In this report, we describe four major and novel findings. First, we have shown that inactive RSK1 interacts directly with the RI subunit while active RSK1 directly interacts with PKAc. This is the first demonstration of an activation-dependent association of RSK1 with different subunits of the PKA holoenzyme. Moreover, by switching the partners that the inactive and active RSK1 associate with, irrespective of its activation state, RSK1 ensures its indirect association with PKA holoenzyme and, therefore, AKAPs. Because B82L cells express only the RIα subunit, in this report we have focused on the interactions of RSK1 with this subunit. However, given the similarity in the structures of the different regulatory subunit isoforms, it is possible that RSK1 may also interact with RII subunits.
A second important finding of our studies is that the associations between inactive RSK1 and RI and between active RSK1 and PKAc modulate the interactions between the subunits of the PKA holoenzyme. Thus, inactive RSK1, by interacting with RI, decreases the associations between PKAc and RI, whereas active RSK1, which associates with PKAc, increases the interactions between PKAc and RI. The increase in interactions between subunits of PKA resulting from the presence of active RSK1 also decreases the ability of cAMP to activate PKA (Fig. ). These findings demonstrate one of the functional significances of the interactions we have unraveled and identify a novel mode of regulating PKA activity.
Although the regions on PKA subunits that RSK1 interacts with are not known, from the structure of the type I (RI-containing) PKA holoenzyme (25
) it is apparent that upon association with PKAc, the RI subunit undergoes major conformation changes; also, the ordering of the linker region on RI is important in the RI/PKAc interactions (25
). Because inactive RSK1, by interacting with RI, decreases the association of RI with PKAc, it is tempting to suggest either that RSK1 binds to the linker region of RI or that the binding of RSK1 to some other region of RI inhibits the ordering of the linker region on RI and, therefore, association with PKAc. On the other hand, active RSK1, by interacting with PKAc, may somehow alter the conformation of the large lobe where the major interactions with RI occur to facilitate the formation of the holoenzyme (25
). Future studies which identify the interacting regions on RSK1 and PKA subunits will provide mechanistic insights into how RSK1 regulates the interactions between PKA subunits.
A third major functional role of the interactions between RSK1 and PKA subunits elucidated by our experiments is that the indirect interactions (via PKA subunits) of RSK1 with AKAPs are essential for the nuclear localization of active RSK1. Hence, when the interactions of RI with AKAPs are disrupted by Ht31, active RSK1 is excluded from the nucleus and accumulates in the cytoplasm (Fig. and ). The resultant increase in cytosolic active RSK1 levels is accompanied by increased phosphorylation of its cytosolic substrates TSC-2 and BAD and an increased ability of EGF to protect against cellular apoptosis. These findings underscore the importance of the RSK1/PKA subunit interactions in regulating the cellular distribution and biological functions of RSK1.
Because AKAPs localize PKA in the proximity of its substrates (2
) the dissociation of PKA from AKAPs has been shown to decrease BAD phosphorylation (20
). However, the dissociation of RSK1 from AKAPs increases its cytosolic content and phosphorylation of BAD (Fig. ). These findings show that the dissociation of proteins from scaffolds such as AKAPs, by increasing their concentrations in other compartments, can augment some signals. On the other hand, the exclusion of active RSK1 from the nucleus by dissociating the RSK1/PKA complex from AKAPs may decrease the phosphorylation of the nuclear substrates of RSK1 such as c-Fos, Mit1, Bub1, and histone 3. RSK1 also phosphorylates CREB on Ser133 (54
). However this site on CREB is also phosphorylated by several other kinases, including Ca2+
-calmodulin-dependent protein kinases, mitogen- and stress-activated protein kinase 1, and mitogen kinase-activated protein kinase-activated protein 2 (13
). Because EGF, via increases in cytosolic free-Ca2+
levels, activates Ca2+
-calmodulin-dependent protein kinases and downstream of mitogen-activated protein kinases can activate mitogen- and stress-activated protein kinase 1 and mitogen kinase-activated protein kinase-activated protein 2 (8
), we were unable to conclusively decipher changes in CREB phosphorylation on S133 when intracellular distribution of RSK1 was altered by the presence of Ht31 (not shown).
Since active RSK1 is colocalized in the nucleus and perinuclear region with PKAc and RI, an intriguing possibility that arises is that the nuclear AKAP7γ that binds RI (7
) or some other, as-yet-unidentified, nuclear RI-specific AKAP may play a central role in the nuclear localization of RSK1. In this context, a recent study has shown that muscle-selective AKAP (mAKAP) binds and localizes PDK1, ERK, and RSK3 to the nuclear periphery and regulates the activation of RSK3 (31
). However, it is unlikely that mAKAP is involved in the interactions we report since mAKAP is mainly expressed in muscle tissue. Secondly, RSK3, directly or indirectly, binds mAKAP (31
) whereas in our experiments, neither RSK2 and nor RSK3 was present in IPs of PKAc (Fig. ). Nevertheless, in tissues such as myocardium that are rich in mAKAP and RSK1, it is possible that the mAKAP may also associate with RSK1 via PKA subunits. The mAKAP-mediated colocalization of RSK1, PKAc, ERK, and PDK1 at the periphery of the nucleus may be important in bringing these kinases in proximity of each other, especially because PDK1 plays an integral role in the activation of these two kinases (5
). In this regard, unlike the colocalization of ERK, PDK1, and RSK3 in the complex with mAKAP (31
), we do not presently know whether ERK or PDK1 is also present in the complex of PKA subunits and RSK1 investigated here. Moreover, although in the studies of Michel et al. (31
) activation of RSK3 by PDK1 released the enzyme from the mAKAP complex, we do not know whether ERK or PDK1 alters the interactions between PKA subunits and RSK1 studied here. Future experiments will address these possibilities.
The PKA subunits and RSK1 also interact with other proteins (see the introduction). Therefore, it is possible that these other interactions may also influence the ability of RSK1 to associate with PKA subunits in the cells and permit the spatial localization of RSK1 at its cellular targets. For instance, the interactions between RI and cytochrome c
oxidase subunit Vc or PAP7 (28
) and between RI and RSK1 may localize RSK1 to the mitochondria to permit phosphorylation of BAD (23
). In this respect, D-AKAP1, which specifically binds RI, also contains a mitochondrial localization sequence and could also permit the colocalization of RSK1 to the mitochondria. Future experimentation will elucidate whether the other proteins that interact with PKA subunits (see the introduction) also modulate the interactions between RSK1 and PKA subunits or regulate the spatiotemporal localization of RSK1 and augment the repertoire of signaling events.
A fourth important point that our studies brought forth is that in certain cell types in which cAMP, by increasing ERK1/2 activity, activates RSK1, both PKAc and active RSK1 reside as a complex. Because these kinases share common phosphorylation sites on certain substrates such as CREB, BAD, and Nur77, the phosphorylation of these substrates by agonists that increase intracellular cAMP content may be erroneously ascribed solely to PKAc. This could account for the ambiguity in the literature concerning the identity of the kinase that actually phosphorylates some of the common substrates of PKA and RSK1 (see examples with respect to Nur77 [21
] and Ser155 [29
] phosphorylation of BAD).
In conclusion, herein, we report novel interactions of inactive and active RSK1 with RI and PKAc subunits of PKA, respectively. The functional significance of these interactions is demonstrated by the following. First, by modulating the interactions between PKAc and RI, the inactive and active RSK1 may alter the ability of cAMP to activate PKA. Indeed, in the presence of active RSK1, the Ka of cAMP for PKA activation is increased. This represents a novel mechanism for regulating PKA activity. Second, the indirect (via PKA subunit) interactions of active RSK1 with AKAPs are essential for its nuclear localization and phosphorylation of its substrates. Thus, when its interactions with AKAPs are disrupted, active RSK1 is excluded from the nucleus and its cytosolic content is augmented, with a resultant increase in phosphorylation of its cytosolic substrates such as BAD that enhance the antiapoptotic actions of EGF. Finally, in certain cell types in which cAMP activates RSK1, because RSK1 and PKAc are in the same complex, the identity of the kinase that phosphorylates their common substrates may be erroneously ascribed to PKAc.