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

通过AKAP/PKA复合物磷酸化Na^+， K^+- atp酶1。我们检测了a激酶锚定蛋白(AKAP)/PKA复合物在大鼠三大唾液腺，即腮腺（PG）、下颌腺（SMG）和舌下腺（SLG）中的表达。PKA的AKAP-150 mRNA和RII调控亚基在PG中明显检测到，但在SMG和SLG.2中几乎检测不到。[γ-^<32 b> P]ATP孵育发现，在加入cAMP后，PG膜上的Na^+、K^+-ATP酶被迅速磷酸化，而SMG和SLG膜上的ATP酶则没有被磷酸化，这表明AKAP/PKA对Na^+、K^+-ATP酶的磷酸化是PG在三种主要唾液腺中的特异性特征。通过cAMP/PKA途径磷酸化Na^+/K^+/2Cl^-共转运体（NKCC1） n端。我们之前已经证明腮腺腺泡细胞中的Na^+，K^+，2Cl^-共转运活性被n端磷酸化显著上调。然而，我们发现在体外系统中，n端不会被PKA直接磷酸化。在不刺激β-肾上腺素能受体的情况下，将cAMP添加到渗透性腺泡中可以模拟n端磷酸化。这一结果表明，n端并没有被PKA直接磷酸化，而是被PKA激活触发的PKA级联磷酸化。通过将包括Thr-208在内的部分n端肽添加到渗透腺泡中，可竞争性地阻断cAMP依赖的n端磷酸化。这一结果表明NKCC1的功能受到n端Thr-208磷酸化的调控。我们克隆了NKCC1基因，制备了转染该基因的大肠杆菌。我们准备在不同的磷酸化位点，即n端和PKA共识位点的Thr-203、-208和-221位点，将其修饰为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.