The critical role of Toll-like receptors (TLRs) as mediators of pathogen recognition by the innate immune system is now firmly established. Such recognition results in the initiation of an inflammatory immune response and subsequent instruction of the adaptive immune system, both of which are designed to rid the host of the invading pathogen. More controversial is the potential role of TLRs in the recognition of endogenous ligands and what effect this might have on the consequent development of autoimmune or other chronic sterile inflammatory disorders. An increasing number of studies implicate TLRs as being involved in the immune response to self-molecules that have in some way been altered from their native state or accumulate in non-physiologic sites or amounts, although questions have been raised about possible contaminants in certain of these studies. In this review, we discuss the evidence for endogenous ligand-TLR interactions with particular emphasis on mammalian chromatin, systemic lupus erythematosus, and atherosclerosis. Overall, the data support the general concept of a role for TLRs in the recognition of endogenous ligands. However, the precise details of the interactions and the extent to which they may contribute to the pathogenesis of human disease remain to be clarified.

In the vascular endothelium, diverse cell surface receptors are coupled to the Ca2+/calmodulin-dependent activation of nitric oxide (NO) synthase. We now report that, in intact cultured endothelial cells, several drugs and agonists are associated with increased serine phosphorylation of the endothelial NO synthase. We biosyntheticaily labeled bovine aortic endothelial cells with [32PJorthophosphoric acid, exposed the cells to various drugs and hormones, and then immunoprecipitated the enzyme from cell extracts using a highly specific anti-peptide antibody. The marked endothelial NO synthase phosphorylation induced by bradykinin is maximal only after 5 min of agonist exposure and is stable for at least 20 min. Basal and agonist-induced phosphorylation of the NO synthase in endothelial cells is completely inhibited by the calmodulin antagonist compound W-7. We prepared subcel- nine-labeled NO synthase is now found in the cytosolic fraction, associated with a marked increase in the level of cytosolic enzyme phosphorylation. We propose that agonist-induced phosphorylation of NO synthase is associated with translocation of the enzyme from membrane to cytosol and may thereby regulate the biological effects of endothelial NO synthesis in situ.Nitric oxide (NO) is a ubiquitous intercellular signaling molecule and is synthesized in diverse mammalian tissues by a family of related NO synthase enzymes. In all tissues thus far characterized, the NO synthase isoforms share a common overall catalytic scheme for the oxidation of L-arginine to form NO plus L-citrulline and appear to share similar cofactor requirements (reviewed in refs. 1-3). The different tissuespecific NO synthase isoforms subserve disparate biological functions in diverse mammalian cells and appear to be encoded by distinct genes (4).The endothelial NO synthase plays an important role in the control of blood pressure and platelet aggregation (5). In bovine endothelial cells, the NO synthase is transiently activated by increases in intracellular calcium induced by the activation of diverse G-protein-coupled cell surface receptors, including bradykinin. Our analysis of the primary structure of the endothelial NO synthase cDNA identified consensus sequences for posttranslational modifications of the enzyme, including N-terminal myristoylation as well as phosphorylation (4). We have recently shown that the endothelialThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.NO synthase undergoes N-terminal myristoylation, and this covalent modification is essential for the association of the enzyme with the particulate subcellular fraction (6). Some myristoylated proteins implicated in intracellular signaling pathways have been shown to regulated by reversible phosphorylation, and phosphorylation further appears to modulate their subcellular localization (reviewed in refs. 7 and 8).The deduced primary stru...

The nitric oxide synthases (NOS) comprise a family of enzymes which differ in primary structure, biological roles, subcellular distribution, and post-translational modifications. The endothelial nitric oxide synthase (ec-NOS) is unique among the NOS isoforms in being modified by N-terminal myristoylation, which is necessary for its targeting to the endothelial cell membrane. The subcellular localization of the ecNOS, but not enzyme myristoylation, is dynamically regulated by agonists such as bradykinin, which promote ecNOS translocation from membrane to cytosol, as well as enhancing enzyme phosphorylation. Using transiently transfected endothelial cells, we now show that a myristoylation-deficient mutant ecNOS undergoes phosphorylation despite restriction to the cytosol, suggesting that phosphorylation may be a consequence rather than a cause of ecNOS translocation. We therefore explored whether other post-translational modifications might regulate ecNOS targeting and now report that ecNOS is reversibly palmitoylated. Biosynthetic labeling of endothelial cells with [3H]palmitic acid followed by immunoprecipitation of ecNOS revealed that the enzyme is palmitoylated; the label is released by hydroxylamine, consistent with formation of a fatty acyl thioester, and authentic palmitate can be recovered from labeled ecNOS following acid hydrolysis. Importantly, pulse-chase experiments in endothelial cells biosynthetically labeled with [3H]palmitate show that bradykinin treatment promotes ecNOS depalmitoylation. We conclude that ecNOS palmitoylation is dynamically regulated by bradykinin and propose that depalmitoylation of the enzyme may result in its cytosolic translocation and subsequent phosphorylation.

Calcium-dependent protein kinases (CDPKs), the most abundant serine/threonine kinases in plants, are found in various subcellular localizations, which suggests that this family of kinases may be involved in multiple signal transduction pathways. A complete analysis to try to understand the molecular basis of the presence of CDPKs in various localizations in the cell has not been accomplished yet. It has been suggested that myristoylation may be responsible for membrane association of CDPKs. In this study, we used a rice CDPK, OSCPK2, which has a consensus sequence for myristoylation at the N-terminus, to address this question. We expressed wild-type OSCPK2 and various mutants in different heterologous systems to investigate the factors that affect its membrane association. The results show that OSCPK2 is myristoylated and palmitoylated and targeted to the membrane fraction. Both modifications are required, myristoylation being essential for membrane localization and palmitoylation for its full association. The fact that palmitoylation is a reversible modification may provide a mechanism for regulation of the subcellular localization. OSCPK2 is the first CDPK shown to be targeted to membranes by an src homology domain 4 (SH4) located at the N-terminus of the molecule.