1Department of Physiology and Graduate Center for Nutritional Sciences, University of Kentucky, Lexington, Kentucky; and 2Center for Cell Signaling, University of Virginia School of Medicine, Charlottesville, Virginia
Submitted 10 March 2005 ; accepted in final form 2 April 2005
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
protein kinase C; signal transduction; adenovirus
CPI-17 is a phosphatase-inhibitory protein that was first isolated from porcine aorta (14). Accumulating evidence suggests that CPI-17 plays an important role in myosin light chain phosphatase inhibition and Ca2+ sensitization regulation. CPI-17 is highly expressed in smooth muscle tissue, especially in the arteries (15, 59). Agonist-, GTPS-, and phorbol ester-induced smooth muscle contraction are associated with an increase in CPI-17 phosphorylation and its phosphatase-inhibitory activity (12, 30, 31, 43). Nitric oxide-induced smooth muscle relaxation is associated with a decrease in CPI-17 phosphorylation and its phosphatase-inhibitory activity (4, 16). In addition, alterations in CPI-17 protein expression level correlate with changes in the Ca2+ sensitization of smooth muscle contraction (32, 44, 46). Phosphorylation of Thr38 in CPI-17 increases its phosphatase inhibitory potency >1,000-fold (15, 50). PKC-
and PKC-
have been reported to mediate histamine-induced CPI-17 phosphorylation in smooth muscle (12). However, in several recent studies, investigators have found that in an isolated enzyme system, several other kinases can phosphorylate CPI-17. Such kinases include integrin-linked kinase (7, 9), protein kinase N (PKN) (23), MYPT1-associated kinase (37), p21-activated kinase (55), protein kinase A/G (8, 9), and, importantly, ROCK (34).
It is well recognized that the RhoA-ROCK pathway plays a critical role in agonist-induced Ca2+ sensitization of smooth muscle contraction (52). A variety of agonists can activate RhoA, a small GTPase, in vascular smooth muscle. The activated RhoA then turns on downstream effectors, including ROCK. ROCK directly phosphorylates MYPT1 at Thr696/Thr853 (17, 18, 29) and consequently inhibits phosphatase activity. In addition, in isolated enzyme systems, it was reported that ROCK can phosphorylate CPI-17 (34). This raises the possibility that phosphorylating CPI-17 is another mechanism by which the RhoA-ROCK pathway inhibits myosin light chain phosphatase in smooth muscle. In agreement with this possibility, inhibition of ROCK by Y-27632 partially but significantly inhibited histamine-, endothelin-, and phenylephrine-induced CPI-17 phosphorylation in permeabilized smooth muscle tissue (30, 31, 43). However, recently it was found that at the concentration used in the studies that showed inhibition of agonist-induced CPI-17 phosphorylation, Y-27632 also inhibited a well-recognized CPI-17 kinase, PKC- (12), and acetylcholine-induced activation of PKC (40). Thus the inhibition of agonist-induced CPI-17 phosphorylation by Y-27632 may reflect the ability of Y-27632 to inhibit PKC-
rather than indicating the involvement of ROCK in CPI-17 phosphorylation. Indeed, PMA-induced CPI-17 phosphorylation by selectively activating PKC was significantly suppressed by Y-27632 (12). Therefore, whether ROCK mediates agonist-induced CPI-17 phosphorylation in intact vascular smooth muscle cells (VSMCs) remains an open question.
To address this unresolved but important issue, we used primary cultured VSMCs and combined molecular biological, biochemical, and pharmacological approaches in the current study. The hypothesis that ROCK activated by RhoA serves as an endogenous kinase mediating some agonist-induced CPI-17 phosphorylation was tested. Our data provide the first solid evidence that the RhoA-ROCK pathway is responsible for thrombin- and U-46619-induced CPI-17 phosphorylation in VSMCs.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Primary cell culture. Rabbit VSMCs were isolated by performing enzymatic dissociation as described previously (47). Briefly, thoracic aortas were removed and washed in Hanks' balanced salt solution containing 100 U/ml penicillin and 100 µg/ml streptomycin. After being cut into four pieces, the vessels were digested in 2 mg/ml collagenase I and then in 2 mg/ml collagenase II plus 0.5 mg/ml elastase for 30 min each at 37°C. The isolated cells were cultured in DMEM supplemented with 10% (vol/vol) fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cultures were maintained at 37°C in a 95% air-5% CO2 humidified atmosphere.
Measurement of VSMC contraction. VSMC contraction was measured by analyzing the thrombin-stimulated transvascular horseradish peroxidase (HRP) diffusion according to a previously published protocol (2, 10, 48). Briefly, VSMCs were plated on collagen-coated Transwell inserts (6 x 104 cells/cm2, 3.0-µm pore size; Costar) and cultured for 5 days in DMEM supplemented with 10% serum. Subsequently, the cells were incubated in serum-free DMEM for 2 days. To establish basal HRP diffusion, HRP (1.5 µg/ml, type VI-A, 44,000 Mr; Sigma) was added to the upper chamber. Medium (60 µl) was collected from the lower chamber after 3 min and kept on ice until the enzymatic activity of HRP had been assayed. After being washed twice for total of 2 h, the cells were stimulated with thrombin (1 U/ml) for 20 min and then HRP was added to the upper chamber to determine the thrombin-stimulated HRP diffusion. To assay HRP enzymatic activity, the collected 60 µl of lower-chamber medium was added to 840 µl of reaction buffer (50 mM NaH2PO4 and 5 mM guaiacol). The reaction was initiated by addition of 100 µl of freshly made H2O2 solution (60 mM in H2O). The absorbance was measured at 470 nm after 15-min incubation at room temperature.
Immunoblot analysis. Proteins from primary cultured rabbit aortic VSMCs were denatured using trichloroacetic acid and separated by SDS-polyacrylamide gel electrophoresis. Specific proteins were detected using immunoblot analysis as described previously (22). The dilutions of specific antibodies were anti-phosphorylated MYPT1 antibody [p-MYPT1 (Thr853)], 1:1,000; total MYPT1, 1:10,000; anti-phosphorylated CPI-17 [p-CPI-17 (Thr38)], 1:2,500; anti-total-CPI-17 protein, 1:20,000; and anti-RhoA, 1:1,000. CPI-17 and MYPT1 phosphorylation levels were quantified using the ratios of phosphorylated vs. total CPI-17 and MYPT1 signals from two sets of parallel immunoblots. Special care was taken to ensure that the immunoblot signals were within linear range.
Recombinant adenovirus construction. The BamHI-EcoRI fragment of pBK-CMV/V14 was generated by performing PCR. This V14 RhoA fragment was then ligated into a modified version of pBluescript KS (pFLAG BamHI) containing an NH2-terminal FLAG epitope tag (DYKDDDDK). The FLAG-V14 RhoA fragment was excised by SpeI and XhoI and ligated into a modified adenoviral shuttle vector (pAdtracgfptre) containing two expression cassettes. One cassette uses the cytomegalovirus (CMV) promoter to drive green fluorescent protein (GFP) expression, and the other uses the tetracycline response element promoter to drive RhoA expression.
C3 DNA was excised from the PRK5-C3 vector using double digestion with EcoRI and ClaI. The cohesive ends of the C3 DNA fragment were filled in by a large fragment of DNA polymerase I (Klenow fragment). The blunt ends of the C3 DNA were then ligated into the Zero Blunt PCR vector (Invitrogen). After double digestion with SpeI and XhoI, the C3 DNA fragment was cloned into the pShuttle-IRES-hrGFP1 vector (Stratagene). This adenoviral vector contains a dicistronic expression cassette in which both C3 and a recombinant GFP are under the common control of a CMV promoter.
The V14RhoA and C3 adenoviruses were constructed using the Adeasy system (4) as described previously (22). Expression of FLAG V14 RhoA was examined using immunoblot analysis with anti-FLAG and anti-RhoA antibodies, respectively. Large quantities of recombinant adenovirus were produced by sequentially infecting human embryonic kidney-293 cells in 100-mm2 dishes and purified using CsCl gradient ultracentrifugation (22). The viral particle infectious units (pfu) were determined using an Adeno-X rapid titer kit (Clontech).
Adenoviral infection. VSMCs were starved in serum-free medium for 24 h and then incubated with Ad/V14 RhoA [multiplicity of infection (MOI) of 500] plus Ad/Tet-on (encoding a tetracycline-regulatory transcription factor) or Ad/C3 (MOI of 200) viruses for 12 h. Subsequently, the cells were cultured in serum-free medium for 36 h to allow V14RhoA and C3 expression. Different concentrations of doxycycline were present for the total 48-h period. The adenoviral transfection conditions were optimized to maximize RhoA or C3 expression and to minimize the cytopathic effect of the adenovirus.
Statistical analysis. Each experiment was repeated a minimum of three times. Data are expressed as means ± SE. Statistical analysis was performed using an unpaired t-test.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
Minimal role of PKC in thrombin- and U-46619-induced CPI-17 phosphorylation in primary cultured VSMCs.
To determine the kinase that mediates agonist-induced CPI-17 phosphorylation, we stimulated VSMCs with thrombin and U-46619. Thrombin and U-46619 induce Ca2+ sensitization in smooth muscle by activating specific receptors: protease-activated receptors and thromboxane A2 receptors, respectively (6, 42). As shown in Fig. 5, we found that thrombin and U-46619 induced marked CPI-17 phosphorylation. We next tried to identify the endogenous kinase that mediates agonist-induced CPI-17 phosphorylation in smooth muscle cells. Because PKC- and PKC-
are well recognized to mediate some agonists, such as histamine-induced CPI-17 phosphorylation in vascular smooth muscle cells, we first tested the role of PKC in thrombin- and U-46619-induced CPI-17 phosphorylation. Interestingly, inhibition of conventional and novel PKCs with GF109203X affected neither thrombin-induced nor U-46619-induced CPI-17 phosphorylation (Fig. 5). As a control, the same GF109203X treatment nearly abolished PMA-induced CPI-17 phosphorylation. Taken together, these results suggest that in contrast to a prominent role in histamine-induced CPI-17 phosphorylation, PKC-
and PKC-
play a minimal role in thrombin- and U-46619-induced CPI-17 phosphorylation.
|
To inhibit RhoA function by ADP-ribosylation, VSMCs were infected with recombinant adenovirus-encoding exoenzyme C3 and then stimulated with thrombin. As shown in Fig. 6, A and B, thrombin-induced CPI-17 phosphorylation was significantly decreased by prior treatment with C3. As a control, a parallel treatment with an empty adenoviral vector did not affect thrombin-induced CPI-17 phosphorylation (Fig. 6A). This suggests that the inhibition of thrombin-induced CPI-17 phosphorylation by C3-containing recombinant adenovirus is not due to a nonspecific effect of the adenovirus. In addition, the upshift of RhoA mobility in SDS-PAGE gel (Fig. 6C) indicated effective ADP ribosylation of RhoA under our experimental conditions (1). In addition to inhibition of thrombin-induced CPI-17 phosphorylation, ADP ribosylation of RhoA inhibited thrombin-induced MYPT1 Thr853 phosphorylation (data not shown).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CPI-17 is a phosphorylation-dependent myosin phosphatase-inhibitory protein. PKC- and PKC-
have been shown to phosphorylate CPI-17 in vitro and in situ (12, 30, 33, 40). Depleting CPI-17 protein by skinning smooth muscle cells eliminates PKC activator-induced Ca2+ sensitization of smooth muscle contraction (12, 36). The response can be reconstituted by the addition of recombinant CPI-17 protein and PKC (36). Furthermore, the inhibition of PKC by GF109203X abolishes histamine-induced CPI-17 phosphorylation in smooth muscle tissue (12). However, in several recent studies, investigators have reported that multiple kinases, including ROCK (34), integrin-linked kinase (7, 9), PKN (23), MYPT1-associated kinase (37), p21-activated kinase (55), and protein kinase A/G (8, 9), can phosphorylate CPI-17 in an isolated enzyme system. This raises the possibility that, in addition to PKC-
and PKC-
, other kinases may be involved in CPI-17 phosphorylation. Consistent with this possibility, inhibition of ROCK by Y-27632 partially inhibits histamine-, endothelin-, and phenylephrine-induced CPI-17 phosphorylation (30, 31, 43). However, the following facts prevent the establishment of a role of ROCK in agonist-induced CPI-17 phosphorylation. First, there is no evidence that selective activation of the RhoA-ROCK pathway indeed phosphorylates CPI-17 in intact smooth muscle cells. Second, at the concentration used in the studies that showed inhibition of agonist-induced CPI-17 phosphorylation, Y-27632 recently was found to inhibit PKC-
, which is well known to phosphorylate CPI-17 (12). In addition, Y-27632 inhibited acetylcholine-induced PKC activation by 45% in smooth muscle cells (40).
To clarify the role of the RhoA-ROCK pathway in agonist-induced CPI-17 phosphorylation, we used molecular biological and biochemical approaches in addition to pharmacological approaches in the present study. Our data clearly show that ROCK is responsible for thrombin- and U-46619-induced CPI-17 phosphorylation in primary cultured VSMCs. First, we selectively activated the RhoA-ROCK pathway by ectopic expression of a constitutively active mutant V14RhoA in VSMCs and found that it was sufficient to induce CPI-17 phosphorylation in a V14RhoA expression level-dependent manner (Fig. 3). Second, V14RhoA-induced CPI-17 phosphorylation was not affected by inhibition of PKC with GF109203X but was abolished by Y-27632 (Fig. 4), suggesting that selective activation of RhoA is sufficient to phosphorylate CPI-17 by activating ROCK in intact smooth muscle cells. Third, we have shown that some agonists, such as thrombin and U-46619, induced CPI-17 phosphorylation in VSMCs (Fig. 5) and that inhibition of RhoA by adenovirus-mediated expression of exoenzyme C3 in VSMCs significantly inhibited thrombin-induced CPI-17 phosphorylation (Fig. 6). Fourth, we have shown that Y-27632, similarly to exoenzyme C3, also significantly diminished thrombin- and U-46619-induced CPI-17 phosphorylation in VSMCs (Fig. 7).
We found that in contrast to the predominant role of PKC in histamine-induced CPI-17 phosphorylation in mature vascular smooth muscle tissue (12), the RhoA-ROCK pathway plays a predominant role in thrombin- and U-46619-induced CPI-17 phosphorylation in primary cultured VSMCs. That different kinases mediate CPI-17 phosphorylation may reflect the different systems used: primary cultured cells vs. mature smooth muscle tissue. In line with this possibility, recent reports have shown that the contractility of cultured VSMCs display a greater dependency than fresh tissue on the RhoA-ROCK pathway (3). Moreover, the phosphorylation site on MYPT1 changes upon culturing (53). Mature VSMCs are in a quiescent contractile phenotype under physiological conditions. They shift to a synthetic phenotype under certain pathological conditions such as restenosis, wound healing, and atherosclerosis. Cultured VSMCs resemble the synthetic phenotype in many aspects. Alternatively, on the basis of the comparable expression levels of smooth muscle marker genes and the ability of the primary cultures of aortic smooth muscle to contract in response to thrombin stimulation, it is also possible that the fact that different kinases mediate CPI-17 phosphorylation may reflect the use of different agonists: thrombin and U-46619 vs. histamine.
The physiological significance of the RhoA-ROCK pathway in the regulation of vascular smooth muscle contraction has been shown in numerous studies. We (21) and others (24, 35) have shown that recombinant RhoA or ROCK induces Ca2+ sensitization of smooth muscle contraction. Inhibiting endogenous RhoA by ADP ribosylation (20, 21, 24) or glycosylation (45), or inhibiting ROCK by Y-27632 (19, 25, 57) or HA-1077 (41, 49), inhibits various G protein-coupled receptor agonist-induced vascular smooth muscle contractions. Moreover, recent data indicate a significant pathological role of the RhoA-ROCK pathway in several cardiovascular diseases, including hypertension, coronary artery spasm, effort-induced angina, atherosclerosis, and restenosis (for review, see Refs. 5, 52).
The molecular mechanisms underlying RhoA-ROCK pathway-induced inhibition of myosin phosphatase remain to be elucidated completely. Our data underscore the significance of phosphorylating CPI-17 as one mechanism mediating ROCK-induced inhibition of myosin phosphatase. ROCK can inhibit myosin phosphatase by at least two mechanisms: phosphorylation of MYPT1 (17, 18) and phosphorylation of CPI-17 (Ref. 34 and the present study). ROCK has been reported to phosphorylate MYPT1 on Thr696 in isolated enzyme systems and in smooth muscle tissue, platelets, and endothelial cells (54), and Thr696 phosphorylation inhibits phosphatase activity (57). Therefore, Thr696 phosphorylation has been thought to be the mechanism mediating RhoA-ROCK pathway-induced phosphatase inhibition. However, this view has been challenged recently on the basis of the failure to detect an increase in Thr696 phosphorylation during the agonist-induced Ca2+ sensitization of smooth muscle contraction (31, 43). Indeed, with the ectopic expression of V14RhoA in smooth muscle, we failed to observe consistent stimulation of MYPT1 phosphorylation on Thr696. In contrast, we consistently observed V14RhoA expression level-dependent MYPT1 phosphorylation at Thr853. Furthermore, inhibition of ROCK by Y-27632 is associated with the inhibition of Thr853 phosphorylation. Although phosphorylation of MYPT1 at Thr853 does not directly affect phosphatase activity (17, 18), Thr853 phosphorylation may cause MYPT1 dissociation from its substrate myosin (58), resulting in the decrease of phosphatase activity toward myosin. Indeed, Thr853 is located at the myosin binding site on MYPT1 (18). In addition, our results clearly show that activation of the RhoA-ROCK pathway causes CPI-17 phosphorylation. Phosphorylated CPI-17 specifically binds and inhibits myosin phosphatase in cells (11). This suggests that at least two mechanisms, directly phosphorylating MYPT1 at Thr853 and phosphorylating CPI-17, can mediate ROCK-induced myosin phosphatase inhibition. Because both the MYPT1 Thr853 phosphorylation and the CPI-17 phosphorylation are observed in parallel using V14RhoA expression or thrombin or U-46619 stimulation, further experiments are required to assess the exact contribution by each of the two mechanisms to the RhoA-ROCK pathway-mediated inhibition of phosphatase activity.
In conclusion, the selective activation of the RhoA-ROCK pathway can phosphorylate CPI-17 in primary culture rabbit aortic smooth muscle cells, and the RhoA-ROCK pathway is primarily responsible for thrombin- and U-46619-induced CPI-17 phosphorylation in primary cultured VSMCs.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Begum N, Duddy N, Sandu O, Reinzie J, and Ragolia L. Regulation of myosin-bound protein phosphatase by insulin in vascular smooth muscle cells: evaluation of the role of Rho kinase and phosphatidylinositol-3-kinase-dependent signaling pathways. Mol Endocrinol 14: 13651376, 2000.
3. Bi D, Nishimura J, Niiro N, Hirano K, and Kanaide H. Contractile properties of the cultured vascular smooth muscle cells: the crucial role played by RhoA in the regulation of contractility. Circ Res 96: 890897, 2005.
4. Bonnevier J and Arner A. Actions downstream of cyclic GMP/protein kinase G can reverse protein kinase C-mediated phosphorylation of CPI-17 and Ca2+ sensitization in smooth muscle. J Biol Chem 279: 2899829003, 2004.
5. Chitaley K, Weber D, and Webb RC. RhoA/Rho-kinase, vascular changes, and hypertension. Curr Hypertens Rep 3: 139144, 2001.[Medline]
6. Coughlin SR. Thrombin signalling and protease-activated receptors. Nature 407: 258264, 2000.[CrossRef][ISI][Medline]
7. Deng JT, Sutherland C, Brautigan DL, Eto M, and Walsh MP. Phosphorylation of the myosin phosphatase inhibitors, CPI-17 and PHI-1, by integrin-linked kinase. Biochem J 367: 517524, 2002.[CrossRef][ISI][Medline]
8. Dubois T, Howell S, Zemlickova E, Learmonth M, Cronshaw A, and Aitken A. Novel in vitro and in vivo phosphorylation sites on protein phosphatase 1 inhibitor CPI-17. Biochem Biophys Res Commun 302: 186192, 2003.[CrossRef][ISI][Medline]
9. Erdodi F, Kiss E, Walsh MP, Stefansson B, Deng JT, Eto M, Brautigan DL, and Hartshorne DJ. Phosphorylation of protein phosphatase type-1 inhibitory proteins by integrin-linked kinase and cyclic nucleotide-dependent protein kinases. Biochem Biophys Res Commun 306: 382387, 2003.[CrossRef][ISI][Medline]
10. Essler M, Amano M, Kruse HJ, Kaibuchi K, Weber PC, and Aepfelbacher M. Thrombin inactivates myosin light chain phosphatase via Rho and its target Rho kinase in human endothelial cells. J Biol Chem 273: 2186721874, 1998.
11. Eto M, Kitazawa T, and Brautigan DL. Phosphoprotein inhibitor CPI-17 specificity depends on allosteric regulation of protein phosphatase-1 by regulatory subunits. Proc Natl Acad Sci USA 101: 88888893, 2004.
12. Eto M, Kitazawa T, Yazawa M, Mukai H, Ono Y, and Brautigan DL. Histamine-induced vasoconstriction involves phosphorylation of a specific inhibitor protein for myosin phosphatase by protein kinase C and
isoforms. J Biol Chem 276: 2907229078, 2001.
14. Eto M, Ohmori T, Suzuki M, Furuya K, and Morita F. A novel protein phosphatase-1 inhibitory protein potentiated by protein kinase C: isolation from porcine aorta media and characterization. J Biochem (Tokyo) 118: 11041107, 1995.[Abstract]
15. Eto M, Senba S, Morita F, and Yazawa M. Molecular cloning of a novel phosphorylation-dependent inhibitory protein of protein phosphatase-1 (CPI17) in smooth muscle: its specific localization in smooth muscle. FEBS Lett 410: 356360, 1997.[CrossRef][ISI][Medline]
16. Etter EF, Eto M, Wardle RL, Brautigan DL, and Murphy RA. Activation of myosin light chain phosphatase in intact arterial smooth muscle during nitric oxide-induced relaxation. J Biol Chem 276: 3468134685, 2001.
17. Feng J, Ito M, Ichikawa K, Isaka N, Nishikawa M, Hartshorne DJ, and Nakano T. Inhibitory phosphorylation site for Rho-associated kinase on smooth muscle myosin phosphatase. J Biol Chem 274: 3738537390, 1999.
18. Feng J, Ito M, Kureishi Y, Ichikawa K, Amano M, Isaka N, Okawa K, Iwamatsu A, Kaibuchi K, Hartshorne DJ, and Nakano T. Rho-associated kinase of chicken gizzard smooth muscle. J Biol Chem 274: 37443752, 1999.
19. Fu X, Gong MC, Jia T, Somlyo AV, and Somlyo AP. The effects of the Rho-kinase inhibitor Y-27632 on arachidonic acid-, GTPS-, and phorbol ester-induced Ca2+-sensitization of smooth muscle. FEBS Lett 440: 183187, 1998.[CrossRef][ISI][Medline]
20. Fujihara H, Walker LA, Gong MC, Lemichez E, Boquet P, Somlyo AV, and Somlyo AP. Inhibition of RhoA translocation and calcium sensitization by in vivo ADP-ribosylation with the chimeric toxin DC3B. Mol Biol Cell 8: 24372447, 1997.
21. Gong MC, Iizuka K, Nixon G, Browne JP, Hall A, Eccleston JF, Sugai M, Kobayashi S, Somlyo AV, and Somlyo AP. Role of guanine nucleotide-binding proteinsras-family or trimeric proteins or bothin Ca2+ sensitization of smooth muscle. Proc Natl Acad Sci USA 93: 13401345, 1996.
22. Guo Z, Su W, Ma Z, Smith GM, and Gong MC. Ca2+-independent phospholipase A2 is required for agonist-induced Ca2+ sensitization of contraction in vascular smooth muscle. J Biol Chem 278: 18561863, 2003.
23. Hamaguchi T, Ito M, Feng J, Seko T, Koyama M, Machida H, Takase K, Amano M, Kaibuchi K, Hartshorne DJ, and Nakano T. Phosphorylation of CPI-17, an inhibitor of myosin phosphatase, by protein kinase N. Biochem Biophys Res Commun 274: 825830, 2000.[CrossRef][ISI][Medline]
24. Hirata K, Kikuchi A, Sasaki T, Kuroda S, Kaibuchi K, Matsuura Y, Seki H, Saida K, and Takai Y. Involvement of rho p21 in the GTP-enhanced calcium ion sensitivity of smooth muscle contraction. J Biol Chem 267: 87198722, 1992.
25. Ishizaki T, Uehata M, Tamechika I, Keel J, Nonomura K, Maekawa M, and Narumiya S. Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases. Mol Pharmacol 57: 976983, 2000.
26. Ito M, Nakano T, Erdodi F, and Hartshorne DJ. Myosin phosphatase: structure, regulation and function. Mol Cell Biochem 259: 197209, 2004.[CrossRef][ISI][Medline]
27. Kandabashi T, Shimokawa H, Miyata K, Kunihiro I, Eto Y, Morishige K, Matsumoto Y, Obara K, Nakayama K, Takahashi S, and Takeshita A. Evidence for protein kinase C-mediated activation of Rho-kinase in a porcine model of coronary artery spasm. Arterioscler Thromb Vasc Biol 23: 22092214, 2003.
28. Kandabashi T, Shimokawa H, Miyata K, Kunihiro I, Kawano Y, Fukata Y, Higo T, Egashira K, Takahashi S, Kaibuchi K, and Takeshita A. Inhibition of myosin phosphatase by upregulated rho-kinase plays a key role for coronary artery spasm in a porcine model with interleukin-1. Circulation 101: 13191323, 2000.
29. Kimura K, Ito M, Amano M, Chihara K, Fukata Y, Nakafuku M, Yamamori B, Feng J, Nakano T, Okawa K, Iwamatsu A, and Kaibuchi K. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science 273: 245248, 1996.[Abstract]
30. Kitazawa T, Eto M, Woodsome TP, and Brautigan DL. Agonists trigger G protein-mediated activation of the CPI-17 inhibitor phosphoprotein of myosin light chain phosphatase to enhance vascular smooth muscle contractility. J Biol Chem 275: 98979900, 2000.
31. Kitazawa T, Eto M, Woodsome TP, and Khalequzzaman M. Phosphorylation of the myosin phosphatase targeting subunit and CPI-17 during Ca2+ sensitization in rabbit smooth muscle. J Physiol 546: 879889, 2003.
32. Kitazawa T, Polzin AN, and Eto M. CPI-17-deficient smooth muscle of chicken. J Physiol 557: 515528, 2004.
33. Kitazawa T, Takizawa N, Ikebe M, and Eto M. Reconstitution of protein kinase C-induced contractile Ca2+ sensitization in Triton X-100-demembranated rabbit arterial smooth muscle. J Physiol 520: 139152, 1999.
34. Koyama M, Ito M, Feng J, Seko T, Shiraki K, Takase K, Hartshorne DJ, and Nakano T. Phosphorylation of CPI-17, an inhibitory phosphoprotein of smooth muscle myosin phosphatase, by Rho-kinase. FEBS Lett 475: 197200, 2000.[CrossRef][ISI][Medline]
35. Kureishi Y, Kobayashi S, Amano M, Kimura K, Kanaide H, Nakano T, Kaibuchi K, and Ito M. Rho-associated kinase directly induces smooth muscle contraction through myosin light chain phosphorylation. J Biol Chem 272: 1225712260, 1997.
36. Li L, Eto M, Lee MR, Morita F, Yazawa M, and Kitazawa T. Possible involvement of the novel CPI-17 protein in protein kinase C signal transduction of rabbit arterial smooth muscle. J Physiol 508: 871881, 1998.
37. MacDonald JA, Eto M, Borman MA, Brautigan DL, and Haystead TA. Dual Ser and Thr phosphorylation of CPI-17, an inhibitor of myosin phosphatase, by MYPT-associated kinase. FEBS Lett 493: 9194, 2001.[CrossRef][ISI][Medline]
38. Malcolm KC, Ross AH, Qiu RG, Symons M, and Exton JH. Activation of rat liver phospholipase D by the small GTP-binding protein RhoA. J Biol Chem 269: 2595125954, 1994.
39. Morishige K, Shimokawa H, Eto Y, Kandabashi T, Miyata K, Matsumoto Y, Hoshijima M, Kaibuchi K, and Takeshita A. Adenovirus-mediated transfer of dominant-negative rho-kinase induces a regression of coronary arteriosclerosis in pigs in vivo. Arterioscler Thromb Vasc Biol 21: 548554, 2001.
40. Murthy KS, Zhou H, Grider JR, Brautigan DL, Eto M, and Makhlouf GM. Differential signalling by muscarinic receptors in smooth muscle: m2-mediated inactivation of myosin light chain kinase via Gi3, Cdc42/Rac1 and p21-activated kinase 1 pathway, and m3-mediated MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation via Rho-associated kinase/myosin phosphatase targeting subunit 1 and protein kinase C/CPI-17 pathway. Biochem J 374: 145155, 2003.[CrossRef][ISI][Medline]
41. Nagumo H, Sasaki Y, Ono Y, Okamoto H, Seto M, and Takuwa Y. Rho kinase inhibitor HA-1077 prevents Rho-mediated myosin phosphatase inhibition in smooth muscle cells. Am J Physiol Cell Physiol 278: C57C65, 2000.
42. Narumiya S and FitzGerald GA. Genetic and pharmacological analysis of prostanoid receptor function. J Clin Invest 108: 2530, 2001.
43. Niiro N, Koga Y, and Ikebe M. Agonist-induced changes in the phosphorylation of the myosin-binding subunit of myosin light chain phosphatase and CPI17, two regulatory factors of myosin light chain phosphatase, in smooth muscle. Biochem J 369: 117128, 2003.[CrossRef][ISI][Medline]
44. Ohama T, Hori M, Sato K, Ozaki H, and Karaki H. Chronic treatment with interleukin-1 attenuates contractions by decreasing the activities of CPI-17 and MYPT-1 in intestinal smooth muscle. J Biol Chem 278: 4879448804, 2003.
45. Otto B, Steusloff A, Just I, Aktories K, and Pfitzer G. Role of Rho proteins in carbachol-induced contractions in intact and permeabilized guinea-pig intestinal smooth muscle. J Physiol 496: 317329, 1996.[Abstract]
46. Ozaki H, Yasuda K, Kim YS, Egawa M, Kanzaki H, Nakazawa H, Hori M, Seto M, and Karaki H. Possible role of the protein kinase C/CPI-17 pathway in the augmented contraction of human myometrium after gestation. Br J Pharmacol 140: 13031312, 2003.[CrossRef][ISI][Medline]
47. Pauly RR, Bilato C, Cheng L, Monticone R, and Crow MT. Vascular smooth muscle cell cultures. Methods Cell Biol 52: 133154, 1997.[ISI][Medline]
48. Sandu OA, Ragolia L, and Begum N. Diabetes in the Goto-Kakizaki rat is accompanied by impaired insulin-mediated myosin-bound phosphatase activation and vascular smooth muscle cell relaxation. Diabetes 49: 21782189, 2000.[Abstract]
49. Sasaki Y, Suzuki M, and Hidaka H. The novel and specific Rho-kinase inhibitor (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinoline)sulfonyl]-homopiperazine as a probing molecule for Rho-kinase-involved pathway. Pharmacol Ther 93: 225232, 2002.[CrossRef][ISI][Medline]
50. Senba S, Eto M, and Yazawa M. Identification of trimeric myosin phosphatase (PP1M) as a target for a novel PKC-potentiated protein phosphatase-1 inhibitory protein (CPI17) in porcine aorta smooth muscle. J Biochem (Tokyo) 125: 354362, 1999.[Abstract]
51. Shimokawa H, Seto M, Katsumata N, Amano M, Kozai T, Yamawaki T, Kuwata K, Kandabashi T, Egashira K, Ikegaki I, Asano T, Kaibuchi K, and Takeshita A. Rho-kinase-mediated pathway induces enhanced myosin light chain phosphorylations in a swine model of coronary artery spasm. Cardiovasc Res 43: 10291039, 1999.[CrossRef][ISI][Medline]
52. Somlyo AP and Somlyo AV. Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase. Physiol Rev 83: 13251358, 2003.
53. Stevenson AS, Matthew JD, Eto M, Luo S, Somlyo AP, and Somlyo AV. Uncoupling of GPCR and RhoA-induced Ca2+-sensitization of chicken amnion smooth muscle lacking CPI-17. FEBS Lett 578: 7379, 2004.[CrossRef][ISI][Medline]
54. Swärd K, Mita M, Wilson DP, Deng JT, Susnjar M, and Walsh MP. The role of RhoA and Rho-associated kinase in vascular smooth muscle contraction. Curr Hypertens Rep 5: 6672, 2003.[ISI][Medline]
55. Takizawa N, Koga Y, and Ikebe M. Phosphorylation of CPI17 and myosin binding subunit of type 1 protein phosphatase by p21-activated kinase. Biochem Biophys Res Commun 297: 773778, 2002.[CrossRef][ISI][Medline]
56. Toullec D, Pianetti P, Coste H, Bellevergue P, Grand-Perret T, Ajakane M, Baudet V, Boissin P, Boursier E, Loriolle F, Duhamel L, Charon D, and Kirilovsky J. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem 266: 1577115781, 1991.
57. Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita T, Tamakawa H, Yamagami K, Inui J, Maekawa M, and Narumiya S. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 389: 990994, 1997.[CrossRef][ISI][Medline]
58. Velasco G, Armstrong C, Morrice N, Frame S, and Cohen P. Phosphorylation of the regulatory subunit of smooth muscle protein phosphatase 1M at Thr850 induces its dissociation from myosin. FEBS Lett 527: 101104, 2002.[CrossRef][ISI][Medline]
59. Woodsome TP, Eto M, Everett A, Brautigan DL, and Kitazawa T. Expression of CPI-17 and myosin phosphatase correlates with Ca2+ sensitivity of protein kinase C-induced contraction in rabbit smooth muscle. J Physiol 535: 553564, 2001.