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Zipper-interacting protein kinase (ZIPK) has been implicated in Ca2؉ -independent smooth muscle contraction, although its specific role is unknown. The addition of ZIPK to demembranated rat caudal arterial strips induced an increase in force, which correlated with increases in LC 20 and MYPT1 phosphorylation. However, because of the number of kinases capable of phosphorylating LC 20 and MYPT1, it has proven difficult to identify the mechanism underlying ZIPK action. Therefore, we set out to identify bona fide ZIPK substrates using a chemical genetics method that takes advantage of ATP analogs with bulky substituents at the N 6 position and an engineered ZIPK capable of utilizing such substrates.32 P-Labeled 6-phenyl-ATP and ZIPK-L93G mutant protein were added to permeabilized rat caudal arterial strips, and substrate proteins were detected by autoradiography following SDS-PAGE. Mass spectrometry identified LC 20 as a direct target of ZIPK in situ for the first time. Tissues were also exposed to 6-phenyl-ATP and ZIPK-L93G in the absence of endogenous ATP, and putative ZIPK substrates were identified by Western blotting. LC 20 was thereby confirmed as a direct target of ZIPK; however, no phosphorylation of MYPT1 was detected. We conclude that ZIPK is involved in the regulation of smooth muscle contraction through direct phosphorylation of LC 20 .Smooth muscle plays an important role in the regulation of diverse physiological processes, including vascular tone, gastrointestinal motility, penile erection, bronchial diameter, and parturitional/postparturitional myometrial contraction. All smooth muscle tissues rely on the Ca 2ϩ /calmodulin-dependent activation of myosin light chain kinase (MLCK) 6 and subsequent phosphorylation of the 20-kDa myosin regulatory light chains (LC 20 ) at Ser-19 to initiate actomyosin cross-bridge cycling and force development (1). On the other hand, relaxation is induced by dephosphorylation of LC 20 by myosin light chain phosphatase (MLCP), a type 1 protein serine/threonine phosphatase (2, 3). Contraction of multiple smooth muscle tissues has frequently been observed in the absence of an increase in cytosolic free Ca 2ϩ concentration in response to a variety of stimuli (4). This phenomenon, commonly referred to as Ca 2ϩ sensitization, involves alteration of the MLCK:MLCP activity ratio in favor of the kinase, which can be achieved by the following mechanisms: (i) activation of MLCK by a mechanism not involving Ca 2ϩ /calmodulin, e.g. phosphorylation of MLCK by proline-directed kinases (5, 6); (ii) an increase in Ca 2ϩ -independent LC 20 kinase activity (7); and (iii) inhibition of MLCP either directly by phosphorylation of inhibitory residues (Thr-697 and/or Thr-855) in the myosin phosphatase targeting subunit 1 (MYPT1) of MLCP (8 -10) or indirectly by phosphorylation of the protein kinase C-potentiated inhibitory protein for heterotrimeric MLCP of 17 kDa (CPI-17) at Thr-38 (11, 12). The observation that intact and permeabilized smooth muscle tissues exhibit Ca 2ϩ -independent contracti...

C. difficile is a Gram-positive spore-forming anaerobic bacterium that is the leading cause of nosocomial diarrhea in the developed world. The pathogenesis of C. difficile infections (CDI) is driven by toxin A (TcdA) and toxin B (TcdB), secreted factors that trigger the release of inflammatory mediators and contribute to disruption of the intestinal epithelial barrier. Neutrophils play a key role in the inflammatory response and the induction of pseudomembranous colitis in CDI. TcdA and TcdB alter cytoskeletal signaling and trigger the release of CXCL8/IL-8, a potent neutrophil chemoattractant, from intestinal epithelial cells; however, little is known about the surface receptor(s) that mediate these events. In the current study, we sought to assess whether toxin-induced CXCL8/IL-8 release and barrier dysfunction are driven by the activation of the P2Y6 receptor following the release of UDP, a danger signal, from intoxicated Caco-2 cells. Caco-2 cells express a functional P2Y6 receptor and release measurable amounts of UDP upon exposure to TcdA/B. Toxin-induced CXCL8/IL-8 production and release were attenuated in the presence of a selective P2Y6 inhibitor (MRS2578). This was associated with inhibition of TcdA/B-induced activation of NFκB. Blockade of the P2Y6 receptor also attenuated toxin-induced barrier dysfunction in polarized Caco-2 cells. Lastly, pretreating mice with the P2Y6 receptor antagonists (MSR2578) attenuated TcdA/B-induced inflammation and intestinal permeability in an intrarectal toxin exposure model. Taken together these data outline a novel role for the P2Y6 receptor in the induction of CXCL8/IL-8 production and barrier dysfunction in response to C. difficile toxin exposure and may provide a new therapeutic target for the treatment of CDI.