE-induced adhesion through beta1 and beta7 integrins promotes survival, IL-6 production, and DNA synthesis. Importantly, we have identified Lyn and Syk as requisite tyrosine kinases and Hck, Btk, and protein kinase C theta as contributory kinases in HC IgE-induced adhesion and spreading, whereas protein kinase C epsilon plays a negative role. Consistent with these results, Lyn, Syk, and Btk are activated in HC IgE-stimulated cells in a slower but more sustained manner, compared with cells stimulated with IgE and Ag. Thus, binding of HC IgEs to Fc epsilon RI induces adhesion of mast cells to fibronectin by modulating cellular activation signals in a unique fashion.     

Differential requirement for adapter proteins Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa and adhesion- and degranulation-promoting adapter protein in FcepsilonRI signaling and mast cell function

The adapter molecule Src homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is essential for FcepsilonRI-mediated signaling, degranulation and IL-6 production in mast cells. To test the structural requirements of SLP-76 in mast cell signaling and function, we have studied the functional responses of murine bone marrow-derived mast cells (BMMCs) expressing mutant forms of SLP-76. We found that the N-terminal tyrosines as well as the central proline-rich region of SLP-76 are required for participation of SLP-76 in FcepsilonRI-mediated signaling and function. The C-terminal SH2 domain of SLP-76 also contributes to optimal function of SLP-76 in mast cells. Another adapter molecule, adhesion- and degranulation-promoting adapter protein (ADAP), is known to bind the SH2 domain of SLP-76, and cell line studies have implicated ADAP in mast cell adhesion and FcepsilonRI-induced degranulation. Surprisingly, we found that mast cells lacking ADAP expression demonstrate no defects in FcepsilonRI-induced adhesion, granule release, or IL-6 production, and that ADAP-deficient mice produce a normal passive systemic anaphylactic response. Thus, failure to bind ADAP does not underlie the functional defects exhibited by SLP-76 SH2 domain mutant-expressing mast cells.     

p56(lck), ZAP-70, SLP-76, and calcium-regulated effectors are involved in NF-kappaB activation by bisperoxovanadium phosphotyrosyl phosphatase inhibitors in human T cells

This study investigates the second messengers involved in NF-kappaB activation by the bisperoxovanadium (bpV) phosphotyrosyl phosphatase inhibitors. We first initiated a time course analysis of bpV-mediated activation of the human immunodeficiency virus type-1 long terminal repeat- and NF-kappaB-driven reporter gene. Our results showed a slower and more transient activation of both kappaB-regulated luciferase-encoding vectors by bpV compounds when compared with the action of tumor necrosis factor-alpha (TNF). Time course analyses of NF-kappaB translocation by shift assay experiments further confirmed these results, hence implying distinct pathways of NF-kappaB activation for bpV compounds and TNF. Attempts to characterize the bpV-dependent signaling cascade revealed that the src family protein tyrosine kinase p56(lck) was critical for NF-kappaB induction by bpV. Furthermore, p56(lck) interaction with the intracytoplasmic tail of CD4 markedly enhanced such induction. Optimal activation of NF-kappaB following bpV treatment necessitated downstream effectors of p56(lck) such as the syk family protein tyrosine kinase ZAP-70 and the molecular adaptor SLP-76. Importantly, reduced NF-kappaB activation was observed when capacitative calcium entry was deficient but also upon pharmacological inhibition of calmodulin and calcineurin. Altogether, these results suggest that induction of NF-kappaB by phosphotyrosyl phosphatase bpV inhibitors necessitates both proximal and distal effectors of T cell activation.     

Nuclear matrix proteins in well and poorly differentiated human breast cancer cell lines

The nuclear matrix, besides providing the structural support of the nucleus, is involved in various cellular functions of the nucleus. Nuclear matrix proteins (NMPs), which are both tissue- and cell type–specific, are altered with transformation and state of differentiation. Furthermore, NMPs have been identified as informative markers of disease states. Here, the NMP profiles from human breast cancer cell lines and breast tumours were analyzed using two-dimension gel electrophoresis. We identified NMPs that are associated with well and poorly differentiated human breast cancer cells in vitro and in vivo. Five NMPs (NMBC 1–5) were found to be exclusive for well-differentiated human breast cancer cells, while one NMP (NMBC-6) was found to be present only in poorly differentiated human breast cancer cells. The identification of these proteins suggests the potential use of nuclear matrix proteins as prognostic indicators. J. Cell. Biochem. 66:9–15, 1997. © 1997 Wiley-Liss, Inc.     

Evidence that phospholipase C-gamma2 interacts with SLP-76, Syk, Lyn, LAT and the Fc receptor gamma-chain after stimulation of the collagen receptor glycoprotein VI in human platelets.

Platelet activation by collagen is mediated by the sequential tyrosine phosphorylation of the Fc receptor gamma-chain (FcR gamma-chain), which is part of the collagen receptor glycoprotein VI, the tyrosine kinase Syk and phospholipase C-gamma2 (PLC-gamma2). In this study tyrosine-phosphorylated proteins that associate with PLC-gamma2 after stimulation by a collagen-related peptide (CRP) were characterized using glutathione S-transferase fusion proteins of PLC-gamma2 Src homology (SH) domains and by immunoprecipitation of endogenous PLC-gamma2. The majority of the tyrosine-phosphorylated proteins that associate with PLC-gamma2 bind to its C-terminal SH2 domain. These were found to include PLC-gamma2, Syk, SH2-domain-containing leucocyte protein of 76 kDa (SLP-76), Lyn, linker for activation of T cells (LAT) and the FcR gamma-chain. Direct association was detected between PLC-gamma2 and SLP-76, and between PLC-gamma2 and LAT upon CRP stimulation of platelets by far-Western blotting. FcR gamma-chain and Lyn were found to co-immunoprecipitate with PLC-gamma2 as well as with unidentified 110-kDa and 75-kDa phosphoproteins. The absence of an in vivo association between Syk and PLC-gamma2 in platelets is in contrast with that for PLC-gamma1 and Syk in B cells. The in vivo function of PLC-gamma2 SH2 domains was examined through measurement of Ca2+ increases in mouse megakaryocytes that had been microinjected with recombinant proteins. This revealed that the C-terminal SH2 domain is involved in the regulation of PLC-gamma2. These data indicate that the C-terminal SH2 domain of PLC-gamma2 is important for PLC-gamma2 regulation through possible interactions with SLP-76, Syk, Lyn, LAT and the FcR gamma-chain.     

Persistent and remodeling hepatic preneoplastic lesions present differences in cell proliferation and apoptosis, as well as in p53, Bcl-2 and NF-kappaB pathways

During rat hepatocarcinogenesis preneoplastic lesions (PNL) emerge which may persist (pPNL) and be sites of progress to cancer or suffer remodeling (rPNL) tending to disappear. Cellular and molecular mechanisms involved in both phenotypes are not sufficiently elucidated. pPNL and rPNL cellular proliferation and apoptosis were evaluated in rats submitted to the resistant hepatocyte (RH) model, and an adjusted growth index (AGI) was established. p53, Bcl-2, and NF-kappaB p65 subunit expression was evaluated by immunohistochemistry in pPNL and rPNL. p65 expression and NF-kappaB activation was evaluated by Western blot assays in whole livers. A lower number of BrdU-stained hepatocyte nuclei/mm(2) and higher number of apoptotic bodies (AB) per mm(2) were observed in remodeling compared to pPNL. Cytoplasmic p53 accumulation is related to increased hepatocarcinoma malignancy. We observed that 71.3% pPNL and 25.4% rPNL (P < 0.05) presented p53 staining in the cytoplasm. Similarly, 67.7% pPNL and 23.1 % rPNL (P < 0.05) presented increased Bcl-2 staining. Thirty-two percent pPNL and 15.6% rPNL (P < 0.05) presented p65 staining. Compared to normal rats, increase (P < 0.05) of hepatic p65 expression and NF-kappaB activation in rats submitted to the RH model was observed. In agreement to previous studies hepatic pPNL and rPNL differ regarding cell proliferation and apoptosis. Moreover, persistence and remodeling involve differences in p53, Bcl-2, and NF-kappaB pathways. These data point to molecular pathways that may direct preneoplastic lesions to spontaneously regress or to progress to cancer. J. Cell. Biochem. 103: 538-546, 2008. (c) 2007 Wiley-Liss, Inc.     

Comparison of CMV, RSV, SV40 viral and Vlambda1 cellular promoters in B and T lymphoid and non-lymphoid cell lines.

Determining the activity of viral and cellular regulatory elements in B or T lymphoid cell lines would facilitate appropriate utilization of the regulatory sequences for gene transfer- and expression-dependent applications. We have compared the activity of the CMV, RSV and SV40 viral promoter/enhancers as well as the Vlambda1 cellular promoter, in three B cell lines (REH, SMS-SB, C3P), three T cell lines (CEM, Jurkat, ST-F10), and two non-lymphoid cell lines (K-562, HeLa) using the luciferase reporter gene. In B cell lines, the activity of the CMV promoter/enhancer construct was the highest ranging from 10- to 113-fold greater than that of SV40. In contrast, in T cell lines the RSV promoter/enhancer activity was 11-65-fold higher than that of SV40. The Vlambda1 promoter activity was close to that of SV40 promoter/enhancer in most of the cell lines tested. We conclude that CMV and RSV promoter/enhancers contain stronger regulatory elements than do the SV40 and Vlambda1 for expression of genes in lymphoid cell lines.     

Murine T cell lines can change CD25 expression patterns as well as proliferation and cell death patterns in response to interleukin 2

T cell subpopulations were obtained from F12.5 and B245/270D T cell lines during long-term culture. Two altered F12.5 subpopulations proliferated more intensively than the original clone. These two subpopulations of F12.5 constantly expressed CD25 (interleukin 2 receptor α chain) at a high level and exogenously added interleukin 2 (IL-2) enhanced cell death for one of these subpopulations. However, the original clone expressed CD25 only after activation and IL-2 inhibited cell death of the original clone. On the other hand, the altered B245/270D subpopulation lost the antigen-specific proliferation ability. This altered cell line under the stimulation culture did not express CD25 even after activation, although the original line expressed CD25. However, the expression pattern of CD25on the altered cell line at resting state was induced similar to that of the original one. These results suggest that an expression pattern of CD25 can be changed during long-term cultures, accompanied with alteration in response to proliferation and cell death. This revised version was published online in August 2006 with corrections to the Cover Date.     

Identification of proteins regulated by interferon-alpha in resistant and sensitive malignant melanoma cell lines

Treatment of patients with malignant melanoma with interferon-alpha achieves a response in a small but significant subset of patients. Currently, although much is known about interferon biology, little is known about either the particular mechanisms of interferon-alpha activity that are crucial for response or why only some patients respond to interferon-alpha therapy. Two melanoma cell lines (MeWo and MM418) that are known to differ in their response to the antiproliferative activity of interferon-alpha, have been used as a model system to investigate interferon-alpha action. Using a proteomics approach based on two-dimensional polyacrylamide gel electrophoresis and mass spectrometry, several proteins induced in response to interferon-alpha have been identified. These include a number of gene products previously known to be type I interferon responsive (tryptophanyl tRNA synthetase, leucine aminopeptidase, ubiquitin cross-reactive protein, gelsolin, FUSE binding protein 2 and hPNPase) as well as a number of proteins not previously reported to be induced by type I interferon (cathepsin B, proteasomal activator 28alpha and alpha-SNAP). Although the proteins upregulated by interferon-alpha were common between the cell lines when examined at the level of Western blotting, the disparity in the basal level of cathepsin B was striking, raising the possibility that the higher level in MM418 may contribute to the sensitivity of this cell line to interferon-alpha treatment.     

Selective repression of YKL-40 by NF-kappaB in glioma cell lines involves recruitment of histone deacetylase-1 and -2

Here we show that in contrast to other cancer types, tumor necrosis factor (TNF)-alpha suppresses YKL-40 expression in glioma cell lines in a nuclear factor kappaB (NF-kappaB) dependent manner. Even though TNF-alpha causes recruitment of p65 and p50 subunits of NF-kappaB to the YKL-40 promoter in all cell types, recruitment of histone deacetylases (HDAC)-1 and -2, and a consequent deacetylation of histone H3 at the YKL-40 promoter occurs only in glioma cells. Importantly, using chromatin immunoprecipitation assays in frozen glioblastoma multiforme tissues, we show that YKL-40 levels decrease consistent with HDAC1 recruitment despite high levels of nuclear p-p65. This study presents a paradigm for NF-kappaB regulation of one of its targets in a strict cell type specific manner.     

Pathogenesis and stress related,as well as metabolic proteins are regulated in tomato stems infected with Ralstonia solanacearum

A comparative proteome analysis was initiated to systematically investigate the physiological response of tomato (Solanum lycopersicum) to infection with Ralstonia solanacearum, causal agent of bacterial wilt. Plants of the susceptible tomato recombinant inbred line NHG3 and the resistant NHG13 were either infected or not infected with R. solanacearum and subsequently used for proteome analysis. Two-dimensional isoelectric focussing/sodium dodecyl-sulphate polyacrylamide gel electrophoresis (2-D IEF/SDS-PAGE) allowed the separation of about 650–690 protein spots per analysis. Twelve proteins were of differential abundance in susceptible plants in response to bacterial infection, while no differences were observed in the resistant genotype. LC-MS/MS analysis of these spots revealed 12 proteins, six of which were annotated as plant and six as bacterial proteins. Among the plant proteins, two represent pathogenesis related (PR) proteins, one stress response protein, one enzyme of carbohydrate and energy metabolism, and one hypothetical protein. A constitutive difference between resistant and susceptible lines was not found.     

Post-translational modification of proteins by 15-carbon and 20-carbon isoprenoids in three mammalian cell lines

Summary A number of cellular proteins, including p21^ras, lamin B, and the G-protein subunits, undeDanvillergo post-translational modification by 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoid moieties derived from pyrophosphate intermediates of the cholesterol biosynthetic pathway. In this study, isoprenylated proteins in three mammalian cell lines (Hela cells, Rat-6 fibroblasts and COS cells) were radiolabeled with an isoprenoid precursor, [³H]mevalonate, and resolved by SDS gel electrophoresis. Groups of proteins with different molecular masses were eluted from the gels and the chain-lengths of the radiolabeled isoprenyl groups, released from the proteins by Raney-nickel-catalyzed desulfurization, were established by gel permeation chromatography. 15-Carbon and 20-carbon isoprenyl groups were found in separate classes of proteins within each cell line. With the exception of p21^ras, which incorporated a 15-carbon group when expressed in COS cells, the proteins in the region of the 21–28 kDa ras-related GTP binding proteins contained mostly 20-carbon isoprenyl chains. In contrast, proteins belonging to the 66–72 kDa nuclear lamin family, as well as unidentified proteins with molecular masses of 41–46 kDa and 53–55 kDa, contained predominantly 15-carbon isoprenyl chains. The chain-lengths of the isoprenoids associated with particular classes of proteins did not vary from one cell line to another, suggesting that the nature of the isoprenoid modification (farnesyl versus geranylgeranyl) is determined by intrinsic structural features of the proteins, rather than the cell type in which the proteins are expressed.Abbreviations MVA Mevalonolactone - SDS Sodium Dodecyl Sulfate - PAGE Polyacrylamide Gel Electrophoresis     

Hypothetical proteins with putative enzyme activity in human amnion, lymphocyte, bronchial epithelial and kidney cell lines

A myriad of predicted proteins have been described based upon nucleic acid sequences but the existence of these structures has not been confirmed at the protein level. The aim of the study was therefore to show expression of hypothetical proteins in several cell lines and to provide the analytical basis for their identification and characterisation. We used two-dimensional gel electrophoresis with in-gel digestion of high protein spots and subsequent MALDI-TOF analysis of cell lysates from human amnion, lymphocyte, bronchial epithelial and kidney cell lines. A pI range from 3 to 10 was selected and second dimension was run using 9-16% gradient gels. A series of structures that have not been described before at the protein level were identified in several cell lines and were assigned to major enzyme systems including proteolysis (proteases, peptidases, ubiquitin), intermediary metabolism and oxidoreductases. We conclude that the proteomic approach used serves as a suitable tool to verify the existence of predicted/hypothetical proteins. The herein identified enzymes may contribute to several pathways/cascades in the human organism. Furthermore, analytical data given are of major relevance as pIs, a prerequisite to find proteins in a map, cannot be predicted from nucleic acid sequences.     

Fludarabine and cladribine induce changes in surface proteins on human B-lymphoid cell lines involved with apoptosis, cell survival, and antitumor immunity

Fludarabine and cladribine are purine analogues used to treat hematological malignancies. Alone or in combination with therapeutic antibodies, they are effective in treating patients with chronic lymphocytic leukemia and non-Hodgkin's lymphoma. However, the mechanisms of action of these drugs are not well understood. Plasma membrane proteins perform a variety of essential functions that can be affected by malignancy and perturbed by chemotherapy. Analysis of surface proteins may contribute to an understanding of the mechanisms of action of purine analogues and identify biomarkers for targeted therapy. The surface of human cells is rich in N-linked glycoproteins, enabling use of a hydrazide-coupling technique to enrich for glycoproteins, with iTRAQ labeling for quantitative comparison. A number of plasma membrane proteins on human leukemia and lymphoma cells were affected by treatment with a purine analogue, including decreases in CD22 (an adhesion and signaling molecule) and increases in CD205 (a "damaged cell marker") and CD80 and CD50 (T-cell interaction molecules). Purine analogues may affect B-cell receptor (BCR) signaling and costimulatory molecules, leading to multiple signals for apoptosis and cell clearance. Fludarabine and cladribine induce differential effects, with some cell survival proteins (ECE-1 and CD100) more abundant after fludarabine treatment. Cell surface proteins induced by fludarabine and cladribine may be targets for therapeutic antibodies.     

Neuronal cell life,death, and axonal degeneration as regulated by the BCL-2 family proteins

Axonal degeneration and neuronal cell death are fundamental processes in development and contribute to the pathology of neurological disease in adults. Both processes are regulated by BCL-2 family proteins which orchestrate the permeabilization of the mitochondrial outer membrane (MOM). MOM permeabilization (MOMP) results in the activation of pro-apoptotic molecules that commit neurons to either die or degenerate. With the success of small-molecule inhibitors targeting anti-apoptotic BCL-2 proteins for the treatment of lymphoma, we can now envision the use of inhibitors of apoptosis with exquisite selectivity for BCL-2 family protein regulation of neuronal apoptosis in the treatment of nervous system disease. Critical to this development is deciphering which subset of proteins is required for neuronal apoptosis and axon degeneration, and how these two different outcomes are separately regulated. Moreover, noncanonical BCL-2 family protein functions unrelated to the regulation of MOMP, including impacting necroptosis and other modes of cell death may reveal additional potential targets and/or confounders. This review highlights our current understanding of BCL-2 family mediated neuronal cell death and axon degeneration, while identifying future research questions to be resolved to enable regulating neuronal survival pharmacologically.Subject terms: Cell biology, Chemical tools, Neuroscience, Neurological disorders