Pyk2 Controls Integrin-Dependent CTL Migration through Regulation of De-Adhesion.

Protein tyrosine kinase 2 (Pyk2) is required for T cell adhesion to ICAM-1; however, the mechanism by which it regulates adhesion remains unexplored. Pyk2 function in murine CTL clones and activated ex vivo CD8(+) T cells was disrupted by pharmacological inhibition, knockdown of expression with small interfering RNA, or expression of the dominant-negative C-terminal domain. We found that Pyk2 is not absolutely required for adhesion of CTL to ICAM-1, but rather delays the initial adhesion.

A role for the protein tyrosine phosphatase CD45 in macrophage adhesion through the regulation of paxillin degradation.

CD45 is a protein tyrosine phosphatase expressed on all cells of hematopoietic origin that is known to regulate Src family kinases. In macrophages, the absence of CD45 has been linked to defects in adhesion, however the molecular mechanisms involved remain poorly defined. In this study, we show that bone marrow derived macrophages from CD45-deficient mice exhibit abnormal cell morphology and defective motility.

Enhanced immunogenicity of a tricomponent mannan tetanus toxoid conjugate vaccine targeted to dendritic cells via Dectin-1 by incorporating β-glucan.

In a previous attempt to generate a protective vaccine against Candida albicans, a β-mannan tetanus toxoid conjugate showed poor immunogenicity in mice. To improve the specific activation toward the fungal pathogen, we aimed to target Dectin-1, a pattern-recognition receptor expressed on monocytes, macrophages, and dendritic cells. Laminarin, a β-glucan ligand of Dectin-1, was incorporated into the original β-mannan tetanus toxoid conjugate providing a tricomponent conjugate vaccine.

Paxillin associates with the microtubule cytoskeleton and the immunological synapse of CTL through its leucine-aspartic acid domains and contributes to microtubule organizing center reorientation.

The cytoskeletal adaptor protein paxillin localizes to the microtubule organizing center (MTOC) in T cells and, upon target cell binding, is recruited to the supramolecular activation complex (SMAC). We mapped the region of paxillin that associates with both the MTOC and SMAC to the leucine-aspartic acid (LD) domains and showed that a protein segment containing LD2-4 was sufficient for MTOC and SMAC recruitment. Examination of the localization of paxillin at the SMAC revealed that paxillin localizes to the peripheral area of the SMAC along with LFA-1, suggesting that LFA-1 may contribute to its recruitment.

Hypophosphorylated and inactive Pyk2 associates with paxillin at the microtubule organizing center in hematopoietic cells.

Pyk2 is a non-receptor tyrosine kinase that regulates cellular adhesion. We generated antibodies to a peptide corresponding to the N-terminus (NT) of Pyk2 and another to a portion of the C-terminal (CT) domain. Only the CT antiserum recovered paxillin-associated Pyk2.

Regulation of the tyrosine kinase Pyk2 by calcium is through production of reactive oxygen species in cytotoxic T lymphocytes.

Pyk2 was identified as a Ca(2+)-dependent kinase, however, the regulation of Pyk2 by Ca(2+) in T cells remains controversial. We found that Ca(2+) mobilization preferentially induced Pyk2 phosphorylation in cytotoxic T lymphocytes (CTL). Furthermore, Pyk2 phosphorylation in CTL was not absolutely Ca(2+) dependent but relied on the strength of T cell receptor stimulation.

Stored Fas ligand, a mediator of rapid CTL-mediated killing, has a lower threshold for response than degranulation or newly synthesized Fas ligand.

CTL lyse target cells through the release of cytolytic granule mediators and expression of the death receptor ligand Fas ligand (FasL). We previously demonstrated that FasL is stored in vesicles distinct from cytolytic granules and is translocated to the cell surface within 15 min of TCR stimulation, followed by a later wave of newly synthesized FasL cell surface expression at 2 h poststimulation. Initial studies suggested that the two FasL responses had different signaling thresholds.

CD45 regulates thymocyte survival during development in fetal thymic organ culture.

The CD45 protein tyrosine phosphatase is essential for T cell development. Its external domain undergoes changes in glycosylation and isoform usage during thymocyte development, the consequences of which remain unknown. The contribution of this complex external domain to T cell development is unknown so we sought to examine the impact of CD45 engagement on T cell development.

CTLs contain and use intracellular stores of FasL distinct from cytolytic granules.

CTL lyse target cells through the release of cytolytic granule contents and cell surface expression of Fas ligand (FasL). Current models suggest that FasL is stored in cytolytic granules and that FasL cell surface expression would be subject to the same controls as degranulation. We demonstrate that murine CTLs undergo two waves of FasL cell surface expression after stimulation.

A novel PKC regulates ERK activation and degranulation of cytotoxic T lymphocytes: Plasticity in PKC regulation of ERK.

Stimulation of cytotoxic T lymphocyte (CTL) degranulation with plate-bound anti-CD3 Ab leads to two phases of ERK activation: an early PKC-independent phase followed by a later sustained PKC-dependent phase. Herein, we show that a novel PKC (nPKC) mediates the late phase of ERK activation, upstream of Ras in murine T cells. In contrast, when CTL are activated with cross-linked anti-CD3 Ab, which does not trigger CTL degranulation, there is a requirement for conventional PKC (cPKC) for ERK activation.

A role for phosphatidylinositol 3-kinase in TCR-stimulated ERK activation leading to paxillin phosphorylation and CTL degranulation.

PI3K is an important regulator of a number of cellular processes. We examined the contribution of PI3K to mouse CTL signaling, leading to degranulation. We show that TCR-triggered, but not phorbol ester and calcium ionophore-induced, CTL degranulation is dependent on PI3K activity.

Tyrosine kinase activity and remodelling of the actin cytoskeleton are co-temporally required for degranulation by cytotoxic T lymphocytes.

In this study, we examined the contribution of the actin cytoskeleton to T-cell receptor (TCR)-initiated signalling in cytotoxic T lymphocytes (CTLs). We demonstrate that cytoskeletal remodelling is required for sustaining TCR-stimulated signals that lead to degranulation by CTLs. Disruption of the actin cytoskeleton in CTLs already undergoing signalling responses results in an almost immediate loss of essentially all protein tyrosine phosphorylation.

Differential Src family kinase activity requirements for CD3 zeta phosphorylation/ZAP70 recruitment and CD3 epsilon phosphorylation.

The current model of T cell activation is that TCR engagement stimulates Src family tyrosine kinases (SFK) to phosphorylate CD3zeta. CD3zeta phosphorylation allows for the recruitment of the tyrosine kinase ZAP70, which is phosphorylated and activated by SFK, leading to the phosphorylation of downstream targets. We stimulated mouse CTLs with plate-bound anti-CD3 and, after cell lysis, recovered proteins that associated with the CD3 complex.

Focal adhesion kinase-related protein tyrosine kinase Pyk2 in T-cell activation and function.

Pyk2 is a protein tyrosine kinase expressed primarily in brain and hematopoietic cells. It becomes activated in response to stimulation through numerous receptors, including integrins, chemokine receptors, and antigen receptors, and is found in association with src-family kinases. Although this enzyme associates with many proteins known to be important for activation and has many characteristics of a scaffolding protein, its function remains elusive.

Phosphatidylinositol-3-kinase regulates PKCtheta activity in cytotoxic T cells.

Protein kinase C (PKC) theta plays a crucial role in T cell activation. We, therefore, examined the regulation of PKCtheta activity in cytotoxic T lymphocytes (CTL). We demonstrated that PMA did not stimulate PKCtheta activation and phospholipase C inhibition did not block anti-CD3-stimulated PKCtheta activation in a CTL clone.

Phosphorylation of Cdc25C by pp90Rsk contributes to a G2 cell cycle arrest in Xenopus cycling egg extracts.

In unfertilized Xenopus eggs, the p42 mitogen activated protein kinase (p42MAPK) pathway is known to maintain cell cycle arrest at metaphase of meiosis II. However, constitutive activation of p42MAPK in post-meiotic, cycling Xenopus egg extracts can lead to either a G2 or M-phase arrest of the cell cycle, depending on the timing of p42MAPK activation. Here, we examined the molecular mechanism by which activation of the p42MAPK pathway during interphase leads to cell cycle arrest in G2.

Beta 1/beta 3 integrin ligation is uncoupled from ERK1/ERK2 activation in cytotoxic T lymphocytes.

beta 3 integrins mediate fibronectin binding and enhanced activation of cytotoxic T lymphocytes (CTL). The intracellular signals initiated by beta 3 integrins in lymphocytes are not well characterized, but in many cell types, beta 1 integrin ligation activates mitogen-activated protein (MAP) kinases. In the present study, we find that fibronectin can synergize with very low levels of CD3 stimulation to activate the extracellular signal-regulated kinase (ERK)1 and ERK2 MAP kinases but that fibronectin alone induces no detectable MAP kinase activation in CTL.

Dynamic association of CD45 with detergent-insoluble microdomains in T lymphocytes.

The receptor-like protein tyrosine phosphatase CD45 is essential for TCR signal transduction. Substrates of CD45 include the protein tyrosine kinases p56(lck) and p59(fyn), both of which have been shown to be enriched in detergent-insoluble microdomains. Here we find that there is a cholesterol-dependent association between CD45 and the raft-associated protein linker for activation of T cells, suggesting that CD45 and linker for activation of T cells may colocalize in lipid rafts.

The protein-tyrosine phosphatase CD45 reaches the cell surface via golgi-dependent and -independent pathways.

CD45 is a receptor protein-tyrosine phosphatase essential for T cell development and lymphocyte activation. It is highly glycosylated, with multiple isoforms and glycoforms expressed on the cell surface depending on the cell type and stage of differentiation. Interestingly, we found two pools of newly synthesized CD45 expressed on plasma membrane, one of which arrived by 5 min after synthesis.

Modulation of Mac-1 (CD11b/CD18)-mediated adhesion by the leukocyte-specific protein 1 is key to its role in neutrophil polarization and chemotaxis.

Leukocyte-specific protein 1 (LSP1) is an intracellular filamentous-actin binding protein which modulates cell motility. The cellular process in which LSP1 functions to regulate motility is not yet identified. In this study, we show that LSP1 negatively regulates fMLP-induced polarization and chemotaxis of neutrophils through its function on adhesion via specific integrins.