RESUMO
Digestion in the stomach depends on acidification of the lumen. Histamine-elicited acid secretion is triggered by activation of the PKA cascade, which ultimately results in the insertion of gastric H,K-ATPases into the apical plasma membranes of parietal cells. Our recent study revealed the functional role of PKA-MST4-ezrin signaling axis in histamine-elicited acid secretion. However, it remains uncharacterized how the PKA-MST4-ezrin signaling axis operates the insertion of H,K-ATPases into the apical plasma membranes of gastric parietal cells. Here we show that MST4 phosphorylates ACAP4, an ARF6 GTPase-activating protein, at Thr545 Histamine stimulation activates MST4 and promotes MST4 interaction with ACAP4. ACAP4 physically interacts with MST4 and is a cognate substrate of MST4 during parietal cell activation. The phosphorylation site of ACAP4 by MST4 was mapped to Thr545 by mass spectrometric analyses. Importantly, phosphorylation of Thr545 is essential for acid secretion in parietal cells because either suppression of ACAP4 or overexpression of non-phosphorylatable ACAP4 prevents the apical membrane reorganization and proton pump translocation elicited by histamine stimulation. In addition, persistent overexpression of MST4 phosphorylation-deficient ACAP4 results in inhibition of gastric acid secretion and blockage of tubulovesicle fusion to the apical membranes. Significantly, phosphorylation of Thr545 enables ACAP4 to interact with ezrin. Given the location of Thr545 between the GTPase-activating protein domain and the first ankyrin repeat, we reason that MST4 phosphorylation elicits a conformational change that enables ezrin-ACAP4 interaction. Taken together, these results define a novel molecular mechanism linking the PKA-MST4-ACAP4 signaling cascade to polarized acid secretion in gastric parietal cells.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Modelos Biológicos , Células Parietais Gástricas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Substituição de Aminoácidos , Animais , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Polaridade Celular , Células Cultivadas , Biologia Computacional , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Bases de Dados de Proteínas , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/genética , Microscopia Eletrônica de Transmissão , Mutação , Células Parietais Gástricas/citologia , Células Parietais Gástricas/ultraestrutura , Fosforilação , Conformação Proteica , Multimerização Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico , Coelhos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Tumor metastasis represents the main causes of cancer-related death. Our recent study showed that chemokine CCL18 secreted from tumor-associated macrophages regulates breast tumor metastasis, but the underlying mechanisms remain less clear. Here, we show that ARF6 GTPase-activating protein ACAP4 regulates CCL18-elicited breast cancer cell migration via the acetyltransferase PCAF-mediated acetylation. CCL18 stimulation elicited breast cancer cell migration and invasion via PCAF-dependent acetylation. ACAP4 physically interacts with PCAF and is a cognate substrate of PCAF during CCL18 stimulation. The acetylation site of ACAP4 by PCAF was mapped to Lys311 by mass spectrometric analyses. Importantly, dynamic acetylation of ACAP4 is essential for CCL18-induced breast cancer cell migration and invasion, as overexpression of the persistent acetylation-mimicking or non-acetylatable ACAP4 mutant blocked CCL18-elicited cell migration and invasion. Mechanistically, the acetylation of ACAP4 at Lys311 reduced the lipid-binding activity of ACAP4 to ensure a robust and dynamic cycling of ARF6-ACAP4 complex with plasma membrane in response to CCL18 stimulation. Thus, these results present a previously undefined mechanism by which CCL18-elicited acetylation of the PH domain controls dynamic interaction between ACAP4 and plasma membrane during breast cancer cell migration and invasion.