The Type and the Localization of cAMP-dependent Protein Kinase Regulate Transmission of cAMP Signals to the Nucleus in Cortical and Cerebellar Granule Cells

Abstract

cAMP signals are received and transmitted by multiple isoforms of cAMP-dependent protein kinases, typically determined by their specific regulatory subunits.In the brain the major regulatory isoform RII␤ and the RII-anchor protein, AKAP150 (rat) or 75 (bovine), are differentially expressed. Cortical neurons express RII␤ and AKAP75; conversely, granule cerebellar cells express predominantly RI␣ and RII␣. Cortical neurons accumulate PKA catalytic subunit and phosphorylated cAMP responsive element binding protein very efficiently into nuclei upon cAMP induction, whereas granule cerebellar cells fail to do so. Down-regulation of RII␤ synthesis by antisense oligonucleotides inhibited cAMPinduced nuclear signaling in cortical neurons. Expression in cerebellar granule cells of RII␤ and AKAP75 genes by microinjection of specific expression vectors, markedly stimulated cAMP-induced transcription of the lacZ gene driven by a cAMP-responsive element promoter.These data indicate that the composition of PKA in cortical and granule cells underlies the differential ability of these cells to transmit cAMP signals to the nucleus.cAMP formed by adenylyl cyclases after stimulation of Gprotein-coupled receptors binds the regulatory subunits (R) of the tetrameric PKA 1 holoenzyme and promotes dissociation of the catalytic subunits (C-PKA). A fraction of C-PKA translocates to the nucleus and stimulates cAMP-dependent gene expression (1-3). Multiple isoforms of PKA are determined by their specific regulatory subunits. Four regulatory subunits (RI␣, RI␤, RII␣, and RII␤) have been cloned. PKA containing RII␤ is the predominant PKA isoform in the brain and is expressed in the cortex, whereas in the brainstem and cerebellum (except Purkinje cells) RII␤ has not been found (4 -6). In mammalian brain, signals triggered by cAMP are targeted to specific effector sites by the tethering of cAMP-dependent protein kinases to intracellular compartments (4,7,8). PKAII is bound to membranes via specific anchor proteins (AKAPs), which bind R subunits. Bovine brain AKAP75 has been studied as prototype of kinase A anchor protein and shares high homology with human AKAP79 and rat AKAP150 (9, 10). These proteins have similar properties, related sequences, and are recognized by the same antibodies (9 -11). AKAP150/75 and RII␤ are co-localized in the dendritic cytoskeleton and perikarya of forebrain neurons; both proteins have not been found in cerebellar granule cells (6).Although the structure and expression pattern of the PKA regulatory subunits and AKAPs are well documented, the functional role of these proteins in the transduction of cAMP signals is still poorly understood. It is not known how the different PKA isoforms in different districts of the central nervous system receive and transmit cAMP signals.We have chosen primary cortical and granule cerebellar neurons as prototype cells with different PKA composition and localization. PKA in cortical neurons is mainly of II␤ type and is membrane-anchored by AKAP150/75. Conversely, granule cerebellar cel...

The Expression of the Thyroid-stimulating Hormone (TSH) Receptor and the cAMP-dependent Protein Kinase RII β Regulatory Subunit Confers TSH-cAMP-dependent Growth to Mouse Fibroblasts

“…Anti-rat PCREB antibodies (23) were purchased from Upstate Biotechnology, Lake Placid, NY. Specific anti-RII␤ antibodies were generated by immunizing rabbits with a synthetic RII␤ peptide (peptide 31-57 from the AUG of the rat sequence), cross-linked to soybean trypsin inhibitor (24). Anti-Rap-1b was purchased from Santa Cruz Biotechnology, Santa Cruz, CA.…”

“…In thyroid cells and in other cells containing a high PKAII/ PKAI holoenzyme ratio (3T3 fibroblasts expressing RII␤), the efficient expression of RII␤ subunit increases the stability and membrane anchoring of PKAII holoenzyme (14,24,42). Membrane-anchoring appears to be an important determinant of the intracellular cAMP gradient, because Ht31, a peptide that inhibits anchoring of PKAII to the membranes, suppressed cAMP gradients in isolated cardiomyocytes (43).…”

“…RII␤ binds to A kinase anchor proteins (AKAPs) with high affinity, is associated to membranes and its affinity for cAMP is lower than that of the RI subunits (14,24,30). Mouse 3T3 fibroblasts contain mostly RI subunits, which form cytosolic PKAI holoenzyme.…”

“…Since PKAII has been associated with efficient transcription of cAMP-induced genes (24,30), we have determined cAMP signaling to the nucleus in the cell lines indicated above, by measuring accumulation of phosphorylated CREB in the nucleus and the transcription of a prototypic cAMP-dependent promoter CRE-CAT (36). The as- FIG.…”

cAMP signaling selectively influences Ras effectors pathways

“…To show that PKA mediates the e ects observed upon TSH stimulation on the Ras-PI3K complex, we transfected NIH3T3 ®broblasts with tagged active Ras (val12-HaRas-AU5), constitutively active TSH receptor or the catalytic subunit of PKA (Porcellini et al, 1995;Bonapace et al, 1990;Paolillo et al, 1999). The extracts were immunoprecipitated with antibodies directed to the AU5 tag present in the Ras protein and blotted with antibodies against the p85 subunit of PI3K.…”