N-terminal phosphorylation of mammalian and piscine AQP12 by PKC and PKA control YPM channel transport in X. laevis oocytes. (A) Schematic representation of an AQP12 monomer inserted in the plasma membrane showing the putative six transmembrane domains, the five connecting loops (A–E), and the amino acid sequence alignment of the first part of the human, rat, and zebrafish N termini indicating the potential PKC and PKA phosphorylation sites. (B) Immunoblots of Ser phosphorylation of immunoprecipitated HsAQP12-HA, RnAQP12-HA, or DrAqp12-HA, and single channel mutants at the PKC and PKA phosphorylation residues, from transiently transfected HEK293T cells by 100 nM of rPKC or rPKA, in the presence or absence of 200 μM ATP. The negative control cells were transfected with an empty pcDNA3 expression vector. Representative blots from two independent experiments are shown. (C) Representative immunoblots of HA-tagged human, rat, and zebrafish AQP12 WT and single or double mutants in TM and YPM using α-HA antibody, and corresponding quantitation in YPM normalized to PDI (lower panels), from oocytes expressing the CCK1R and the different constructs and treated with vehicle or 10 nM of CCK-8. Data (mean ± SEM, n = 3 independent experiments) were statistically analyzed by one-way ANOVA, followed by Tukey’s multiple comparisons test. *P < 0.05; **P < 0.01; with respect to untreated oocytes, with respect to CCK-treated oocytes (in parenthesis), or as indicated in brackets. (D) Proposed model of the CCK1R signaling pathways controlling the trafficking of mammalian and piscine AQP12 to the YPM of X. laevis oocytes. TM, total membrane. Source data are available for this figure: SourceData F7.