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Involvement of protein phosphatase-1-mediated MARCKS translocation in myogenic differentiation of embryonic muscle cells

Sang Soo Kim^*, Jung Hwa Kim^*, Seung-Hye Lee, Sung Soo Chung, Ok-Sun Bang, Dongeun Park and Chin Ha Chung

NRL of Protein Biochemistry, School of Biological Sciences, Seoul National University, 56-1 Shinreem-dong, Kwanak-gu, Seoul 151-742, Korea
^* These authors contributed equally to this work

View larger version (34K): [in a new window]   Fig. 1. Effects of protein phosphatase inhibitors on MARCKS translocation and myoblast fusion. Myoblasts were treated with 25 nM okadaic acid (lane O) or 0.5 µM tautomycin (lane T) at the time of medium change, as described in Materials and Methods. The cells that had been cultured for 24 hours in the presence of okadaic acid or tautomycin were freed of the drug by changing the medium (lanes FO and FT, respectively). These cells were further cultured for the next 48 and 24 hours, respectively. Lane C indicates the cells cultured for 72 hours without any treatment. Total cell lysates (A) and membrane fractions (B) were prepared and subjected to SDS-PAGE followed by immunoblot analysis using an anti-MARCKS antibody. The arrows indicate MARCKS. The cells cultured as above were subjected to Giemsa staining to determine the degree of cell fusion (C). Cells containing more than three nuclei were regarded as fused cells.

View larger version (36K): [in a new window]   Fig. 2. Changes in the dephosphorylating activity against 32P-labeled MARCKS during myogenic differentiation. (A) Soluble extracts were prepared from myoblasts that had been cultured for the indicated periods, and aliquots of them (30 µg/lane) were incubated with 32P-labeled MARCKS (0.3 µg) for 30 minutes at 30°C. The extracts from the 72 hour cultured cells were also incubated as above but in the presence of 100 nM okadaic acid (lane 72O) or 5 nM tautomycin (lane 72T). As a control (lane C), 32P-labeled MARCKS was incubated as above but without any addition. The samples were then subjected to SDS-PAGE followed by autoradiography. Note that the rate of dephosphorylation reaction was linear up to 30 minutes when incubated with the extracts of the cells cultured for 48 hours in the absence of okadaic acid. The arrows indicate 32P-labeled MARCKS. (B) The same extracts were subjected to immunoblot analysis using an anti-phospho-MARCKS antibody. The arrowhead shows phospho-MARCKS. (C) The cells were cultured in the absence () and presence of 25 nM okadaic acid () or 0.5 µM tautomycin ([UNK]) for the indicated period, and the degree of cell fusion was determined as in Fig. 1.

View larger version (31K): [in a new window]   Fig. 3. Identification of protein phosphatase responsible for dephosphorylation of MARCKS in cultured myoblasts. (A) Effects of increasing concentrations of tautomycin and okadaic acid on dephosphorylation of 32P-labeled MARCKS. Soluble extracts were prepared from the cells cultured for 48 hours and subjected to the phosphatase assay in the presence of increasing concentrations of tautomycin () and okadaic acid ([UNK]) as described in Materials and Methods. The resulting samples were subjected to SDS-PAGE followed by autoradiography. The bands corresponding to 32P-labeled MARCKS were quantified using a densitometer. The intensity of the band seen without any treatment was expressed as 100% and the others were expressed as relative values. Each of the data points represents mean±s.e. of triplicate determinations. (B) Effect of immunoprecipitation with anti-PP-1C IgG on dephosphorylation of 32P-labeled MARCKS. Soluble extracts were prepared from the cells cultured for 48 hours and incubated with 10 µg of preimmune IgG (lane b) or anti-PP-1C IgG (lane c) at 4°C for 2 hours. After incubation, the samples were treated with protein A-Sepharose and centrifuged. The resulting supernatants were then subjected to the phosphatase activity assay followed by SDS-PAGE and autoradiography. Lane a represents 32P-labeled MARCKS incubated alone. (C) Effects of protein phosphatase inhibitors on dephosphorylation of 32P-labeled MARCKS. Soluble extracts were prepared and assayed for their phosphatase activity as above but in the absence (lane a) or presence of 20 nM protein phosphatase inhibitor-2 (lane b), 100 nM protein phosphatase-2A inhibitor (lane c), or both (lane d). Lane e represents 32P-labeled MARCKS incubated alone. The arrows indicate the phosphorylated MARCKS.

View larger version (34K): [in a new window]   Fig. 4. Changes in the protein level of PP-1C during myogenic differentiation. Soluble extracts were prepared from the cells that had been cultured for the indicated periods. They (30 µg each) were then subjected to immunoblot analysis using an anti-PP-1C antibody. Extracts of A431 cells were used as a control (lane C). The arrow indicates PP-1C.

View larger version (63K): [in a new window]   Fig. 5. Transient overexpression and subcellular localization of MARCKS and its mutant forms in cultured myoblasts. Myoblasts that had been cultured for 24 hours were transfected with the cDNAs for MARCKS (Wt) and its mutant forms (N/S and A2G2) and further incubated for 48 hours in the absence (a) or presence of 100 nM okadaic acid (b). After incubation, total cell lysates were prepared and subjected to immunoblot analysis using anti-MARCKS (A) or anti-Myc antibody (B). Lanes C represent the mock transfected with empty vector. Total cell lysates were also separated into soluble and membranous fractions, which were then subjected to immunoblot analysis using anti-Myc antibody (C). Letters S and P represent the supernatant (soluble) and pelleted (membrane) fractions, respectively. The arrowhead indicates the endogenous MARCKS, and the arrows show the Myc-tagged MARCKS proteins.

View larger version (114K): [in a new window]   Fig. 6. Effect of overexpression of N/S and A2G2 on myoblast fusion and colocalization of MARCKS with filamentous actin. Myoblasts cultured for 24 hours were transfected with the pcDNAs for Myc-tagged, wild-type MARCKS (A-C), N/S (D-F) and A2G2 (G-I). These cells were further cultured for the next 48 hours, followed by immunostaining with anti-Myc antibody and then with FITC-conjugated anti-IgG antibody, as described in Materials and Methods. They were also subjected to rhodamine-phalloidin staining (C,F,I). The middle panels (B,E,H) show the overlapped views of the left and right panels for colocalization of MARCKS and F-actin. The extents of membrane fusion were 82%, 93% and 27% for the cells transfected with the pcDNAs for Myc-tagged wild-type MARCKS, N/S and A2G2, respectively. Magnification, 400x (A-F); 500x (G-I).

View larger version (13K): [in a new window]   Fig. 7. Effects of the N/S and A2G2 mutations on accumulation of creatine kinase. Myoblasts that had been cultured for 24 hours were transfected with recombinant pcDNAs for Myc-tagged wild-type MARCKS, N/S and A2G2. After incubation for 48 hours, the cells were stained with anti-Myc antibody (FITC) and anti-creatine kinase antibody (PE) followed by FACS analysis, as described in Materials and Methods. The level of creatine kinase (CK) in the cells transfected with pcDNA for the wild-type MARCKS was expressed as 100% and the others were expressed as relative values. Each of the data represent the mean±s.e. of triplicate determinations.