This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 24/3/464    most recent 01.ATV.0000116028.42230.4cv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hirano, K. Articles by Kanaide, H. Search for Related Content PubMed PubMed Citation Articles by Hirano, K. Articles by Kanaide, H. Related Collections Other Vascular biology

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2004;24:464.)
© 2004 American Heart Association, Inc.

Vascular Biology

Transduction of the N-Terminal Fragments of MYPT1 Enhances Myofilament Ca²⁺ Sensitivity in an Intact Coronary Artery

Katsuya Hirano; Dmitry N. Derkach; Mayumi Hirano; Junji Nishimura; Shosuke Takahashi; Hideo Kanaide

From the Division of Molecular Cardiology, Research Institute of Angiocardiology (K.H., D.N.D., M.H., J.N., H.K.), the Department of Anesthesia and Critical Care Medicine (S.T.), and the Kyushu University COE Program on Lifestyle-Related Diseases (H.K.), Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

Correspondence to Dr Hideo Kanaide, Professor, Division of Molecular Cardiology, Research Institute of Angiocardiology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail kanaide{at}molcar.med.kyushu-u.ac.jp

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Objective— The region of the 110 kDa regulatory subunit (MYPT1) of smooth muscle myosin phosphatase involved in the regulation of contraction was determined under physiological conditions.

Methods and Results— Using HIV Tat protein-mediated protein transduction, the N-terminal fragments of MYPT1 were introduced to the intact porcine coronary arterial strips. Pre-incubation with 3 µmol/L TAT-MYPT1^1–374, a construct containing the Tat peptide and the residues 1 to 374 of MYPT1, for 15 minutes augmented (2.4-fold) the subsequent contraction induced by adding 1.25 mmol/L of extracellular Ca²⁺ under 118 mmol/L K⁺ depolarization, with no augmentation of the [Ca²⁺]_i elevation. The deletion of the Tat peptide, MYPT1^1–374, abolished the augmenting effect. TAT-MYPT1^1–296 demonstrated a weaker but significant augmentation (1.7-fold). However, TAT-MYPT1^1–171, TAT-MYPT1^39–374, TAT-MYPT1^39–296, and TAT-MYPT1^297–374 had no augmenting activity. The myosin light chain phosphorylation level as a function of extracellular Ca²⁺ concentrations was shifted to the left in the strips pretreated with TAT-MYPT1^1–374 compared with the control.

Conclusions— Region 1 to 296 was the minimal region involved in the enhancement of contraction, and region 297 to 374 played a supplemental role. These results suggested that the interaction mainly between catalytic subunit and MYPT1 play a critical role in the regulation of the endogenous myosin phosphatase in intact smooth muscle.

Key Words: myosin • phosphatase • smooth muscle • contraction • protein transduction

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
The increase in the vascular tone plays an important role in the pathophysiology of the vascular diseases such as hypertension and vasospasm. The tone of the vascular smooth muscle is primarily regulated by Ca²⁺-dependent reversible phosphorylation of the 20 kDa myosin light chain (MLC).^1–3 The enhanced MLC phosphorylation for a given level of Ca²⁺ enhances the myofilament Ca²⁺ sensitivity, and thus increases vascular tone. The level of MLC phosphorylation is determined by the balance between the activities of MLC kinase (MLCK) and MLC phosphatase (MLCP).^1,4 Because the activity of MLCK is primarily determined by the Ca²⁺ level, the decrease in the MLCP activity is considered to play a key role in the enhancement of the Ca²⁺ sensitivity.^3,4 MLCP is composed of three subunits: the 38 kDa catalytic subunit (PP1c), the 110 kDa regulatory myosin phosphatase target subunit (MYPT1), and the 20 kDa regulatory subunit (M20).^4–9 MYPT1 plays an important role in targeting MLCP to myosin filaments, enhancing substrate specificity toward myosin, and regulating enzymatic activity.⁴ The phosphorylation of T696 (in human MYPT1) was shown to be associated with an inhibition of the MLCP activity, and is thus considered to be one of the important mechanisms for the regulation of the MLCP activity.⁴ This phosphorylation is catalyzed by several kinases including Rho kinase, Zip-like kinase, and myotonic dystrophy protein kinase and integrin-linked kinase.^10–13

Primarily, the interaction between PP1c and MYPT1 increases the activity of PP1c toward MLC while inhibiting the activity toward phosphorylase.^14–16 The enhancement of the activity toward MLC requires the N-terminal part of MYPT1.¹⁵ It is conceivable that the increased activity toward MLC can be attributed to the interactions among the N-terminal part of MYPT1, PP1c, substrate MLC, and myosin filament. The N-terminal 38 residues, especially the PP1c-binding motif (K/R-V/I-x-F),¹⁷ and the C-terminal half of the ankyrin repeats domain play a critical role in binding to PP1c.^14,15 The phosphorylated MLC was shown to bind to the C-terminal half of the ankyrin repeats.¹⁵ Both N-terminal⁷ and C-terminal¹⁸ region of MYPT1 have been shown to bind to myosin. The essential region of MYPT1 for myosin binding is thus still controversial. The requirement of an acidic cluster to obtain an increased PP1c activity toward MLC remains controversial.^15,16 Nevertheless, precisely which interaction plays an important role in the regulation of MLCP, and therefore in MLC phosphorylation and smooth muscle contraction, under physiological conditions remains to be elucidated.

It was reported that the covalent conjugation of protein transduction domain (PTD) of HIV transcription factor Tat¹⁹ allows the conjugated protein to enter the cells with intact plasma membrane,^20–22 in a quantitative manner within 10 minutes.²³ This technique is thus anticipated to be a powerful tool to investigate the physiological role of the functional proteins under physiological conditions. In the present study, using the Tat PTD-mediated protein transduction technique, we introduced the N-terminal fragments of MYPT1 into the intact smooth muscle of the isolated arterial strips and thus determined which part of the MYPT1 is important in the regulation of MLCP under physiological conditions.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
The N-terminal fragments of MYPT1 (Figure I, available online at http://atvb.ahajournals.org) were introduced into porcine coronary artery medial strips as fusion proteins tagged with Tat PTD at their N-terminus. The recombinant proteins were expressed in bacteria and purified with a Ni²⁺-loaded Hi-Trap chelating column (Pharmacia Biotech, Tokyo, Japan). The arterial strips without endothelium were prepared as previously described.^24,25 The effects of N-terminal fragments of MYPT1 on [Ca²⁺]_i, isometric force, and MLC phosphorylation were examined as previously described.^24–27 All data were expressed as the mean±SD. The unpaired Student t test and an analysis of variance (ANOVA) evaluated statistical significance; P<0.05 were considered significant.

See Methods (available online at http://atvb.ahajournals.org) for expanded information.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Transduction of Tat PTD-Tagged Recombinant Proteins Into Intact Strips of Porcine Coronary Artery
The Tat PTD-mediated transduction of protein into the intact arterial strips was verified as shown in online supplementary Figures II and III (available online at http://atvb.ahajournals.org). First, the transduction of green fluorescence protein (GFP) was observed under a confocal microscopy, which demonstrated a specific fluorescence with a pattern of smooth muscle fibers similar to that observed with the fura-2 fluorescence in the fura-2-loaded strips, as we previously reported.²⁸ Furthermore, the transduction of all Tat PTD-tagged MYPT1 fragments was detected by immunoblot analysis using anti-(His)₆ antibody. In both experiments, transduction of the proteins without PTD was not detected.

Enhancement of the Ca²⁺-Induced Contraction by Transduction of the N-Terminal Fragments of MYPT1 in the Intact Strips
The strips were stimulated with 118 mmol/L K⁺ in the presence of 1.25 mmol/L extracellular Ca²⁺, and the contractile response was recorded as a reference response (Figure 1a). The strips were then exposed to the Ca²⁺-free PSS with and then without 2 mmol/L EGTA for 5 minutes each and stimulated with 118 mmol/L K⁺ in the Ca²⁺-free PSS for 15 minutes. The stimulation with 118 mmol/L K⁺ in the absence of extracellular Ca²⁺ did not induce any contraction, whereas the subsequent addition of extracellular Ca²⁺ induced a contraction (Figure 1a). In the vehicle-treated strips, the tension (111.7%±9.2%, n=9) obtained by the addition of 1.25 mmol/L of Ca²⁺ was similar (P>0.05) to the reference response. Pretreatment with 3 µmol/L TAT-MYPT1^1–374 during the 15-minute stimulation with 118 mmol/L K⁺ in the Ca²⁺-free PSS augmented the subsequent contraction (Figure 1b). This effect of TAT-MYPT1^1–374 was reversible and reproducible (Figure 1e). Tension induced by replenishing 1.25 mmol/L Ca²⁺ was 267.4%±22.1% (n=10) in the strips treated with 3 µmol/L TAT-MYPT1.^1–374 The protein was removed from the bathing solution when the extracellular Ca²⁺ was added, because the continuous presence of elution buffer at 10-fold dilution, regardless of the presence or absence of the proteins, inhibited the tension development (data not shown). Imidazole in the elution buffer was considered to be responsible for inhibition of contractility. However, removal of imidazole by dialysis caused precipitation of the protein. We thus used this experimental protocol to examine the effect of TAT-proteins on the contraction. Pretreatment with MYPT1^1–374 without Tat PTD demonstrated no significant augmenting effect (Figure 1c). Pretreatment with 3 µmol/L TAT-GFP did not have any augmenting effect on the contraction (Figure 1d). The simultaneous measurement of [Ca²⁺]_i and tension revealed that the augmentation of tension by TAT-MYPT1^1–374 was not associated with the augmentation of [Ca²⁺]_i elevation (Figure 2). The replenishment of 1.25 mmol/L Ca²⁺ produced a similar [Ca²⁺]_i elevation to the reference response in TAT-MYPT1^1–374-treated and vehicle-treated strips (Figure 2c).

View larger version (29K): [in this window] [in a new window]   Figure 1. Enhancement of contraction by the transduction of the N-terminal fragment of MYPT1 in the intact medial strips of the porcine coronary artery. Representative traces of tension development in the strips treated with the elution buffer (vehicle) (a), 3 µmol/L TAT-MYPT11–374 (b, e), 3 µmol/L MYPT11–374 (c), and 3 µmol/L TAT-GFP (d). The strips were contracted with 118 mmol/L K+ in the 1.25 mmol/L Ca2+-containing PSS. They were then exposed to the Ca2+-free PSS with 2 mmol/L EGTA for 5 minutes and without EGTA for 5 minutes. The strips were then stimulated with 118 mmol/L K+ in the Ca2+-free PSS, and the contraction was initiated by replenishing 1.25 mmol/L extracellular Ca2+. The proteins were added during the 15 minutes of stimulation with 118 mmol/L K+ in the Ca2+-free PSS and removed when the extracellular Ca2+ was replenished. This protocol was repeated (e) to demonstrate the reversibility and reproducibility of the effect of TAT-MYPT1.1–374

View larger version (26K): [in this window] [in a new window]   Figure 2. Effect of TAT-MYPT11–374 on the [Ca2+]i elevation and tension development in intact medial strips of the porcine coronary artery. Shown are the representative recording of [Ca2+]i and tension in the strips treated with TAT-MYPT11–374 (a) and vehicle (b). The contraction was initiated by the same protocol as that shown in Figure 1. C, Summary of [Ca2+]i and tension obtained at the peak of the contraction. The levels of [Ca2+]i and tension obtained in the normal PSS (5.9 mmol/L K+, 1.25 mmol/L Ca2+) and those obtained with the first contraction induced by 118 mmol/L K+ were assigned to be 0% and 100%, respectively. The resting level of [Ca2+]i is indicated by the broken lines. Data are the mean±SD (n=3). *Significantly different (P<0.05); NS indicates not significantly different.

The augmentation of the contraction by TAT-MYPT1^1–374 was dependent on the incubation time and the concentration, as shown in online supplementary Figure IV (available online at http://atvb.ahajournals.org). The level of tension at rest (in the normal PSS containing 5.9 mmol/L K⁺, 1.25 mmol/L Ca²⁺) and that obtained with the first contraction induced by 118 mmol/L K⁺ was assigned to be 0% and 100%, respectively. In control experiment with buffer alone, the addition of 1.25 mmol/L Ca²⁺ during 118 mmol/L K⁺ depolarization induced a similar contraction to that seen with the first reference contraction. A significant enhancement (P<0.05) was obtained after 5-minute incubation, while reaching a maximal effect within 10 to 15 minutes of incubation (Figure IVa). The incubation time required to obtain a significant enhancement of contraction was similar to that required for the intracellular transduction of TAT-GFP, as observed under fluorescence microscopy (Figure II). However, the significant enhancement of the Ca²⁺-induced contraction required TAT-MYPT1^1–374 concentrations of 3 µmol/L and higher (Figure IVb).

Determination of the MYPT1^1–374 Region Required for the Enhancement of Ca²⁺-Induced Contraction
The effect of various fragments of MYPT1 on the Ca²⁺-induced contraction was examined to determine the essential region for the enhancement of contraction (Figure 3). We compared the effects of truncation mutants at a fixed concentration (3 µmol/L) and at incubation time (15 minutes). TAT-MYPT1^1–296, which lacks a region containing an acidic cluster, still enhanced the Ca²⁺-induced contraction (Figure 3a). The extent of tension obtained with 1.25 mmol/L Ca²⁺ (183.4%±11.0%, n=9) was significantly smaller than that obtained with TAT-MYPT1^1–374. TAT-MYPT1^1–171, which lacks a C-terminal half of the ankyrin repeats, had no significant effect (Figure 3b), and the extent of tension obtained with 1.25 mmol/L Ca²⁺ was 99.4%±10.4% (n=9). The deletion of N-terminal 38 residues containing the PP1c-binding motif, TAT-MYPT1^39–374 and TAT-MYPT1^39–296, abolished the augmenting effects of TAT-MYPT^1–374 and TAT-MYPT1^1–296, respectively (Figure 3c and 3d). However, TAT-MYPT1^297–374, containing the acidic cluster but not the N-terminal ankyrin repeats, did not enhance the Ca²⁺-induced contraction (Figure 3e). As a result, region 1 to 296 was the minimal sequence required for augmentation of the Ca²⁺-induced contraction in the intact tissue.

View larger version (27K): [in this window] [in a new window]   Figure 3. Effects of truncation mutants of MYPT1 on the Ca2+-induced contraction in intact medial strips of the porcine coronary artery. Representative traces of tension development observed in the strips treated with 3 µmol/L TAT-MYPT11–296 (a), 3 µmol/L TAT-MYPT11–171 (b), 3 µmol/L TAT-MYPT139–374 (c), 3 µmol/L TAT-MYPT139–296 (d), and 3 µmol/L TAT-MYPT1297–374 (e). The experimental protocol was the same as that shown in Figure 1. Summary of the effects of various constructs of MYPT1 (f) on the contraction induced by replenishing 0.125 mmol/L, 0.25 mmol/L, 0.5 mmol/L, and 1.25 mmol/L Ca2+ after the treatment with 3 µmol/L proteins as indicated in the key or vehicle (control). The level of tension obtained in the normal PSS (5.9 mmol/L K+, 1.25 mmol/L Ca2+) and that obtained with the first contraction induced by 118 mmol/L K+ were assumed to be 0% and 100%, respectively. Data are the mean±SD (n=7).

The effects of the MYPT1 fragments on the Ca²⁺-induced contraction were quantitatively evaluated by inducing contraction with various concentration of Ca²⁺ (Figure 3f). In the vehicle-treated strips, a significant contraction developed by adding 0.125 mmol/L and higher concentrations of Ca²⁺, and the extent of tension obtained with 1.25 mmol/L of Ca²⁺ was similar to the reference response. TAT-MYPT1^1–374 augmented the contraction seen with 0.125 mmol/L of Ca²⁺ and higher concentrations. TAT-MYPT1^1–296 demonstrated a weaker enhancement, while the other constructs, MYPT1^1–374, TAT-MYPT1^1–171, TAT-MYPT^297–374, TAT-MYPT^39–374, and TAT-MYPT1^39–296 did not demonstrate significant enhancement (Figure 3f).

The correlation between in vivo effect of MYPT1 fragments on the contraction and the in vitro effect on the activity of the isolated MLCP holoenzyme was studied. TAT-MYPT1^1–374 significantly inhibited the activity of the isolated MCLP toward MLC, whereas TAT-MYPT1^1–171 and TAT-GFP also demonstrated an inhibitory effect (data not shown). However, the inhibitory effect seen with TAT-MYPT1^1–374 was greater than that seen with TAT-MYPT1^1–171 and TAT-GFP.

Enhancement of MLC Phosphorylation by TAT-MYPT1^1–374
The effect of TAT-MYPT1^1–374 transduction on the MLC phosphorylation was examined under the same experimental conditions as those used in the tension measurements. The resting level of MLC phosphorylation (21±1.8%, n=7) obtained in the normal PSS (5.9 mmol/L K⁺, 1.25 mmol/L Ca²⁺) decreased significantly (P<0.05) to 7.3±3.9% (n=7) after exposing the strips to the Ca²⁺-free PSS and just before replenishing the extracellular Ca²⁺ in the vehicle-treated strips (Figure 4a and 4b). The subsequent Ca²⁺ replenishment induced a concentration-dependent increase in MLC phosphorylation, as determined at the peak of contraction (3 to 4 minutes after initiation of contraction) (Figure 4a and 4b). The level of MLC phosphorylation obtained with replenishment of 1.25 mmol/L Ca²⁺ was 43.8±5.5% (n=7), and this value was similar to that obtained with the reference contraction (41±2.7%, n=7). However, when the strips were pretreated with 3 µmol/L TAT-MYPT1,^1–374 the level of MLC phosphorylation obtained just before the Ca²⁺ replenishment did not significantly differ from that obtained in the control strips. The MLC phosphorylation levels obtained with 0.125 mmol/L and 0.25 mmol/L Ca²⁺ were significantly higher than the control levels, whereas the MLC phosphorylation obtained with 0.5 mmol/L and 1.25 mmol/L Ca²⁺ did not significantly differ from the control. The analysis at the earlier time point also revealed that the phosphorylation level obtained with 0.5 mmol/L and 1.25 mmol/L Ca²⁺ in the TAT-MYPT1^1–374-treated strips was similar to the control (data not shown). The concentration-dependent increases in MLC phosphorylation as a function of the extracellular Ca²⁺ obtained in the TAT-MYPT1^1–374-treated strips shifted to the left of that obtained in the vehicle-treated strips (Figure 4b). The tension obtained with 0.5 mmol/L and 1.25 mmol/L Ca²⁺ in the TAT-MYPT1^1–374-treated strips was thus not associated with an increase in MLC phosphorylation.

View larger version (28K): [in this window] [in a new window]   Figure 4. Enhancement of MLC phosphorylation by TAT-MYPT11–374 in intact medial strips of the porcine coronary artery. Representative photos (a) showing MLC immunoblot analysis in the strips treated with 3 µmol/L TAT-MYPT11–374 and vehicle (control). The samples were obtained at the peak of contraction during the tension measurement with the experimental protocol the same as in Figure 1, except for the fact that the contraction was initiated by replenishing 0 mmol/L (just before replenishing Ca2+), 0.125 mmol/L, 0.25 mmol/L, 0.5 mmol/L, and 1.25 mmol/L Ca2+. The resting level (resting) was determined in the normal PSS (5.9 mmol/L K+, 1.25 mmol/L Ca2+) before incubation with proteins. MLC indicates unphosphorylated MLC; MLC-P, monophosphorylated MLC. The extent of MLC phosphorylation obtained at the peak of contraction (b) induced by replenishment of different concentrations of Ca2+ in the strips treated with 3 µmol/L TAT-MYPT11–374 and the vehicle (control). *P<0.05; NS indicates not significantly different. Data are the mean±SD (n=7).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We herein demonstrated that Tat PTD-mediated protein transduction was successfully applied to intact arterial strips. It was reported earlier that Clostridium botulinum exoenzyme C3 tagged with Tat PTD inhibited the urotensin-induced contraction in the rat aorta.²⁹ In the present study, the introduction of the protein into the cells by Tat PTD was clearly proved by the observation of GFP fluorescence in the TAT-GFP-treated strips, and by immunoblot detection of Tat PTD-tagged MYPT1 fragments in the extract of the strips. The introduction of protein is also supported by the observation that the MYPT1 fragments enhanced the Ca²⁺-induced contraction only when tagged with Tat PTD. The time needed to obtain a significant enhancement of contraction suggested that the transduction of functional protein into the intact arterial strips takes place within 10 minutes, which is consistent with previous reports.^22,23

Treatment with TAT-MYPT1^1–374 enhanced the Ca²⁺-induced contraction with no effect on the [Ca²⁺]_i elevation, suggesting that the Ca²⁺ sensitivity of the contractile apparatus was potentiated. The following two control experiments excluded the possibilities that TAT-MYPT1^1–374 acted from outside of the cells and that Tat PTD per se enhanced the contraction. First, the deletion of Tat PTD abolished the augmenting effect of TAT-MYPT1^1–374 (Figure 1b). Second, TAT-GFP and some of the truncation mutants of MYPT1 had no effect on the contraction (Figures 1d and 3). We thus conclude that MYPT1^1–374 was introduced into the cells and enhanced the myofilament Ca²⁺ sensitivity in the intact muscle. This observation is consistent with previous observations of Triton X-100–permeabilized muscle.²⁶ However, the enhanced contraction seen in the strips pretreated with TAT-MYPT1 fragments was not sustained. According to the experimental protocol, the strips were no longer exposed to the TAT-MYPT1 fragments during the contraction initiated by replenishing extracellular Ca²⁺. The decline of the contraction may reflect the leak of the transduced protein from the strips. Indeed, the augmenting effect seen with TAT-MYPT1 fragments was reversible and reproducible (Figure 1e).

The enhancement of Ca²⁺ sensitivity by TAT-MYPT1^1–374 was associated with an increase in MLC phosphorylation, especially in the contractions induced by replenishing Ca²⁺ at levels less than 0.5 mmol/L. The Ca²⁺–MLC phosphorylation curve was thus shifted to the left (Figure 4). The level of MLC phosphorylation is determined mainly by the balance between MLCK activity and MLCP activity.⁴ It is conceivable that this balance shifted to the kinase-dominated state by the introduction of TAT-MYPT1^1–374, namely either the MLCP activity decreased or the MLCK activity increased. The concentration of intracellular TAT-MYPT1^1–374 was estimated to be in µmol/L order by quantitative immunoblot analysis with a purified recombinant protein as reference (data not shown), and this estimation is consistent with the concentration of endogenous MYPT1 (3 µmol/L) as reported in the literature.⁴ We thus suggest that the introduced TAT-MYPT1^1–374 served as a dominant negative mutant against endogenous MYPT1 and inhibited the MLCP activity. The dominant negative effect of TAT-MYPT1^1–374 may be supported by its inhibitory effect on the isolated MLCP. However, in vitro phosphatase assay also demonstrated a lesser inhibitory effect of TAT-MYPT1^1–171 and TAT-GFP on MLCP. We thus suggest that the in vitro effect of TAT-MYPT1^1–171 and TAT-GFP was insufficient to exert an inhibitory effect in vivo, while the additional inhibition seen with TAT-MYPT1^1–374 correlated to the dominant negative effect seen in vivo.

However, the contraction induced by replenishing 0.5 mmol/L and higher concentrations of Ca²⁺ was not associated with an increase in MLC phosphorylation (Figure 4). This enhancement of contraction was thus suggested to be independent of MLC phosphorylation. The Ca²⁺ sensitivity of the contractile apparatus can be regulated in a manner either dependent on or independent of MLC phosphorylation.^30–34 It is thus possible that the transduced MYPT1 might modulate such MLC phosphorylation-independent mechanism and thereby enhance the Ca²⁺ sensitivity. The precise mechanism for enhancement of contraction seen with high Ca²⁺ concentrations remains to be determined.

The present study suggests that region 1 to 296 is the minimal sequence required for a significant enhancement of Ca²⁺ sensitivity in intact artery. Region 1 to 296 contains PP1c-binding motif and ankyrin repeats,^4,14,15,17 and the C-terminal half of the ankyrin repeats are involved in PP1c binding and substrate binding.¹⁵ It is thus suggested that the introduced MYPT1 fragments primarily impaired the endogenous interaction between PP1c and MYPT1 mediated by region 1 to 296 and inhibited the endogenous MLCP. A dissociation of PP1c from the holoenzyme was reported to play an important role in inhibiting the myosin phosphatase activity.^35,36 However, the augmenting effect of TAT-MYPT1 fragments was demonstrated to be reversible and reproducible (Figure 1e). It is thus unlikely that exogenous MYPT1 fragments caused substantial dissociation of PP1c from the holoenzyme and thereby inhibited the myosin phosphatase activity. MYPT1 fragments are rather suggested to cause subtle changes in protein interactions involved in the regulation of MLCP activity.

Both the present study and our previous study²⁶ demonstrated MYPT1^1–374 to enhance contraction. However, there is a discrepancy in the region of MYPT1^1–374 that played an essential role in enhancing contraction between two studies. In the previous study, the Triton X-100–permeabilized strips of the porcine interlobular renal artery were used, and the recombinant proteins tagged with (His)₆ were introduced. The analysis of the various truncated fragments determined region 304 to 374 to be necessary and sufficient for enhancement of contraction, thus suggesting this region to play an essential role in regulation of endogenous MLCP in the Triton X-100–permeabilized strips. The region 1 to 296 played no significant role in the permeabilized strips. However, the present study found that region 1 to 296 was necessary for enhancing the contraction and thus played an essential role in regulating endogenous MLCP, whereas region 297 to 374 played supplemental role. As a result, the relative importance of two regions 1 to 296 and 297 to 374 within MYPT1^1–374 seems to differ with experimental conditions. Both regions play an important role in intact strips, although region 1 to 296 is essential. However, in the permeabilized preparation, region 297 to 374 plays an essential role. One possible explanation for this discrepancy is the effect of Triton X-100 on the protein interaction. The region 1 to 296 contains the ankyrin repeats domain,⁷ which is predicted to form a protein-interacting platform consisting of repeated -helixes and ß-turns.³⁷ The region 297 to 374 contains an acidic cluster and is thus considered to mediate ionic interaction. Triton X-100, a nonionic detergent, may disturb some of the nonionic interactions. We thus speculate that the interaction mediated by region 1 to 296 is more susceptible to Triton X-100 than is the interaction mediated by the acidic cluster; therefore, the functional importance of the acidic cluster may be magnified in the permeabilized muscle. However, this possibility remains to be examined.

In conclusion, we herein demonstrate that the introduction of the N-terminal fragment of MYPT1 using Tat PTD enhanced the myofilament Ca²⁺ sensitivity by primarily inhibiting the endogenous MLCP activity toward MLC and possibly other substrates. The protein interactions mediated by region 1 to 296 of MTPT1, such as the interaction between PP1c and MPYT1, are thus suggested to play a critical role in the regulation of MLCP in intact muscle. The interactions mediated by the acidic cluster are thus suggested to play a supplemental role under physiological conditions. We therefore propose that Tat-mediated protein transduction is a powerful tool for investigating the physiological role of functional protein in the cells with an intact plasma membrane.

	Acknowledgments

 
Acknowledgments

We thank Brian Quinn for linguistic comments and help with the manuscript, and Dr David J. Hartshorne (University of Arizona) for his help with in vitro phosphatase assay. This study was supported in part by the 21^st Century COE Program and grants-in-aid for Scientific Research (no. 13470149, 14657174, 14570675, and 15590758) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, by the Research Grant for Cardiovascular Diseases (13C-4) from the Ministry of Health, Labor, and Welfare, Japan, and by grants from the Japan Space Forum and the Naito Foundation.

	Footnotes

 
Dr Hirano and Dr Derkach contributed equally to this study.

Received November 18, 2003; accepted December 22, 2003.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Hartshorne DJ. Biochemistry of the contractile process in smooth muscle. In: Johnson LR, ed. Physiology of the Gastrointestinal Tract, second ed. New York: Raven Press; 1987: 423–482.
2. Kamm KE, Stull JT. Regulation of smooth muscle contractile elements by second messengers. Ann Rev Physiol. 1989; 51: 299–313.[CrossRef][Medline] [Order article via Infotrieve]
3. Somlyo AP, Somlyo AV. Signal transduction and regulation in smooth muscle. Nature. 1994; 372: 231–236.[CrossRef][Medline] [Order article via Infotrieve]
4. Hartshorne DJ, Ito M, Erdodi F. Myosin light chain phosphatase: subunit composition, interactions and regulation. J Muscle Res Cell Motil. 1998; 19: 325–341.[CrossRef][Medline] [Order article via Infotrieve]
5. Alessi D, MacDougall LK, Sola MM, Ikebe M, Cohen P. The control of protein phosphatase-1 by targeting subunits. The major myosin phosphatase in avian smooth muscle is a novel form of protein phosphatase-1. Eur J Biochem. 1992; 210: 1023–1035.[Medline] [Order article via Infotrieve]
6. Chen YH, Chen MX, Alessi DR, Campbell DG, Shanahan C, Cohen P, Cohen PT. Molecular cloning of cDNA encoding the 110 kDa and 21 kDa regulatory subunits of smooth muscle protein phosphatase 1M. FEBS Lett. 1994; 356: 51–55.[CrossRef][Medline] [Order article via Infotrieve]
7. Shimizu H, Ito M, Miyahara M, Ichikawa K, Okubo S, Konishi T, Naka M, Tanaka T, Hirano K, Hartshorne DJ. Characterization of the myosin-binding subunit of smooth muscle myosin phosphatase. J Biol Chem. 1994; 269: 30407–30411.[Abstract/Free Full Text]
8. Haystead CM, Gailly P, Somlyo AP, Somlyo AV, Haystead TA. Molecular cloning and functional expression of a recombinant 72.5 kDa fragment of the 110 kDa regulatory subunit of smooth muscle protein phosphatase 1M. FEBS Lett. 1995; 377: 123–127.[CrossRef][Medline] [Order article via Infotrieve]
9. Hartshorne DJ, Hirano K. Interactions of protein phosphatase type 1, with a focus on myosin phosphatase. Mol Cell Biochem. 1999; 190: 79–84.[CrossRef][Medline] [Order article via Infotrieve]
10. MacDonald JA, Borman MA, Muranyi A, Somlyo AV, Hartshorne DJ, Haystead TA. Identification of the endogenous smooth muscle myosin phosphatase-associated kinase. Proc Natl Acad Sci U S A. 2001; 98: 2419–2424.[Abstract/Free Full Text]
11. Muranyi A, Zhang R, Liu F, Hirano K, Ito M, Epstein HF, Hartshorne DJ. Myotonic dystrophy protein kinase phosphorylates the myosin phosphatase targeting subunit and inhibits myosin phosphatase activity. FEBS Lett. 2001; 493: 80–84.[CrossRef][Medline] [Order article via Infotrieve]
12. Kiss E, Muranyi A, Csortos C, Gergely P, Ito M, Hartshorne DJ, Erdodi F. Integrin-linked kinase phosphorylates the myosin phosphatase target subunit at the inhibitory site in platelet cytoskeleton. Biochem J. 2002; 365: 79–87.[CrossRef][Medline] [Order article via Infotrieve]
13. Kimura K, Ito M, Amano M, Chihara K, Fukata Y, Nakafuku M, Yamamori B, Feng J, Nakano T, Okawa K, Iwamatsu A, Kaibuchi K. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science. 1996; 273: 245–248.[Abstract]
14. Johnson DF, Moorhead G, Caudwell FB, Cohen P, Chen YH, Chen MX, Cohen PT. Identification of protein-phosphatase-1-binding domains on the glycogen and myofibrillar targetting subunits. Eur J Biochem. 1996; 239: 317–325.[Medline] [Order article via Infotrieve]
15. Hirano K, Phan BC, Hartshorne DJ. Interactions of the subunits of smooth muscle myosin phosphatase. J Biol Chem. 1997; 272: 3683–3688.[Abstract/Free Full Text]
16. Tanaka J, Ito M, Feng J, Ichikawa K, Hamaguchi T, Nakamura M, Hartshorne DJ, Nakano T. Interaction of myosin phosphatase target subunit 1 with the catalytic subunit of type 1 protein phosphatase. Biochemistry. 1998; 37: 16697–16703.[CrossRef][Medline] [Order article via Infotrieve]
17. Cohen PTW. Protein phosphatase 1 - targeted in many directions. J Cell Sci. 2002; 115: 241–256.[Abstract/Free Full Text]
18. Johnson D, Cohen P, Chen MX, Chen YH, Cohen PT. Identification of the regions on the M110 subunit of protein phosphatase 1M that interact with the M21 subunit and with myosin. Eur J Biochem. 1997; 244: 931–939.[Medline] [Order article via Infotrieve]
19. Wadia JS, Dowdy SF. Protein transduction technology. Curr Opin Biotechnol. 2002; 13: 52–56.[CrossRef][Medline] [Order article via Infotrieve]
20. Lindgren M, Hallbrink M, Prochiantz A, Langel U. Cell-penetrating peptides. Trends Pharmacol Sci. 2000; 21: 99–103.[CrossRef][Medline] [Order article via Infotrieve]
21. Schwarze SR, Dowdy SF. In vivo protein transduction: intracellular delivery of biologically active proteins, compounds and DNA. Trends Pharmacol Sci. 2000; 21: 45–48.[CrossRef][Medline] [Order article via Infotrieve]
22. Schwarze SR, Hruska KA, Dowdy SF. Protein transduction: unrestricted delivery into all cells? Trends Cell Biol. 2000; 10: 290–295.[CrossRef][Medline] [Order article via Infotrieve]
23. Nagahara H, Vocero-Akbani AM, Snyder EL, Ho A, Latham DG, Lissy NA, Becker-Hapak M, Ezhevsky SA, Dowdy SF. Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27^Kip1 induces cell migration. Nature Med. 1998; 4: 1449–1452.[CrossRef][Medline] [Order article via Infotrieve]
24. Hirano K, Kanaide H, Abe S, Nakamura M. Effects of diltiazem on calcium concentrations in the cytosol and on force of contractions in porcine coronary arterial strips. Br J Pharmacol. 1990; 101: 273–280.[Medline] [Order article via Infotrieve]
25. Derkach DN, Ihara E, Hirano K, Nishimura J, Takahashi S, Kanaide H. Thrombin causes endothelium-dependent biphasic regulation of vascular tone in the porcine renal interlobar artery. Br J Pharmacol. 2000; 131: 1635–1642.[CrossRef][Medline] [Order article via Infotrieve]
26. Zhou Y, Hirano K, Sakihara C, Nishimura J, Kanaide H. NH₂-terminal fragments of the 130 kDa subunit of myosin phosphatase increase the Ca²⁺ sensitivity of porcine renal artery. J Physiol. 1999; 516: 55–65.[Abstract/Free Full Text]
27. Persechini A, Kamm KE, Stull JT. Different phosphorylated forms of myosin in contracting tracheal smooth muscle. J Biol Chem. 1986; 261: 6293–6299.[Abstract/Free Full Text]
28. Kuroiwa M, Aoki H, Kobayashi S, Nishimura J, Kanaide H. Mechanism of endothelium-dependent relaxation induced by substance P in the coronary artery of the pig. Br J Pharmacol. 1995; 116: 2040–2047.[Medline] [Order article via Infotrieve]
29. Sauzeau V, Le Mellionnec E, Bertoglio J, Scalbert E, Pacaud P, Loirand G. Human urotensin II-induced contraction and arterial smooth muscle cell proliferation are mediated by RhoA and Rho-kinase. Circ Res. 2001; 88: 1102–1104.[Abstract/Free Full Text]
30. Morgan KG, Gangopadhyay SS. Invited review: cross-bridge regulation by thin filament-associated proteins. J Appl Physiol. 2001; 91: 953–962.[Abstract/Free Full Text]
31. Walsh MP, Horowitz A, Clement-Chomienne O, Andrea JE, Allen BG, Morgan KG. Protein kinase C mediation of Ca²⁺-independent contractions of vascular smooth muscle. Biochem Cell Biol. 1996; 74: 485–502.[Medline] [Order article via Infotrieve]
32. Winder SJ, Allen BG, Clement-Chomienne O, Walsh MP. Regulation of smooth muscle actin-myosin interaction and force by calponin. Acta Physiol Scand. 1998; 164: 415–426.[Medline] [Order article via Infotrieve]
33. Murphy RA, Rembold CM, Hai CM. Contraction in smooth muscle: what is latch? Prog Clin Biol Res. 1990; 327: 39–50.[Medline] [Order article via Infotrieve]
34. Hori M, Karaki H. Regulatory mechanisms of calcium sensitization of contractile elements in smooth muscle. Life Sci. 1998; 62: 1629–1633.[CrossRef][Medline] [Order article via Infotrieve]
35. Gong MC, Fuglsang A, Alessi D, Kobayashi S, Cohen P, Somlyo AV, Somlyo AP. Arachidonic acid inhibits myosin light chain phosphatase and sensitizes smooth muscle to calcium. J Biol Chem. 1992; 267: 21492–21498.[Abstract/Free Full Text]
36. Shin HM, Je HD, Gallant C, Tao TC, Hartshorne DJ, Ito M, Morgan KG. Differential association and localization of myosin phosphatase subunits during agonist-induced signal transduction in smooth muscle. Circ Res. 2002; 90: 546–553.[Abstract/Free Full Text]
37. Michaely P, Bennett V. The ANK repeat: a ubiquitous motif involved in macromolecular recognition. Trends Cell Biol. 1992; 2: 127–129.[CrossRef][Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				K. Hirano, N. Nomoto, M. Hirano, F. Momota, A. Hanada, and H. Kanaide Distinct Ca2+ Requirement for NO Production between Proteinase-Activated Receptor 1 and 4 (PAR1 and PAR4) in Vascular Endothelial Cells J. Pharmacol. Exp. Ther., August 1, 2007; 322(2): 668 - 677. [Abstract] [Full Text] [PDF]

				T. Yufu, K. Hirano, D. Bi, M. Hirano, J. Nishimura, Y. Iwamoto, and H. Kanaide Rac1 Regulation of Surface Expression of Protease-Activated Receptor-1 and Responsiveness to Thrombin in Vascular Smooth Muscle Cells Arterioscler. Thromb. Vasc. Biol., July 1, 2005; 25(7): 1506 - 1511. [Abstract] [Full Text] [PDF]

				N. Shiga, K. Hirano, M. Hirano, J. Nishimura, H. Nawata, and H. Kanaide Long-Term Inhibition of RhoA Attenuates Vascular Contractility by Enhancing Endothelial NO Production in an Intact Rabbit Mesenteric Artery Circ. Res., May 13, 2005; 96(9): 1014 - 1021. [Abstract] [Full Text] [PDF]

				D. Bi, J. Nishimura, N. Niiro, K. Hirano, and H. Kanaide Contractile Properties of the Cultured Vascular Smooth Muscle Cells: The Crucial Role Played by RhoA in the Regulation of Contractility Circ. Res., April 29, 2005; 96(8): 890 - 897. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 24/3/464    most recent 01.ATV.0000116028.42230.4cv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hirano, K. Articles by Kanaide, H. Search for Related Content PubMed PubMed Citation Articles by Hirano, K. Articles by Kanaide, H. Related Collections Other Vascular biology