The present study identifies the Z-disc protein myopodin as a novel direct target of PKA, CaMKII, and calcineurin signaling in the heart. These enzymes regulate the phosphorylation state of myopodin, thereby controlling the subcellular localization of myopodin in adult cardiac myocytes (Fig. ). These results support the idea that myopodin serves as a messenger in a unique intracellular signaling pathway, whereby changes in Z-disc dynamics may translate into altered nuclear function during cardiac development and remodeling.
FIG. 9. Model for the regulation of myopodin's subcellular localization in cardiac myocytes. (A) Under normal conditions, myopodin is tethered to the Z-disc of differentiated cardiac myocytes, where it is part of a signal transduction unit. The complex contains (more ...)
Recently, it has been suggested that Z-disc proteins, in addition to their structural function, participate in intracellular signaling pathways (13
). Work from several groups provided evidence that the Z-disc of cardiac myocytes may serve as a mechanosensor during myocyte differentiation and cardiac remodeling (13
). Specifically, it was proposed that the Z-disc is a signal transduction unit that communicates with the nucleus. In this scenario the Z-disc would sense an increase in the mechanical load of the heart and respond by sending a signal to the nucleus. This signal would in turn alter gene expression of muscle-specific proteins, leading to cardiac hypertrophy and remodeling (36
). Clearly, such pathways would require messengers that can shuttle between the Z-disc and the nucleus. Several proteins meet these criteria and therefore could function as potential signal transducers between the two compartments. For example, LIM domain-containing proteins, like FHL2 and FHL3 (12
), zyxin (20
), and the muscle LIM protein (MLP) (23
), can shuttle between the cytosol and the nucleus of myocytes in a differentiation- and stress-dependent manner. MLP, also called CRP3 (cystein-rich protein), and other members of the CRP protein family (CRP1 and CRP2) have also been proposed to function as stress and damage sensors at the actin cytoskeleton (18
). Upon activation, CRPs translocate into the nucleus, where they can activate the expression of muscle-specific genes, resulting in muscle repair (18
). However, the molecular mechanisms regulating the intracellular shuttling of LIM domain proteins have yet to be elucidated.
Myopodin does not contain a LIM domain, but it shares several key features with LIM proteins. Similar to LIM proteins, myopodin directly binds to α-actinin, which anchors myopodin at the Z-disc. More importantly, myopodin also shuttles between the Z-disc and the nucleus in a differentiation- and stress-dependent fashion (43
), thus making it a good candidate as a signaling intermediary between both compartments. We recently demonstrated that serine/threonine phosphorylation-dependent binding of myopodin to 14-3-3 is necessary and sufficient for the interaction between myopodin and importin α (11
), but the protein kinase(s) which phosphorylates myopodin remained unknown. The present study shows that myopodin is part of a multiprotein signal transduction unit that also includes PKA and CaMKII. Both kinases can phosphorylate the 14-3-3-binding motifs of myopodin, thereby enabling the binding of myopodin to 14-3-3. Similar to the biochemical inhibition of 14-3-3 binding (11
), the pharmacological inhibition of PKA and CaMKII abrogates the nuclear import of myopodin in myoblasts. Moreover, in adult cardiac myocytes the activation of PKA induces the release of myopodin from the Z-disc and results in its nuclear import. It should be noted that the differences between the in vitro and in vivo phosphorylation data imply that myopodin harbors additional PKA and CaMKII phosphorylation sites, which remain to be identified. These sites may also participate in the regulation of the nuclear import of myopodin. Such a function could explain the observed differential effects of PKA and CaMKII inhibition on the subcellular localization of myopodin. The observed discrepancy between the in vitro and vivo phosphorylation experiments using PKA and CaMKII might be due to the fact that both kinases act cooperatively on the two 14-3-3-binding sites or in combination with other unidentified protein kinases. Clearly, future studies will be required to test this hypothesis.
This study also reveals that 14-3-3 proteins can modulate the binding of myopodin to its interacting proteins in several ways. 14-3-3 binding enables not only the interaction between myopodin and importin α in a cooperative fashion (11
) but also competitively abrogates the interaction between myopodin and α-actinin. In undifferentiated myoblasts that do not express α-actinin (26
), myopodin is imported into the nucleus (43
). In contrast, when α-actinin is overexpressed in these cells, myopodin is sequestered in the cytoplasm, where it colocalizes with α-actinin. However, this α-actinin-mediated cytoplasmic anchorage of myopodin is overridden when myopodin is mutated, in that it can bind 14-3-3 constitutively. These results support the idea that α-actinin serves as a cytoplasmic anchor of myopodin, which is antagonized by 14-3-3 binding, resulting in the nuclear import of myopodin. This idea is in keeping with the results of the binding assays showing that increasing amounts of 14-3-3 decrease the binding of α-actinin to myopodin and vice versa, thereby explaining the negative effect of α-actinin on the nuclear import of myopodin. Such an α-actinin-mediated cytoplasmic anchoring mechanism in striated myocytes has been proposed before in that it regulates the subcellular localization of CRPs (3
). Of note, the present study is the first to identify such a mechanism on a molecular level.
Several lines of evidence support the notion that signaling events leading to the development of cardiac hypertrophy can be modulated by mAKAP signaling units that contain PKA, the cAMP-responsive guanine nucleotide exchange factor EPAC, and phosphodiesterase 4 (PDE4) (29
). In the current study we identified mAKAP (19
) as another component of the myopodin signaling complex, thereby linking mAKAP signaling to the Z-disc. In addition to PKA, AKAPs can also bind to PDEs (29
), enzymes that degrade cyclic nucleotides like cAMP. cAMP is synthesized by adenylate cyclases at the plasma membrane and diffuses into the cytosol to activate downstream effectors like PKA (29
). By anchoring PDEs at specific sites, the cellular action of cAMP can be controlled spatially and temporally (17
), and the coanchoring of PKA and PDEs enables a tight regulation of PKA signaling. The cAMP-specific PDE4 subfamily is highly expressed in the heart (30
). PDE4 consists of about 16 distinct isoforms, and the mAKAP-anchored isoform is PDE4D3 (7
). A previous yeast two-hybrid screen using PDE4D3 as bait identified a novel protein named myomegalin (42
). We have now identified myomegalin as myopodin-interacting protein. Hence, myopodin can interact with two PDE4D3-anchoring proteins, mAKAP and myomegalin. Furthermore, myopodin exists in a complex with PKA via mAKAP and serves as a PKA substrate. Together these findings raise the intriguing possibility that in the heart, cAMP signaling tightly regulates not only the phosphorylation of myopodin but also its subcellular localization. Consistently, the increase of cAMP levels in adult cardiac myocytes by simultaneous activation of adenylate cyclases and inhibition of PDEs lead to the nuclear import of myopodin and 14-3-3β.
The present study has also identified myopodin as a novel direct target of calcineurin. In adult cardiac myocytes, a fraction of the calcineurin pool is anchored at the Z-disc via interactions with calsarcins/α-actinin (14
) or MLP (16
). Thus far, only a few substrates of calcineurin have been described, including the NFAT family of transcription factors (6
). Interestingly, NFATc is also localized at the Z-disc of resting myocytes. Upon electric stimulation it translocates into the nucleus, where it activates prohypertrophic gene programs (27
). Of note, the nuclear translocation and full transcriptional activity of NFAT requires its dephosphorylation by calcineurin (6
) as well as the activation of the mAKAP signaling complex (34
) and the release of NFAT from 14-3-3 binding (4
). The experiments described herein imply that myopodin not only directly interacts with calcineurin but also indirectly via the calcineurin-binding proteins mAKAP (34
) and calsarcins/α-actinin (14
). Calcineurin dephosphorylates myopodin and thereby abrogates the myopodin-14-3-3 interaction. Conversely, the inhibition of calcineurin by CsA causes the release from the Z-disc and the nuclear import of myopodin. Hence, the dephosphorylation by calcineurin is required for the anchorage of myopodin at the Z-disc. In concert, these results suggest that calcineurin is a general regulator of signaling protein targeting. It will be interesting to see whether in addition to NFAT and myopodin the subcellular localization of other proteins in the heart or elsewhere is also controlled by calcineurin. Of note, similar to NFAT (15
), myopodin only interacts with the activated form of CnA. However, in contrast to NFAT and most other known calcineurin target proteins, myopodin lacks a consensus (PxIxIT) calcineurin docking site (15
). Clearly, future studies will be required to map the calcineurin-binding site(s) in myopodin.
Altogether, we identified a novel Z-disc signal transduction unit that communicates with the nucleus of cardiac myocytes by regulating the phosphorylation state of myopodin. Our results highlight how phosphorylation and dephosphorylation events can dynamically modulate the composition of large multiprotein signaling complexes (Fig. ). Such complexes contain signaling and scaffolding proteins as well as mobile signal mediators like myopodin, whose access to or release from these complexes is tightly regulated by external or internal cues (35
). The regulated presence or absence of a mediator can either alter the function of the complex or influence a target outside the complex. The identification of myopodin as a direct target of PKA, CaMKII, and calcineurin defines a novel intracellular signaling pathway, whereby changes in Z-disc dynamics may alter nuclear function during cardiac development and remodeling and may open new therapeutic modalities for the prevention or treatment of cardiac failure by the modulation of myopodin signaling.