Ca2+-independent Smooth Muscle Contraction

A NOVEL FUNCTION FOR INTEGRIN-LINKED KINASE*

Jing Ti Deng, Jacquelyn E. Van Lierop, Cindy Sutherland, and Michael P. WalshDagger

From the Smooth Muscle Research Group and Canadian Institutes of Health Research Group in Regulation of Vascular Contractility, Department of Biochemistry and Molecular Biology, University of Calgary Faculty of Medicine, Calgary, Alberta T2N 4N1, Canada

Received for publication, December 22, 2000, and in revised form, February 7, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha 1-adrenergic agonists, or stretch (1, 2). Ca2+ originates from the extracellular space and/or intracellular stores, principally the sarcoplasmic reticulum (3). Ca2+ binds to calmodulin (CaM)1 (4), which activates myosin light chain kinase (MLCK) (5), resulting in phosphorylation of myosin II, specifically at Ser19, and sometimes to a small extent at Thr18 (6-8), 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 (1, 2). Several agonists that act via seven-transmembrane domain-containing, G protein-coupled receptors elicit a contractile response without a change in [Ca2+]i (9). 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 (10). 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 (11, 12); (ii) the small GTPase RhoA, which activates Rho-associated kinase (ROK), which in turn phosphorylates the myosin-targeting subunit of MLCP, inhibiting phosphatase activity (13, 14); or (iii) arachidonic acid, which inhibits MLCP activity either directly (15) or via activation of an atypical PKC isoenzyme (16) or ROK (17). Of these signal transduction pathways, the RhoA-ROK pathway, appears to be the most important quantitatively (9).

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 (18). 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 (6, 19); 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 (18).

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials-- [gamma -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 confirmed by analytical high performance liquid chromatography and amino acid analysis. Chicken gizzard myosin II (20), LC20 (21), MLCK (22), and the 61-kDa constitutively active fragment of MLCK (18) were purified as described. Anti-MLCK was generously provided by Dr. David Hartshorne (University of Arizona). Phosphorylation site-specific antibodies to LC20 were generously provided by Drs. Y. Sasaki and M. Seto (Asahi Chemical Industry Co., Ltd., Shizuoka, Japan); antibody pLC1 recognizes exclusively LC20 phosphorylated at Ser19, and antibody pLC2 recognizes exclusively LC20 phosphorylated at both Ser19 and Thr18 (18). All other reagents were analytical grade or better and were purchased from VWR Canlab or Sigma.

Purification of Ca2+-independent Myosin LC20 Kinase-- Chicken gizzard myofilaments were prepared as follows. All procedures were carried out at 0-4 °C. Fresh gizzards (100 g) were trimmed, minced in a meat grinder, and homogenized in 500 ml of buffer A (20 mM imidazole-HCl, pH 6.9, 60 mM KCl, 1 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol (DTT), 1 µg/ml leupeptin, 0.5 mM phenylmethylsulfonyl fluoride) with the aid of a Brinkman Polytron (setting 6). The homogenate was centrifuged at 20,000 × g for 20 min. The supernatant was discarded. The pellet was resuspended in 500 ml of buffer A plus 0.5% Triton X-100, homogenized with the aid of a Brinkman Polytron (setting 5), and centrifuged as before. The pellet was resuspended in 500 ml of buffer A plus 0.5% Triton X-100, homogenized, and centrifuged as before. The pellet was resuspended in 500 ml of buffer A plus 0.3% Triton X-100, homogenized, and centrifuged as before. The pellet was resuspended in 500 ml of buffer A, homogenized, and centrifuged as before. The pellet (myofilament preparation) was resuspended in 500 ml of buffer B (20 mM Tris-HCl, pH 7.5, 80 mM KCl, 30 mM MgCl2, 1 mM EGTA, 1 mM DTT, 1 µg/ml leupeptin, 0.5 mM phenylmethylsulfonyl fluoride), stirred gently for 30 min, and centrifuged as before. This extraction with 30 mM MgCl2 removes almost all the MLCK (18). The pellet was resuspended in 473.7 ml of buffer B, and 31.3 ml of buffer C (buffer B plus 4 M NaCl) were added slowly with stirring. The mixture was stirred gently for 30 min and centrifuged as before. The supernatant, which contained the Ca2+-independent myosin LC20 kinase, was dialyzed overnight against buffer C (20 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 0.1 M NaCl, 1 mM DTT) to lower the NaCl concentration. The dialysate was centrifuged as before to remove particulate material, and the supernatant was subjected to chromatography at a flow rate of 40 ml/h on a column of DEAE-Sephacel (1.5 × 20 cm) previously equilibrated with buffer C. Fractions containing Ca2+-independent kinase activity were identified by assaying myosin phosphorylation in the absence of Ca2+ as previously described (18). The flow-through fractions, which contained the Ca2+-independent LC20 kinase, were dialyzed overnight against buffer D (20 mM NaHepes, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 0.1 M NaCl, 1 mM DTT). The dialysate was applied at a flow rate of 60 ml/h to a column of SP-Sepharose Fast Flow (1.5 × 15 cm) previously equilibrated with buffer D, and the column was washed with 100 ml of buffer D. Bound proteins, including the Ca2+-independent LC20 kinase, were eluted with a linear [NaCl] gradient (0.1-0.6 M) at 40 ml/h, collecting 10-ml fractions. Fractions containing Ca2+-independent kinase were pooled, dialyzed overnight against buffer D, and applied to a Mono Q FPLC column, eluting bound proteins with a linear [NaCl] gradient (0.1-0.4 M in buffer D). Fractions of 1 ml were collected and assayed for Ca2+-independent MLCK activity.

In-gel Kinase Assay-- Protein samples were mixed with an equal volume of 2 × SDS gel sample buffer (50 mM Tris-HCl, pH 6.8, 1% SDS, 30% glycerol, and 0.01% bromphenol blue) and incubated at 20 °C for 2 h prior to electrophoresis in SDS gels with a 10-20% acrylamide gradient (23) with LC20 throughout the 0.75-mm thick gel (5 µg/ml running gel solution). Gels were washed at room temperature with 25 mM Tris-HCl, pH 7.5, 60 mM KCl, 10 mM MgCl2, 10 mM DTT, 10 mM EGTA, 2.5% Triton X-100 for 2 h to remove the SDS and then washed for a further 2 h in 20 mM Tris-HCl, pH 7.5, 60 mM KCl, 10 mM MgCl2, 10 mM DTT, 10 mM EGTA, 0.1% Tween 80 (kinase assay buffer). The buffer was replaced with 25 ml of fresh kinase assay buffer, and LC20 phosphorylation was initiated by the addition of 60 µM ATP containing 100 µCi of [gamma -32P]ATP. After incubation at 20 °C for 3 h, the gel was washed extensively with 5% trichloroacetic acid, 1% sodium pyrophosphate until the radioactivity in the wash solution was negligible. The gel was stained, destained, dried, and exposed to x-ray film (23).

Cloning of Chicken Gizzard ILK cDNA-- A partial ILK cDNA was cloned from chicken gizzard mRNA by reverse transcriptase-polymerase chain reaction using primers (5'-TGTGGCTGGACAACACAGAG-3' and 5'-GAGAGGTCAGCAAAGGGCAC-3') corresponding to nucleotides 53-72 and 1168-1187, respectively, within the coding region of the human cDNA (24). Full-length ILK cDNA was cloned by 5'- and 3'-rapid amplification of cDNA ends (25). The full-length cloned cDNA was sequenced by the Sanger dideoxynucleotide chain termination method (26).

Mass Spectrometric Analysis-- Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed on the trypsin-digested 59-kDa kinase band from the Mono Q column (see Fig. 2B), and one of the peptides (1594 Da) was directly sequenced by MALDI-TOF MS in the post-source decay mode by Borealis Biosciences Inc. (Toronto, Ontario, Canada).

Expression and Purification of Glutathione S-Transferase (GST)-ILK-- Full-length chicken ILK cDNA was inserted into the EcoRI/BamHI site of the vector pGEX-2T (Amersham Pharmacia Biotech). The recombinant plasmid pGEX-2T-ILK and the empty vector as a negative control were used to transform competent BL21(DE3)pLysS cells. Expression was induced with 0.1 mM isopropyl beta -D-thiogalactopyranoside for 3 h. GST-ILK was purified from the bacterial lysate by glutathione-Sepharose 4B affinity chromatography, and the GST was cleaved off by thrombin treatment following the manufacturer's instructions (Amersham Pharmacia Biotech).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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The Ca2+-independent Myosin Light Chain Kinase Is Integrin-linked Kinase-- We showed previously that myofilaments isolated from chicken gizzard smooth muscle contain endogenous Ca2+-independent kinase activity toward myosin LC20, phosphorylation occurring at Ser19 and Thr18 (18). These myofilaments also retain the full complement of cellular MLCK (18). To estimate the relative activities of these two myofilament-associated kinases, we examined the effect of synthetic peptide inhibitors of MLCK, corresponding to the autoinhibitory domain of MLCK, on the phosphorylation of endogenous myosin in the presence of Ca2+ and CaM. These peptides are the most highly selective inhibitors of MLCK (27-29). SM-1 corresponds to the autoinhibitory sequence of MLCK (residues 783-804 of chicken gizzard MLCK), and AV25 is a similar peptide corresponding to residues 783-807 of chicken gizzard MLCK with three substitutions (Trp800 was replaced by Leu to effectively eliminate interaction of the peptide with CaM, and Ser787 and Thr803 were replaced by Ala to eliminate phosphorylatable residues). The two peptides proved to be equipotent inhibitors of MLCK, so data obtained from their use were combined. As shown in Fig. 1, most of the MLCK activity in myofilaments can be accounted for by Ca2+/CaM-dependent MLCK, but a significant proportion was found to be insensitive to inhibition by the autoinhibitory domain of MLCK (Fig. 1A). For comparison, purified MLCK was completely inhibited by the MLCK inhibitory peptides, with half-maximal inhibition at 1 µM peptide (Fig. 1B). [Ala9]autocamtide 2, a peptide inhibitor of Ca2+/CaM-dependent protein kinase II (CaM kinase II), an enzyme known to phosphorylate myosin in vitro (30), had no effect on Ca2+-independent LC20 phosphorylation in myofilaments but potently inhibited the phosphorylation of caldesmon in myofilaments by exogenous CaM kinase II, as expected (18). CaM kinase II, or a constitutively active proteolytic fragment of CaM kinase II, is therefore not involved in myosin phosphorylation in myofilaments. On the other hand, the Ca2+-independent phosphorylation of myosin in myofilaments was inhibited by staurosporine, a relatively nonselective kinase inhibitor, with half-maximal inhibition at ~0.2 µM (18). Staurosporine also inhibited microcystin-induced, Ca2+-independent contraction of skinned smooth muscle (18). The Ca2+-independent phosphorylation of myosin in myofilaments was insensitive to the PKC-selective inhibitor chelerythrine and the ROK inhibitor HA-1077 (18).


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Fig. 1.   Smooth muscle myofilaments contain two distinct myosin light chain kinase activities. A, phosphorylation of the endogenous LC20 in chicken gizzard myofilaments was quantified in the presence of the indicated concentrations of the Ca2+/CaM-dependent MLCK inhibitory peptides AV25 or SM-1 (the effects of the two related peptides were identical, and therefore data have been combined). Conditions were as follows: 25 mM Tris-HCl, pH 7.5, 60 mM KCl, 10 mM MgCl2, 10 mM DTT, 0.1 mM CaCl2, 10 µM okadaic acid, 10 µg/ml CaM, 0.63 mg/ml myofilament protein (0.111 mg/ml myosin), 0-100 µM peptide, and 1 mM [gamma -32P]ATP. Reactions (60 µl) were started with ATP, incubated at 30 °C for 75 s, and stopped by the addition of an equal volume of 2× SDS gel sample buffer and boiled. Samples were electrophoresed in 10-20% polyacrylamide gradient gels and autoradiographed. LC20 phosphorylation was quantified by scanning densitometry, and results are expressed as mol of Pi incorporated/mol of LC20 (urea/glycerol gel electrophoresis (18) indicated that the level of LC20 phosphorylation in the absence of peptide was 1.0 mol of Pi/mol of LC20). Numbers beside the symbols indicate n values. B, the activity of purified Ca2+/CaM-dependent MLCK was assayed at the indicated concentrations of AV25 or SM-1 (again effects were identical, and therefore data have been combined). Conditions were as follows: 25 mM Tris-HCl, pH 7.5, 60 mM KCl, 10 mM MgCl2, 10 mM DTT, 0.1 mM CaCl2, 0.1% Tween 80, 10 µM microcystin-LR, 10 µg/ml CaM, 10 µg/ml MLCK, 0.5 mg/ml myosin, 0-100 µM peptide, and 0.2 mM [gamma -32P]ATP (300 cpm/pmol). Reactions (30 µl) were started with ATP, incubated at 30 °C for 75 s, and stopped by spotting 20 µl on P81 paper. LC20 phosphorylation was quantified as described (18). The inset shows a replot of the data on a log scale to show the peptide concentration dependence of the inhibition more clearly.

The MLCK activity that was resistant to the MLCK inhibitor peptides was partially purified from myofilaments by first removing MLCK by extraction with 30 mM Mg2+ (18). The Ca2+-independent kinase was then extracted with 0.25 M NaCl and further purified by sequential chromatography on columns of DEAE-Sephacel, SP-Sepharose, and Mono Q. The elution profile of Ca2+-independent MLCK activity from the final column is shown in Fig. 2A. An in-gel kinase assay in the absence of Ca2+, with isolated LC20 as substrate, showed that this activity resides in a 59-kDa protein (Fig. 2B). This band, which was barely visible in a Coomassie Blue-stained gel of the corresponding Mono Q fractions (Fig. 2C), was cut out of the gel, digested with trypsin, and analyzed by MALDI-TOF MS (Table I) (31). Comparison of the measured masses of the tryptic peptides with predicted masses from the protein sequence data bases revealed a single match to ILK (24). Peptides 1-11 perfectly matched predicted tryptic peptides from human ILK, and together the 11 peptides accounted for 31% of the complete sequence. To confirm its identity as ILK, one of the tryptic peptides (peptide 6) was selected and sequenced directly by MALDI-TOF MS in the postsource decay mode (31). Its sequence was confirmed as GMAFLHTLEPLIPR, corresponding to residues 304-317 of human ILK. From the mass spectrometric analysis, there was no evidence of a second protein in the 59-kDa band used for trypsin digestion.


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Fig. 2.   Elution profile of Ca2+-independent MLCK from Mono Q. A, fractions eluted from the Mono Q column were assayed for MLCK activity in the absence of Ca2+ with intact myosin as substrate. Phosphorylation of LC20 was analyzed by SDS-PAGE and autoradiography. The autoradiogram is shown. B, fractions eluted from the Mono Q column were subjected to an in-gel kinase assay to identify the Ca2+-independent MLCK. Isolated LC20 was incorporated throughout the gel by the addition to the gel buffers prior to polymerization. Mono Q fractions were then subjected to SDS-PAGE, following which the proteins in the gel were renatured and [gamma -32P]ATP was added to the bathing solution. Incorporation of 32P into LC20 was identified by autoradiography. The arrow indicates the Ca2+-independent MLCK of 59 kDa. Numbers to the right (in kDa) indicate the positions of molecular mass marker proteins. C, Coomassie Brilliant Blue-stained SDS gel of Mono Q fractions. The faintly staining band at 59 kDa in fractions 11-15 (indicated by the arrow) correlates with Ca2+-independent MLCK activity. This band was recognized by anti-ILK (see Fig. 3). The stronger staining band of the same molecular weight in fractions 16-21 is a distinct protein (it was not recognized by anti-ILK).

                              
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Table I
MALDI-TOF MS analysis of Ca2+-independent myosin light chain kinase

Identification of the Ca2+-independent MLCK as ILK was confirmed by Western blotting with an antibody to human ILK. Fig. 3 shows a correlation of Ca2+-independent MLCK activity (eluted from the Mono Q column) in the in-gel kinase assay (Fig. 3A) and immunoreactivity with anti-ILK (Fig. 3B), both at 59 kDa.


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Fig. 3.   Correlation between in-gel kinase activity and ILK immunoreactivity. Mono Q fractions corresponding to the elution of Ca2+-independent MLCK activity were subjected to the in-gel kinase assay (A), and the same fractions were immunoblotted with anti-ILK (B). Lanes 1-10 correspond to successive fractions in the peak of Ca2+-independent MLCK activity (identified by solution assay with intact myosin as substrate; cf. Fig. 2A).

ILK Is Not Contaminated with a MLCK Fragment-- Ca2+/CaM-dependent MLCK can be proteolytically degraded to a constitutively active fragment that retains the catalytic domain but lacks the autoinhibitory and CaM-binding domains (32). To rule out the possibility that ILK purified through the Mono Q column is contaminated with a constitutively active fragment of Ca2+/CaM-dependent MLCK, pooled ILK fractions from the Mono Q column were subjected to Western blotting with an antibody that recognizes both intact MLCK (130 kDa) and the 61-kDa catalytically active fragment of Ca2+/CaM-dependent MLCK (18). Fig. 4 shows that the ILK preparation contains no trace of Ca2+/CaM-dependent MLCK fragments (lane 3), whereas both intact Ca2+/CaM-dependent MLCK (lane 1) and the 61-kDa constitutively active tryptic fragment (lane 2) are clearly recognized by the antibody. Furthermore, Ca2+-independent phosphorylation of myosin by ILK purified through the Mono Q column was unaffected by AV25, supporting the conclusion that the ILK preparation is not contaminated with proteolyzed MLCK (Fig. 5).


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Fig. 4.   ILK purified through the Mono Q column is not contaminated with MLCK. Intact MLCK (1.3 µg; lane 1), a constitutively active fragment of MLCK (61 kDa) generated by trypsin digestion (0.61 µg; lane 2), and pooled ILK-containing fractions from the Mono Q column (6 µg of protein; lane 3) were subjected to Western blotting with anti-MLCK.


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Fig. 5.   Phosphorylation of myosin by Mono Q-purified Ca2+-independent myosin LC20 kinase is unaffected by AV25. Smooth muscle myosin was incubated under phosphorylating conditions in the absence (lanes 1 and 2) and presence (lanes 3 and 4) of pooled ILK-containing fractions from the Mono Q column (178.5 µl/ml) and in the absence and presence of 50 µM AV25, as described previously (18). At the end of the reaction, samples (40 µl) were subjected to SDS-PAGE and autoradiography. The autoradiogram is shown. Numbers to the right indicate molecular mass markers.

ILK Phosphorylates Myosin at Ser19 and Thr18 of LC20-- Mono Q-purified ILK catalyzed mono- and diphosphorylation of isolated LC20 (Fig. 6A, lanes 2 and 3) and intact myosin (Fig. 6A, lane 5). Isolated LC20 was phosphorylated to a stoichiometry of 1.64 mol of Pi/mol of LC20 (36.2% monophosphorylated LC20 and 63.8% diphosphorylated LC20) and intact myosin to 1.14 mol of Pi/mol of myosin (45.4% monophosphorylated LC20 and 5.7% diphosphorylated LC20). The sites of phosphorylation were identified as Ser19 and Thr18 using phosphorylation site-specific antibodies (Fig. 6B).


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Fig. 6.   Ca2+-independent MLCK catalyzes mono- and diphosphorylation of isolated LC20 and intact myosin at Ser19 and Thr18. A, LC20 (lanes 1-3) or myosin (lanes 4 and 5) was incubated under phosphorylating conditions in the absence (lanes 1 and 4) or presence of Mono Q-purified Ca2+-independent MLCK (lanes 2, 3, and 5). Two different kinase preparations were used in lanes 2 and 3. Unphosphorylated and mono- and diphosphorylated forms of LC20 (designated P0-, P1-, and P2-LC20, respectively) were separated by urea/glycerol gel electrophoresis (18). The two fastest migrating bands in lanes 4 and 5 are CaM and the 17-kDa light chain of myosin. B, intact myosin was incubated under phosphorylating conditions in the absence (lanes 1 and 3) or presence of Mono Q-purified Ca2+-independent MLCK (lanes 2 and 4) prior to SDS-PAGE and Western blotting with antibody pLC1 that recognizes only LC20 phosphorylated at Ser19 (lanes 1 and 2) or antibody pLC2 that recognizes only LC20 phosphorylated at both Ser19 and Thr18 (lanes 3 and 4).

ILK Exists in Two Distinct Subcellular Fractions in Smooth Muscle-- While a portion of the ILK in chicken gizzard smooth muscle was recovered in the cytosolic fraction following homogenization of the tissue in the absence of detergent (Fig. 7, lane 2), a substantial fraction remained in the Triton-insoluble fraction composed of myofilaments and cytoskeleton (Fig. 7, lane 6), consistent with its retention in Triton-skinned smooth muscle, where it phosphorylates myosin in situ in the absence of Ca2+ and elicits a Ca2+-independent contraction (18). From in-gel kinase assays, ~73% of total ILK activity was recovered in the Triton-insoluble fraction.


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Fig. 7.   Subcellular fractionation indicates two distinct populations of ILK in smooth muscle. Fractions obtained during the purification of ILK as described under "Experimental Procedures" were analyzed by Western blotting with anti-ILK. Lane 1, chicken gizzard total homogenate; lane 2, the homogenate supernatant (cytosolic fraction prepared in the absence of detergent); lanes 3-5, three successive washes of the pellet with Triton X-100 (0.5, 0.5, and 0.3%, respectively) following centrifugation of the homogenate; lane 6, the final detergent-washed pellet or myofilament preparation; lane 7, pooled fractions from the DEAE-Sephacel column; lane 8, pooled fractions from the SP-Sepharose column; lane 9, pooled fractions from the Mono Q columns. Samples of 30 µl were applied to each lane.

Chicken ILK Is Very Similar to Human ILK-- ILK cDNA was cloned from chicken gizzard mRNA by reverse transcription-polymerase chain reaction (33) and sequenced (26). The nucleotide and deduced amino acid sequences of chicken ILK are shown in Fig. 8 and are available from GenBankTM under accession number AF296130. A comparison of the chicken and human ILK amino acid sequences is shown in Fig. 9. The deduced amino acid sequence of chicken ILK is 90.7% identical and 95.4% similar to the human ILK sequence (GenBankTM accession number U40282), with 21 conservative and 21 nonconservative substitutions in the 452-residue protein. Compared with many other protein-serine/threonine kinases, ILK contains a number of amino acid substitutions in highly conserved subdomains within the catalytic domain (34). The GXGXXG sequence in subdomain I of most protein kinases is NENHSG in human ILK and NENQSG in the chicken enzyme. Conserved residues in subdomain VIB (HDRL) are PRHA in human ILK and PRHH in chicken ILK, and conserved residues in subdomain VII (DFG) are MAD in both ILKs. On the basis of these amino acid substitutions, it has been suggested that ILK may not be a functional protein kinase (35, 36). The fact that we isolated the Ca2+-independent MLCK activity and then identified it as ILK confirms that ILK is indeed an active protein-serine/threonine kinase.


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Fig. 8.   The nucleotide sequence and deduced amino acid sequence of chicken ILK. The cloned ILK cDNA was 1934 nucleotides in length, and the encoded protein consists of 452 amino acids. The asterisk denotes a stop codon. These sequences are available from GenBankTM under accession number AF296130.


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Fig. 9.   Comparison of the amino acid sequences of chicken and human ILK. The upper sequence corresponds to chicken ILK, and the lower sequence corresponds to human ILK.

Reexamination of the mass spectrometric analysis of chicken ILK based on the deduced chicken sequence identified nine more peptide matches covering an additional 18% of the sequence (Table II).

                              
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Table II
Further MALDI-TOF MS analysis of Ca2+-independent myosin light chain kinase

Expression of Chicken ILK-- Full-length chicken ILK was expressed in E. coli as a GST fusion protein and purified from a bacterial cell lysate by glutathione-Sepharose affinity chromatography (Fig. 10A). Purified GST-ILK was shown to phosphorylate LC20 in intact smooth muscle myosin in the absence of Ca2+ (Fig. 10B).


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Fig. 10.   Expression and characterization of chicken ILK. A, Coomassie Blue-stained gel of purified GST-ILK expressed in E. coli (lane 3) and of purified ILK (lane 2) and GST (lane 4) following digestion of GST-ILK with thrombin and purification. Molecular mass marker proteins are shown in lane 1 with their masses in kDa indicated to the left. B, phosphorylation of myosin by bacterially expressed ILK. Intact myosin (0.5 mg/ml) was incubated at 30 °C for 60 min in 25 mM Tris-HCl, pH 7.5, 60 mM KCl, 10 mM MgCl2, 10 mM DTT, 0.1% Tween 80, 10 mM EGTA, 10 µM microcystin, and 0.2 mM ATP (containing 100 µCi/ml [gamma -32P]ATP) in the absence (lane 1) or presence (lane 2) of purified GST-ILK. LC20 phosphorylation was analyzed by SDS-PAGE and autoradiography. The autoradiogram shows the ILK-dependent phosphorylation of LC20 in intact myosin.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The mechanism underlying the phenomenon of Ca2+ sensitization of smooth muscle contraction has received a great deal of attention recently (9). Ca2+ sensitization refers to the process whereby agonist-induced 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 resultant cross-bridge cycling. Inhibition of MLCP per se cannot induce an increase in LC20 phosphorylation, but rather alters the balance of kinase/phosphatase activity in favor of the kinase. The question then arises as to which kinase is responsible for LC20 phosphorylation following inhibition of MLCP. An obvious candidate is Ca2+/CaM-dependent MLCK. In this case, in the absence of agonist, the activity of MLCP would presumably mask the basal activity of MLCK that exists at the prevailing [Ca2+]i, and agonist-induced inhibition of MLCP would unmask this basal MLCK activity, leading to an increase in LC20 phosphorylation and contraction. However, resting [Ca2+]i in smooth muscle cells, which is in the range of 130-140 nM (37, 38), would be insufficient to induce significant activation of MLCK (39). This raises the possibility that a distinct, Ca2+-independent kinase may be responsible for the observed increase in LC20 phosphorylation and contraction at constant [Ca2+]i, i.e. Ca2+ sensitization. In support of this possibility, we demonstrated recently that the phosphatase inhibitor microcystin can trigger LC20 phosphorylation and contraction of skinned smooth muscle in the absence of Ca2+, phosphorylation occurring at Ser19 and Thr18, Ser19 being the principal site of phosphorylation by Ca2+/CaM-dependent MLCK (18). This Ca2+-independent phosphorylation was catalyzed by a kinase distinct from MLCK, since it was found to be Ca2+/CaM-independent, resistant to the selective MLCK peptide inhibitors AV25 and SM-1, and could be separated from MLCK. In this study, we show that, under the conditions used in the experiments of Fig. 1, a significant proportion of the myosin phosphorylating activity was resistant to the MLCK-selective peptide inhibitors AV25 and SM-1.

Using other selective kinase inhibitors, we have also shown that this kinase is not CaM kinase II, PKC, or ROK (18). It is also distinct from a kinase that was identified in preparations of MLCP that phosphorylated both the myosin targeting subunit of MLCP and myosin LC20, since that kinase was shown to be sensitive to chelerythrine (40). It is, however, sensitive to the nonselective kinase inhibitor staurosporine; the myofilament-associated, Ca2+-independent kinase activity was half-maximally inhibited at ~0.2 µM and the partially purified kinase at ~0.3 µM staurosporine. As shown in Figs. 4 and 5, we confirmed that the kinase preparation is not contaminated with a fragment of MLCK that has lost the requirement for Ca2+ and CaM due to partial proteolysis. Consistent with its ability to phosphorylate myosin in skinned smooth muscle strips, this kinase is retained in myofilament preparations, suggesting its association with a component of the myofilaments.

The principal objective of this work was to isolate and identify this Ca2+-independent myosin LC20 kinase, starting from myofilaments prepared from chicken gizzard, a rich source of smooth muscle tissue. The bulk of MLCK was first extracted from the myofilaments by treatment with 30 mM Mg2+, following which the Ca2+-independent kinase was extracted with high [NaCl]. The extracted kinase was then further purified by sequential chromatography on columns of DEAE-Sephacel, SP-Sepharose, and Mono Q. The partially purified kinase phosphorylated isolated LC20 and intact myosin at Ser19 and Thr18 of LC20 in a Ca2+-independent manner (Figs. 5 and 6). The kinase activity was associated with a 59-kDa protein, as shown by the ability of fractions from the Mono Q column to phosphorylate myosin in the absence of Ca2+ in solution and to phosphorylate isolated LC20 in a Ca2+-independent manner in the in-gel kinase assay (Fig. 2). Tryptic digestion, peptide mass fingerprinting by mass spectrometry, and de novo sequencing of a selected tryptic peptide, followed by data base searching, identified the kinase as ILK (Tables I and II). Its identity was confirmed by Western blotting with antibodies to ILK (Fig. 3).

ILK was originally discovered, through yeast two-hybrid screening, as an enzyme that binds to the cytoplasmic domain of beta -integrins and plays a role in integrin-mediated signal transduction (24, 41). Since integrins are plasma membrane proteins, the identification of the Ca2+-independent LC20 kinase as ILK raised the question of how a plasma membrane-associated kinase would have access to a substrate (myosin) located in the myofilaments. Using a subcellular fractionation approach, we identified two distinct populations of ILK, one with the expected properties of integrin-associated ILK and the other that was retained in myofilament preparations (Fig. 7). Presumably, the latter population is responsible for the observed Ca2+-independent phosphorylation of myosin in myofilaments and skinned smooth muscle preparations. It is noteworthy that ILK contains four ankyrin repeat motifs in the N-terminal domain and that the myosin-targeting subunit of MLCP, which anchors the phosphatase to the myosin filaments, contains seven N-terminal ankyrin repeats, and this region of the molecule is involved in the interaction with myosin (42). It is feasible, therefore, that ILK may associate with the myosin filaments of smooth muscle via its ankyrin repeats. In support of this possibility, purified myosin contains a low level of Ca2+-independent LC20 kinase activity that is resistant to the MLCK inhibitor AV25 (Fig. 5, lanes 1 and 2). It remains possible, however, that ILK may be associated with a protein(s) other than myosin in the myofilaments.

Gohla et al. (43) recently demonstrated that receptors for contractile agonists such as endothelin-1, angiotensin II, and vasopressin are coupled to both Gq/11 and G12/13, the latter being upstream of RhoA and ROK. Such agonists, therefore, appear to induce contraction via both activation of Ca2+/CaM-dependent MLCK (downstream of Gq/11) and inhibition of MLCP (downstream of G12/13). The possibility arises that ILK may also play a role in these contractile responses, contributing to the overall increase in LC20 phosphorylation (Fig. 11).


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Fig. 11.   Proposed mechanism of regulation of smooth muscle contraction via inhibition of MLCP via the RhoA/ROK pathway and phosphorylation of myosin by ILK. Contractile agonists such as endothelin-1, angiotensin II, and vasopressin act via receptors that are coupled to both Gq/11 and G12/13 heterotrimeric G proteins (43). Gq/11 activates phospholipase Cbeta (PLCbeta ), which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to generate inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol. Inositol 1,4,5-trisphosphate diffuses to the sarcoplasmic reticulum (SR), where it interacts with the inositol 1,4,5-trisphosphate receptor, a Ca2+ release channel in the sarcoplasmic reticulum membrane, thereby triggering Ca2+ release from the sarcoplasmic reticulum. The increase in [Ca2+]i activates CaM-dependent MLCK and cross-bridge cycling. G12/13, on the other hand, probably activates the guanine nucleotide exchange factor p115 RhoGEF (GEF) (48). Activated guanine nucleotide exchange factor triggers the dissociation of a guanine nucleotide dissociation inhibitor from the small GTPase RhoA, replacement of GDP by GTP on RhoA, and translocation of RhoA to the plasma membrane, where it inserts into the membrane via its geranylgeranyl moiety (indicated as a green extension) (9). Activated RhoA-GTP activates ROK, which phosphorylates the myosin binding regulatory or targeting subunit of MLCP, resulting in inhibition of the phosphatase activity (13, 14). This alters the MLCP/ILK activity ratio in favor of the kinase, resulting in an increase in myosin phosphorylation and contraction. If this occurs without an increase in [Ca2+]i, which would occur if the agonist is coupled to G12/13 but not Gq/11, as in the case of thromboxane A2, for example (9), then ILK could account entirely for the increase in myosin phosphorylation. This mechanism would then account for Ca2+ sensitization of contraction. On the other hand, if the agonist is coupled to both G12/13 and Gq/11, as in the case of endothelin-1, for example (43), then both ILK and Ca2+/CaM-dependent MLCK would account for the increase in myosin phosphorylation. Not depicted in this figure are other mechanisms of inhibition of MLCP involving PKC/CPI-17 or arachidonic acid, which are referred to in the Introduction.

Two recent reports have suggested that ILK may not be a functional protein-serine/threonine kinase, based on a number of amino acid substitutions in highly conserved regions within the catalytic domain and the inability to detect kinase activity in immunoprecipitates of ILK (35, 36). The rationale based on ILK sequence differences is unconvincing, since other active protein kinases have been identified with amino acid substitutions in highly conserved regions. For example, the kinases Mik1 and Vps15p lack all three glycine residues in the GXGXXG motif (44, 45). Furthermore, we expressed ILK in E. coli and detected Ca2+-independent kinase activity toward intact myosin, as shown in Fig. 10. However, our ability to detect this activity required denaturation followed by careful renaturation of the expressed kinase. Furthermore, the activity of the bacterially expressed ILK was low relative to that of the smooth muscle enzyme, suggesting that the kinase expressed in bacteria does not fold uniformly into the active conformation and may require post-translational modification. Based on these observations and the fact that we first detected the Ca2+-independent kinase activity in smooth muscle myofilaments and then identified the kinase as ILK, we conclude that ILK is indeed a bona fide protein-serine/threonine kinase.

ILK is widespread in terms of its tissue distribution, being expressed in most mammalian cells, with highest expression levels in cardiac and skeletal muscle, and its sequence is highly conserved across species. Regulation of nonmuscle motility involves myosin phosphorylation (9), similar to the regulation of smooth muscle contraction. The possibility arises, therefore, that ILK may also play a role in regulation of nonmuscle motile processes such as cell migration, chemotaxis, cytokinesis, and fibroblast contraction mediating orientation of collagen fibers in connective tissue. Indeed, Kolodney et al. (46) recently demonstrated that contraction of chick embryo fibroblasts in response to fetal bovine serum stimulation involves the Ca2+-independent phosphorylation of myosin II.

Finally, it is likely that myofilament-associated ILK is subject to regulation by as yet unidentified mechanisms that will be the subject of future investigations. ILK is known to be activated by phosphatidylinositol 3,4,5-trisphosphate (47), consistent with the presence of a phosphoinositide-binding domain resembling a pleckstrin homology domain in the center of the molecule (41). However, we observed no effect of phosphatidylinositol 3,4,5-trisphosphate on the phosphorylation of myosin by partially purified smooth muscle ILK (results not shown).

The results of this study provide the first indication of a novel role for ILK in Ca2+ sensitization of smooth muscle contraction, whereby signaling pathways that lead to inhibition of MLCP unmask the activity of myofilament-associated ILK, which phosphorylates myosin and activates contraction in a Ca2+-independent manner (Fig. 11). ILK is also likely to contribute to the increase in myosin phosphorylation that occurs in response to activation of receptors, e.g. the endothelin-1 receptor, which are coupled to both Gq/11 and G12/13 heterotrimeric GTP-binding proteins. Given the widespread tissue distribution of ILK (24), it is also conceivable that ILK may be involved in regulation of nonmuscle motility.

    ACKNOWLEDGEMENT

We are very grateful to Dr. S. Dedhar for critical reading of the manuscript and valuable discussions.

    FOOTNOTES

* This work was supported by a grant from the Canadian Institutes of Health Research.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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF296130.

Dagger An Alberta Heritage Foundation for Medical Research Medical Scientist and recipient of a Canada Research Chair in Biochemistry. To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Calgary Faculty of Medicine, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, Canada. Tel.: 403-220-3021; Fax: 403-270-2211; E-mail: walsh@ucalgary.ca.

Published, JBC Papers in Press, February 8, 2001, DOI 10.1074/jbc.M011634200

    ABBREVIATIONS

The abbreviations used are: CaM, calmodulin; CaM kinase II, Ca2+- and calmodulin-dependent protein kinase II; DTT, dithiothreitol; ILK, integrin-linked kinase; LC20, 20-kDa light chain subunit of myosin II; MALDI-TOF MS, matrix-assisted laser desorption/ionization-time of flight mass spectrometry; MLCK, myosin light chain kinase; MLCP, myosin light chain phosphatase; PKC, protein kinase C; ROK, Rho-associated kinase; PAGE, polyacrylamide gel electrophoresis.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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