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. 2014 Apr 8;53(16):2701–2709. doi: 10.1021/bi5001728

Figure 3.

Figure 3

Autoinhibition of MLCP through Thr696 phosphorylation. (A) Activity of thiophospho-MLCP. Specific activity of untreated T853A MLCP (U-853A) and T853A MLCP thio-phosphorylated at Thr696 (tP-853A) determined using P-MLC20(3–26) at the indicated concentration. Vm and Km values were obtained by curve fitting (n = 3). (B) Interference with autoinhibition by the anti-P-T696-MYPT1 antibody. WT MLCP was preincubated for 90 min at 30 °C under the indicated conditions in the absence or presence of ROCK (100 milliunits) and then subjected to the MLCP assay using 32P-labeled MLC20 (1 μM). The anti-P-T696 antibody was added 30 min prior to the assay (n = 3). (C) Thio-phosphorylation-dependent inhibition of MLCP activity. Untreated and thio-phosphorylated WT, DEE > QQQ (DEE), and RK > QQ (RK) MLCPs were subjected to the MLCP assay using P-MLC20(3–26), and the relative activities against untreated enzymes are shown. p < 0.05 (n = 6). The number indicates the relative extent of thio-phosphorylation of MYPT1, which was determined by immuno-dot blotting using PNBM and the antibody for the alkylated thiophosphate, as described in Experimental Procedures.