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- Experimental procedures
- Supporting Information
Dystrobrevins are a family of proteins related to and associated with dystrophin. Together with the transmembrane dystroglycan and sarcoglycans, and the cytoplasmic proteins dystrophin and syntrophin, dystrobrevin is a component of the macromolecular membrane complex known as the dystrophin-associated glycoprotein complex (DGC). The DGC was first isolated and characterized from skeletal muscle, where, by linking the extracellular matrix with the actin cytoskeleton, it stabilizes the sarcolemma during muscle contraction and relaxation cycles [1, 2]. Mutations of the coding genes for some of the DGC components result in several forms of muscular dystrophies .
DGCs have also been widely studied in nonmuscle tissues, such as brain, lung, and kidney [4, 5]. Various DGCs can originate from the assembly of dystrophin, dystrobrevin, and syntrophin-specific isoforms. Different cellular compartmentalization and/or cell type expression derive from this diverse assembly, and add functional complexity to the DGC. Besides its structural role, the DGC is now thought to function as a scaffold that concentrates and organizes interrelated signaling molecules at the plasma membrane .
Dystrobrevin shares sequence homology with the C-terminus of dystrophin, but does not bind dystroglycan. It contains two EF-hands, a zinc-binding domain (ZZ), syntrophin-binding sites, and two coiled-coil domains that mediate the association between dystrobrevin and dystrophin . Two separate genes, both of which yield multiple transcripts, code for two dystrobrevin isoforms, α and β [7, 8]. Whereas α-dystrobrevin is a ubiquitous isoform, β-dystrobrevin (β-DB) is expressed in nonmuscle tissues, including brain [4, 5].
Besides dystrophin and syntrophin, a number of dystrobrevin-interacting partners have so far been identified. These include: α-sarcoglycan ; intermediate filament proteins such as syncoilin  and desmuslin ; dysbindin , a subunit of biogenesis of lysosome-related organelles complex-1 and the product of a potential susceptibility gene for schizophrenia [13, 14]; DAMAGE, an α1-dystrobrevin-associated protein that is highly expressed in brain ; pancortin, an extracellular matrix protein that has been proposed to play a role in neuronal differentiation ; kinesin heavy chain Kif5 , a motor protein involved in axonal and dendritic transport of vesicles, organelles, and protein complexes ; the regulatory subunits RIα and RIIβ of cAMP-dependent kinase [protein kinase A (PKA)] , and α-catulin, a protein recruited to the α(1D)-adrenergic receptor signalosome by the C-terminal domain of α1-dystrobrevin . Moreover, we have recently demonstrated and characterized the interaction between β-DB and the high mobility group-domain proteins, iBRAF and BRAF35, suggesting an involvement of β-DB in the regulation of chromatin dynamics . Through exploration of the activity of these proteins, a multifaceted function of dystrobrevin in distinct cell compartments emerges. By these multiple interactions, dystrobrevin can organize molecular platforms and link them to the cytoskeleton and intracellular organelles.
How the interactions of dystrobrevin with its binding partners are spatially and temporally regulated is still not understood. Post-translational modifications such as phosphorylation may be among the regulatory factors. We found, indeed, that dystrobrevin is a substrate of PKA, and that PKA phosphorylation regulates its binding affinity for the cargo-binding domain of kinesin heavy chain (Kif5A) .
In this study, we used biochemical, molecular and proteomic analyses to investigate which serine/threonine residues of β-DB were phosphorylated by PKA. Among the phosphorylated residues identified, we found that Thr11 is the regulatory residue for the β-DB–kinesin interaction. As our previous observations indicated that β-DB behaves, similarly to A kinase anchoring proteins (AKAPs), as a platform for phosphatases and kinases , we investigated whether β-DB is a substrate for protein kinases other than PKA. We found that Thr11 is also phosphorylated by protein kinase C (PKC), suggesting that it may be a key residue for docking and undocking of the vesicular or multiprotein complex cargoes from the motor protein.
- Top of page
- Experimental procedures
- Supporting Information
In this study, we have demonstrated that β-DB is a phosphorylation substrate for both PKA and PKC, and have shown that Thr11 is a key residue for regulating the binding of β-DB to kinesin heavy chain. Thr11 is a target for both kinases, suggesting that phosphorylation at this site may occur following the activation of different signaling pathways.
Besides being a component of the DGCs in muscle and nonmuscle tissues, dystrobrevin is now thought to participate, through its multiple interactions, to protein networks connecting different cell compartments, also playing a role in signaling. Numerous dystrobrevin-binding proteins, which may specify dystrobrevin function, have been identified so far [9-12, 15-17, 19, 20]. How these multiple associations take place in the cell may depend on several factors, such as the dystrobrevin isoform (α, β, and their spliced variants) [25-28], the cell type , the stage of development of the tissue , and the binding affinity for the different partners . For instance, as we reported previously, both α-dystrobrevin and β-DB can bind to the cargo-binding domain of kinesin heavy chain, but with different binding affinities, as measured by SPR .
The function of dystrobrevins and the interaction with their binding partners can also be regulated by post-translational modifications such as phosphorylation. It is known that α-dystrobrevin, which was first isolated and characterized in the torpedo electric organ neuromuscular junctions, is a tyrosine-phosphorylated protein . The phosphorylated tyrosines are located in the unique α1-dystrobrevin C-terminal domain , a region that is absent in other α-dystrobrevin isoforms as well as in β-DB [7, 8]. The function of α-dystrobrevin tyrosine phosphorylation has been investigated in the skeletal muscle of transgenic mice . The findings revealed that the effectiveness of α1-dystrobrevin in stabilizing the neuromuscular junction depends, in part, on its ability to serve as a tyrosine kinase substrate, and that α-dystrobrevin tyrosine phosphorylation may be a key regulatory point for synaptic remodeling . Moreover, both α-dystrobrevin and β-DB are in vitro substrates of PKA . β-DB phosphorylation specifically reduces the binding to kinesin heavy chain but not to other dystrobrevin-binding partners, such as dysbindin .
Here, we have identified, by MS, two β-DB residues that are phosphorylated in vitro by PKA, Thr11 and Thr424. We first analyzed the functional role of phosphorylation on Thr424. We had previously demonstrated that deletion of the coiled-coil or the C-terminal region decreased the kinetics of the binding of β-DB to kinesin, suggesting that factors affecting β-DB tertiary structure may play a role in regulating its associations . As Thr424 is located next to the first coiled-coil domain, we wondered whether Thr424 phosphorylation was involved in the regulation of β-DB–kinesin association, and found that it was not. Nonetheless, Thr424 is adjacent to motifs involved in the interaction with other partners (syntrophin and dystrophin), and it is therefore conceivable that phosphorylation on this residue may influence the functionality of this region. In this context, our preliminary data (not shown) suggest that Thr424 phosphorylation does not directly affect the binding to syntrophin; further studies are needed to reveal the function of this phosphorylation site.
We previously reported that the binding site for kinesin heavy chain is located in the N-terminal region of β-DB (amino acids 1–236) and that phosphorylation influences the interaction between the two proteins. We subjected the N-terminal deletion mutant β-DB1–352 to MS analysis to detect β-DB phosphorylated residues. Despite a sequence coverage of 83%, we failed to identify any phosphorylated peptide. We overcame this problem by analyzing the phosphorylated short products derived from β-DB1–236, which allowed us to restrict our search to the first 22 amino acids of the molecule. In this peptide, a putative PKA consensus sequence (Lys-Arg-X-Thr/Ser) precedes the threonine at position 11. By the use of a synthetic peptide (amino acids 1–23), we finally identified Thr11 as the PKA target in the N-terminus of β-DB, and by site-directed mutagenesis we demonstrated that phosphorylation of Thr11 regulates the interaction of β-DB with kinesin. Indeed, when Thr11 was mutated into aspartic acid, which mimics a phosphorylated residue, the ability of β-DB to bind kinesin from rat brain extract was reduced by ~ 80% in comparison with wild-type or T11A mutated β-DB. Phosphorylation on Thr11 may function as a mechanism to undock β-DB-bound cargoes transported by the kinesin molecular motor to specific cell compartments. So, it appears that the high-affinity interaction of β-DB with kinesin is a highly dynamic one, as it can be regulated by very fast events such as phosphorylation. The modification of the molecule induced by phosphorylation on Thr11 does not affect binding to molecules that involves regions other than the N-terminus, such as syntrophin (Fig. 7) or dysbindin , suggesting that the release of the motor protein does not affect the binding of β-DB to other partners.
Besides being phosphorylated by PKA, we found that β-DB is also an in vitro substrate for PKC phosphorylation. This finding supports our suggestion that β-DB behaves as an AKAP-like protein. AKAPs assemble and organize kinases and phosphatases on the same molecular platform, and they are themselves enzymatic substrates . β-DB phosphorylation by PKC resulted in a reduction of the binding to kinesin similar to that following phosphorylation by PKA. We found that the synthetic peptide corresponding to amino acids 1–23 of β-DB, which contains Thr11, was phosphorylated by PKC, suggesting that Thr11 may be a key residue modified by the activation of different signaling pathways. Moreover, we identified other threonines that are phosphorylated in vitro by PKC (Thr69, Thr179, and Thr212), all of which are in the EF-hand-like domains of β-DB. From the crystallography structure of the homologous dystrophin region , we can infer information about the tertiary structure of the N-terminal region of dystrobrevin. Thr69 is located between the first and the second α-helical structures of the EF1-hand-like domain, whereas Thr179 and Thr212 are located at the end of the second and fifth α-helical structures of the EF2-hand-like domain, respectively . Phosphorylation at these sites could alter the conformation of β-DB and, as a consequence, its association with partners that bind to the N-terminal region. So, even if β-DB is the target of different kinases, phosphorylation on diverse residues could result in different signaling pathways/cascades, depending on the β-DB-specific binding partners in different cell types.
Phosphorylation plays an essential role in the regulation of a large number of processes, from cell metabolism to higher-level functions of learning and memory. Phosphorylation events are controlled through the compartmentalization of protein kinases and phosphatases provided by scaffold and anchor proteins. When extracellular or intracellular stimuli reach the subcellular compartment and activate signaling components, scaffold proteins themselves may be the targets of phosphorylation/dephosphorylation processes. The fact that dystrobrevin is phosphorylated by both PKA and PKC suggests that, like other scaffold proteins, dystrobrevin can play a role not only as a stationary anchor but also as a dynamic signaling component. We have not yet investigated the possible implications of Thr11 and Thr424 phosphorylation on β-DB functions in terms of, for instance, affecting cell localization or protein lifetime. During our experiments, we observed that, in solution, the phosphorylated dystrobrevin is slightly unstable as compared with the unphosphorylated one (not shown). If this is true, phosphorylation could be also regarded as a mechanism to regulate protein lifetime. Further studies are necessary to test this hypothesis.
The results reported in this article contribute to our understanding of how dystrobrevin molecular interactions may be regulated, and add new evidence to the previously suggested hypothesis [17, 19, 22] of dystrobrevin involvement in trafficking and signaling mechanisms.