Koch M, Korf H-W
Dr. Senckenbergische Anatomie, Institut für Anatomie II, Fachbereich Medizin der J.W. Goethe-Universität Frankfurt am Main, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany.
Cell Tissue Res. 2002 Dec;310(3):331-8. doi: 10.1007/s00441-002-0633-9. Epub 2002 Oct 2.
The rat pineal organ is an established model to study signal transduction cascades that are activated by norepinephrine (NE) and cause increases in cAMP levels and stimulation of protein kinase A (PKA). PKA type II catalyzes the phosphorylation of the transcription factor cAMP-response-element-binding protein (CREB) which is essential for the transcriptional induction of the arylalkylamine- N-acetyltransferase (AANAT), the rate limiting enzyme of melatonin biosynthesis. Moreover, PKA may control protein levels and enzyme activity via two PKA-dependent phosphorylation sites in the AANAT molecule. Despite the functional importance of PKA very little is known about the distribution of its isoenzymes and of A-kinase anchor proteins (AKAPs) that target the PKA to specific membrane areas and organelles by binding to the regulatory (R) subunits of PKA. We have addressed this problem by demonstrating the R subunits alpha and beta of PKA type I and II and two AKAPs (150 and 95) in NE-stimulated and untreated rat pinealocytes by immunoblot and immunocytochemistry. The immunoreactions (IR) of all four R subunits were confined to granules evenly distributed in the pinealocyte cytoplasm. Immunoreactions of RIIalpha and RIIbeta were stronger than those of RIalpha and RIbeta. AKAP 150-IR was concentrated at the cell periphery; AKAP 95-IR was restricted to the nucleus. Amount and subcellular distribution of the immunoreactions of all proteins investigated did not change upon NE stimulation. A substantial colocalization was observed between RII-subunits and AKAP 150-IR, suggesting that, in rat pinealocytes, AKAP 150 primarily anchors the R subunits of PKA II.
大鼠松果体是研究由去甲肾上腺素（NE）激活并导致cAMP水平升高和蛋白激酶A（PKA）激活的信号转导级联反应的成熟模型。II型PKA催化转录因子cAMP反应元件结合蛋白（CREB）的磷酸化，这对于芳基烷基胺-N-乙酰基转移酶（AANAT）的转录诱导至关重要，AANAT是褪黑素生物合成的限速酶。此外，PKA可能通过AANAT分子中的两个PKA依赖性磷酸化位点控制蛋白质水平和酶活性。尽管PKA具有重要的功能，但关于其同工酶和A激酶锚定蛋白（AKAP）的分布知之甚少，AKAP通过与PKA的调节（R）亚基结合将PKA靶向特定的膜区域和细胞器。我们通过免疫印迹和免疫细胞化学方法，在NE刺激和未处理的大鼠松果体细胞中证实了I型和II型PKA的R亚基α和β以及两种AKAP（150和95），从而解决了这个问题。所有四个R亚基的免疫反应（IR）都局限于均匀分布在松果体细胞质中的颗粒。RIIα和RIIβ的免疫反应比RIα和RIβ更强。AKAP 150-IR集中在细胞周边；AKAP 95-IR局限于细胞核。在NE刺激后，所有研究蛋白质的免疫反应的量和亚细胞分布均未改变。观察到RII亚基与AKAP 150-IR之间有大量共定位，这表明在大鼠松果体细胞中，AKAP 150主要锚定PKA II的R亚基。
