Dynamic regulation of cell-cell adhesion by the coordinated formation and dissolution of E-cadherin-based adherens junctions is crucial for tissue homeostasis. The actin-binding protein cortactin interacts with E-cadherin and enables F-actin accumulation at adherens junctions. Here, we were interested to study the broader functional interactions of cortactin in adhesion complexes. In line with literature, we demonstrate that cortactin binds to E-cadherin, and that a posttranslational modification of cortactin, RhoA-induced phosphorylation by protein kinase D1 (PKD1; also known as PRKD1) at S298, impairs adherens junction assembly and supports their dissolution. Two new S298-phosphorylation-dependent interactions were also identified, namely, that phosphorylation of cortactin decreases its interaction with β-catenin and the actin-binding protein vinculin. In addition, binding of vinculin to β-catenin, as well as linkage of vinculin to F-actin, are also significantly compromised upon phosphorylation of cortactin. Accordingly, we found that regulation of cell-cell adhesion by phosphorylation of cortactin downstream of RhoA and PKD1 is vitally dependent on vinculin-mediated protein interactions. Thus, cortactin, unexpectedly, is an important integration node for the dynamic regulation of protein complexes during breakdown and formation of adherens junctions.

Trauma is a major cause of death worldwide. The post-traumatic immune response culminates in the release of pro-inflammatory mediators, translating in the infiltration of neutrophils (PMNs) at injury sites. The extent of this inflammation is determined by multiple factors, such as PMN adhesion to the endothelium, transendothelial migration, endothelial barrier integrity as well as PMN swarming, mass infiltration and activation. This process is initiated by secondary lipid mediators, such as leukotriene B4 (LTB4). We here provide evidence that Protein kinase D1 (PRKD1) in endothelial cells is implicated in all these processes. Endothelial PRKD1 is activated by pro-inflammatory stimuli and amplifies PMN-mediated inflammation by upregulation of cytokine and chemokines as well as adhesion molecules, such as ICAM-1, VCAM-1 and E-selectin. This induces enhanced PMN adhesion and trans-migration. PRKD1 activation also destabilizes endothelial VE-cadherin adhesion complexes and thus the endothelial barrier, fostering PMN infiltration. We even describe a yet unrecognized PRKD1-dependant mechanism to induce biosynthesis of the PMN-swarming mediator LTB4 directed via intercellular communication through small extracellular vesicles (sEVs) and enhanced CXCL8 secretion from activated endothelial cells. These endothelial sEVs transfer the LTB4 biosynthesis enzyme LTA4 hydrolase (LTA4H) to prime PMNs, while initiating biosynthesis also requires additional signals, like CXCL8. We further demonstrate the respective LTA4H-positive sEVs in the serum of polytrauma patients, peaking 12 h post injury. Therefore, PRKD1 is a key regulator in the coordinated communication of the endothelium with PMNs and a vital signaling node during post-traumatic inflammation.