RESUMEN
Mutations in with-no-lysine kinase (WNK) 1, WNK4, Kelch-like 3 (KLHL3), and Cullin3 result in an inherited hypertensive disease, pseudohypoaldosteronism type II. WNK activates the Na-Cl cotransporter (NCC), increasing sodium reabsorption in the kidney. Further, KLHL3, an adapter protein of Cullin3-based E3 ubiquitin ligase, has been recently found to bind to WNK, thereby degrading them. Insulin and vasopressin have been identified as powerful activators of WNK signaling. In this study, we investigated effects of Akt and PKA, key downstream substrates of insulin and vasopressin signaling, respectively, on KLHL3. Mass spectrometry analysis revealed that KLHL3 phosphorylation at S433. Phospho-specific antibody demonstrated defective binding between phosphorylated KLHL3 and WNK4. Consistent with the fact that S433 is a component of Akt and PKA phosphorylation motifs, in vitro kinase assay demonstrated that Akt and PKA can phosphorylate KLHL3 at S433, that was previously reported to be phosphorylated by PKC. Further, forskolin, a representative PKA stimulator, increased phosphorylation of KLHL3 at S433 and WNK4 protein expression in HEK293 cells by inhibiting the KLHL3 effect that leads to WNK4 degradation. Insulin also increased phosphorylation of KLHL3 at S433 in cultured cells. In conclusion, we found that Akt and PKA phosphorylated KLHL3 at S433, and phosphorylation of KLHL3 by PKA inhibited WNK4 degradation. This could be a novel mechanism on how insulin and vasopressin physiologically activate the WNK signal.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Proteínas Portadoras/metabolismo , Colforsina/farmacología , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Insulina/farmacología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Microfilamentos , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Vasopresinas/farmacología , Proteína Quinasa Deficiente en Lisina WNK 1RESUMEN
A 45-year-old man complaining of cough, dyspnea, and difficulty in swallowing was referred to our hospital. Chest CT scan showed a mediastinal mass compressing the trachea. He was diagnosed with poorly differentiated lung carcinoma by percutaneous needle biopsy. Bronchoscopy and upper gastrointestinal endoscopy revealed a tracheoesophageal fistula (TEF). Long-lasting febrile neutropenia made it impossible to continue chemotherapy, but a course of radiotherapy (total 61 Gy) was completed. The next endoscopy revealed closure of the TEF. Chemoradiotherapy (CRT) has been reported to close TEF in esophageal cancer, but the risk of a CRT-induced worsening of the fistula has dissuaded physicians from using CRT to treat TEF in lung cancer patients. CRT may serve as a palliative treatment for TEF in lung cancer as well as esophageal cancer.