Regulation of ion-transporters by A-kinase regulated by via anchoring protein

a激酶锚定蛋白（AKAPs）在腮腺（PGs）中的存在在cAMP存在的情况下，膜结合蛋白激酶A （PKA）可使分离的基底外侧膜囊（BLMVs）中的Na^+、K^+- atp酶和Na^+-K^+- 2cl ^-共转运蛋白磷酸化，提示AKAPs参与了这些磷酸化。RT-PCR和Western blot分析显示，pg中AKAP-150的mrna和蛋白含量显著高于AKAP-95和AKAP-220。用抗RII抗体将blmv的AKAP-150与RII亚基共免疫沉淀。这些结果表明，AKAP-150在blmv中具有功能性，并与内源性RII亚基相关。通过AKAP/PKA复合物磷酸化Na^+， K^+- atp酶1。HT-31肽抑制RII与AKAP的结合导致Na^+， K^+- atpase的膜结合依赖性磷酸化减少。AKAP/PKA磷酸化Na^+， K^+- atp酶与Na^+， K^+…More - atp酶活性降低相关cAMP/PKA途径磷酸化Na^+-K^+- 2cl ^-共转运体n端的机制1。异丙肾上腺素可上调布美他尼敏感的Na^+- k ^+- 2cl ^-协同转运活性。其活性的增加伴随着Na^+- k ^+- 2cl ^-共转运蛋白的磷酸化和高亲和力布美他尼构建位点的增加。异丙肾上腺素刺激腺泡细胞后，Na^+- k ^+- 2cl ^-共转运蛋白磷酸化位点位于n端。与外源PKA或AKAP/PKA一起体外培养BLM时，Na^+- k ^+- 2cl ^-共转运体未发生n端磷酸化，其活性和与布美他胺的结合均未发生变化。当腺泡细胞被洋地黄苷轻度渗透时，Na^+- k ^+- 2cl ^-共转运体的n端在加入cAMP后明显磷酸化。这些结果表明Na^+- k ^+- 2cl ^-共转运体受其n端磷酸化调控，而PKA不是直接磷酸化共转运体n端，而是通过PKA触发的蛋白磷酸化级联作用。少

Existence of A-kinase anchoring proteins (AKAPs) in parotid glands (PGs)1. Membrane proteins including Na^+, K^+-ATPase and Na^+-K^+-2Cl^- cotransporter in isolated basolateral membrane vesicles (BLMVs) were phosphorylated by membrane-bound protein kinase A (PKA) in the presence of cAMP, suggesting the involvement of AKAPs in these phosphorylations.2. mRNAs and proteins of AKAP-150 were detected in PGs significantly by RT-PCR and Western blot analysis, whereas AKAP-95 and AKAP-220 were less.3. AKAP-150 of BLMVs was co-immunoprecipitated with the RII subunit by anti-RII antibody. These results indicate that AKAP-150 in BLMVs is functional and associated with the endogenous RII subunit.Phosphorylation of Na^+, K^+-ATPase via AKAP/PKA complex1. Inhibition of the RII binding to AKAP by HT-31 peptide resulted in a decrease in membrane-bound PKA-dependent phosphorylation of Na^+, K^+-ATPase.2. Phosphorylation of Na^+, K^+-ATPase by the AKAP/PKA was associated with a decrease in the Na^+, K^+ … More -ATPase activityMechanism of phosphorylation of Na^+-K^+-2Cl^- cotransporter N-terminus via cAMP/PKA pathway1. Bumetanide-sensitive Na^+-K^+-2Cl^- cotransport activity in rat PG acini was upregulated with isoproterenol. The increase in its activity was accompanied by phosphorylation of the Na^+-K^+-2Cl^- cotransporter protein and by an increase in the number of high affinity bumetanide building sites.2. The phosphorylation site of Na^+-K^+-2Cl^- cotransporter was on its N-terminus by isoproterenol-stimulation of acinar cells.3. N-terminus phosphorylation never occurred by in vitro incubation of the Na^+-K^+-2Cl^- cotransporter in BLM preparation with exogenous PKA or AKAP/PKA, and neither its activity nor its bumetanide binding were changed.4. When acinar cells were mildly permeabilized with digitonin, N-terminus of the Na^+-K^+-2Cl^- cotransporter was clearly phosphorylated upon the addition of cAMP. These results indicated that Na^+-K^+-2Cl^- cotransporter was regulated by the phosphorylation on its N-terminus and that PKA did not directly phosphorylate the cotransporter N-terminus but via protein phosphorylation cascade triggered by PKA. Less