Figure 6.

Phosphorylation of ACAP4 by MST4 elicited relocation of proton pump H,K-ATPase to the apical membrane of parietal cells. A, Western blot analyses of ACAP4, ezrin, and H,K-ATPase (α subunit) of subcellular fractions derived from resting (R, cimetidine) and stimulated (S, histamine) gastric glands. Note that stimulation enriches the protein level of H,K-ATPase (α subunit) in the P1 fraction. B, Western blot analyses of ACAP4, Na,K-ATPase (α subunit), and H,K-ATPase (α subunit) of subcellular fractions derived from resting and stimulated gastric glands infected with GFP–ACAP4 adenovirus (wild-type, ACAP4-T545A, and ACAP4-T545D). C, quantification of the α subunit of H,K-ATPase proteins from P1 (plasma membrane–enriched) and P3 (tubulovesicle-enriched) fractions. The measurements are expressed as the P1/P3 ratio. All data are given as means ± S.E. (error bars) of four preparations. Cim., cimetidine; Hist., histamine. *, p < 0.05; **, p < 0.01. D, working model illustrating how histamine stimulation–elicited PKA–MST4 signaling induces ezrin and ACAP4 conformational change, which enables the association of ACAP4 with ezrin and provides a spatial cue for tubulovesicle translocation. In non-secreting parietal cells, ACAP4 resides on tubulovesicles and relocates apically upon histamine stimulation. Histamine stimulation induces MST4 activation that phosphorylates ACAP4 at Thr⁵⁴⁵. In addition, phosphorylation of ACAP4 promotes its association with ezrin phosphorylated at Ser⁶⁶ by PKA (11, 18). Phosphorylation of Ser⁶⁶ elicits a conformational change of ezrin, which promotes docking and anchoring of ACAP4-containing tubulovesicles at the apical membrane for subsequent fusion of H,K-ATPase–containing vesicles to the apical membrane for the proton pump.