Inhibition of non-receptor tyrosine kinase Src induces phosphoserine 256-independent aquaporin-2 membrane accumulation.

KEY POINTS: Aquaporin-2 (AQP2) is crucial for water homeostasis, and vasopressin (VP) induces AQP2 membrane trafficking by increasing intracellular cAMP, activating PKA and causing phosphorylation of AQP2 at serine 256, 264 and 269 residues and dephosphorylation of serine 261 residue on the AQP2 C-terminus. It is thought that serine 256 is the master regulator of AQP2 trafficking, and its phosphorylation has to precede the change of phosphorylation state of other serine residues. We found that Src inhibition causes serine 256-independent AQP2 membrane trafficking and induces phosphorylation of serine 269 independently of serine 256. This targeted phosphorylation of serine 269 is important for Src inhibition-induced AQP2 membrane accumulation; without serine 269, Src inhibition exerts no effect on AQP2 trafficking. This result helps us better understand the independent pathways that can target different AQP2 residues, and design new strategies to induce or sustain AQP2 membrane expression when VP signalling is defective. ABSTRACT: Aquaporin-2 (AQP2) is essential for water homeostasis. Upon stimulation by vasopressin, AQP2 is phosphorylated at serine 256 (S256), S264 and S269, and dephosphorylated at S261. It is thought that S256 is the master regulator of AQP2 trafficking and membrane accumulation, and that its phosphorylation has to precede phosphorylation of other serine residues. In this study, we found that VP reduces Src kinase phosphorylation: by suppressing Src using the inhibitor dasatinib and siRNA, we could increase AQP2 membrane accumulation in cultured AQP2-expressing cells and in kidney collecting duct principal cells. Src inhibition increased exocytosis and inhibited clathrin-mediated endocytosis of AQP2, but exerted its effect in a cAMP, PKA and S256 phosphorylation (pS256)-independent manner. Despite the lack of S256 phosphorylation, dasatinib increased phosphorylation of S269, even in S256A mutant cells in which S256 phosphorylation cannot occur. To confirm the importance of pS269 in AQP2 re-distribution, we expressed an AQP2 S269A mutant in LLC-PK1 cells, and found that dasatinib no longer induced AQP2 membrane accumulation. In conclusion, Src inhibition causes phosphorylation of S269 independently of pS256, and induces AQP2 membrane accumulation by inhibiting clathrin-mediated endocytosis and increasing exocytosis. We conclude that S269 can be phosphorylated without pS256, and pS269 alone is important for AQP2 apical membrane accumulation under some conditions. These data increase our understanding of the independent pathways that can phosphorylate different residues in the AQP2 C-terminus, and suggest new strategies to target distinct AQP2 serine residues to induce membrane expression of this water channel when VP signalling is defective.

Chocolate consumption modulates cytokine production in healthy individuals.

Epidemiological studies suggest that chocolate increases the incidence and severity of acne. Here we demonstrate that chocolate consumption primes human blood mononuclear cells from volunteers to release more interleukin-1ß (IL-1ß) and IL-10 upon stimulation with Propionibacterium acne or Staphylcoccus aureus, the two microorganisms involved in the pathogenesis of acne. In contrast, production of the Th17-derived cytokine IL-22 was inhibited by chocolate. Modulation of inflammation could represent an important mechanism through which chocolate consumption influences acne.

The AT-hook protein D1 is essential for Drosophila melanogaster development and is implicated in position-effect variegation.

We have analyzed the expression pattern of the D1 gene and the localization of its product, the AT hook-bearing nonhistone chromosomal protein D1, during Drosophila melanogaster development. D1 mRNAs and protein are maternally contributed, and the protein localizes to discrete foci on the chromosomes of early embryos. These foci correspond to 1.672- and 1.688-g/cm(3) AT-rich satellite repeats found in the centromeric heterochromatin of the X and Y chromosomes and on chromosomes 3 and 4. D1 mRNA levels subsequently decrease throughout later development, followed by the accumulation of the D1 protein in adult gonads, where two distributions of D1 can be correlated to different states of gene activity. We show that the EP473 mutation, a P-element insertion upstream of D1 coding sequences, affects the expression of the D1 gene and results in an embryonic homozygous lethal phenotype correlated with the depletion of D1 protein during embryogenesis. Remarkably, decreased levels of D1 mRNA and protein in heterozygous flies lead to the suppression of position-effect variegation (PEV) of the white gene in the white-mottled (w(m4h)) X-chromosome inversion. Our results identify D1 as a DNA-binding protein of known sequence specificity implicated in PEV. D1 is the primary factor that binds the centromeric 1.688-g/cm(3) satellite repeats which are likely involved in white-mottled variegation. We propose that the AT-hook D1 protein nucleates heterochromatin assembly by recruiting specialized transcriptional repressors and/or proteins involved in chromosome condensation.