The immunomodulatory drug FTY720 is phosphorylated in vivo, and the resulting FTY720 phosphate as a ligand for sphingosine-1-phosphate receptors is responsible for the unique biological effects of the compound. So far, phosphorylation of FTY720 by murine sphingosine kinase (SPHK) 1a had been documented. We found that, while FTY720 is also phosphorylated by human SPHK1, the human type 2 isoform phosphorylates the drug 30-fold more efficiently, because of a lower K m of FTY720 for SPHK2. Similarly, murine SPHK2 was more efficient than SPHK1a. Among splice variants of the human SPHKs, an N-terminally extended SPHK2 isoform was even more active than SPHK2 itself. Further SPHK superfamily members, namely ceramide kinase and a "SPHK-like" protein, failed to phosphorylate sphingosine and FTY720. Thus, only SPHK1 and 2 appear to be capable of phosphorylating FTY720. Using selective assay conditions, SPHK1 and 2 activities in murine tissues were measured. While activity of SPHK2 toward sphingosine was generally lower than of SPHK1, FTY720 phosphorylation was higher under conditions favoring SPHK2. In human endothelial cells, while activity of SPHK1 toward sphingosine was 2-fold higher than of SPHK2, FTY720 phosphorylation was 7-fold faster under SPHK2 assay conditions. Finally, FTY720 was poorly phosphorylated in human blood as compared with rodent blood, in line with the low activity of SPHK1 and in particular of SPHK2 in human blood. To conclude, both SPHK1 and 2 are capable of phosphorylating FTY720, but SPHK2 is quantitatively more important than SPHK1.FTY720 is an immunomodulatory drug, which is highly efficacious in models of transplantation and of autoimmune diseases (1). It was recently found to be effective in kidney transplantation in humans (2). FTY720 elicits a lymphopenia resulting from the reversible redistribution of lymphocytes from the circulation to secondary lymphoid organs, without leading to general immunosuppression (3, 4). Conversion of FTY720 to its monophosphate appears to be essential for the effects of the drug on lymphocyte homing, since FTY720 phosphate acts as an agonist at four of the five G-protein-coupled receptors for sphingosine-1-phosphate (S1P) 1 (5, 6); it is assumed that at least one S1P receptor is critical to the lymphopenic response induced by FTY720 treatment (2). More recently, FTY720 was found to stimulate multidrug transporterdependent T-cell chemotaxis to lymph nodes (7); in this instance, FTY720 phosphate as the active metabolite is hypothesized to be responsible for stimulation of efflux activity of the lipid transporter Abcb1. FTY720 has been reported to be phosphorylated ex vivo by rodent lymphoid tissues (5) and whole blood of several species (6), and is rapidly phosphorylated in vivo (5, 6). After oral application of FTY720 to rats, the blood levels of the monophosphate exceeded those of the parent compound 2-4 fold (5). FTY720 was shown to be a substrate for recombinant murine sphingosine kinase 1a (muSPHK1a) (5). Studies with chiral analogs of FTY720 (namely the R-an...

The paracaspase MALT1 plays an important role in immune receptor-driven signaling pathways leading to NF-κB activation. MALT1 promotes signaling by acting as a scaffold, recruiting downstream signaling proteins, as well as by proteolytic cleavage of multiple substrates. However, the relative contributions of these two different activities to T and B cell function are not well understood. To investigate how MALT1 proteolytic activity contributes to overall immune cell regulation, we generated MALT1 protease-deficient mice (Malt1PD/PD) and compared their phenotype with that of MALT1 knockout animals (Malt1−/−). Malt1PD/PD mice displayed defects in multiple cell types including marginal zone B cells, B1 B cells, IL-10–producing B cells, regulatory T cells, and mature T and B cells. In general, immune defects were more pronounced in Malt1−/− animals. Both mouse lines showed abrogated B cell responses upon immunization with T-dependent and T-independent Ags. In vitro, inactivation of MALT1 protease activity caused reduced stimulation-induced T cell proliferation, impaired IL-2 and TNF-α production, as well as defective Th17 differentiation. Consequently, Malt1PD/PD mice were protected in a Th17-dependent experimental autoimmune encephalomyelitis model. Surprisingly, Malt1PD/PD animals developed a multiorgan inflammatory pathology, characterized by Th1 and Th2/0 responses and enhanced IgG1 and IgE levels, which was delayed by wild-type regulatory T cell reconstitution. We therefore propose that the pathology characterizing Malt1PD/PD animals arises from an immune imbalance featuring pathogenic Th1- and Th2/0-skewed effector responses and reduced immunosuppressive compartments. These data uncover a previously unappreciated key function of MALT1 protease activity in immune homeostasis and underline its relevance in human health and disease.

FTY720, a potent immunomodulatory drug in phase 2/3 clinical trials, induces rapid and reversible sequestration of lymphocytes into secondary lymphoid organs, thereby preventing their migration to sites of inflammation. As prerequisite for its function, phosphorylation of FTY720 to yield a potent agonist of the sphingosine-1-phosphate receptor S1P 1 is required in vivo, catalyzed by an as-yet-unknown kinase. Here, we report on the generation of sphingosine kinase 2 (SPHK2) knockout mice and demonstrate that this enzyme is essential for FTY720 phosphate formation in vivo. Consequently, administration of FTY720 does not induce lymphopenia in SPHK2-deficient mice. After direct dosage of FTY720 phosphate, lymphopenia is only transient in this strain, indicating that SPHK2 is constantly required to maintain FTY720 phosphate levels in vivo. IntroductionNaive T cells regularly circulate between the bloodstream and lymphatic tissue in search for foreign antigen, as well as for tumor and autoantigen. Their activation in secondary lymphoid organs followed by regulated egress back into the circulation to reach sites of inflammation is a prerequisite for any adaptive immune response in the T-cell compartment. Recently, one of the G protein-coupled receptors for sphingosine-1-phosphate (S1P), namely the S1P 1 receptor, was shown to be crucial for the tempo-spatial trafficking of T cells into and out of the secondary peripheral lymphoid organs. 1 The importance of S1P 1 in lymphocyte trafficking became clear through studies with FTY720, an analog of sphingosine. FTY720, after phosphorylation in vivo to FTY720 phosphate (FTY720-P), induces a reversible sequestration of lymphocytes into lymph nodes and Peyer patches. 2,3 FTY720-P thereby acts as a functional antagonist of the S1P 1 receptor, thus inducing aberrant internalization and consequently rendering T cells unresponsive to the obligatory egress signal provided by S1P. 1,[4][5][6] FTY720 has emerged as a potent immunomodulatory agent with usefulness in the control of organ transplant rejection and for treatment of autoimmune diseases. In animals, FTY720 is efficacious in prolonging graft survival, as well as in models of multiple sclerosis, acute lung injury, autoimmune diabetes, atherosclerosis, and renal ischemia-reperfusion injury. 7 Promising results have been obtained from human trials on FTY720 for indications in renal transplantation and multiple sclerosis. 7,8 Since FTY720 prodrug activation is essential for its action on T (and B) cells, understanding how the drug gets phosphorylated in vivo is of high interest, in particular for the design of novel analogs with altered pharmacologic properties.FTY720 is known to be phosphorylated in vitro by the 2 mammalian sphingosine kinases SPHK1 and 2, with SPHK2 being considerably more efficient. [9][10][11] As shown by a recent study in SPHK1-deficient mice, 12 this enzyme appears to be dispensable for the action of FTY720 in vivo, as Sphk1-null mice are still rendered lymphopenic by the drug.In this study, we describe th...

Serine 657 in protein kinase C-␣ (PKC␣) is a site of phosphorylation on expression of the recombinant protein in mammalian cells. To define the function of this phosphorylation, PKC␣ species with mutations of this site were investigated. The alanine mutant, S657A PKC␣, displayed slow phosphate accumulation in pulsechase experiments, indicating a rate-limiting role in the initial phase of phosphorylation. Consistent with this, the aspartic acid mutant, S657D PKC␣, showed an increased rate of phosphate accumulation. Both the S657D and S657A PKC␣ mutants were slow to accumulate as fully phosphorylated forms during a second phase of phosphorylation. This latter property is shown to correlate with an increased phosphatase sensitivity and decreased protein kinase activity for these two PKC␣ mutants. It is further shown that once fully phosphorylated, the S657D PKC␣ mutant displays WT PKC␣ properties with respect to thermal stability and phosphatase sensitivity in vitro and in vivo; in contrast, the S657A PKC␣ mutant remains sensitive. The properties of the Ser-657 site PKC␣ mutants define functional roles for this phosphorylation in both the accumulation of phosphate on PKC␣ as well as in its agonist-induced dephosphorylation. These results are discussed in the context of a working model of PKC␣ behavior, providing insight into the workings of other kinases with equivalent sites of phosphorylation.