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Protein clustering is a ubiquitous event in diverse cellular processes. Self‐association of proteins triggers recruitment of downstream proteins to regulate cellular signaling. To investigate the interactions in detail, chemical biology tools to identify proteins recruited to defined assemblies are required. Here, we exploit an identification of proteins recruited in artificial granules (IPRAG) platform that combines intracellular Y15‐based supramolecule construction with a proximity labeling method. We validated the IPRAG tool using Nck1 as a target bait protein. We constructed Nck1‐tethering granules, labeled the recruited proteins with biotin, and analyzed them by LC‐MS/MS. As a result, we successfully identified proteins that directly or indirectly interact with Nck1. [ABSTRACT FROM AUTHOR]

The gap junction plays roles not only in electrical coupling of cardiomyocytes but also in intercellular transport of biologically active substances. Furthermore, the gap junction participates in decision making on cell survival versus cell death in various types of cells, and a part of reperfusion injury in the heart has been indicated to be gap junction mediated. The contribution of gap junction communication (GJC) and/or mitochondrial 'hemichannels' to protective signaling during the trigger phase of ischemic preconditioning (IPC) is suggested by observations that IPC failed to protect the heart when GJC was blocked during IPC. Although ischemia suppresses both electrical and chemical GJC, chemical GJC persists for a considerable time after electrical GJC is lost. IPC facilitates the ischemia-induced suppression of chemical GJC, whereas IPC delays the reduction of electrical GJC after ischemia. The inhibition of GJC during sustained ischemia and reperfusion by GJC blockers mimics the effect of IPC on myocardial necrosis. IPC induces distinct effects on the interaction of connexin-43 with protein kinases, and the phosphorylation of connexin-43 at Ser368 by PKC[e[P.sub.s]ilon] is a primary mechanism of inhibition of chemical GJC by IPC. Several lines of evidence support the notion that the modulation of GJC is a part of the mechanism of IPC-induced protection against myocardial necrosis and arrhythmias, though what percentage of IPC protection is attributable to the inhibition of GJC during ischemia-reperfusion still remains unclear. connexin; ischemia doi: 10.1152/ajpheart.00879.2009.

Ischemic preconditioning (PC) suppresses chemical coupling of cardiomyocytes via gap junctions (GJs) during ischemia, which is an adjunct mechanism of protection. The aim of this study was to characterize roles of protein kinases in PC-induced GJ modulation. In isolated rat hearts, ventricular tissues were sampled before and after ischemia with or without PC, and intercalated disc-rich fractions were separated for immunoprecipitation and immunoblotting. Levels of protein kinase C (PKC)-[epsilon], p38mitogen-activated protein kinase (MAPK)-[alpha], and Src coimmunoprecipitated with connexin-43 (Cx43) were increased after ischemia, whereas p38MAPK[beta] was not detected in the Cx43 immunoprecipitates. PC did not modify the level of Cx43-Src complex after ischemia. However, PC enhanced Cx43-PKC[epsilon] complex formation, which was abolished by PKC[epsilon] translocation inhibitory peptide (TIP). In contrast, PC reduced Cx43-p38MAPK[alpha] complex level and p38MAPK activity in the Cx43 immunoprecipitates after ischemia. The effect of PC on Cx43-p38MAPK[alpha] interaction was mimicked by SB-203580, a p38MAPK inhibitor. PC reduced permeability of GJs to Lucifer yellow in the myocardium at 25 min after ischemia, and this effect was abolished by PKC[epsilon]-TIP. SB-203580 increased the GJ permeability at 15 min after ischemia compared with that in untreated controls, but the difference became insignificant 25 min after ischemia. In conclusion, PC has distinct effects on interaction of GJ Cx43 with PKC[epsilon], p38MAPK[alpha], and Src during ischemia. Suppression of GJ permeability during ischemia by PC is primarily achieved by enhanced interaction of Cx43 with PKC[epsilon], which overwhelms the counterbalancing effect of reduced Cx43-p38MAPK[alpha] interaction. connexin-43; protein kinase C; p38 mitogen-activated protein kinase; Src