In vivo roles for myosin phosphatase targeting subunit-1 phosphorylation sites T694 and T852 in bladder smooth muscle contraction

Abstract

Key points: Force production and maintenance in smooth muscle is largely controlled by myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. MYPT1 is the regulatory subunit of MLCP that biochemically inhibits MLCP activity via T694 or T852 phosphorylation in vitro. Here we separately investigated the contribution of these two phosphorylation sites in bladder smooth muscles by establishing two single point mutation mouse lines, T694A and T852A, and found that phosphorylation of MYPT1 T694, but not T852, mediates force maintenance via inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. Our findings reveal the role of MYPT1 T694/T852 phosphorylation in vivo in regulation of smooth muscle contraction.

Abstract: Force production and maintenance in smooth muscle is largely controlled by different signalling modules that fine tune myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. To investigate the regulation of MLCP activity in vivo, we analysed the role of two phosphorylation sites on MYPT1 (regulatory subunit of MLCP) that biochemically inhibit MLCP activity in vitro. MYPT1 is constitutively phosphorylated at T694 by unidentified kinases in vivo, whereas the T852 site is phosphorylated by RhoA-associated protein kinase (ROCK). We established two mouse lines with alanine substitution of T694 or T852. Isolated bladder smooth muscle from T852A mice displayed no significant changes in RLC phosphorylation or force responses, but force was inhibited with a ROCK inhibitor. In contrast, smooth muscles containing the T694A mutation showed a significant reduction of force along with reduced RLC phosphorylation. The contractile responses of T694A mutant smooth muscle were also independent of ROCK activation. Thus, phosphorylation of MYPT1 T694, but not T852, is a primary mechanism contributing to inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. The constitutive phosphorylation of MYPT1 T694 may provide a mechanism for regulating force maintenance of smooth muscle.

Figure 1

Phosphorylation pattern of MYPT1 at T694 and T852 in bladder smooth muscle Bladder smooth muscle strips from P0 or E18.5 mice were treated with different substances. The resultant muscle samples were subjected to Western blot assay for MYPT1, CPI-17 as well as RLC phosphorylation. A and B, time courses of phosphorylation of MYPT1, RLC and CPI-17 of bladder smooth muscle (from P0 mice) that received stimulation of 124 mm KCl or 200 μm BCh. Total MYPT1, CPI-17 and RLC proteins were used for their corresponding loading control. At least three independent experiments were performed and showed similar results. C, MYPT1 phosphorylation in response to calyculin A (CLA), which causes a maximal increase in MYPT1 phosphorylation by preventing dephosphorylation. Bladder strips from mice (E18.5 or P0) were treated with 1 μm calyculin A for 20 min and then quickly frozen followed by sampling for Western blot analysis. D and H, Western blot analysis showing effects of ROCK and PKC inhibitors on phosphorylation of MYPT1 and CPI-17. Bladder strips from P0 mice were preincubated for 15 min with 10 μm Y27632 or 3 μm GF109203X followed by 124 mm KCl or 30 μm CCh containing Y27632 or GF109203X as indicated. Total actin was used as a loading control. Quantification is shown in E, F, G, I, J and K, and the data are analysed with Student's paired t test; n = 3–5, *P < 0.05, **P < 0.01, ***P < 0.001 (compared with vehicle at the same time), ^†P < 0.05, ^††P < 0.01, ^†††P < 0.001 (compared with value at rest).

Figure 2

Generation of Mypt1 knock-in mutant mice A, schematic representation of Mypt1 knock-in strategy of T694A (top) and T852A (bottom). B, Southern blot assay for positive ES clones with homologous recombination. The extracted genomic DNA digested with BamHI, electrophoresed on a 1% agarose gel, transferred to nitrocellulose and probed with ³²P-labelled 5′ and 3′ probe as indicated in A. The wild-type allele generates a 18 kb fragment while the targeted allele generates a 7.7 kb (T694A) or 12.5 kb (T852A) fragment for the 5′ probe; and a 10.7 kb (T694A) or 7.7 kb (T852A) fragment for the 3′ probe. C, gross appearance of mutant embryos (E18.5). Top panel (left): 100% of T694A heterozygous littermates have the severe omphalocoele phenotype. Top panel (right): magnification of the area containing umbilicus from T694A and control pups. The appearance of T852A homozygotes was varied: mice with severe (50%) and moderate (7.1%) omphalocoele and normal appearance (42.1%) were observed as shown in the bottom panel. Arrows indicate omphalocoele phenotypes. D, PCR products of cDNA containing the T694 site or T852 site from the mutant mice were cut by HincII (T694A) or AccI (T852A) in an expected pattern. The arrows show the bands containing mutation that cannotbe cut by the enzymes. ‘1/2’ indicates half loading. E, sequencing for PCR products from cDNA isolated from T694A heterozygous (T694A/+) mice, T852A homozygous (homo) mice and wild-type (+/+) mice. Correct mutations at T694 and T852 are shown. F, Western blot assay showing disappearance of MYPT1 phosphorylation at the T694 site and T852 site in smooth muscle tissues from Mypt1^T694A/− mice and Mypt1^T852A/− mice, respectively. G, Western blot assay showing the T694A mutation does not affect MYPT1 expression but reduces T694 phosphorylation of MYPT1. β-Actin was used as an internal control. H, RT-PCR for Mypt1 LZ– (with exon 23) and Mypt1 LZ+ (without exon 23) transcripts of bladder tissues from E18.5 mice. Smooth muscle tissues such as aorta and jejunum from adult mouse were used as positive controls which respectively enriched LZ+ and LZ– isoforms.

Figure 3

Morphology and weight of bladders from T694A mice A, gross morphology of bladders from Mypt1^+/+, Mypt1^+/−, Mypt1^T694A/− and Mypt1^T694A/+ mice at E18.5; no obvious alteration was found among these bladders. Scale bars = 1 mm. B, the body weight (left) (n = 24–42), bladder wet weight (middle) (n = 14–27) and bladder to body weight ratio (right) (n = 12–20) did not alter in Mypt1^T694A/+ mice, but slightly decreased in Mypt1^+/− compared to Mypt1^+/+ mice. The T694A mutation significantly rescued the bladder to body weight ratio in Mypt1^+/−. Asterisks, Mypt1^T694A/– vs. Mypt1^+/−; daggers, Mypt1^+/− vs. Mypt1^+/+. ***P < 0.001; ^†††P < 0.001 (Student's t test). C, haematoxylin & eosin-stained sections of bladders showed normal mucosa layer, submucosa and smooth muscle layers comprising the bladder walls among all lines with different genotypes. D, comparable thickness of smooth muscle layers from all four genotypes (Mypt1^+/+, n = 8; Mypt1^+/−, n = 6; Mypt1^T694A/−, n = 4; Mypt1^T694A/+, n = 8). Scale bars = 400 μm.

Figure 4

Morphology and weight of bladders from T852A mice A, gross morphology of bladders from Mypt1^+/+, Mypt1^+/− and Mypt1^T852A/− mice at E18.5. Scale bars = 1 mm. B, quantification of body weight (left), bladder wet weight (middle) and bladder to body weight ratio (right) from T852A mutant and control mice. The T852A mutation slightly rescued bladder weight and bladder to body weight ratio decreased in Mypt1^+/−. Asterisks, Mypt1^T852A/– vs. Mypt1^+/−; daggers, Mypt1^+/– vs. Mypt1^+/+. *P < 0.05, **P < 0.01; ^†††P < 0.001 (Student's t test). C, haematoxylin & eosin-stained sections of bladders showed normal bladder morphology among all lines with different genotypes. D, comparable thickness of smooth muscle layers from Mypt1^+/+, Mypt1^+/− and Mypt1^T852A/− mice (all n = 4). Scale bars = 400 μm.

Figure 5

Expression of contractile and regulatory proteins in MYPT1 mutant bladder Bladder smooth muscle samples were prepared from E18.5 mice and subjected to SDS-PAGE (A and D) and Western blot assay (B and E). There were no apparent differences in the whole protein expression patterns, including the contraction-related proteins myosin heavy chain (MHC), caldesmon (CaD), RLC and 17 kDa essential light chain (LC17), between T694A mutant mice and control mice (A) and T852A mutant mice and control mice (D). Western blot assays (B and E) and quantitative results (C and F) are shown for contractile and regulatory proteins for bladder tissue from Mypt1^T694A/− (B and C) and Mypt1^T852A/− (E and E) and their control bladders. Total actin stained with Coomassie brilliant blue was used as a loading control and the values are expressed relative to those obtained in tissues from Mypt1^+/−; n = 3–4, P > 0.05.

Figure 6

Bladder smooth muscle contraction of MYPT1 T694A mutants evoked by KCl and CCh Bladder smooth muscle strips from E18.5 mice were isolated and subjected to force measurement responding to different stimuli. A and C, representative force traces evoked by depolarization or 30 μm CCh in Mypt1^T694A/− and Mypt1^+/− bladder smooth muscles. B and D, maximal force evoked by KCl or CCh in bladder smooth muscle from E18.5 mice of Mypt1^T694A/− (KCl, n = 10; CCh, n = 6), Mypt1^+/– (KCl, n = 16; CCh, n = 10), Mypt1^T694A/+ (KCl, n = 9; CCh, n = 7) and Mypt1^+/+ (KCl, n = 10; CCh, n = 12). All P > 0.05. E and F, quantification of dynamic alteration of sustained force by KCl or 30 μm CCh in bladder from mutant groups (Mypt1^T694A/− and Mypt1^T694A/+) compared to their corresponding control groups (Mypt1^+/– and Mypt1^+/+) at the indicated time points after stimulation. The values are represented as percentages of the peak force. Asterisks, Mypt1^T694A/− vs. Mypt1^+/−; daggers, Mypt1^T694A/+ vs. Mypt1^+/+. *P < 0.05, **P < 0.01, ***P < 0.001; ^†P < 0.05, ^††P < 0.01, ^†††P < 0.001 (Student's t test); n values are identical to those of B and D. G, dose–response effects of CCh on sustained force development of bladders from Mypt1^T694A/−, Mypt1^T694A/+, Mypt1^+/– and Mypt1^+/+ mice. Values are expressed as percentages of the maximal force by 100 μm CCh at 5 min. Asterisks, Mypt1^T694A/− vs. Mypt1^+/−; n = 3–7, *P < 0.05, **P < 0.01.

Figure 7

MYPT1 T694A mutation decreases RLC phosphorylation in bladder tissue A and C, after stimulation with 124 mm KCl, bladder muscles were quickly frozen for sample preparation. Phosphorylated RLC (p-RLC) was measured by Western blot analysis of glycerol/urea PAGE gels. RLC, non-phosphorylated; p-RLC, monophosphorylated. B, quantification of A shows a decreased RLC phosphorylation in mutant muscle after 3 min compared to control muscle; n = 7–9, *P < 0.05 (Student's t test). The p-RLC level was expressed as the percentage of the total RLC. D, quantification of C shows lower level of phosphorylation of RLC in Mypt1^T694A/+ smooth muscle at 1 min and 3 min after KCl treatment compared to Mypt1^+/+ but comparable at 10 s and 20 s; n = 6–13, *P < 0.05, **P < 0.01 (Student's t test). E, representative force tracing of bladder strips evoked by 1 μm calyculin A (CLA). F, quantification of calyculin A-induced contraction. Values are expressed as percentages of the maximal force by 124 mm KCl; n = 3–4, P > 0.05.

Figure 8

MYPT1 T852A mutation slightly decreases sustained phase in bladder A and C, representative force traces evoked by depolarization or 30 μm CCh in Mypt1^T852A/− and Mypt1^+/− bladder smooth muscles from E18.5 mice. B and D, maximal force evoked by depolarization or 30 μm CCh in Mypt1^T852A/− and Mypt1^+/− bladder smooth muscle (n = 8–10). E and F, quantification of the sustained force in Mypt1^T852A/−, Mypt1^+/−, Mypt1^+/+ and Mypt1^T852A/T852A smooth muscle by KCl or 30 μm CCh. Consistently, bladder smooth muscle slightly reduced sustained contraction evoked by KCl (daggers, Mypt1^T852A/T852A vs. Mypt1^+/+; ^††P < 0.01, ^†P < 0.05). Values are as of the peak force. G, dose–response effect of CCh on contraction of bladders from different genotypes of mice at E18.5. Values of the sustained force evoked by CCh at 5 min are expressed as percentages of the maximal force at 100 μm CCh. H and I, quantification of the sustained phase evoked by KCl or 200 μm BCh in bladders from Mypt1^T852A/T852A and Mypt1^+/+ mice (P0). Data were from six measurements and each from different bladders. Mypt1^T852A/T852A vs. Mypt1^+/+, ^†P < 0.05 (Student's t test). J, quantification of phosphorylation of RLC for bladder muscles from Mypt1^T852A/− and Mypt1^+/− mice (E18.5) in response to 124 mm KCl at 15 s and thereafter. The p-RLC level was expressed as the percentage of the total RLC; n = 4–7, P > 0.05.

Figure 9

The MYPT1 T694A mutation does not confer resistance to force reduction by the ROCK inhibitor A and D, representative force traces evoked by KCl (A) or CCh (D) in the presence of ROCK inhibitor Y27632 (A) or H1152 (D) or vehicle in Mypt1^T694A/− and Mypt1^+/− bladder smooth muscles. B, C, E and F, quantitative summary of the effects of Y27632 (10 μm) (B and C) and H1152 (0.1 μm) (E and F) on peak (B and E) and sustained (5 min) (C and F) force evoked by KCl (B and C) or 30 μm CCh (E and F) in bladders from T694A mutant mice and control mice (E18.5). Force values are expressed relative to the peak force recorded in the presence of KCl (B and C) or CCh (E and F) (line at 100%). *P < 0.05, **P < 0.01, ***P < 0.001 (Student's paired t test).

Figure 10

The MYPT1 T852A mutation does not confer resistance to force reduction by the ROCK inhibitor A, D and G, representative force traces evoked by KCl (A) or 10 μm CCh (D and G) in the presence of ROCK inhibitor Y27632 (A), H1152 (D and G) or vehicle in T852A mutant and control bladder smooth muscles from E18.5 (A) or adult mice (D and G). B, E, C, F, H and I, quantitative summary of the effects of Y27632 (10 μm) (B and C) and H1152 (0.1 μm) (E, F, H and I) on peak (B, E and H) and sustained (5 min) (C, F and I) tension evoked by KCl (B, C, E and F) or CCh (H and I) in bladders from T852A mutant mice and control mice. Force values are expressed relative to the peak force recorded in the presence of KCl (B, C, E and F) or CCh (H and I) (line at 100%). *P < 0.05, **P < 0.01, ***P < 0.001 (Student's paired t test).

Figure 11

Depolarization-induced cofilin phosphorylation was attenuated by ROCK inhibitor Y-27632 A, bladder strips from P0 mice were preincubated for 15 min with 10 μm Y27632 followed by 124 mm KCl containing Y27632 as indicated. Cofilin phosphorylation was analysed by Western blot assay, and total cofilin was used for loading control. B, quantification of the data and analysed with Student's paired t test; n = 5, *P < 0.05 (compared with vehicle at the same time). C, Western blot analysis shows preincubation with Y27632 attenuates cofilin phosphorylation both in T694A mutant and control bladder strips at 5 min after 124 mm KCl stimulation.