MAKAP as a Model for the Regulation of cAMP Signaling

The scaffold protein mAKAP is an example of an AKAP that is well characterized. Moreover, mAKAP provides an example of how AKAPs function to link multiple signaling pathways and to control local cAMP levels. mAKAPß is a 230-kDa protein present at the nuclear envelope in both cardiac and skeletal myocytes (Kapiloff et al. 1999; Michel et al. 2005). The alternatively spliced-form mAKAPa is present in neurons. mAKAP complexes can contain PKA, the phosphodiesterase PDE4D3, the calcium-activated calcium channel ryanodine receptor, the protein phosphatases PP2A, and calcineurin Aß, nesprin-1a, Epacl, extracellular signalregulated kinase 5 (ERK5), and its upstream activator MEK5, 3-phosphoinositide-dependent kinase-1 (PDK1), and p90RSK (Kapiloff et al. 1999; Marx et al. 2000; Dodge et al. 2001; Ruehr et al. 2003; Pare et al. 2004, 2005; Dodge-Kafka et al. 2005; Michel et al. 2005). Due to mAKAPß's large size and its association with the scaffold protein nesprin-1a, additional mAKAPß-binding partners likely will be identified.

The mAKAP signalosome is unique in that it regulates local cAMP levels through two conjoined negative feedback loops comprised of different signaling pathways (Fig. 2). These pathways converge on the associated phosphodiesterase, either stimulating or abating its activity, allowing for the precise tuning of the surrounding cAMP concentration. Phosphorylation of PDE4D3 at serine 13 enhances the binding affinity of the phosphodiesterase for mAKAP, while phosphorylation of serine 54 increases the catalytic activity of the phosphodiesterase (Sette and Conti 1996; Carlisle Michel et al. 2004). This complex interaction between PDE

Hypertrophic Cicut Expression

Fig. 2 Diagram depicting the mAKAPP singalsome. The P-receptor stimulates the release of the Gas subunit from the trimeric G-protein, initiating the activation of the adenylyl cyclase. The resulting increase in cAMP production stimulates the activation of PKA in the complex and phosphorylation of the two currently identified targets, PDE4D3 and RyR2. Phosphorylation of RyR2 increases the release of calcium, promoting the activation of calcineurin AP and dephospho-rylation of the transcription factor NFAT. The now dephosphorylated NFAT translocates into the nucleus, where it promotes hypertrophic gene expression. The second target for PKA, PDE4D3, is responsible for turning off this pathway by decreasing the concentration of cAMP and promoting the formation of the PKA holoenzyme. However, stimulation of the map kinase ERK5 results in a decrease in cAMP hydrolysis by phosphorylating PDE4D3, lengthening the activation of PKA in the complex. Prolonged increases in cAMP concentration activate Epac within the complex, inhibiting ERK5

Hypertrophic Cicut Expression

Fig. 2 Diagram depicting the mAKAPP singalsome. The P-receptor stimulates the release of the Gas subunit from the trimeric G-protein, initiating the activation of the adenylyl cyclase. The resulting increase in cAMP production stimulates the activation of PKA in the complex and phosphorylation of the two currently identified targets, PDE4D3 and RyR2. Phosphorylation of RyR2 increases the release of calcium, promoting the activation of calcineurin AP and dephospho-rylation of the transcription factor NFAT. The now dephosphorylated NFAT translocates into the nucleus, where it promotes hypertrophic gene expression. The second target for PKA, PDE4D3, is responsible for turning off this pathway by decreasing the concentration of cAMP and promoting the formation of the PKA holoenzyme. However, stimulation of the map kinase ERK5 results in a decrease in cAMP hydrolysis by phosphorylating PDE4D3, lengthening the activation of PKA in the complex. Prolonged increases in cAMP concentration activate Epac within the complex, inhibiting ERK5

and kinase activity sets up a unique feedback mechanism allowing for the temporal control of kinase activity. In a myocyte, one expects that under basal conditions, mAKAPP-bound PKA activity will be minimal due to the hydrolysis of ambient cAMP by bound PDE4D3. Upon P-adrenergic stimulation, cAMP concentrations increase, activating PKA. PDE4D3 is phosphorylated on both serine residues, increasing the binding affinity for mAKAPP and PDE4D3 catalytic activity. These events form a negative feedback loop that inhibits the accumulation of local cAMP, resulting in the attenuation of PKA signaling.

Additional regulation of local cAMP levels is contributed by further phosphorylation of mAKAP-bound PDE4D3. Phosphorylation of serine 579 by the MAP-kinase ERK5 inhibits PDE4D3 catalytic activity (Hoffmann et al. 1999). In contrast to the effects of PKA phosphorylation, ERK5 phosphorylation promotes the local accumulation of cAMP around the mAKAPp signalosome, potentiating PKA activation (Dodge-Kafka et al. 2005). Furthermore, ERK5 is targeted to the mAKAP complex through direct interaction with PDE4D3, and mutation of the ERK-binding domain on PDE4D3 blocks ERK5 association with the complex (Dodge-Kafka et al. 2005). Due to its phosphorylation by both PKA and ERK5, PDE4D3 serves as the fulcrum for the two negative feedback loops. Therefore, by tethering these three enzymes into the same signaling complex, mAKAPp permits the precise tuning of cAMP levels by both Gs-coupled and MAP-kinase signaling pathways.

mAKAP-associated PDE4D3 also directly binds to Epacl. This cAMP effector regulates the activity of the small G-protein Rapl, which in turn regulates ERK5 in the mAKAP complex. As cAMP concentrations climb due to ERK5 and adenylate cyclase activation, Epacl, which has a 100-fold lower affinity for cAMP than PKA, is activated. Epacl activation results in Rapl activation, which subsequently leads to MEK5 and ERK5 inhibition. As a result, in the presence of very high levels of cAMP, PDE4D3 is not inhibited by the ERK signaling pathway, and cAMP degradation is favored.

Another important target of mAKAP-associated PKA is the ryanodine receptor (RyR). The RyR is responsible for the bulk release of calcium ions from intracellular stores and plays an important role in excitation-contraction coupling. Importantly, the cardiac RyR2 isoform is a substrate for PKA phosphorylation. Phosphorylation of RyR2 serine 2808 increases the channel's sensitivity to calcium and the probability of channel opening (Marx 2003). A subset of nuclear RyR2 can be co-immunoprecipitated with mAKAPp from cardiac myocytes, and p-AR stimulation of primary myocyte cultures increases the phosphorylation of mAKAPP-associated RyR2 (Kapiloff et al. 200l). These findings suggest that within the local context of the mAKAPp signalosome, PKA-mediated RyR2 phosphorylation could potentate the release of calcium. Importantly, PKA-phosphorylated RyR2 may be de-phosphorylated by the associated phosphatase PP2A (Marx et al. 2000; Kapiloff et al. 200l). This complex formation and regulation of the RyR2 by the mAKAPp complex may have important clinical applications, as Marx and colleagues have shown that PKA phosphorylation of RyR2 is increased in heart failure due to the decreased expression of PDE4D3 and local phosphatases as well as sustained P-adrenergic signaling (Marx et al. 2000; Lehnart et al. 2005).

One function of the calcium ion released by mAKAPP-associated RyR2 may be the activation of the associated calcium/calmodulin-dependent phosphatase calcineurin Ap. In vivo, calcineurin Ap is important for the activation of NFATc (nuclear factor of activated T-cells) transcription factors and is required for the induction of cardiac hypertrophy (Lehnart et al. 2005). The functional significance of mAKAPp-orchestrated stimulation of calcineruin was demonstrated by Pare et al. (2005), who found activation of RyR2 in the complex led to stimulation of the associated phoshpatase and induction of cardiac hypertrophy. Furthermore, myocytes lacking mAKAPp expression demonstrated a significant reduction of p-adrenergic-stimulation of cardiac hypertrophy. Importantly, regulation of the localized cAMP concentration by the bound phosphodiesterase would affect the calcium release from the RyR2 and the activation of calcineurin AP and induction of cardiac disease.

0 0

Post a comment