In T cells, cAMP-dependent protein kinase (PKA) type I colocalizes with the T cell receptor–CD3 complex (TCR/CD3) and inhibits T cell function via a previously unknown proximal target. Here we examine the mechanism for this PKA-mediated immunomodulation. cAMP treatment of Jurkat and normal T cells reduces Lck-mediated tyrosine phosphorylation of the TCR/CD3 ζ chain after T cell activation, and decreases Lck activity. Phosphorylation of residue Y505 in Lck by COOH-terminal Src kinase (Csk), which negatively regulates Lck, is essential for the inhibitory effect of cAMP on ζ chain phosphorylation. PKA phosphorylates Csk at S364 in vitro and in vivo leading to a two- to fourfold increase in Csk activity that is necessary for cAMP-mediated inhibition of TCR-induced interleukin 2 secretion. Both PKA type I and Csk are targeted to lipid rafts where proximal T cell activation occurs, and phosphorylation of raft-associated Lck by Csk is increased in cells treated with forskolin. We propose a mechanism whereby PKA through activation of Csk intersects signaling by Src kinases and inhibits T cell activation.

We have previously described critical and nonredundant roles for the phosphoinositide 3-kinase p110␦ during the activation and differentiation of naive T cells, and p110␦ inhibitors are currently being developed for clinical use. However, to effectively treat established inflammatory or autoimmune diseases, it is important to be able to inhibit previously activated or memory T cells. In this study, using the isoform-selective inhibitor IC87114, we show that sustained p110␦ activity is required for interferon-␥ production. Moreover, acute inhibition of p110␦ inhibits cytokine production and reduces hypersensitivity responses in mice. Whether p110␦ played a similar role in human T cells was unknown. Here we show that IC87114 potently blocked T-cell receptorinduced phosphoinositide 3-kinase signaling by both naive and effector/memory human T cells. Importantly, IC87114 reduced cytokine production by memory T cells from healthy and allergic donors and from inflammatory arthritis patients. These studies establish that previously activated memory T cells are at least as sensitive to p110␦ inhibition as naive T cells and show that mouse models accurately predict p110␦ function in human T cells. There is therefore a strong rationale for p110␦ inhibitors to be considered for therapeutic use in T-cell-mediated autoimmune and inflammatory diseases. (Blood. 2010;115:2203-2213)

In resting peripheral T cells, Csk is constitutively present in lipid rafts through an interaction with the Csk SH2-binding protein, PAG, also known as Cbp. Upon triggering of the T cell antigen receptor (TCR), PAG/Cbp is rapidly dephosphorylated leading to dissociation of Csk from lipid rafts. However, tyrosine phosphorylation of PAG/Cbp resumes after 3-5 min, at which time Csk reassociates with the rafts. Cells overexpressing a mutant Csk that lacks the catalytic domain, but displaces endogenous Csk from lipid rafts, have elevated basal levels of TCR--chain phosphorylation and spontaneous activation of an NFAT-AP1 reporter from the proximal interleukin-2 promoter as well as stronger and more sustained responses to TCR triggering than controls. We suggest that a transient release from Csk-mediated inhibition by displacement of Csk from lipid rafts is important for normal T cell activation.Activation of the Src family kinases Lck and Fyn after engagement of the T cell antigen receptor is an initiating event in T cell activation and leads to phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) 1 within the TCR complex (1). The subsequent recruitment of the tandem SH2 domain containing tyrosine kinase ZAP-70 to phosphorylated ITAMs generates an activated immune receptor signaling complex that is able to initiate downstream events leading to a functional T cell response (2, 3). The control and fine-tuning of the proximal signaling is not only essential for an effective T cell response to antigen, but also for avoiding exaggerated T cell activation and autoimmunity. Thus a balance must be maintained to avoid hypo-as well as hyper-reactivity and immunopathology. The TCR signaling machinery appears to be controlled by setting a threshold for activation to avoid too easy triggering. Suppression of the catalytic activity of Lck and Fyn by phosphorylation of a C-terminal residue (Tyr 505 in Lck, Tyr 528 in Fyn T ) by the C-terminal Src kinase, Csk, appears to be an important means of negative regulation of TCR signaling (4 -6). Complexed with Csk via binding to its SH3 domain is also a protein tyrosine phosphatase, PEP, that dephosphorylates the activating phosphorylation site (Tyr 394 in Lck, Tyr 416 in Fyn T ) (7,8). Recent discoveries indicate that the assembly of TCR signaling complexes occurs in specific membrane subdomains with high cholesterol and glycolipid contents, called glycosphingolipid-enriched microdomains or lipid rafts (9 -11). Key components, including Lck and LAT, are targeted to rafts by virtue of their lipid modifications, whereas other proteins such as the -chain can localize via interaction with raft components only after TCR engagement (11-13). Lipid rafts serve to concentrate and promote specific protein-protein interactions and the tyrosine phosphorylation of signaling intermediates by the Src family kinases during the proximal phases of immunoreceptor signaling via the TCR as well as via the B cell and Fc receptors (14 -16).The ubiquitously expressed, cytosolic Csk ty...

Background:The ULK complex regulates autophagy, but how it interacts with the basal autophagy apparatus is unknown. Results: ULK1, -2, ATG13, and FIP200 bind to ATG8 family proteins via LIR (LC3 interacting region) motifs. Conclusion: ATG8 family proteins act as scaffolds anchoring the ULK complex on autophagosomes. Significance: We define sequence requirements for the LIR motifs and suggest how the ULK complex interacts with autophagosomes.

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Myotubularin is the archetype of a family of highly conserved protein-tyrosine phosphatase-like enzymes. The myotubularin gene, MTM1, is mutated in the genetic disorder, X-linked myotubular myopathy. We and others have previously shown that myotubularin utilizes the lipid second messenger, phosphatidylinositol 3-phosphate (PI(3)P), as a physiologic substrate. We demonstrate here that the myotubularin-related protein MTMR2, which is mutated in the neurodegenerative disorder, type 4B Charcot-Marie-Tooth disease, is also highly specific for PI(3)P as a substrate. Furthermore, the MTM-related phosphatases MTMR1, MTMR3, and MTMR6 also dephosphorylate PI(3)P, suggesting that activity toward this substrate is common to all myotubularin family enzymes. A direct comparison of the lipid phosphatase activities of recombinant myotubularin and MTMR2 demonstrates that their enzymatic properties are indistinguishable, indicating that the lack of functional redundancy between these proteins is likely to be due to factors other than the utilization of different physiologic substrates. To this end, we have analyzed myotubularin and MTMR2 transcripts during induced differentiation of cultured murine C2C12 myoblasts and find that their expression is divergently regulated. In addition, myotubularin and MTMR2 enhanced green fluorescent protein fusion proteins exhibit overlapping but distinct patterns of subcellular localization. Finally, we provide evidence that myotubularin, but not MTMR2, can modulate the levels of endosomal PI(3)P. From these data, we conclude that the developmental expression and subcellular localization of myotubularin and MTMR2 are differentially regulated, resulting in their utilization of specific cellular pools of PI(3)P. Myotubularin (MTM1)1 is a dual specificity protein-tyrosine phosphatase (PTP)-like enzyme that is mutated in X-linked myotubular myopathy, a severe congenital disorder in which muscle cell development is compromised (1-3). Myogenesis in affected individuals is arrested at a late stage of differentiation/ maturation following myotube formation, and the muscle cells have characteristic large centrally located nuclei (1). The MTM1 protein is the first characterized member of one of the largest families of dual specificity PTPs yet identified (reviewed in Refs. 4 and 5). The MTM family includes at least eight putative catalytically active proteins as well as four forms that are predicted to be enzymatically inactive (4 -7). The inactive MTM proteins contain substitutions at specific residues that are required for catalysis by PTP superfamily enzymes and may function as interaction modules (4 -8). Phylogenetic analysis of MTM family proteins indicates that they can be further divided into at least four distinct subgroups, which include the catalytically active MTM1/MTMR1/MTMR2, MTMR3/MTMR4, and MTMR6/MTMR7/MTMR8 enzymes, as well as the SBF1/LIP-STYX/MTMR10/3-PAP inactive forms (4 -7). Our laboratory and others have previously shown that myotubularin specifically dephosphorylates the D3 position ...

Continuous antigen stimulation of CD4 + CD25 -T cells leads to generation of adaptive CD4 + CD25 + FOXP3 + regulatory T (T R ) cells. Here, we show that highly suppressive adaptive CD8 + CD25 + FOXP3 + T cells can be generated in the same manner by continuous antigen stimulation in the presence of CD14 + monocytes. During the course of stimulation, acquisition of immunosuppressive properties develops in parallel with up-regulation and expression of cytotoxic molecules. The CD8 + T R cells inhibit CD4 + and CD8 + T cell proliferation and cytokine production, but do not alter the expression of granzyme A and granzyme B or perforin in CD8 + effector T cells. Although, the CD8 + T R cells express prostaglandin E 2 , IL-10 and TGF-b, the mechanism of suppression was independent of these soluble factors. In contrast to adaptive CD4 + T R cells, the CD8 + T R cells suppress mainly by a contact-dependent mechanism as evident from transwell experiments. However, neither blocking antibodies to CTLA-4, CD80 nor CD86 could reverse CD8 + T R -mediated suppression, indicating that other mechanism(s) must be employed by these cells. IntroductionCD4 + regulatory T (T R ) cells maintain self-tolerance to autoantigens and are involved in the pathogenesis of various clinical conditions such as autoimmune diseases, chronic viral infections and cancer. Several subsets of CD4 + T R cells have been characterized [1,2]. Whereas naturally occurring CD4 + CD25 + FOXP3 + T R cells are generated in the thymus and suppress effector T cells in a cell contact-dependent manner [3][4][5], adaptive CD4 + CD25 + FOXP3 + T R cells are induced from naive T cells in the periphery and the suppressive activity is independent of cell contact [2,[6][7][8].Suppressive T cells are not strictly confined to the CD4 + T cell compartment and CD8 + T R cells have been characterized in both clinical and experimental conditions [9][10][11][12]. The CD8 + T R cells share phenotypic features with CD4 + T R cells, and as their counterpart, CD8 + T R cells can be generated both in the thymus and in the periphery [13][14][15][16][17][18].In humans, two subsets of adaptive CD8 + CD28 -T R cells exist. Type 1 CD8 + T R cells are generated by stimulation of naive T cells with allogeneic antigenpresenting cells (APC) [19][20][21] properties appears in parallel with up-regulation and expression of cytotoxic molecules. Although the adaptive CD8 + T R cells express prostaglandin E 2 (PGE 2 ), IL-10 and TGF-b, the suppressive mechanism appears to be cell contact-dependent. This is in contrast to adaptive CD4 + T R cells that inhibit T cell immune responses by secretion of humoral factors. Figure 1. Generation of adaptive CD8 + T R cells by continuous antigen stimulation. (A) CD25 + cell-depleted PBMC were stimulated with SEB (4 days). CD8 + CD25 + T cells were added in increasing concentrations to CD25 -cells stimulated with SEB from the autologous blood donor. Proliferation of responding T cells was assessed by CFSE proliferation assay. Representative data are shown (n = 2...

Activation of T and natural killer (NK) cells leads to the tyrosine phosphorylation of pp36 and to its association with several signaling molecules, including phospholipase Cγ-1 and Grb2. Microsequencing of peptides derived from purified rat pp36 protein led to the cloning, in rat and man, of cDNA encoding a T- and NK cell–specific protein with several putative Src homology 2 domain–binding motifs. A rabbit antiserum directed against a peptide sequence from the cloned rat molecule recognized tyrosine phosphorylated pp36 from pervanadate-treated rat thymocytes. When expressed in 293T human fibroblast cells and tyrosine-phosphorylated, pp36 associated with phospholipase Cγ-1 and Grb2. Studies with GST–Grb2 fusion proteins demonstrated that the association was specific for the Src homology 2 domain of Grb-2. Molecular cloning of the gene encoding pp36 should facilitate studies examining the role of this adaptor protein in proximal signaling events during T and NK cell activation.