Ca2+-independent Smooth Muscle Contraction

Smooth muscle contraction follows an increase in cytosolic Ca2+ concentration, activation of myosin light chain kinase, and phosphorylation of the 20-kDa light chain of myosin at Ser19. Several agonists acting via G protein-coupled receptors elicit a contraction without a change in [Ca2+]i via inhibition of myosin light chain phosphatase and increased myosin phosphorylation. We showed that microcystin (phosphatase inhibitor)-induced contraction of skinned smooth muscle occurred in the absence of Ca2+ and correlated with phosphorylation of myosin light chain at Ser19 and Thr18 by a kinase distinct from myosin light chain kinase. In this study, we identify this kinase as integrin-linked kinase. Chicken gizzard integrin-linked kinase cDNA was cloned, sequenced, expressed in E. coli, and shown to phosphorylate myosin light chain in the absence of Ca2+ at Ser19 and Thr18. Subcellular fractionation revealed two distinct populations of integrin-linked kinase, including a Triton X-100-insoluble component that phosphorylates myosin in a Ca2+-independent manner. These results suggest a novel function for integrin-linked kinase in the regulation of smooth muscle contraction via Ca2+-independent phosphorylation of myosin, raise the possibility that integrin-linked kinase may also play a role in regulation of nonmuscle motility, and confirm that integrin-linked kinase is indeed a functional protein-serine/threonine kinase.AF296130 Smooth muscle contraction follows an increase in cytosolic Ca2+ concentration, activation of myosin light chain kinase, and phosphorylation of the 20-kDa light chain of myosin at Ser19. Several agonists acting via G protein-coupled receptors elicit a contraction without a change in [Ca2+]i via inhibition of myosin light chain phosphatase and increased myosin phosphorylation. We showed that microcystin (phosphatase inhibitor)-induced contraction of skinned smooth muscle occurred in the absence of Ca2+ and correlated with phosphorylation of myosin light chain at Ser19 and Thr18 by a kinase distinct from myosin light chain kinase. In this study, we identify this kinase as integrin-linked kinase. Chicken gizzard integrin-linked kinase cDNA was cloned, sequenced, expressed in E. coli, and shown to phosphorylate myosin light chain in the absence of Ca2+ at Ser19 and Thr18. Subcellular fractionation revealed two distinct populations of integrin-linked kinase, including a Triton X-100-insoluble component that phosphorylates myosin in a Ca2+-independent manner. These results suggest a novel function for integrin-linked kinase in the regulation of smooth muscle contraction via Ca2+-independent phosphorylation of myosin, raise the possibility that integrin-linked kinase may also play a role in regulation of nonmuscle motility, and confirm that integrin-linked kinase is indeed a functional protein-serine/threonine kinase.AF296130 calmodulin Ca2+- and calmodulin-dependent protein kinase II dithiothreitol integrin-linked kinase 20-kDa light chain subunit of myosin II matrix-assisted laser desorption/ionization-time of flight mass spectrometry myosin light chain kinase myosin light chain phosphatase protein kinase C Rho-associated kinase polyacrylamide gel electrophoresis Smooth muscle contraction is activated primarily by an increase in cytosolic free Ca2+ concentration ([Ca2+]i) in response to membrane depolarization, a variety of blood-borne agonists such as angiotensin II and α1-adrenergic agonists, or stretch (1Somlyo A.P. Somlyo A.V. Nature. 1994; 372: 231-236Crossref PubMed Scopus (1727) Google Scholar, 2Allen B.E. Walsh M.P. Trends Biochem. Sci. 1994; 19: 362-368Abstract Full Text PDF PubMed Scopus (160) Google Scholar). Ca2+ originates from the extracellular space and/or intracellular stores, principally the sarcoplasmic reticulum (3Missiaen L. De Smedt H. Droogmans G. Himpens B. Casteels R. Pharmacol. Ther. 1992; 56: 191-231Crossref PubMed Scopus (98) Google Scholar). Ca2+ binds to calmodulin (CaM)1 (4Chin D. Means A.R. Trends Cell Biol. 2000; 10: 322-328Abstract Full Text Full Text PDF PubMed Scopus (1124) Google Scholar), which activates myosin light chain kinase (MLCK) (5Gallagher P.J Herring B.P. Stull J.T. J. Muscle Res. Cell Motil. 1997; 18: 1-16Crossref PubMed Scopus (174) Google Scholar), resulting in phosphorylation of myosin II, specifically at Ser19, and sometimes to a small extent at Thr18 (6Ikebe M. Hartshorne D.J. J. Biol. Chem. 1985; 260: 10027-10031Abstract Full Text PDF PubMed Google Scholar, 7Colburn J.C. Michnoff C.H. Hsu L-C. Slaughter C.A. Kamm K.E. Stull J.T. J. Biol. Chem. 1988; 263: 19166-19173Abstract Full Text PDF PubMed Google Scholar, 8Seto M. Sasaki Y. Sasaki Y. Pflügers Arch. 1990; 415: 484-489Crossref PubMed Scopus (35) Google Scholar), of the pair of 20-kDa light chain subunits (LC20). This simple phosphorylation reaction triggers cross-bridge cycling and the development of force or shortening of the muscle (1Somlyo A.P. Somlyo A.V. Nature. 1994; 372: 231-236Crossref PubMed Scopus (1727) Google Scholar, 2Allen B.E. Walsh M.P. Trends Biochem. Sci. 1994; 19: 362-368Abstract Full Text PDF PubMed Scopus (160) Google Scholar). Several agonists that act via seven-transmembrane domain-containing, G protein-coupled receptors elicit a contractile response without a change in [Ca2+]i (9Somlyo A.P. Somlyo A.V. J. Physiol. 2000; 522: 177-185Crossref PubMed Scopus (1074) Google Scholar). This generally results from inhibition of myosin light chain phosphatase (MLCP), a myosin-associated type 1 protein-serine/threonine phosphatase that dephosphorylates LC20 phosphorylated by MLCK (10Hartshorne D.J. Ito M. Erdödi F. J. Muscle Res. Cell Motil. 1998; 19: 325-341Crossref PubMed Scopus (344) Google Scholar). MLCP inhibition occurs via signaling pathways that involve (i) protein kinase C (PKC) and its substrate protein CPI-17 (17-kDa phosphatase inhibitor and substrate of protein kinase C), which becomes a very potent inhibitor of type 1 phosphatase after phosphorylation by PKC (11Li L. Eto M. Lee M.R. Morita F. Yazawa M. Kitazawa T. J. Physiol. 1998; 508: 871-881Crossref PubMed Scopus (157) Google Scholar, 12Kitazawa T. Eto M. Woodsome T.P. Brautigan D.L. J. Biol. Chem. 2000; 275: 9897-9900Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar); (ii) the small GTPase RhoA, which activates Rho-associated kinase (ROK), which in turn phosphorylates the myosin-targeting subunit of MLCP, inhibiting phosphatase activity (13Kimura K. Ito M. Amano M. Chihara K. Fukata Y. Nakafuku M. Yamamori B. Feng J. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 273: 245-248Crossref PubMed Scopus (2426) Google Scholar, 14Swärd K. Dreja K. Susnjar M. Hellstrand P. Hartshorne D.J. Walsh M.P. J. Physiol. 2000; 522: 33-49Crossref PubMed Scopus (184) Google Scholar); or (iii) arachidonic acid, which inhibits MLCP activity either directly (15Gong M.C. Fuglsang A. Alessi D. Kobayashi S. Cohen P. Somlyo A.V. Somlyo A.P. J. Biol. Chem. 1992; 267: 21492-21498Abstract Full Text PDF PubMed Google Scholar) or via activation of an atypical PKC isoenzyme (16Gailly P. Gong M.C. Somlyo A.V. Somlyo A.P. J. Physiol. 1997; 500: 95-109Crossref PubMed Scopus (102) Google Scholar) or ROK (17Feng J. Ito M. Kureishi Y. Ichikawa K. Amano M. Isaka N. Okawa K. Iwamatsu A. Kaibuchi K. Hartshorne D.J. Nakano T. J. Biol. Chem. 1999; 274: 3744-3752Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). Of these signal transduction pathways, the RhoA-ROK pathway, appears to be the most important quantitatively (9Somlyo A.P. Somlyo A.V. J. Physiol. 2000; 522: 177-185Crossref PubMed Scopus (1074) Google Scholar). All of these signaling pathways terminate in inhibition of MLCP and an increase in myosin phosphorylation, which accounts for the contractile response. The kinase responsible for the increase in myosin phosphorylation without a change in [Ca2+]i could be MLCK itself that is partially activated at the prevailing [Ca2+]i, or it could be a distinct kinase capable of phosphorylating myosin at Ser19 in a Ca2+-independent manner. We recently obtained evidence for the presence in smooth muscle of a Ca2+-independent kinase that can phosphorylate LC20 at Ser19 and Thr18 (18Weber L.P. Van Lierop J.E. Walsh M.P. J. Physiol. 1999; 516: 805-824Crossref PubMed Scopus (133) Google Scholar). The addition of the phosphatase inhibitor microcystin-LR to Triton X-100-demembranated (skinned) rat tail arterial smooth muscle strips in the absence of Ca2+elicited a contraction that reached a steady-state level of force comparable with that evoked in skinned muscle by Ca2+ or in intact muscle by K+ depolarization. This Ca2+-independent contraction correlated with phosphorylation of myosin at Ser19 and Thr18 of LC20. The kinase responsible for this phosphorylation was not MLCK for the following reasons: (i) its activity was independent of Ca2+ and CaM; (ii) it did not bind to CaM in either the presence or absence of Ca2+; (iii) its activity was insensitive to AV25, a synthetic peptide inhibitor of MLCK corresponding to the autoinhibitory (pseudosubstrate) domain of MLCK; (iv) it phosphorylated Ser19 and Thr18 of LC20, whereas MLCK only phosphorylates Thr18 at high kinase concentrations and only after Ser19 has been fully phosphorylated (6Ikebe M. Hartshorne D.J. J. Biol. Chem. 1985; 260: 10027-10031Abstract Full Text PDF PubMed Google Scholar, 19Ikebe M. Hartshorne D.J. Elzinga M. J. Biol. Chem. 1986; 261: 36-39Abstract Full Text PDF PubMed Google Scholar); and (v) the activity could be separated from MLCK. The kinase appeared to be associated with the myofilaments, since Ca2+-independent kinase activity directed toward endogenous myosin LC20 was recovered in myofilament preparations. We were able to separate Ca2+-independent kinase activity from MLCK by differential extraction from myofilaments and by affinity chromatography on a column of CaM-Sepharose (18Weber L.P. Van Lierop J.E. Walsh M.P. J. Physiol. 1999; 516: 805-824Crossref PubMed Scopus (133) Google Scholar). We report here the identification of this Ca2+-independent kinase as integrin-linked kinase (ILK) and provide evidence for two distinct pools of this enzyme in smooth muscle, one of which is associated with the myofilaments and is responsible for the Ca2+-independent phosphorylation of myosin. ILK could account, at least in part, for the phosphorylation of myosin and contraction that occurs in smooth muscle in response to agonists that trigger a contractile response without a change in [Ca2+]i. Furthermore, ILK could play a role in the contractile response to agonists that induce both an increase in [Ca2+]i and inhibition of MLCP. These results also raise the possibility that ILK may play an important role in regulation of nonmuscle motile processes such as cell migration, chemotaxis, and cytokinesis. Finally, this work confirms that ILK is indeed a functional protein-serine/threonine kinase. [γ-32P]ATP (>5000 Ci/mmol) was purchased from Amersham Pharmacia Biotech, Triton X-100 was from Roche Molecular Biochemicals, Tween 80 was from Fisher, anti-ILK was from Upstate Biotechnology Inc., and molecular mass marker proteins were from New England Biolabs. MLCK inhibitor peptides AV25 (AKKLAKDRMKKYMARRKLQKAGHAV) and SM-1 (AKKLSKDRMKKYMARRKWQKTG) were synthesized in the Peptide Synthesis Core Facility at the University of Calgary. The purity of the peptides (>95%) was by high chromatography and Chicken gizzard myosin II A. Hartshorne D.J. Science. PubMed Scopus Google Scholar), LC20 Biochem. PubMed Scopus Google Scholar), MLCK C.A. Walsh M.P. Biochem. J. PubMed Scopus Google Scholar), and the of MLCK (18Weber L.P. Van Lierop J.E. Walsh M.P. J. Physiol. 1999; 516: 805-824Crossref PubMed Scopus (133) Google Scholar) were as was by Hartshorne of to LC20 were by Y. Sasaki and M. at Ser19, and LC20 phosphorylated at both Thr18 (18Weber L.P. Van Lierop J.E. Walsh M.P. J. Physiol. 1999; 516: 805-824Crossref PubMed Scopus (133) Google Scholar). All were or and were purchased from or Chicken gizzard myofilaments were as All were at were in a and in of 1 1 1 dithiothreitol 1 with the of a The was at for The was The was in of Triton with the of a and as The was in of Triton and as The was in of Triton and as The was in of and as The was in of 80 1 1 1 for and as This extraction with the MLCK (18Weber L.P. Van Lierop J.E. Walsh M.P. J. Physiol. 1999; 516: 805-824Crossref PubMed Scopus (133) Google Scholar). The was in of and of C were with The was for and as The which the Ca2+-independent myosin LC20 kinase, was C 1 1 to the The was as to and the was to chromatography at a of on a column of with Ca2+-independent kinase activity were by myosin phosphorylation in the absence of (18Weber L.P. Van Lierop J.E. Walsh M.P. J. Physiol. 1999; 516: 805-824Crossref PubMed Scopus (133) Google Scholar). The which the Ca2+-independent LC20 kinase, were 1 1 The was at a of to a column of with and the column was with of D. including the Ca2+-independent LC20 kinase, were with a at Ca2+-independent kinase were and to a proteins with a in of 1 were and for Ca2+-independent MLCK were with an of gel and and at for to electrophoresis in with a Walsh M.P. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar) with the gel gel were at with Triton X-100 for to the and for a in Tween 80 The was with of kinase and LC20 phosphorylation was by the addition of of at for the gel was with acid, the in the was The gel was and to Walsh M.P. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). ILK cDNA was from gizzard by chain reaction and corresponding to and the of the cDNA L. G. J. J.C. S. Nature. 1996; PubMed Scopus Google Scholar). ILK cDNA was by and of cDNA PubMed Scopus Google Scholar). The cDNA was by the chain F. Science. PubMed Scopus Google Scholar). laser mass spectrometry was on the kinase from the column and one of the peptides was directly by in the by ILK cDNA was of the Pharmacia The and the as a were to was with for was from the by affinity and the was by following the Pharmacia We showed that myofilaments from gizzard smooth muscle endogenous Ca2+-independent kinase activity toward myosin LC20, phosphorylation at Ser19 and Thr18 (18Weber L.P. Van Lierop J.E. Walsh M.P. J. Physiol. 1999; 516: 805-824Crossref PubMed Scopus (133) Google Scholar). These myofilaments also the of MLCK (18Weber L.P. Van Lierop J.E. Walsh M.P. J. Physiol. 1999; 516: 805-824Crossref PubMed Scopus (133) Google Scholar). the of these two we the of synthetic peptide of corresponding to the autoinhibitory domain of on the phosphorylation of endogenous myosin in the presence of Ca2+ and These peptides the most of MLCK M. Biochem. Res. 1990; PubMed Scopus Google Scholar, B. Arch. Biochem. 1990; PubMed Scopus Google Scholar, Means A.R. B.E. Science. 1992; PubMed Scopus Google Scholar). SM-1 to the autoinhibitory of MLCK of gizzard and AV25 is a peptide corresponding to of gizzard MLCK with was by to of the peptide with and and were by to The two peptides to be of obtained from were shown in most of the MLCK activity in myofilaments can be for by a was to be insensitive to inhibition by the autoinhibitory domain of MLCK 1 MLCK was by the MLCK with inhibition at 1 peptide a peptide inhibitor of protein kinase II kinase an enzyme to phosphorylate myosin in D.J. Biochem. 1990; PubMed Scopus Google Scholar), on Ca2+-independent in myofilaments the phosphorylation of in myofilaments by CaM kinase II, as (18Weber L.P. Van Lierop J.E. Walsh M.P. J. Physiol. 1999; 516: 805-824Crossref PubMed Scopus (133) Google Scholar). CaM kinase II, or a of CaM kinase II, is not in myosin phosphorylation in the the Ca2+-independent phosphorylation of myosin in myofilaments was by a kinase with inhibition at L.P. Van Lierop J.E. Walsh M.P. J. Physiol. 1999; 516: 805-824Crossref PubMed Scopus (133) Google Scholar). also Ca2+-independent contraction of skinned smooth muscle (18Weber L.P. Van Lierop J.E. Walsh M.P. J. Physiol. 1999; 516: 805-824Crossref PubMed Scopus (133) Google Scholar). The Ca2+-independent phosphorylation of myosin in myofilaments was insensitive to the inhibitor and the ROK inhibitor (18Weber L.P. Van Lierop J.E. Walsh M.P. J. Physiol. 1999; 516: 805-824Crossref PubMed Scopus (133) Google Scholar). The MLCK activity that was to the MLCK inhibitor peptides was partially from myofilaments by MLCK by extraction with (18Weber L.P. Van Lierop J.E. Walsh M.P. J. Physiol. 1999; 516: 805-824Crossref PubMed Scopus (133) Google Scholar). The Ca2+-independent kinase was with and by chromatography on of and The of Ca2+-independent MLCK activity from the column is shown in A. kinase in the absence of with LC20 as showed that this activity in a protein This which was in a gel of the corresponding C), was of the with and by J.T. Biol. PubMed Scopus Google Scholar). of the of the peptides with from the protein revealed a to ILK L. G. J. J.C. S. Nature. 1996; PubMed Scopus Google Scholar). peptides from and the peptides for of the confirm its as one of the peptides was and directly by in the J.T. Biol. PubMed Scopus Google Scholar). was as corresponding to of the mass was evidence of a protein in the for of Ca2+-independent myosin light chain in a of the Ca2+-independent MLCK as ILK was by with an to a of Ca2+-independent MLCK activity from the in the kinase and with anti-ILK both at MLCK can be to a that the domain the autoinhibitory and M. S. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the possibility that ILK the column is with a of ILK from the column were to with an that both intact MLCK and the of MLCK (18Weber L.P. Van Lierop J.E. Walsh M.P. J. Physiol. 1999; 516: 805-824Crossref PubMed Scopus (133) Google Scholar). that the ILK of MLCK whereas both intact MLCK and the by the Furthermore, Ca2+-independent phosphorylation of myosin by ILK the column was by AV25, the that the ILK is not with MLCK of myosin by Ca2+-independent myosin LC20 kinase is by Smooth muscle myosin was phosphorylating in the absence 1 and and presence and of from the column and in the absence and presence of AV25, as (18Weber L.P. Van Lierop J.E. Walsh M.P. J. Physiol. 1999; 516: 805-824Crossref PubMed Scopus (133) Google Scholar). the of the were to and The is to molecular mass ILK and of and and intact myosin LC20 was phosphorylated to a of of of LC20 LC20 and and intact myosin to of of myosin LC20 and The of phosphorylation were as Ser19 and Thr18 phosphorylation a of the ILK in gizzard smooth muscle was recovered in the cytosolic following of the in the absence of a in the of myofilaments and with its in smooth muscle, it phosphorylates myosin in in the absence of Ca2+ and a Ca2+-independent contraction (18Weber L.P. Van Lierop J.E. Walsh M.P. J. Physiol. 1999; 516: 805-824Crossref PubMed Scopus (133) Google Scholar). kinase of ILK activity was recovered in the ILK cDNA was from gizzard by chain reaction Lee J.E. M. K. M. K. A. 1998; Scholar) and F. Science. PubMed Scopus Google Scholar). The and of ILK shown in and from of the and ILK is shown in The of ILK is and to the ILK with and in the with protein-serine/threonine ILK a of in the domain T. Science. 1988; PubMed Scopus Google Scholar). The in of most protein is in ILK and in the in in ILK and in and in in both the of these it has been that ILK may not be a functional protein kinase A. A. M. A. P. R. Alessi Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, C.A. 1999; 18: PubMed Scopus Google Scholar). The that we the Ca2+-independent MLCK activity and it as ILK confirms that ILK is indeed an protein-serine/threonine of the of and The to and the to of the mass of ILK on the peptide an of the of Ca2+-independent myosin light chain in a ILK was expressed in E. as a protein and from a cell by affinity chromatography was shown to phosphorylate LC20 in intact smooth muscle myosin in the absence of Ca2+ The the of of smooth muscle contraction has a of recently (9Somlyo A.P. Somlyo A.V. J. Physiol. 2000; 522: 177-185Crossref PubMed Scopus (1074) Google Scholar). Ca2+ to the contraction of smooth muscle occurs without a change in [Ca2+]i and appears to involve signal transduction pathways that terminate in inhibition of MLCP, an increase in myosin LC20 phosphorylation, and cross-bridge of MLCP induce an increase in LC20 phosphorylation, the of activity in of the kinase. The as to which kinase is responsible for LC20 phosphorylation following inhibition of MLCP. is MLCK. In this in the absence of the activity of MLCP the activity of MLCK that at the prevailing [Ca2+]i, and inhibition of MLCP this MLCK to an increase in LC20 phosphorylation and [Ca2+]i in smooth muscle which is in the of J. Physiol. 1986; PubMed Google Scholar, Science. PubMed Scopus Google Scholar), be to induce activation of MLCK Herring B.P. A. J. Stull J.T. J. Biol. Chem. Full Text PDF PubMed Google Scholar). This the possibility that a Ca2+-independent kinase may be responsible for the increase in and contraction at [Ca2+]i, In of this we recently that the phosphatase inhibitor microcystin can trigger and contraction of skinned smooth muscle in the absence of phosphorylation at Ser19 and Ser19 the of phosphorylation by MLCK (18Weber L.P. Van Lierop J.E. Walsh M.P. J. Physiol. 1999; 516: 805-824Crossref PubMed Scopus (133) Google Scholar). This Ca2+-independent phosphorylation was by a kinase distinct from since it was to be to the MLCK peptide AV25 and and could be separated from MLCK. In this study, we the in the of a of the myosin phosphorylating activity was to the peptide AV25 and kinase we also shown that this kinase is not CaM kinase II, or ROK (18Weber L.P. Van Lierop J.E. Walsh M.P. J. Physiol. 1999; 516: 805-824Crossref PubMed Scopus (133) Google Scholar). is also distinct from a kinase that was in of MLCP that phosphorylated both the myosin subunit of MLCP and myosin LC20, since that kinase was shown to be to K. Ito M. Hartshorne D.J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). to the kinase inhibitor the Ca2+-independent kinase activity was at and the partially kinase at shown in and we that the kinase is not with a of MLCK that has the for Ca2+ and CaM to with its to phosphorylate myosin in skinned smooth muscle this kinase is in myofilament its with a component of the The of this work was to and identify this Ca2+-independent myosin LC20 kinase, from myofilaments from a of smooth muscle The of MLCK was from the myofilaments by with following which the Ca2+-independent kinase was with high The kinase was by chromatography on of and The partially kinase phosphorylated LC20 and intact myosin at Ser19 and Thr18 of LC20 in a Ca2+-independent and The kinase activity was associated with a as shown by the of from the column to phosphorylate myosin in the absence of Ca2+ in and to phosphorylate LC20 in a Ca2+-independent in the kinase peptide mass by mass and of a by the kinase as ILK and was by with to ILK ILK was as an enzyme that binds to the domain of and a role in signal transduction L. G. J. J.C. S. Nature. 1996; PubMed Scopus Google Scholar, S. B. Trends Cell Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). membrane the identification of the Ca2+-independent LC20 kinase as ILK the of a kinase to a substrate in the a fractionation we two distinct populations of one with the of ILK and the that was in myofilament the is responsible for the Ca2+-independent phosphorylation of myosin in myofilaments and skinned smooth muscle preparations. is that ILK in domain and that the myosin-targeting subunit of MLCP, which the phosphatase to the myosin and this of the is in the with myosin K. K. Ito M. J. Nakano T. Hartshorne D.J. 1996; PubMed Scopus Google Scholar). is that ILK may with the myosin of smooth muscle via its In of this myosin a level of Ca2+-independent LC20 kinase activity that is to the MLCK inhibitor AV25 1 and that ILK may be associated with a myosin in the A. G. S. Res. 2000; PubMed Scopus Google Scholar) recently that receptors for contractile agonists such as angiotensin II, and to both and the of and agonists, to induce contraction via both activation of MLCK of and inhibition of MLCP of The possibility that ILK may also play a role in these contractile to the increase in LC20 phosphorylation that ILK may not be a functional protein-serine/threonine kinase, on a of in the domain and the to kinase activity in of ILK A. A. M. A. P. R. Alessi Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, C.A. 1999; 18: PubMed Scopus Google Scholar). The on ILK is since protein been with in the and in the K. N. R. M. M. D. Full Text PDF PubMed Scopus Google Scholar, Full Text PDF PubMed Scopus Google Scholar). Furthermore, we expressed ILK in E. and Ca2+-independent kinase activity toward intact myosin, as shown in to this activity by of the expressed kinase. Furthermore, the activity of the expressed ILK was to that of the smooth muscle that the kinase expressed in not the and may on these and the that we the Ca2+-independent kinase activity in smooth muscle myofilaments and the kinase as we that ILK is indeed a protein-serine/threonine kinase. ILK is in of its expressed in most with in and muscle, and its is of nonmuscle myosin phosphorylation (9Somlyo A.P. Somlyo A.V. J. Physiol. 2000; 522: 177-185Crossref PubMed Scopus (1074) Google Scholar), to the regulation of smooth muscle The possibility that ILK may also play a role in regulation of nonmuscle motile processes such as cell migration, chemotaxis, and contraction of in G. J. Physiol. 1999; PubMed Scopus Google Scholar) recently that contraction of in response to the Ca2+-independent phosphorylation of myosin Finally, it is that ILK is to regulation by as that be the of ILK is to be activated by M. L. J. S. Sci. S. A. 1998; PubMed Scopus Google Scholar), with the presence of a domain a domain in the of the S. B. Trends Cell Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). we of on the phosphorylation of myosin by partially smooth muscle ILK not The results of this provide the of a novel role for ILK in Ca2+ of smooth muscle signaling pathways that to inhibition of MLCP the activity of which phosphorylates myosin and activates contraction in a Ca2+-independent ILK is also to to the increase in myosin phosphorylation that occurs in response to activation of the which to both and the of ILK L. G. J. J.C. S. Nature. 1996; PubMed Scopus Google Scholar), it is also that ILK may be in regulation of nonmuscle We very to S. for of the and

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