MKBP, a novel member of the small heat shock protein family, binds and activates the myotonic dystrophy protein kinase

Abstract

Muscle cells are frequently subjected to severe conditions caused by heat, oxidative, and mechanical stresses. The small heat shock proteins (sHSPs) such as alphaB-crystallin and HSP27, which are highly expressed in muscle cells, have been suggested to play roles in maintaining myofibrillar integrity against such stresses. Here, we identified a novel member of the sHSP family that associates specifically with myotonic dystrophy protein kinase (DMPK). This DMPK-binding protein, MKBP, shows a unique nature compared with other known sHSPs: (a) In muscle cytosol, MKBP exists as an oligomeric complex separate from the complex formed by alphaB-crystallin and HSP27. (b) The expression of MKBP is not induced by heat shock, although it shows the characteristic early response of redistribution to the insoluble fraction like other sHSPs. Immunohistochemical analysis of skeletal muscle cells shows that MKBP localizes to the cross sections of individual myofibrils at the Z-membrane as well as the neuromuscular junction, where DMPK has been suggested to be concentrated. In vitro, MKBP enhances the kinase activity of DMPK and protects it from heat-induced inactivation. These results suggest that MKBP constitutes a novel stress-responsive system independent of other known sHSPs in muscle cells and that DMPK may be involved in this system by being activated by MKBP. Importantly, since the amount of MKBP protein, but not that of other sHSP family member proteins, is selectively upregulated in skeletal muscle from DM patients, an interaction between DMPK and MKBP may be involved in the pathogenesis of DM.

Figure 1

MKBP specifically interacts with the kinase domain of DMPK. (a) Shown are molecular structures of the two major DMPK isoforms, tentatively designated as DMPK-1 (Jansen et al., 1992) and DMPK-2 (Sasagawa et al., 1994), and the corresponding regions used in two-hybrid screening. (b) Specificity of the interaction of MKBP with DMPK detected in the yeast two-hybrid assay. The growth on −ULWH plates, which lack uracil, leucine, tryptophan, and histidine, indicates the interaction (lower panels). The cotransfected plasmid vectors are indicated (DNA-binding domain fusion/Activation domain fusion). D2-1 represents a plasmid clone obtained during screening. MKBP (full) represents a plasmid in which the extra sequence preceding the initiation codon in D2-1 was deleted. 3-aminotriazole was added to suppress the background growth of yeast. The same results were also confirmed by β-galactosidase filter assay (data not shown). (c) In vivo interaction between DMPK and MKBP detected in COS1 cells. Immune complexes precipitated with anti-T7 epitope antibody (top, middle), or total cell extracts (bottom) were assayed for DMPK and MKBP content by immunoblotting. MKBP was detected with the anti-MKBP antiserum c-2 (see Fig. 3). (d) Blot overlay assay showing the direct interaction between recombinant DMPK (1–541) and MKBP. Histidine-tagged DMPK (1–541) (0.5 μg) purified from E. coli (lanes 2–6) or molecular mass markers (total protein amount = ∼5 μg) (lane 1) were blotted onto a membrane after SDS-PAGE separation and assayed for MKBP binding by overlaying purified GST–MKBP or GST. The binding of the overlaid protein was detected by immunostaining with anti-GST antibody. Note that the molecular mass markers did not bind GST-MKBP (lane 1).

Figure 2

Deduced amino acid sequence of human MKBP and alignment with four human sHSPs. Black boxes indicate amino acid identity. The α-crystallin domain is indicated by the double-headed arrow. The amino acid sequence used to generate the anti-MKBP antibody, MKC148, is underlined. The table shows the percent amino acid identity among sHSPs in the alignment of the α-crystallin domain as calculated by the DNASIS program (Hitachi Software Engineering Co., Ltd., Tokyo, Japan). The amino acid residues of the sHSPs used for comparison are as follows; 66–148 for MKBP, 87–169 for HSP27, 64–144 for αA-crstallin, 67–149 for αB-crystallin, and 66–148 for p20. The sequence data for MKBP are available from GenBank/EMBL/DDBJ under accession number D8961.

Figure 3

MKBP is highly expressed in skeletal muscle and heart. (a) Northern blot of MKBP mRNA in adult human tissues. Each lane contained 2 μg of poly (A)⁺ RNA isolated from the indicated tissues. An x-ray film was exposed at −70°C for 14 d with an intensifying screen. (b) Western blot analysis showing the expression of three members of the sHSP family in muscle cells. Identical blots on which the extracts of human skeletal muscle (lane 1), mouse hindlimb skeletal muscle (lane 2), and mouse cardiac muscle (lane 3) were transferred after SDS-PAGE separation were probed with antibodies against three kinds of sHSPs. c-2 depleted indicates serum from which anti-MKBP IgG was specifically depleted using the antigen. (c) Characterization of another anti-MKBP antibody, MKC148, raised against a COOH-terminal sequence of MKBP. The extract of human skeletal muscle was analyzed using the indicated antisera. MKC148 was used for immunohistochemical analysis in Fig. 6. (d) Two-dimensional PAGE analysis of human skeletal muscle. The blot membrane was probed with the indicated mixture of antisera. Arrowheads indicate the positions of HSP27 (pI = 5.95) used as a PI marker. The positions of αB-crystallin and MKBP are indicated by open triangles. The arrow indicates a nonspecific spot stained by the anti–αB-crystallin antibody (see b).

Figure 6

MKBP localizes to the Z-membrane and neuromuscular junction in human skeletal muscle. Shown are cross sections (a–c and g–i) and longitudinal sections (d–f) of wild-type (wt; a, c, and d–f) and DM (b and g–i) skeletal muscle. Sections were stained with MKC148 (mk; a, b, d, and g), anti-MKBP IgG-depleted MKC148 (co; c), antidesmin antibody (des; e), or α-bungarotoxin (BT; h). f and i represent the superimposed images of d and e, and g and h, respectively. Bar: (a–c and g–i) 25 μm; (d–f) 5 μm.

Figure 4

MKBP exists in an oligomeric complex separate from the complex composed of αB-crystallin and HSP27 in muscle cells. (a) Size fractionation of oligomeric complexes formed by each member of the sHSP family in mouse cardiac muscle (top) and rat skeletal muscle (bottom). The soluble fraction from each muscle cell type was gel-filtered through Superose 12 and analyzed by immunoblotting. c-2 was used to detect MKBP. (b) Interactions between three members of the sHSP family as monitored by the yeast two-hybrid assays. The region of the cDNA corresponding to the complete open reading frame of each sHSP was subcloned into pGBT9 (for fusion with the DNA-binding domain of GAL4) or pGAD424 (for fusion with the activation domain of GAL4) and subjected to assays. The same results were also confirmed by β-galactosidase filter assay (data not shown).

Figure 5

MKBP is a unique stress-responsive protein. (a) Northern blot analysis of total RNA from differentiated C2C12 cells extracted at the indicated times after heat treatment (44°C 15 min). Three identical blots were hybridized with each sHSP probe as indicated at the top. The same membranes were rehybridized with a probe for glyceraldehyde- 3 -phosphate dehydrogenase (GAPDH) to confirm that the amount of RNA in each lane were the same. (b) Heat-inducible redistribution of MKBP from the cytoplasm to the insoluble fraction in rat skeletal muscle. After incubation at the indicated temperature for 20 min, the freshly excised hindlimb muscle from 11-wk-old SD rats was homogenized and fractionated into soluble (S) and insoluble (P) fractions by centrifugation (130,000 g, 40 min). T indicates the total homogenate before centrifugation. Each sample was separated in 12% SDS-PAGE and analyzed by immunoblotting (top). Each membrane was stained with Coomassie brilliant blue (CBB) after immunoblot analysis (bottom).

Figure 5

MKBP is a unique stress-responsive protein. (a) Northern blot analysis of total RNA from differentiated C2C12 cells extracted at the indicated times after heat treatment (44°C 15 min). Three identical blots were hybridized with each sHSP probe as indicated at the top. The same membranes were rehybridized with a probe for glyceraldehyde- 3 -phosphate dehydrogenase (GAPDH) to confirm that the amount of RNA in each lane were the same. (b) Heat-inducible redistribution of MKBP from the cytoplasm to the insoluble fraction in rat skeletal muscle. After incubation at the indicated temperature for 20 min, the freshly excised hindlimb muscle from 11-wk-old SD rats was homogenized and fractionated into soluble (S) and insoluble (P) fractions by centrifugation (130,000 g, 40 min). T indicates the total homogenate before centrifugation. Each sample was separated in 12% SDS-PAGE and analyzed by immunoblotting (top). Each membrane was stained with Coomassie brilliant blue (CBB) after immunoblot analysis (bottom).

Figure 7

MKBP enhances the kinase activity of DMPK. (a) Coomassie blue staining of the recombinant proteins used (0.8 μg of His-DMPK [1–541] and 0.4 μg of each sHSP) after separation on 12% SDS-PAGE. (b) In vitro kinase assay using sHSPs as substrates. 50 ng (40 nM) of His-DMPK (1–541) was incubated in kinase assay buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM MgCl_2,, and 0.01% leupeptin) containing 10 μM [γ-³²P]ATP (185 GBq/mmol) for 30 min at 30°C in the presence (second through fourth lanes) or absence (first lane) of sHSPs (1 μg) as indicated at the top. Reactions were stopped by boiling in sample buffer and analyzed by 12% SDS-PAGE and autoradiography. The position of each recombinant protein is indicated. While DMPK autophosphorylation was observed, no sHSPs were phosphorylated. (c) The effect of MKBP on the phosphorylation of MBP by DMPK. 20 μg of MBP were phosphorylated by His-DMPK (1–541) under the same conditions as b in the presence of sHSP or GST. Shown are the results of autoradiography demonstrating the incorporation of ³²P into MBP. The additives and their concentrations are indicated. The data are quantitatively analyzed in d. (e) The effect of MKBP on the phosphorylation of MBP by PKCα. Two micrograms of MBP were phosphorylated by PKCα (10 ng) in kinase buffer containing 0.5 mM CaCl₂, 25 μg/ml phosphatidyl serine, 50 ng/ml TPA, and 10 μM [γ-³²P]ATP (1.85 GBq/mmol) for 10 min at 30°C with various concentrations of MKBP. The data are quantitatively analyzed in f. (g) A Lineweaver-Burk plot derived from the result on MBP dose dependence of ³²P incorporation. The intercept on the vertical axis gives 1/V _max, whereas that on the horizontal axis gives −1/K _m. The estimated K _m values for MBP by DMPK (1–541) are 2.4 and 6.4 mg/ml in the absence and presence of 25 μg/ml MKBP, respectively.

Figure 8

MKBP protects DMPK from heat-induced inactivation. His–DMPK (1–541) or PKCα was incubated at 43°C with or without the indicated additives (50 μg/ml) for 15 min for DMPK and for 11 min for PKCα. After heat treatment, the activity of each kinase was examined by monitoring ³²P incorporation into MBP.

Figure 9

MKBP is specifically upregulated in skeletal muscle from DM patients. Skeletal muscle biopsy samples from normal, DM, and PM adults (6.75 μg/lane) were analyzed by immunoblotting after 12% SDS-PAGE separation. The antibodies used are indicated on the left. Protein amounts in each lane were confirmed to be equal by Coomassie brilliant blue staining of the same blot membrane after immunostaining (data not shown). The results as quantified densitometrically are shown in the lower histogram. Similar results were obtained with additional samples from five DM patients (data not shown).