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

AKAP/PKA复合体对Na~+，K~+-ATPase的磷酸化作用1.观察A-激酶锚定蛋白(AKAP)/PKA复合体在大鼠腮腺(PG)、颌下腺(SMG)和舌下腺(SLG)三个主要涎腺中的表达。PKA的AKAP-150mRNA和RII调节亚基在PG中明显检测到，而在SMG和SLG中几乎检测不到。[γ-^&lt；32&gt；[P]ATP显示，加入cAMP后，PG细胞膜上的Na+，K+-ATPase迅速被磷酸化，而SMG和SLG细胞膜上的ATPase则不能，这表明AKAP/PKA对Na+，K+-ATPase的磷酸化是三种主要唾液腺中PG所特有的。2.在不刺激β-肾上腺素能受体的情况下，通过将cAMP添加到通透性腺泡上来模拟N-末端的磷酸化。这一结果表明N-末端不被PKA直接磷酸化，但被PKA激活所触发的PKA级联明显地磷酸化。3.将包括Thr-208在内的部分N-末端多肽加入到通透性腺泡中，竞争性地阻断了cAMP依赖的N-末端磷酸化。这一结果表明NKCC1的功能受N端Thr-208的磷酸化调控。4.克隆了NKCC1基因，并制备了转入该基因的大肠杆菌。我们将从苏氨酸在不同的磷酸化位点，即N-末端的-203，-208和-221，以及在PKA的共识位点，制备修饰成ALA的质粒。

Phosphorylation of Na^+, K^+-ATPase via AKAP/PKA complex1.We have examined the expression of A-kinase anchoring protein (AKAP)/PKA complex in the three major salivary glands, i.e., the parotid gland (PG), submandibular gland (SMG), and sublingual gland (SLG), of the rat. AKAP-150 mRNA and RII regulatory subunit of PKA were clearly detected in the PG, but they were hardly detectable in either the SMG or SLG.2.Incubation with [γ-^<32>P]ATP revealed that Na^+,K^+-ATPase in the PG membranes was quickly phosphorylated upon the addition of cAMP, whereas the ATPases in the membranes from SMG and SLG were not, suggesting phosphorylation of Na^+,K^+-ATPase by AKAP/PKA are characteristics specific to PG among the three major salivary glands.Phosphorylation of Na^+/K^+/2Cl^- cotransporter (NKCC1) N-terminus via cAMP/PKA pathway1.We have previously shown that Na^+,K^+,2Cl^--cotransport activity in parotid acinar cells is dramatically upregulated by N-terminus phosphorylation. However we showed here that N-terminus was not phosphorylated by PKA directly in vitro system.2.The phosphorylation of N-terminus was mimicked by the addition of cAMP to permeabilized acini without stimulation of β-adrenergic receptors. This result indicates that the N-terminus was not phosphorylated by PKA directly, however it was clearly phosphorylated by PKA cascade triggered by PKA activation.3.The cAMP dependent phosphorylation of the N-terminus was blocked competitively by the addition of partial N-terminus peptides including Thr-208 into permeabilized acini. This result indicated that functions of NKCC1 were regulated by the phosphorylation of Thr-208 on the N-terminus.4.We carried out cloning of NKCC1 gene and prepared E.Coli transfected its gene. We are going to prepare the plasmids modified to Ala from Thr at various phosphorylation sites, i.e. Thr-203, -208 and -221 in N-terminus and at PKA consensus site.