Phosphorylation sites in members of the protein kinase A (PKA), PKG, and PKC kinase subfamily are conserved. Thus, the PKB kinase PDK1 may be responsible for the phosphorylation of PKC isotypes. PDK1 phosphorylated the activation loop sites of PKCζ and PKCδ in vitro and in a phosphoinositide 3-kinase (PI 3-kinase)–dependent manner in vivo in human embryonic kidney (293) cells. All members of the PKC family tested formed complexes with PDK1. PDK1-dependent phosphorylation of PKCδ in vitro was stimulated by combined PKC and PDK1 activators. The activation loop phosphorylation of PKCδ in response to serum stimulation of cells was PI 3-kinase–dependent and was enhanced by PDK1 coexpression.

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Generation of a barrier in multi-layered epithelia like the epidermis requires restricted positioning of functional tight junctions (TJ) to the most suprabasal viable layer. This positioning necessitates tissue-level polarization of junctions and the cytoskeleton through unknown mechanisms. Using quantitative whole-mount imaging, genetic ablation, and traction force microscopy and atomic force microscopy, we find that ubiquitously localized E-cadherin coordinates tissue polarization of tension-bearing adherens junction (AJ) and F-actin organization to allow formation of an apical TJ network only in the uppermost viable layer. Molecularly, E-cadherin localizes and tunes EGFR activity and junctional tension to inhibit premature TJ complex formation in lower layers while promoting increased tension and TJ stability in the granular layer 2. In conclusion, our data identify an E-cadherin-dependent mechanical circuit that integrates adhesion, contractile forces and biochemical signaling to drive the polarized organization of junctional tension necessary to build an in vivo epithelial barrier.

In chronic kidney disease (CKD), serum concentrations of fibroblast growth factor 23 (FGF23) increase progressively as glomerular filtration rate declines, while renal expression of the FGF23 coreceptor Klotho decreases. Elevated circulating FGF23 levels are strongly associated with mortality and with left ventricular hypertrophy (LVH), which is a major cause of cardiovascular death in CKD patients. The cardiac FGF23/FGF receptor (FGFR) system and its role in the development of LVH in humans have not been addressed previously. We conducted a retrospective case–control study in 24 deceased patients with childhood-onset end-stage renal disease (dialysis: n = 17; transplanted: n = 7), and 24 age- and sex-matched control subjects. Myocardial autopsy samples of the left ventricle were evaluated for expression of endogenous FGF23, FGFR isoforms, Klotho, calcineurin and nuclear factor of activated T-cells (NFAT) by immunohistochemistry, immunofluorescence microscopy, qRT-PCR and western blotting. The majority of patients presented with LVH (67%). Human cardiomyocytes express full-length FGF23, and cardiac FGF23 is excessively high in patients with CKD. Enhanced myocardial expression of FGF23 in concert with Klotho deficiency strongly correlates with the presence of LVH. Cardiac FGF23 levels associate with time-averaged serum phosphate levels, up-regulation of FGFR4 and activation of the calcineurin-NFAT signaling pathway, an established mediator of cardiac remodelling and LVH. These changes are detected in patients on dialysis but not in those with a functioning kidney transplant. Our results indicate a strong association between LVH and enhanced expression levels of FGF23, FGFR4 and calcineurin, activation of NFAT and reduced levels of soluble Klotho in the myocardium of patients with CKD. These alterations are not observed in kidney transplant patients.

The cytoskeletal protein vinculin contributes to the mechanical link of the contractile actomyosin cytoskeleton to the extracellular matrix (ECM) through integrin receptors. In addition, vinculin modulates the dynamics of cell adhesions and is associated with decreased cell motility on two-dimensional ECM substrates. The effect of vinculin on cell invasion through dense three-dimensional ECM gels is unknown. Here, we report how vinculin expression affects cell invasion into three-dimensional collagen matrices. Cell motility was investigated in vinculin knockout and vinculin expressing wild-type mouse embryonic fibroblasts. Vinculin knockout cells were 2-fold more motile on two-dimensional collagen-coated substrates compared with wild-type cells, but 3-fold less invasive in 2.4 mg/ml three-dimensional collagen matrices. Vinculin knockout cells were softer and remodeled their cytoskeleton more dynamically, which is consistent with their enhanced two-dimensional motility but does not explain their reduced three-dimensional invasiveness. Importantly, vinculin-expressing cells adhered more strongly to collagen and generated 3-fold higher traction forces compared with vinculin knockout cells. Moreover, vinculin-expressing cells were able to migrate into dense (5.8 mg/ml) three-dimensional collagen matrices that were impenetrable for vinculin knockout cells. These findings suggest that vinculin facilitates three-dimensional matrix invasion through up-regulation or enhanced transmission of traction forces that are needed to overcome the steric hindrance of ECMs.