Edward A. Clark

Caspase‐mediated activation and induction of apoptosis by the mammalian Ste20‐like kinase Mst1

Mst1 is a ubiquitously expressed serine–threonine kinase, homologous to the budding yeast Ste20, whose physiological regulation and cellular function are unknown. In this paper we show that Mst1 is specifically cleaved by a caspase 3‐like activity during apoptosis induced by either cross‐linking CD95/Fas or by staurosporine treatment. CD95/Fas‐induced cleavage of Mst1 was blocked by the cysteine protease inhibitor ZVAD‐fmk, the more selective caspase inhibitor DEVD‐CHO and by the viral serpin CrmA. Caspase‐mediated cleavage of Mst1 removes the C‐terminal regulatory domain and correlates with an increase in Mst1 activity in vivo, consistent with caspase‐mediated cleavage activating Mst1. Overexpression of either wild‐type Mst1 or a truncated mutant induces morphological changes characteristic of apoptosis. Furthermore, exogenously expressed Mst1 is cleaved, indicating that Mst1 can activate caspases that result in its cleavage. Kinase‐dead Mst1 did not induce morphological alterations and was not cleaved upon overexpression, indicating that Mst1 must be catalytically active in order to mediate these effects. Mst1 activates MKK6, p38 MAPK, MKK7 and SAPK in co‐transfection assays, suggesting that Mst1 may activate these pathways. Our findings suggest the existence of a positive feedback loop involving Mst1, and possibly the SAPK and p38 MAPK pathways, which serves to amplify the apoptotic response.

The Role of CD40 and CD80 Accessory Cell Molecules in Dendritic Cell-Dependent HIV-1 Infection

We investigated the role of blood dendritic cells (DCs) in transmission of HIV-1 from infected to uninfected CD4+ T cells, and the accessory molecules involved. DCs promoted transmission from infected to uninfected CD4+ cells, but DCs themselves were not infectable. DC-mediated transmission was blocked by MAb to CD4 and MHC class II, but strongly increased by MAb to CD40 on DCs or CD28 on T cells. The DC-dependent infection was inhibitable by anti-CD80 and a soluble fusion protein of the CD80 ligand, CTLA4; soluble CTLA4 immunoglobulin also blocked infection augmented by cross-linking CD40. These data suggest a linkage between CD40-CD40L and CD28-CD80 counterreceptors on DCs and T cells, and spread of HIV infection in vivo.

ROLE OF DENDRITIC AND FOLLICULAR DENDRITIC CELLS IN HIV INFECTION AND PATHOGENESIS

Dendritic cells (DCs) and follicular dendritic cells (FDCs) play important roles in HIV-mediated pathogenesis. Recent studies have defined new DC subsets and shown that DCs in lymphoid mucosa can both harbor virus and stimulate its spread into CD4(+) T cells. FDCs harbor unspliced HIV mRNA and immune complexed virus, but not actively infectious virus, yet these cells are a key regulated reservoir of virions.

Cell-Cell Interactions Regulate Dendritic Cell-Dependent HIV-1 Production in CD4+ T Lymphocytes

We investigated the role of blood dendritic cells (DC) in transmission of HIV-1 from infected to uninfected CD4+ T cells, and the accessory molecules involved. DC promoted transmission from infected to uninfected CD4+ cells, but blood DC themselves were not infectable. DC-mediated transmission was blocked by mAb to CD4 and MHC class II, but strongly increased by mAb to CD40 on DC or CD28 on T cells. The DC-dependent infection was inhibitable by anti-CD80 and a soluble fusion protein of the CD80 ligand, CTLA4; soluble CTLA4Ig also blocked infection augmented by crosslinking CD40. We also demonstrated that mAb to CD40 up-regulate the expression of CTLA4 ligands CD80 and B70/B7-2 (CD86) on DC. These data suggest that the dialog between CD40-CD40 ligand (CD40L) and CD28-CD80 counter-receptors on DC and T cells may be linked to HIV infection in vivo.

The Interdependence of Lymphocyte, Stromal Cell, and Follicular Dendritic Cell Maturation

If asked to pick a cell type as a biological metaphor for an independent free-spirited entity, many biologists might pick the lymphocyte. Unlike most other cell types in the body, lymphocytes can and must be able to move about the body, a dynamic circulating surveillance system. The fast-moving lymphocyte- on the go- has been an obvious center of attention for decades now; where lymphocytes go, what they do, what they need in order to live and function are major issues for immunologists. By contrast, attached, relatively sessile cells seem to have fewer options, be less dynamic, less flexible, less interesting. One widely held assumption has been that those cells coming in contact with lymphocytes, by-in-large play nurturing or minor roles, hence the term “accessory” cell, accessory being defined in one dictionary as “aiding or contributing in a secondary way”. They are viewed as “aides”, even valets or butlers, for “executive” lymphocytes such as CD4+ T cells which have the really important jobs.

Accessory Molecules that Influence Signaling Through B Lymphocyte Antigen Receptors

The B lymphocyte antigen receptor complex, commonly called the B cell receptor (BCR) complex, consists of surface immunoglobulin (sIg) and heterodimers of the Igα (CD79a, mb1) and Igβ (CD79b, B29) phosphoglycoproteins. Recent reviews detail pertinent findings on the BCR complex.1 The structure of the BCR complex and some cell-surface molecules that influence signaling via the BCR are shown in Figure 1. The clonotypic Ig receptor has only a very short cytoplasmic tail and therefore must rely on the invariant members of the BCR complex to transmit signals to the cytosol after receptor crosslinking. Igα/Igβ associate with sIgM and sIgD and are both necessary and sufficient for the expression of sIg. This heterodimer is analogous to the TCR CD3e/δ or CD3e/γ heterodimers,2 which, like Igα/Igβ, (1) contain subunits with a single extracellular Ig-like domain; (2) are phosphorylated on tyrosine (CD3e and CD3ζ) after crosslinking of antigen receptors; and (3) contain within their cytoplasmic tail a single antigen receptor homology 1 (ARH1) motif, D/E-X7-D/E-X2-Y-X2-L-X7-Y-X2-L/I (X = any amino acid). Regions within the transmembrane domain of sIgM are required for the release of [Ca2+]i or internalization of bound antigen.3 Mutations within the transmembrane domain of sIgM that inhibit the activation of new protein tyrosine phosphorylation (PTP) and release of [Ca2+]i also uncouple sIgM from Igα/Igβ.4 However, even though such a mutant sIgM does not associate with Igα/Igβ, when crosslinked it still induces some new PTP.4 Both Sanchez et al.4 and Kim et al.5 reported that surface chimeric fusion proteins expressing the cytoplasmic tails of Igα vs. Igβ differ in their ability to transmit signals: the Igα but not the Igβ tail can induce new PTP, results consistent with studies suggesting that Igα and not Igβ strongly associates with the protein tyrosine kinase (PTK) p53/56Lyn (Lyn).6 Matsuuchi et al.7 found that sIgM expression could be reconstituted in a pituitary cell line with Igα/Igβ coexpression, but that Igα/Igβ were not sufficient to reconstitute a complete signal through sIgM. Thus, IgM interaction with Igα/Igβ is critical for signaling but other factors may also be required.