Svetlana P. Sidorenko

Phospholipase C-gamma1 interacts with conserved phosphotyrosyl residues in the linker region of Syk and is a substrate for Syk.

Antigen receptor ligation on lymphocytes activates protein tyrosine kinases and phospholipase C-gamma (PLC-gamma) isoforms. Glutathione S-transferase fusion proteins containing the C-terminal Src-homology 2 [SH2(C)] domain of PLC-gamma1 bound to tyrosyl phosphorylated Syk. Syk isolated from antigen receptor-activated B cells phosphorylated PLC-gamma1 on Tyr-771 and the key regulatory residue Tyr-783 in vitro, whereas Lyn from the same B cells phosphorylated PLC-gamma1 only on Tyr-771. The ability of Syk to phosphorylate PLC-gamma1 required antigen receptor ligation, while Lyn was constitutively active. An mCD8-Syk cDNA construct could be expressed as a tyrosyl-phosphorylated chimeric protein tyrosine kinase in COS cells, was recognized by PLC-gamma1 SH2(C) in vitro, and induced tyrosyl phosphorylation of endogenous PLC-gamma1 in vivo. Substitution of Tyr-525 and Tyr-526 at the autophosphorylation site of Syk in mCD8-Syk substantially reduced the kinase activity and the binding of this variant chimera to PLC-gamma1 SH2(C) in vitro; it also failed to induce tyrosyl phosphorylation of PLC-gamma1 in vivo. In contrast, substitution of Tyr-348 and Tyr-352 in the linker region of Syk in mCD8-Syk did not affect the kinase activity of this variant chimera but almost completely eliminated its binding to PLC-gamma1 SH(C) and completely eliminated its ability to induce tyrosyl phosphorylation of PLC-gamma1 in vivo. Thus, an optimal kinase activity of Syk and an interaction between the linker region of Syk with PLC-gamma1 are required for the tyrosyl phosphorylation of PLC-gamma1.

The dual-function CD150 receptor subfamily: the viral attraction.

The CD150 subfamily within the CD2 family is a growing group of dual-function receptors that have within their cytoplasmic tails a characteristic signaling motif. The ITSM (immunoreceptor tyrosine-based switch motif) enables these receptors to bind to and be regulated by small SH2 domain adaptor proteins, including SH2D1A (SH2-containing adaptor protein SH2 domain protein 1A) and EAT-2 (EWS-activated transcript 2). A major signaling pathway through the prototypic receptor in this subfamily, CD150, leads to the activation of interferon-γ, a key cytokine for viral immunity. As a result, many viruses have designed strategies to usurp or alter CD150 functions. Measles virus uses CD150 as a receptor and Molluscum contagiosum virus encodes proteins that are homologous to CD150. Thus, viruses use CD150 subfamily receptors to create a favorable environment to elude detection and destruction. Understanding the CD150 subfamily may lead to new strategies for vaccine development and antiviral therapies.

Protein Kinase C μ (PKCμ) Associates with the B Cell Antigen Receptor Complex and Regulates Lymphocyte Signaling

We have identified a Ser/Thr kinase associated with the B cell receptor (BCR) complex as protein kinase C mu (PKC mu). PKC mu activity is up-regulated after cross-linking the BCR and CD19 on B cells, and PKC mu co-precipitates with Syk and phospholipase C-gamma 1/2 (PLC gamma 1/2). In vitro phosphorylation of fusion proteins showed that both Syk and PLC gamma 1 are potential substrates of PKC mu in vivo. Analysis of mutants of the chicken B cell line DT40 deficient in either Syk, Lyn, Btk, or PLC gamma 2 revealed that BCR-induced activation of PKC mu, like activation of PLC gamma 2, requires Syk and is partially regulated by Btk, but is Lyn independent. PKC mu can down-regulate the ability of Syk to phosphorylate PLC gamma 1 in vitro. Thus, PKC mu may function in a negative feedback loop regulating BCR-initiated signaling cascades.

Regulation of lymphocyte activation by the cell-surface molecule CD22

Accessory molecules play an important role in the regulation of lymphocyte activation mediated by the B-cell antigen receptor (BCR). CD22 is one such accessory molecule expressed on B-lineage cells. Here, Che-Leung Law and colleagues review current knowledge on the structure-function relationship between CD22 and the BCR, discuss the role of CD22 as a cell-adhesion molecule and suggest models for potential in vivo functions of CD22.

Immunodeficiency virus cDNA synthesis in resting T lymphocytes is regulated by T cell activation signals and dendritic cells

Abstract: We explored the relationship between T cell activation signals and dendritic cells (DC) in the replication cycle of immunodeficiency viruses. First we analyzed the effect of two cell cycle inhibitors (mimosine and aphidicolin) on SIV reverse transcription, circularization, and integration in macaque resting T cells stimulated with anti‐CD3 mAb at the time of infection. The formation of SIV LTR circles was blocked by the G1 inhibitor mimosine. The G1/S inhibitor aphidicolin neither affected circularization nor integration of SIV DNA. Therefore, the induction of SIV LTR circle production is likely to be mediated by signaling events normally regulating the G1 to S transition. We further characterized DC‐dependent HIV‐expression in human T cells. We examined the effect of ligating two novel receptors, IPO‐3 and Bgp95, on DC‐dependent HIV‐1 expression. Activation of DCs through IPO‐3 receptors, and to a lesser extent Bgp95 ligation, upregulated HIV spread in these cells. The mechanisms by which IPO‐3 vs. Bgp95 increase HIV‐1 levels appear to be different. In particular, IPO‐3 ligation alone on T cells also increased HIV‐1 levels. Activation of T cells via defined surface receptors or with DCs is required for establishing HIV/SIV cDNA synthesis in T cells.

CD150 regulates JNK1/2 activation in normal and Hodgkin's lymphoma B cells.

The CD150 receptor is expressed on thymocytes, activated and memory T cells, B cells, platelets, natural killer T cells, and mature dendritic cells, and is also detected on tumor cells of Hodgkins lymphoma (HL) and diffuse large B‐cell lymphoma with an activated B cell phenotype. Here, we report that the level of CD150 expression is elevated during B cell differentiation toward plasma cells. In primary tonsillar B cells and HL cell lines, CD150 signaling regulates the phosphorylation of three types of mitogen‐activated protein kinases (MAPKs): extracellular signal‐regulated kinase 1/2 (ERK1/2), p38 MAPK, and Jun N‐terminal kinase 1/2 (JNK1/2). CD150 induced ERK1/2 activation in primary tonsillar B cells and in two HL cell lines. CD150 mediated activation of JNK1/2 p54 and JNK2‐γ kinase isoforms in all CD150+ B cell lines we tested. CD150 associated with the serine/threonine kinase hematopoetic progenitor kinase 1 (HPK1) regardless of CD150 tyrosine phosphorylation or binding of the SH2D1A adaptor protein to CD150, and HPK1 overexpression enhanced CD150‐mediated JNK1/2 phosphorylation. CD150 ligation inhibited cell proliferation of all studied HL cell lines and induced apoptosis in L1236 HL cells that did not depend on JNK activity. As signaling through CD150 modulates MAPK activity in HL tumor cells, CD150 may contribute to regulation of tumor cell maintenance in low‐rate proliferating HLs.

FCRL6 receptor: Expression and associated proteins

FCRL6 receptor is a more recently identified representative of the FCRL family. We generated a panel of mouse mAbs to baculovirus-derived recombinant FCRL6 protein. The clone 7B2 was found to specifically recognize a 63kDa protein expressed preferentially on the surface of CD8 T and CD56 NK cells in human peripheral blood and spleen. The clone 7B2 reacts with FCRL6 in Western blotting, FACS, and immunohistochemistry. In the T cell lineage, FCRL6 functions in antigen-experienced cells. Mitogenic stimulation of PB leukocytes in vitro resulted in an abrogation of the FCRL6 gene expression. We found a significant decrease in the FCRL6 gene expression in peripheral T cells of patients with certain autoimmune and blood diseases, and its upregulation at the late stages of HIV infection. Study of the FCRL6 association with signaling molecules showed its ability to recruit SHP-1, SHP-2, SHIP-1, and SHIP-2 phosphatases, and also adaptor protein Grb2 through phosphorylated cytoplasmic tyrosines. The current results demonstrate inhibitory potential of FCRL6 and suggest its possible involvement in modulation of CTL effector functions in various immune disorders.

SLAMF1/CD150 in hematologic malignancies: Silent marker or active player?

SLAMF1/CD150 receptor is a founder of signaling lymphocyte activation molecule (SLAM) family of cell-surface receptors. It is widely expressed on cells within hematopoietic system. In hematologic malignancies CD150 cell surface expression is restricted to cutaneous T-cell lymphomas, few types of B-cell non-Hodgkins lymphoma, near half of cases of chronic lymphocytic leukemia, Hodgkins lymphoma, and multiple myeloma. Differential expression among various types of hematological malignancies allows considering CD150 as diagnostical and potential prognostic marker. Moreover, CD150 may be a target for antibody-based or measles virus oncolytic therapy. Due to CD150 signaling properties it is involved in regulation of malignant cell fate decision and tumor microenvironment in Hodgkins lymphoma and chronic lymphocytic leukemia. This review summarizes evidence for the important role of CD150 in pathogenesis of hematologic malignancies.

The interplay of CD150 and CD180 receptor pathways contribute to the pathobiology of chronic lymphocytic leukemia B cells by selective inhibition of Akt and MAPK signaling

Cell surface expression of CD150 and CD180 receptors in chronic lymphocytic leukemia (CLL) associates with mutational IGHV status and favourable prognosis. Here we show a direct correlation between cell surface expression and colocalization of these receptors on CLL B cells. In the absence of CD150 and CD180 on the cell surface both receptors were expressed in the cytoplasm. The CD150 receptor was colocalized with markers of the endoplasmic reticulum, the Golgi apparatus and early endosomes. In contrast, CD180 was detected preferentially in early endosomes. Analysis of CD150 isoforms differential expression revealed that regardless of CD150 cell surface expression the mCD150 isoform with two ITSM signaling motifs was a predominant CD150 isoform in CLL B cells. The majority of CLL cases had significantly elevated expression level of the soluble sCD150, moreover CLL B cells secrete this isoform. CD150 or CD180 crosslinking on CLL B cells alone led to activation of Akt, mTORC1, ERK1/2, p38MAPK and JNK1/2 networks. Both CD150 and CD180 target the translation machinery through mTOR independent as well as mTOR dependent pathways. Moreover, both these receptors transmit pro-survival signals via Akt-mediated inhibition of GSK3β and FOXO1/FOXO3a. Unexpectedly, coligation CD150 and CD180 receptors on CLL B cells led to mutual inhibition of the Akt and MAPK pathways. While CD150 and CD180 coligation resulted in reduced phosphorylation of Akt, ERK1/2, c-Jun, RSK, p70S6K, S6RP, and 4E-BP; it led to complete blocking of mTOR and p38MAPK phosphorylation. At the same time coligation of CD150 and CD40 receptors did not result in Akt and MAPK inhibition. This suggests that combination of signals via CD150 and CD180 leads to blocking of pro-survival pathways that may be a restraining factor for neoplastic CLL B cells propagation in more than 50% of CLL cases where these receptors are coexpressed.