CREB Phosphorylation

An important question is which of the potentially NE-inducible protein kinase(s) mediate(s) CREB phosphorylation in rat pinealocytes. To investigate this problem we analyzed the NE-induced CREB-phosphorylation after inhibition of several protein kinases. We used KN-62 for the inhibition of calcium-calmodulin-dependent protein kinase (CaMK; 60), chelerythrin for protein kinase C (PKC; 73), PD98059 for the inhibition of mitogen-activated-protein-kinase-kinase type 1 (MEK1; 56) and the antagonists Rp-8-CPT-cAMPS (28,42) and H-89 (15) for the inhibition of the cyclic AMP-dependent protein kinase (PKA; 29). Immunocytochemistry and densitometric analyses of unstimulated controls and preparations treated with NE alone or NE plus the protein kinase antagonist revealed that only the PKA antagonist Rp-8-CPT-cAMPS reduced the NE-induced CREB phosphorylation in a dose-dependent manner (Figure 4). The PKA isozyme responsible for CREB phosphorylation in the rat pineal organ was analyzed using different pairs of cAMP analogs (26) that complement each other for the activation of either PKA type I (8-AHA-cAMP and 8-PIP-cAMP) (9,42,48) or type II (Sp-cDBIMPS and N6-PHE-cAMP) (10,26,42). Only cAMP analog combinations which selectively activate PKA type II elevated pCREB immunoreactivity in rat pinealocytes, whereas PKA type I-directed analog pairs were ineffective. This implies that PKA type II mediates the phosphorylation of CREB in the rat pineal organ (Maronde et al., unpublished observations).

Northern blot and reverse transcriptase polymerase chain reaction (RT-PCR) were employed to assess for the possible transcriptional regulation of PKA subunits in the rat pineal organ. The Northern blots showed a 3.2 kb signal for RIa with two additional signals at 3.0kb and 1.7kb as well as a 3.2kb signal for RIIß (Figure 5A/B; Maronde et al., unpublished results). These signals were similar to those described in the bovine pineal gland (42). Transcript levels of both RIa and RIIß did not vary between midday (12:00) and midnight (24:00). RIß and RIIa transcripts could not be detected by Northern blotting. The expression of RIIa, however, was revealed by RT-PCR as was the expression of RIa and RIIß. In order to elucidate whether NE affects

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Figure 4. Semi-quantification of the amount of nuclear pCREB in rat pinealocytes treated with NE (0.1 ^M) and different protein kinase inhibitors for 30minutes. Among the different protein kinase inhibitors tested (CaMK, KN-62,1 ^M; PKC, chelerythrin 1^M; MEK1, PD98059, 50^M; PKA, H-89, 10 |iM and Rp-8-CPT-cAMPS, 1 mM) only the PKA-antagonists inhibit CREB phosphorylation. Shown are the mean SUMDEN-SCORR-values (see Exp. Procedures) ± SEM of 4 replicates (Co = control).

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Figure 5. Expression of the cyclic AMP-dependent protein kinase regulatory subunits RI a, RII a ard RII P in rat pinealocytes. Rats were kept under 12L: 12D. A and B show Northern blots of pineal RNA extracts obtained six hours after lights on (left lanes, respectively) and six hours after lights off (right lanes, respectively) probed with a RIa- (A) or a RIIP- (B) specific probe. C Cultured rat pineal organs were left untreated (Co) or treated for six hours with NE (NE 6h). RT-PCR-reaction products obtained from pineal RNA extracts were amplified with either RIIa - (arrowhead 3) or RII P- (arrowhead 2)-specific primers and a GAPDH (arrowhead 1)-specific primer as a loading control.

PKA subunit transcription, RIa-, RIIa-, and RIIP-mRNA levels were investigated in pinealocytes stimulated with NE for 6 hours. Transcript levels of RIa and RIIa did not change, but a slight upregulation of the RIIP transcript was detected after 6 h of NE treatment (Figure 5C) conforming to observations in other/ systems (68).

By use of specific antibodies protein levels of PKA subunits were analyzed in rat pineal glands or pinealocytes kept in vitro (43) and in pineal glands directly dissected out of the animal (ex vivo; Figure 6). All types of preparations revealed the presence of a signal at 40kDa corresponding to the catalytic subunit (C), detected a major 49 kDa signal corresponding to the regulatory subunit RIa, a double 53/55 kDa signal corresponding to the regulatory subunit RIIa, and a single 52 kDa signal corresponding to the regulatory subunit RIIp. No specific signal was found with the antiserum against the regulatory subunit RI (data not shown). None of the detectable subunits showed significant variation in the course of an LD cycle of 12: 12 (Figure 6). Similar results were obtained with the pineal organ of C3H mice (von Gall et al., unpublished observations).

To examine possible effects of NE on the various PKA subunit proteins immunoblots were prepared from isolated rat pinealocytes that were either left untreated or stimulated for 30min or 6 hours with 1 |iM NE (43). The 40kDa signal of the catalytic subunit (C) and the 52kDa signal of RIIP slightly decreased upon NE treatment after 30min and 6 hours. The RI signal was reduced after 30min of NE-treatment, but reached control levels after 6 hours. These small changes may represent degradation of the subunits occurring after dissociation, since both regulatory and catalytic subunits are more sensitive to proteolysis in the monomeric state (26). The 55/53 kDa double band of RIIa increased slightly after 30min and 6h as compared to controls. In corresponding immunoblots pCREB was undetectable in the controls, strongly induced after 30min and still elevated after 6h, whereas total CREB did not vary considerably (43). This comparison indicates that CREB phosphorylation in the rodent pineal organ is unrelated to the protein levels of PKA subunits.

Recent findings with rat cerebellum and cortex (71) as well as with FRTL-5 thyroid cell lines (23) suggested that PKA type I and soluble PKA type II are mainly involved in phosphorylation of cytosolic proteins, whereas particulate (anchored) PKA type II mediates phosphorylation of nuclear proteins like CREB. In the rat pineal gland RIa was found mostly in the soluble fraction whereas both RIIa and RIIP were preferentially found in the particulate fraction, suggesting that the anchored form of PKA type II (which mediates phosphorylation of nuclear proteins) predominates in the rat pineal organ (Maronde et al., unpublished results). The functional role of PKA I in the pineal gland is not clear. It might keep cytosolic target proteins, like AANAT, phos-phorylated (18,35) or mediate the CAMP-induced inhibition of proteasomal degradation of AANAT (27).

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