Mice homozygous for a single tyrosine mutation in LAT (linker for activation of T cells) exhibited an early block in T cell maturation but later developed a polyclonal lymphoproliferative disorder and signs of autoimmune disease. T cell antigen receptor (TCR)-induced activation of phospholipase C-gamma1 (PLC-gamma1) and of nuclear factor of activated T cells, calcium influx, interleukin-2 production, and cell death were reduced or abrogated in T cells from LAT mutant mice. In contrast, TCR-induced Erk activation was intact. These results identify a critical role for integrated PLC-gamma1 and Ras-Erk signaling through LAT in T cell development and homeostasis.

Two apoptosis-linked genes, named ALG-2 and ALG-3, were identified by means of a functional selection strategy. ALG-2 codes for a Ca(2+)-binding protein required for T cell receptor-, Fas-, and glucocorticoid-induced cell death. ALG-3, a partial complementary DNA that is homologous to the familial Alzheimer's disease gene STM2, rescues a T cell hybridoma from T cell receptor- and Fas-induced apoptosis. These findings suggest that ALG-2 may mediate Ca(2+)-regulated signals along the death pathway and that cell death may play a role in Alzheimer's disease.

Overexpression of the familial Alzheimer's disease gene Presenilin 2 (PS2) in nerve growth factor-differentiated PC12 cells increased apoptosis induced by trophic factor withdrawal or beta-amyloid. Transfection of antisense PS2 conferred protection against apoptosis induced by trophic withdrawal in nerve growth factor-differentiated or amyloid precursor protein-expressing PC12 cells. The apoptotic cell death induced by PS2 protein was sensitive to pertussis toxin, suggesting that heterotrimeric GTP-binding proteins are involved. A PS2 mutation associated with familial Alzheimer's disease was found to generate a molecule with enhanced basal apoptotic activity. This gain of function might accelerate the process of neurodegeneration that occurs in Alzheimer's disease, leading to the earlier age of onset characteristic of familial Alzheimer's disease.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that has novel dual actions. S1P is the ligand for a family of G protein-coupled receptors known as S1PRs that mediate various physiological functions. Growth factors rapidly activate sphingosine kinase type 1 (SPHK1) resulting in phosphorylation of sphingosine to form S1P, which plays important roles in cell growth regulation and protection from apoptosis. However, little is known of the mechanism(s) by which SPHK activity is regulated. Using a yeast two-hybrid screening approach, we cloned a 3-kb cDNA encoding a SPHK1-interacting protein (SKIP). BLAST analysis revealed that SKIP corresponded to the C-terminal region of a larger (ϳ7 kb) cDNA that encoded a protein with a high degree of similarity to a family of protein kinase A anchor proteins (AKAP). In confirmation of the yeast two-hybrid assay, glutathione S-transferase (GST)-SPHK1 specifically pulled down SKIP, whereas GST did not. Moreover, immunoprecipitation of in vitro translated SPHK1 and SKIP revealed that SKIP and SPHK1 are tightly associated. Furthermore, SKIP overexpression in NIH 3T3 fibroblasts reduced SPHK1 activity and interfered with its biological functions. The apoptoticsparing effect of SPHK1 against serum deprivation was reduced when co-transfected with SKIP. In addition, SPHK1-enhanced cell proliferation was also abolished by SKIP, with a corresponding decrease in activation of ERK. Taken together, these results indicate that SKIP is a novel protein likely to play a regulatory role in the modulation of SPHK1 activity.

ALG-3, a truncated mouse homologue of the chromosome 1 familial Alzheimer's disease gene PS2, rescues T hybridoma 3DO cells from T-cell receptor-induced apoptosis by inhibiting Fas ligand induction and Fas signaling. Here we show that ALG-3 transfected 3DO cells express a COOH-terminal PS2 polypeptide. Overexpression of PS2 in ALG-3 transfected 3DO cells reconstitutes sensitivity to receptor-induced cell death, suggesting that the artificial PS2 polypeptide functions as a dominant negative mutant of PS2. ALG-3 and antisense PS2 protect PC12 cells from glutamate-induced apoptosis but not from death induced by hydrogen peroxide or the free radical MPP ؉ . Thus, the PS2 gene is required for some forms of cell death in diverse cell types, and its function is opposed by ALG-3.

Sphingosine kinase catalyzes the phosphorylation of sphingosine to form sphingosine 1-phosphate (SPP), a novel lipid mediator with both intra-and extracellular functions. Based on sequence identity to murine sphingosine kinase (mSPHK1a), we cloned and characterized the first human sphingosine kinase (hSPHK1). The open reading frame of hSPHK1 encodes a 384 amino acid protein with 85% identity and 92% similarity to mSPHK1a at the amino acid level. Similar to mSPHK1a, when HEK293 cells were transfected with hSPHK1, there were marked increases in sphingosine kinase activity resulting in elevated SPP levels. hSPHK1 also specifically phosphorylated D-erythro-sphingosine and to a lesser extent sphinganine, but not other lipids, such as D,L-threo-dihydrosphingosine, N,N-dimethylsphingosine, diacylglycerol, ceramide, or phosphatidylinositol. Northern analysis revealed that hSPHK1 was widely expressed with highest levels in adult liver, kidney, heart and skeletal muscle. Thus, hSPHK1 belongs to a highly conserved unique lipid kinase family that regulates diverse biological functions.z 2000 Federation of European Biochemical Societies.

The apoptosis-linked gene product, ALG-2, is a member of the family of intracellular Ca 2؉ -binding proteins and a part of the apoptotic machinery controlled by T-cell receptor (TCR), Fas, and glucocorticoid signals. To explore the physiologic function of ALG-2 in T-cell development and function, we generated mice harboring a null mutation in the alg-2 gene. The alg-2 null mutant mice were viable and fertile and showed neither gross developmental abnormality nor immune dysfunction. Analyses of apoptotic responses of ALG-2-deficient T cells demonstrated that ALG-2 deficiency failed to block apoptosis induced by TCR, Fas, or dexamethasone signals. These findings indicate that ALG-2 is physiologically dispensable for apoptotic responses induced by the above signaling pathways and suggest that other functionally redundant proteins might exist in mammalian cells.Apoptosis is a unique biological process of multicellular organisms and one of the driving forces underlying tissue/ organ remodeling during animal development. This process also plays an important role during immune system development and function (1). Under physiological conditions, apoptotic response of lymphocytes is elicited by specialized molecular networks comprising of multiple signaling pathways and appears to be pivotal for shaping lymphocyte repertoires and preventing autoimmune diseases (8). Although large numbers of molecules involved in such networks have been identified recently (28), regulation and coordination between these molecules during immune system development and function remain elusive.ALG-2 is a 22-kDa Ca 2ϩ -binding protein belonging to the penta-EF (PEF) hand protein family that contains the Ca 2ϩ -binding helix-loop-helix structure (14,15,25). The PEF hand protein family includes peflin, sorcin, and grancalcin, as well as the large and small subunits of calpains (2,10,15,24). ALG-2 is ubiquitously expressed in mouse tissues, with its highest level of expression detected in thymus and liver. Recent data from T-cell lines indicate that ALG-2 protein plays a critical role for T-cell receptor (TCR)-, Fas-, and glucocorticoid-induced apoptosis, because depletion of ALG-2 in these cells blocks apoptosis whereas its overexpression significantly promotes apoptosis induced by these signals (5, 25). Although it is unclear how ALG-2 affects apoptosis of lymphocytes, evidence indicates that ALG-2 becomes rapidly associated with ALG-2-interacting protein-1 (AIP-1), a proapoptotic protein in a Ca 2ϩ -dependent manner, suggesting that the regulation is likely controlled by a secondary messenger, Ca 2ϩ (17,26). Detailed biochemical analysis also places ALG-2 downstream of the ICE/Ced-3 signaling cascade activated by TCR, Fas, and dexamethasone stimulation (11).To study the function of ALG-2 under physiological conditions, we generated ALG-2-deficient mice by using the gene targeting approach. Our data indicate that the general development and survival of mutant mice, as well as their immune system development and differentiation, appear to be nor...

The lysophospholipids, lysophosphatidic acid, sphingosine-1-phosphate, and sphingosylphosphorylcholine (SPC), are bioactive lipid molecules that regulate diverse biological processes. Although the specific G protein-coupled receptors for lysophosphatidic acid and sphingosine-1-phosphate have been well-characterized, much less is known of the SPC receptors. It has been reported that ovarian cancer G protein-coupled receptor 1 (OGR1) is a high affinity receptor for SPC, and its closely related homologue GPR4 is a high affinity receptor for SPC with low affinity for lysophosphatidylcholine (LPC). However, in a functional assay to examine the specificity of ligand binding, we found that neither SPC nor LPC, or other related lysophospholipids, induced internalization of GPR4 from the plasma membrane. In agreement, these lysolipids also did not induce translocation of beta-arrestin2-GFP from the cytosol to the plasma membrane in GPR4 expressing cells. However, when these cells were cotransfected with G protein-coupled receptor kinase 2, in the absence of added ligands, beta-arrestin2-GFP accumulated in cytoplasmic vesicles, reminiscent of vesicular labeling usually observed after agonist stimulation of GPCRs. In addition, neither SPC nor LPC stimulated the binding of GTPgammaS to membranes prepared from GPR4 expressing cells and did not activate ERK1/2. Surprisingly, enforced expression of GPR4 inhibited activation of ERK1/2 induced by several stimuli, including SPC, sphingosine-1-phosphate, and even EGF. Collectively, our results suggest that SPC and LPC are not the ligands for GPR4 and that this receptor may constitutively inhibit ERK1/2 activation.