IL-1β is a potent proinflammatory cytokine of the innate immune system that is involved in host defense against infection. However, increased production of IL-1β plays a pathogenic role in various inflammatory diseases, such as rheumatoid arthritis, gout, sepsis, stroke, and transplant rejection. To prevent detrimental collateral damage, IL-1β release is tightly controlled and typically requires two consecutive danger signals. LPS from Gram-negative bacteria is a prototypical first signal inducing pro–IL-1β synthesis, whereas extracellular ATP is a typical second signal sensed by the ATP receptor P2X7 that triggers activation of the NLRP3-containing inflammasome, proteolytic cleavage of pro–IL-1β by caspase-1, and release of mature IL-1β. Mechanisms controlling IL-1β release, even in the presence of both danger signals, are needed to protect from collateral damage and are of therapeutic interest. In this article, we show that acetylcholine, choline, phosphocholine, phosphocholine-modified LPS from Haemophilus influenzae, and phosphocholine-modified protein efficiently inhibit ATP-mediated IL-1β release in human and rat monocytes via nicotinic acetylcholine receptors containing subunits α7, α9, and/or α10. Of note, we identify receptors for phosphocholine-modified macromolecules that are synthesized by microbes and eukaryotic parasites and are well-known modulators of the immune system. Our data suggest that an endogenous anti-inflammatory cholinergic control mechanism effectively controls ATP-mediated release of IL-1β and that the same mechanism is used by symbionts and misused by parasites to evade innate immune responses of the host.

Protein kinases of the microtubule affinity-regulating kinase (MARK) family were originally discovered because of their ability to phosphorylate certain sites in tau protein (KXGS motifs in the repeat domain). This type of phosphorylation is enhanced in abnormal tau from Alzheimer brain tissue and causes the detachment of tau from microtubules. MARK-related kinases (PAR-1 and KIN1) occur in various organisms and are involved in establishing and maintaining cell polarity. Herein, we report the ability of MARK2 to affect the differentiation and outgrowth of cell processes from neuroblastoma and other cell models. MARK2 phosphorylates tau protein at the KXGS motifs; this results in the detachment of tau from microtubules and their destabilization. The formation of neurites in N2a cells is blocked if MARK2 is inactivated, either by transfecting a dominant negative mutant, or by MARK2 inhibitors such as hymenialdisine. Alternatively, neurites are blocked if the target KXGS motifs on tau are rendered nonphosphorylatable by point mutations. The results suggest that MARK2 contributes to the plasticity of microtubules needed for neuronal polarity and the growth of neurites. INTRODUCTIONThe establishment of neuronal polarity and the generation of cell processes require the interplay between signaling mechanisms (from extracellular cues to the cytoplasm and to the nucleus), which enable the cell to decide when and where to grow a neurite, and mechanochemical elements (cytoskeleton, motors, and membranes) that allow the neurite to push outward. The actin network in the cell cortex tends to resist gross shape changes, and consequently actin-disassembly drugs facilitate neurite outgrowth (Edson et al., 1993;Knowles et al., 1994;Bradke and Dotti, 1999), whereas microtubules provide the core for a growing cell process, and therefore microtubule-disassembly poisons prevent the outgrowth (Baas and Ahmad, 1993;Rochlin et al., 1996). In addition microtubules must be dynamically unstable to allow growth cone formation, and therefore both microtubulestabilizing and -destabilizing drugs can inhibit neurite outgrowth (Liao et al., 1995;Tanaka et al., 1995;Jordan and Wilson, 1998;Kaverina et al., 1998;Waterman-Storer and Salmon, 1999;Goode et al., 2000;Kabir et al., 2001). In this study, we focus on the neuronal microtubule-associated protein tau, its role in neurite outgrowth, and its regulation by phosphorylation. Tau is a mixture of six splicing isoforms ( Figure 1; Lee et al., 1988;Goedert et al., 1989) that become largely axonal during development (Binder et al., 1985;Hirokawa et al., 1996). The accepted role of tau is that of a microtubule stabilizer (Cleveland et al., 1977;Drubin and Kirschner, 1986;Butner and Kirschner, 1991;Gustke et al., 1994;Panda et al., 1999), although other roles such as a regulator of axonal traffic Stamer et al., 2002), anchor for kinases and phosphatases (Lee et al., 1998;Liao et al., 1998;Sontag et al., 1999), or membrane linker (Brandt et al., 1995) have recently emerged. Tau strongly promotes neurite...

contributed equally to this work MARK, a kinase family related to PAR-1 involved in establishing cell polarity, phosphorylates microtubuleassociated proteins (tau/MAP2/MAP4) at KXGS motifs, causes detachment from microtubules, and their disassembly. The sites are prominent in tau from Alzheimer's disease brains. We studied the activation of MARK and identi®ed the upstream kinase, MARKK, a member of the Ste20 kinase family. It phosphorylates MARK within the activation loop (T208 in MARK2). A fraction of MARK in brain tissue is doubly phosphorylated (at T208/S212), reminiscent of the activation of MAP kinase; however, the phosphorylation of the second site in MARK (S212) is inhibitory. In cells the activity of MARKK enhances microtubule dynamics through the activation of MARK and leads to phosphorylation and detachment of tau or equivalent MAPs from microtubules. Overexpression of MARK eventually leads to microtubule breakdown and cell death, but in neuronal cells the primary effect is to allow the development of neurites during differentiation.

Background: The causal relationship between Tau hyperphosphorylation and aggregation in neuropathology is still under debate.Results: Tau highly phosphorylated in cells increases oligomerization without pronounced aggregation. Oligomers cause reduction of dendritic spines but not cell death.Conclusion: Hyperphosphorylation does not drive Tau fibrillization but contributes to synaptotoxicity.Significance: Pathways and effects of Tau hyperphosphorylation are distinct from those of aggregation.

Highlights d Tracheal chemosensory cells recognize virulence-associated formyl peptides d This activates a TRPM5-dependent pathway, triggering acetylcholine release d Acetylcholine released from chemosensory cells activates mucociliary clearance d Mice with genetic impairment of this pathway are more susceptible to infection

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MARK/Par-1, a kinase family with diverse functions particularly in inducing cell polarity, can phosphorylate microtubule-associated proteins in their repeat domain and cause their detachment from microtubules, and thereby microtubule destabilization. Because of its role in abnormal phosphorylation of the Tau protein in Alzheimer disease, we searched for regulatory kinases. MARK family kinases can be activated by phosphorylation of a conserved threonine (Thr-208 in MARK2), and inactivated by phosphorylation of a serine (Ser-212), both in the activation loop of the catalytic domain. Activation is achieved by the kinases MARKK/TAO1 or LKB1, although the inactivating kinase was unknown. We show here that GSK3␤ serves the role of the inhibitory kinase. Because GSK3␤ can also phosphorylate Tau at sites outside the repeat domain, the activation of GSK3␤, and concomitant inactivation of MARK can shift the pattern of pathological phosphorylation of Tau protein in Alzheimer disease.

Here we report the crystal structure of the catalytic and UBA domain of another isoform, MARK1. Although the crystal packing of the two isoforms are unrelated, the overall conformations of the molecules are similar. Notably, the UBA domain has the same unusual conformation as in MARK2, and it binds at the same site. Remarkable differences occur in the catalytic domain at helix C, the catalytic loop, and the activation segment.The Ser/Thr kinase MARK 2 has been identified by the ability to phosphorylate tau at certain serine residues in the microtubule binding repeats (1). Phosphorylation of tau and other microtubule-associated proteins (MAP2, MAP4) at the KXGS motifs by MARK reduces the affinity to microtubules and leads to microtubule disassembly. Hyperphosphorylation of tau followed by aggregation to paired helical filaments is one of the hallmarks of Alzheimer disease. MARK orthologues KIN1 in fission yeast and Par-1 in Drosophila and Caenorhabditis elegans are involved in the development of cell polarity (2, 3). In neurons, MARK is required for neurite outgrowth and differentiation (4).There are four isoforms of MARK in the human kinome that form a subfamily of the Snf1/AMP-activated protein kinase family of kinases within the calcium/calmodulin-dependent protein kinase group (5). MARK kinases are relatively large; the longest isoform, MARK1, comprises 795 amino acids (Fig. 1). The catalytic domain is flanked by an N-terminal header of about 60 amino acids and a linker of about 20 amino acids that includes a four-residue motif (adjacent to the catalytic domain) that may serve as a common docking site (CD domain) for regulatory binding partners in analogy to MAP kinases (6). The linker connects the catalytic domain to a bona fide UBA (ubiquitin-Associated) domain, a globular domain of 40 amino acids consisting of three ␣-helices. The UBA domain is followed by a long spacer and a globular tail (NMR structure, see Protein Data Bank code 1UL7) that comprises the KA1 domain (kinasesassociated domain 1) with the characteristic ELKL motif at the C terminus. The functions of the putative UBA and KA1 domains are not well understood. The fact that most of the AMP-activated protein kinase-related kinases, including the yeast homologue Snf1, possess a UBA or UBA-like domain (7, 8) suggests a conserved function in structural stabilization or regulation of kinase activity.Like many other kinases, MARK is regulated by phosphorylation of the activation loop (T-loop). MARKK/TAO-1 (9) and the tumor suppressor kinase LKB1/Par-4 (10, 11) activate MARK by phosphorylation of the T-loop. A fraction of MARK isolated from brain tissue is doubly phosphorylated at Thr-208 and Ser-212 (MARK2 numbering used throughout); phosphorylation of the second site, however, is inhibitory (9). There are a number of other mechanisms that seem to be able to regulate MARK, including phosphorylation of the spacer by atypical * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be ...