Diacylglycerol kinase-zeta localization in skeletal muscle is regulated by phosphorylation and interaction with syntrophins

Abstract

Syntrophins are scaffolding proteins that link signaling molecules to dystrophin and the cytoskeleton. We previously reported that syntrophins interact with diacylglycerol kinase-zeta (DGK-zeta), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show syntrophins and DGK-zeta form a complex in skeletal muscle whose translocation from the cytosol to the plasma membrane is regulated by protein kinase C-dependent phosphorylation of the DGK-zeta MARCKS domain. DGK-zeta mutants that do not bind syntrophins were mislocalized, and an activated mutant of this sort induced atypical changes in the actin cytoskeleton, indicating syntrophins are important for localizing DGK-zeta and regulating its activity. Consistent with a role in actin organization, DGK-zeta and syntrophins were colocalized with filamentous (F)-actin and Rac in lamellipodia and ruffles. Moreover, extracellular signal-related kinase-dependent phosphorylation of DGK-zeta regulated its association with the cytoskeleton. In adult muscle, DGK-zeta was colocalized with syntrophins on the sarcolemma and was concentrated at neuromuscular junctions (NMJs), whereas in type IIB fibers it was found exclusively at NMJs. DGK-zeta was reduced at the sarcolemma of dystrophin-deficient mdx mouse myofibers but was specifically retained at NMJs, indicating that dystrophin is important for the sarcolemmal but not synaptic localization of DGK-zeta. Together, our findings suggest syntrophins localize DGK-zeta signaling complexes at specialized domains of muscle cells, which may be critical for the proper control of lipid-signaling pathways regulating actin organization. In dystrophic muscle, mislocalized DGK-zeta may cause abnormal cytoskeletal changes that contribute to disease pathogenesis.

Figure 1.

DGK-ζ and syntrophins associate in skeletal muscle. (A) DGK-ζ migrates as a doublet of ∼116 kDa in immunoblots of mouse skeletal muscle homogenates. (B) Beads charged with GST alone or with a GST-DGK-ζ fusion protein were incubated with skeletal muscle extracts. GST-DGK-ζ, but not GST, captured a significant portion of syntrophin in the offered extract. Input, 5% of starting material. (C) DGK-ζ and syntrophins are coimmunoprecipitated from muscle cell extracts. Lysates of C2 myotubes were immunoprecipitated with an antibody to DGK-ζ or with control IgG. The immunoprecipitates were analyzed by immunoblotting for DGK-ζ and syntrophins. With longer exposures, DGK-ζ was detectable in the input lane. Input, 5% of starting material.

Figure 2.

Immunofluorescence localization of DGK-ζ in skeletal muscle and colocalization with syntrophins. (A-C) Transverse sections of mouse TA muscle were fixed and stained with affinity-purified antibodies to DGK-ζ and FITC-conjugated secondary antibodies. At low magnification (A) DGK-ζ immunoreactivity was observed in a subset of muscle fibers. (B) Specificity of the DGK-ζ immunolabeling was confirmed by preincubation of the antibody with its immunogenic peptide, which completely eliminated the signal. (C) Higher magnification revealed diffuse cytoplasmic and strong sarcolemmal labeling. (D-D″) Mouse TA muscle was double labeled for DGK-ζ (D) and syntrophins (D′). Syntrophins were visualized with mAb 2101 and Texas Red-conjugated secondary antibodies. (D″) Merged image shows syntrophins and DGK-ζ colocalize at the sarcolemma. The arrows indicate regions where DGK-ζ staining is absent and a corresponding reduction in syntrophin staining. (E and E′) In mouse TA muscle, DGK-ζ is absent or greatly reduced in type IIB fibers. (F) DGK-ζ is expressed in all fibers of the soleus muscle (bottom left), but not in the adjacent plantaris muscle (top right). The asterisks indicate DGK-ζ-negative fibers stained for myosin IIB. Bars, 100 μm (A) and 25 μm (C-F).

Figure 3.

DGK-ζ is reduced on the sarcolemma of mdx myofibers and is associated with central nuclei. Sections of TA muscle from normal (A) and mdx (B) mice were stained for DGK-ζ and photographed with identical exposure times. (C) In mdx muscle, occasionally groups of small caliber fibers (arrows) with increased DGK-ζ expression were observed. (D-D″) The increased DGK-ζ immunoreactivity in small caliber fibers coincided with increased syntrophin expression. (E-E″) In mdx TA muscle fibers, DGK-ζ was associated with central nuclei revealed by Hoechst stain (E′ and E′′). Bar, 25 μm.

Figure 4.

DGK-ζ is specifically targeted to NMJs in type IIB fibers and retained at NMJs in mdx muscle. TA muscles were labeled with an antibody to DGK-ζ and with α-BgTx, which labels AChRs. (A and A′) In most fibers, DGK-ζ was present on the extrajunctional sarcolemma and was concentrated at NMJs (arrows). (B and B′) In type IIB fibers (indicated by asterisks), DGK-ζ was restricted to junctional regions (arrows). (C-C″) In mdx myofibers, DGK-ζ is absent from extrajunctional regions but remains concentrated at synaptic sites. Higher magnification of the boxed region in C″ (bottom right) shows DGK-ζ extends beyond the AChR-rich regions. Bar, 25 μm.

Figure 5.

Reciprocal interactions regulate the localization of DGK-ζ and α1-syn in muscle cells. (a) C2 myoblasts were singly (A-C) or doubly transfected (D-E″) with cDNAs encoding the indicated epitope-tagged proteins. The cells were fixed and labeled with antibodies specific for each epitope tag. (b) Representative profile plots of the fluorescence intensity of transfected C2 myoblasts labeled for the indicated constructs showing cytoplasmic (A), nuclear (B), complete membrane localization (C), and partial membrane localization (D). The profile plot in D is taken from the cell shown in Figure 6a, B. The vertical lines indicate transition points in the fluorescence intensity between cellular compartments. (c) The graph shows the percentage of cells with membrane localized protein in C2 myoblasts expressing the indicated constructs. Membrane localization was quantified as described in MATERIALS AND METHODS. Gray bars indicate the percentage of cells with any membrane localization and black bars indicate complete membrane localization. The data are the average of three independent experiments. Error bars indicate SEM. n, nucleus.

Figure 6.

MARCKS domain-phosphorylation regulates DGK-ζ localization. C2 myoblasts expressing the indicated constructs were fixed and visualized as described in Figure 5. The arrow in D indicates nuclear DGK-ζ staining.

Figure 7.

DGK-ζ colocalizes with actin, syntrophin, and Rac1 in membrane ruffles and at the leading edge of lamellipodia. C2 myoblasts were stained with an antibody to DGK-ζ and with either phalloidin to detect F-actin (A-A″), or with antibodies to syntrophin (B-B″) or Rac1 (C-C″). (A-A″) Small arrows indicate membrane ruffles and lamellipodia leading edges, vertical arrowheads actin stress fibers and horizontal arrowheads cortical actin. Bar, 20 μm.

Figure 8.

ERK phosphorylation regulates the association of DGK-ζ with the cytoskeleton. (A) C2 myoblast lysates were fractionated as described in MATERIALS AND METHODS, and equal amounts of protein from each fraction were analyzed by SDS-PAGE and immunoblotting for DGK-ζ and syntrophin. (B) Schematic of DGK-ζ showing the domain organization, the sequence of the proline-rich region near the ankyrin repeats (I-IV) and the ERK docking-site consensus sequence. The N-terminal (open box), C1 (ellipses), MARCKS, and catalytic domains are indicated. (C) COS-7 cells were transfected with DGK-ζ constructs in which all the Ser and Thr residues in the proline-rich sequence were mutated to Ala except the one shown above the corresponding lane of the immunoblot. The presence of a slightly higher shifted band in the lanes marked by an asterisk indicates that the residue is phosphorylated. (D) COS-7 cells were transfected with cDNAs encoding wild-type DGK-ζ (wt), a mutant in which all the Ser/Thr residues shown in B were changed to alanines (AAA), or mutants in which the three Ser residues indicated by asterisks were changed to Asp (DDD) or Asn (NNN). The lysates were analyzed by SDS-PAGE and immunoblotting for DGK-ζ. (E) COS-7 cells were transfected with cDNAs encoding DGK-ζ and either Raf-ER or a control vector. 24 h later the cells were treated for 10 h with 1 μM estrogen or control vehicle. (F) COS-7 cells expressing DGK-ζ, Raf-ER and either the ERK phosphatase MKP3 or a control vector were treated with estrogen or vehicle as described above and then analyzed for DGK-ζ.

Figure 9.

Expression of DGK-ζ^M1-FLAG in C2 cells induces F-actin-rich membrane ruffles and large vesicles. (A-C) Representative images of C2 myoblasts expressing DGK-ζ^M1-FLAG. (A and inset) DGK-ζ was concentrated in membrane ruffles (arrows). (B) DGK-ζ was localized on the perimeter of large invaginations of the plasma membrane (large arrow) and vesicles ranging in size from 2 to 5 μm (small arrows). In some cells, vesicles as large as the nucleus were observed (C, arrow). (D-D″) DGK-ζ^M1-FLAG-induced membrane ruffles (small arrows) and invaginations (large arrow) were enriched in F-actin, which colocalized with DGK-ζ. (E-G) Representative images of C2 myotubes expressing the indicated constructs. 4,6-Diamidino-2-phenylindole-stained nuclei are shown in blue. Vesicles were present in myotubes expressing DGK-ζ^M1-FLAG (E, arrows), α1-Syn^ΔPDZ (F), but not wild-type DGK-ζ (G). n, nucleus. Bars, 20 μm.

Figure 10.

Model depicting the regulation of DGK-ζ localization in muscle cells. (A) PKC-dependent phosphorylation of the MARCKS domain regulates the association of DGK-ζ and syntrophin with the plasma membrane. (B) Syntrophin interaction and PKC-dependent phosphorylation regulate the nuclear-cytoplasmic shuttling of DGK-ζ. (C) ERK-dependent phosphorylation of DGK-ζ negatively regulates its association with the cytoskeleton.