Distribution of regulatory subunits of protein kinase A and A kinase anchor proteins (AKAP 95, 150) in rat pinealocytes.
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.
Koch
M
M
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.
Korf
H-W
HW
D054758:A Kinase Anchor Proteins; D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology / Q000648:ultrastructure; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D008297:Male; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D010870:Pineal Gland / Q000166:cytology / Q000201:enzymology*; D021122:Protein Subunits; D051381:Rats; D017208:Rats, Wistar
D054758:A Kinase Anchor Proteins; C086194:Akap8 protein, rat; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D021122:Protein Subunits; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases