©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Intrasteric Regulation of Myosin Light Chain Kinase (*)

Joanna K. Krueger , Roanna C. Padre , James T. Stull (§)

From the (1)Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9040

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Ca/calmodulin activates myosin light chain kinase by reversal of an autoinhibited state. The effects of substitution mutations on calmodulin activation properties implicate 4 of the 8 basic residues between the catalytic core and the calmodulin-binding domain in maintaining autoinhibition. These residues are further amino-terminal to the basic residues comprising the previously proposed pseudosubstrate sequence and suggest involvement of the connecting region in intrasteric autoinhibition.

The pseudosubstrate model for autoinhibition proposes that basic residues within the autoinhibitory region mimic basic residues in the substrate and bind to defined acidic residues within the catalytic core. Charge reversal mutations of these specific acidic residues, however, had little or no effect on the K value for regulatory light chain. From a total of 20 acidic residues on the surface of the substrate binding lobe of the catalytic core, 7 are implicated in binding directly or indirectly to the autoinhibitory domain but not to the light chain. Only 2 acidic residues near the catalytic site may bind to the autoinhibitory domain and the arginine at P-3 in the light chain. Exposure of these 2 residues upon calmodulin binding may be necessary and sufficient for light chain phosphorylation.


INTRODUCTION

Activation of smooth/nonmuscle myosin light chain kinase by Ca/calmodulin results in phosphorylation of myosin regulatory light chain that plays important roles in initiation of smooth muscle contraction, endothelial cell retraction, secretion, and other cellular processes (Stull et al., 1995). The smooth/nonmuscle myosin light chain kinase contains a catalytic core homologous to that of other protein kinases and a carboxyl-terminal regulatory domain consisting of both an inhibitory sequence and a calmodulin-binding sequence (Kemp et al., 1994; Stull et al., 1995). Initially, inspection of the linear sequence within the regulatory domain revealed a similar number and sequential arrangement of 4 basic residues with those shown to be important substrate determinants in a synthetic peptide containing residues 11-23 of the myosin regulatory light chain (see Fig. 1). Thus, Kemp et al.(1987) proposed that the regulatory domain contained a pseudosubstrate inhibitory sequence whereby 4 specific basic residues in myosin light chain kinase mimic the basic substrate determinants in the light chain peptide substrate. Binding of the pseudosubstrate sequence to the active site inhibited activity. Intrasteric inhibition involves an autoinhibitory sequence that folds back on the catalytic site to inhibit kinase activity as opposed to an allosteric mechanism whereby a conformational change induced at a site distinct from the active site would be responsible for regulation of enzyme activity (Kemp and Pearson, 1991). The sequence comprising the pseudosubstrate region was later expanded to include overlap with the complete amino terminus of the light chain (Faux et al., 1993). However, these additional residues(1, 2, 3, 4, 5, 6, 7, 8, 9, 10) are not important for substrate binding and thus are not part of the consensus phosphorylation sequence (Kemp and Pearson, 1990).


Figure 1: Sequence comparison of the regulatory domain of rabbit myosin light chain kinase with myosin light chain. Boxedresidues in the myosin light chain kinase sequence are those basic residues that were previously noted to be similar in sequential arrangement to the basic residues in the light chain (boxed) that form part of the consensus phosphorylation sequence (Kemp et al., 1987). The proposed pseudosubstrate sequence was later extended to include all residues (Ser-Val) that overlap the amino terminus of the light chain (Knighton et al., 1992). Circledresidues identify basic residues within the regulatory domain implicated in this study to bind to the catalytic core. The catalytic core ends with Leu.



Proteolysis studies have supported the hypothesis that myosin light chain kinase contains an autoinhibitory sequence (Walsh et al., 1982; Ikebe et al., 1987; Pearson et al., 1988). Limited tryptic cleavage of the chicken smooth muscle myosin light chain kinase results in a nonactivatable form (64 kDa) that becomes constitutively active upon further digestion (61 kDa). Because of differences in the reported cleavage sites, there is disagreement on whether the inhibited form contains the pseudosubstrate sequence (Pearson et al., 1988; Ikebe et al., 1989).

Recent mutational analyses have identified several acidic residues in the catalytic core that may bind the inhibitory sequence but not the light chain. When the charge of these residues was reversed by mutation, the respective K()values decreased with no significant effect on the K or V values for the regulatory light chain (Gallagher et al., 1993). It was proposed that a lowered K value reflected a weakened binding of the inhibitory region to the catalytic core. In this series of mutations, only 2 acidic residues, located near the catalytic site in the cleft between the two lobes of the kinase, were identified as binding to both the inhibitory region and the arginine residue at the P-3 position in the light chain substrate.

A three-dimensional model has been proposed for the catalytic core of smooth muscle myosin light chain kinase, and a portion of the bound autoinhibitory region that includes the expanded pseudosubstrate sequence (Knighton et al., 1992). This model is based on the known crystal structure of the catalytic subunit of cAMP-dependent protein kinase in complex with its inhibitory peptide, PKI. A substrate binding groove has been defined utilizing the position of the PKI peptide in the crystal structure of this complex (Kemp et al., 1994). It is predicted that specific acidic residues lining this substrate-binding groove of the myosin light chain kinase catalytic core form salt bridges with basic residues in both the autoinhibitory sequence and the regulatory light chain substrate. Since the role of these acidic residues was not examined in a previous study (Gallagher et al., 1993), we have experimentally tested the predictions of this pseudosubstrate model using selected site mutagenesis to identify interacting residues. A modified molecular mechanism for autoinhibition is proposed.


EXPERIMENTAL PROCEDURES

Oligonucleotide-directed Mutagenesis

A 1,660-base pair 5`-BamHI-XbaI-3` cDNA fragment, representing the carboxyl-terminal half of rabbit smooth/nonmuscle myosin light chain kinase (Gallagher et al., 1991), was subcloned into M13 bacteriophage. Mutagenesis was performed using the oligonucleotide-directed in vitro mutagenesis system (Amersham Corp.) and oligonucleotides designed to produce mutant cDNAs having the desired substitutions. For each mutant cDNA, the desired nucleotide substitutions were verified by DNA sequencing (Sanger et al., 1977).

Expression of Wild-type and Mutant Smooth Muscle Myosin Light Chain Kinases

All wild-type and mutant myosin light chain kinases were expressed in COS cells. In each case, two clones of each mutant were subcloned into a pCMV expression vector as described previously (Gallagher et al., 1991). Subsequent expression was accomplished by transfection into COS cells using DEAE-dextran and chloroquine (Herring et al.,1990).

Myosin Light Chain Kinase Assays

COS cell lysates, prepared as described by Gallagher et al.(1991), were used to determine recombinant wild-type and mutant myosin light chain kinase activity (Gallagher et al., 1991). The quantity of wild-type and mutant smooth muscle myosin light chain kinases present in COS cell lysates was determined by immunoblotting with purified smooth muscle myosin light chain kinase as a standard and probing with antiserum raised against the carboxyl-terminal telokin portion of rabbit smooth muscle myosin light chain kinase (Gallagher and Herring, 1991). The Ca/calmodulin-dependent activity of wild-type and mutant smooth muscle myosin light chain kinases was measured by P incorporation into myosin regulatory light chain purified from chicken gizzards (Blumenthal and Stull, 1980). Ca/calmodulin-independent background activity of the extracts was measured as radioactivity incorporated in the presence of 3 mM EGTA. V and K values were determined from Lineweaver-Burke double-reciprocal plots after performing kinase assays under varying regulatory light chain concentrations.

To examine the calmodulin activation properties of mutant myosin light chain kinases in COS cell lysates, Ca activation assays were performed as described by Herring(1991). The relative K values of the mutant kinases within the COS cell extracts were measured at 1 µM calmodulin with varying Ca concentrations from 75 nM to 100 µM with a Ca/EGTA buffer system (Potter and Gergely, 1975). The added calmodulin is in great excess of the endogenous calmodulin contributed by the COS cell lysate and therefore provides controlled conditions to establish the relative concentrations of Ca/calmodulin required for kinase activation. The free Ca concentration was the determinant of the actual Ca/calmodulin concentration. To assess the quantitative changes in the calmodulin activation properties (K) of mutant myosin light chain kinases, the ratio of activities at Ca concentrations that resulted in less than maximal activity to the maximal activity measured at 100 µM Ca was determined as described previously (Miller et al., 1983; Fitzsimons et al., 1992). The ratio of activities at a specific Ca concentration that is less than that required for maximal activity increases quantitatively as the K value for a mutant myosin light chain decreases relative to the wild-type enzyme. Although the ratio of activities does not allow determination of the absolute value of K, it may be used to calculate the -fold change in K relative to wild-type myosin light chain kinase that has an average K value of 1 nM (Miller et al., 1983; Stull et al., 1990; Fitzsimons et al., 1992). It is proposed that a decrease in the K value (<1 nM) reflects a more easily activated kinase due to a weakening of the binding between the inhibitory region and the catalytic core.


RESULTS

Properties of Myosin Light Chain Kinase Mutated in the Connection Region

To further define the boundaries of myosin light chain kinase inhibitory sequence, we created charge reversal and alanine substitution point mutations at basic residues amino-terminal to the proposed pseudosubstrate region of myosin light chain kinase (Fig. 1) (Kemp et al., 1987). Full-length cDNA was constructed in the vector pCMV for transient expression in COS cells. Immunoblot analysis of COS cell lysates demonstrated that all of the mutant myosin light chain kinases were full-length and expressed at levels (5-20 µg/ml) similar to previously obtained results (Fitzsimons et al.,1992; Gallagher et al., 1993). All active mutant myosin light chain kinases were dependent on Ca for activity.

The Ca/calmodulin activation properties of the wild-type and mutant kinases were determined by performing assays at a high calmodulin concentration with a Ca/EGTA buffer used to vary the free Ca concentration and hence the Ca/calmodulin concentration (Miller et al., 1983). Lys is 21 residues amino-terminal to basic residues (Arg-Lys) in the calmodulin binding domain that form the core of the proposed pseudosubstrate structure (Fig. 1). Both K953D and K953A mutants required significantly less Ca for half-maximal activation (0.26 and 0.42 µM, respectively) relative to wild-type kinase (0.65 µM) with corresponding decreases in K (0.16 and 0.53 nM from 1.0 nM) ( and Fig. 2A). These mutations that produced a 6.3- and 1.9-fold decrease in K values were not accompanied by any significant changes in catalytic properties (). In contrast to these results, mutant K956E had no significant effects on Ca activation or catalytic properties ().


Figure 2: Calcium activation curves for mutant myosin light chain kinases. Myosin light chain kinase mutants were expressed in COS cells, and activity was measured in cell lysates at 1 µM calmodulin at different Ca concentrations. The data were normalized to the percent maximal activity. Symbols represent a mean value of at least three independent assays, each performed in duplicate. Data are presented without error bars for clarity. A, mutations within the regulatory domain of myosin light chain kinase are as follows: , wild-type; , K953D; , K956E; , K961E; , K962E; , R967E. B, mutations within the catalytic core of myosin light chain kinase are as follows: , wild-type; , E858Q; , D896K; , E900R; , D911K/D914K. [Ca] values are 0.65 ± 0.02, 0.13 ± 0.02, 0.57 ± 0.05, 0.57 ± 0.03, and 0.78 ± 0.09 nM, respectively.



The K961E and K961A mutants required significantly less Ca for half-maximal activation (0.30 and 0.31 µM) with 5- and 3.2-fold decreases in K values, respectively (). The charge reversal mutant K961E selectively changed the activation properties of the kinase with no change in V or K values (). In contrast, the charge reversal and alanine substitution mutations of Lys had little effect on activation and catalytic properties. The V and K values were not changed for the charge reversal mutation. The Kvalue for light chain was not measured for the alanine substitution, but the specific activity of the kinase was not different for wild-type enzyme. The charge reversal mutation K962E resulted in a modest decrease in [Ca] from 0.65 to 0.47 mM with only a 1.6-fold decrease in K. By comparing these relative K values with that previously reported (Fitzsimons et al., 1992) for the double mutant KK961/962EE (0.20 nM), it is evident that of these 2 residues, Lys is more sensitive to substitution.

Individual substitution of basic residues Lys and Arg with oppositely charged acidic residues showed a marked decrease in K value (0.03 and 0.09 nM, respectively) relative to the 1 nM value for wild-type kinase (). These values can be compared with the previously reported values (Fitzsimons et al., 1992) for the individual alanine mutants K965A and R967A (0.11 and 0.25 nM) as well as the double mutants K965A/R967A and K965E/R967D (0.04 nM and inactive, respectively). None of the mutations caused significant changes in V or K values except for K965E in which the 33-fold decrease in K was associated with lower V and K values () (Fitzsimons et al., 1992).

Properties of Myosin Light Chain Kinase Mutated in the Catalytic Core

Acidic residues Glu, Asp, Glu, and Asp (corresponding residues for myosin light chain kinase from chicken smooth muscle are Glu, Asp, Glu, and Asp, respectively) within the myosin light chain kinase catalytic core are predicted by the proposed pseudosubstrate model to be involved in binding both the inhibitory sequence in the kinase and the regulatory light chain (Knighton et al.,1992). This hypothesis was tested by charge reversal mutations of these residues. Three neighboring acidic residues, Asp, Asp, and Asp, were chosen as controls for the charge reversal mutation studies. These latter residues are found in the vicinity of those proposed to bind to both the autoinhibitory sequence and the regulatory light chain but are not predicted to bind to either light chain or the autoinhibitory sequence.

The resulting mutant kinases were characterized with respect to catalytic and activation properties. One of the charge reversal mutants, E858K, was catalytically inactive () even though it was expressed at high levels and comigrated with the 152-kDa wild-type recombinant myosin light chain kinase (data not shown). A more conservative mutation, E858Q, resulted in the expression of an active kinase with V, K, and V/K ratio values similar to those of the wild-type kinase. E858Q did, however, require significantly less Ca (0.13 µM) for half-maximal activation relative to the wild-type kinase (0.65 µM) with a 7-fold decrease in the K value (, Fig. 2B). These results are consistent with the binding of this residue to or near the inhibitory domain but not to the light chain.

All of the other catalytic core mutants exhibited no significant changes in [Ca] and K values from that of the wild-type kinase (, Fig. 2B). These results are consistent with the interpretation that these acidic residues do not bind to basic residues in the autoinhibitory domain.

Minor yet statistically significant increases in the K values for regulatory light chain were observed for D896K, D898K, E900R, D911K/D914K, D911R, D913K, and D914K mutants (19.8, 19.3, 11.6, 33.7, 10.2, 9.6, and 16.0 µM, respectively, versus 4.7 µM for wild-type kinase). In several cases (E900R, D911R, D913K, and D914K) the changes were only about 2-fold and could result from nonspecific electrorepulsion rather than perturbation of specific salt bridge bonds between the respective residues in the catalytic core and the light chain. The V values were similar to wild-type kinase except for D913K, which was lower (8.7 µmol of P incorporated per min/mg). The changes in the V/K ratios for the charge reversal mutants were also modest (). The largest change was noted in the double mutant D911K/D914K (5.8-fold). However, single mutations in these residues resulted in only 1.7- and 2.4-fold changes.


DISCUSSION

As a result of previous mutational and deletion analysis of the myosin light chain kinase regulatory domain, some residues immediately amino-terminal to the calmodulin-binding sequence were implicated in autoinhibition (Shoemaker et al., 1990; Ito et al., 1991; Fitzsimons et al., 1992; Yano et al., 1993). Our data indicate that basic residues even further amino-terminal to the originally proposed pseudosubstrate sequence and close to the carboxyl terminus of the catalytic core could be involved in maintaining myosin light chain kinase in an inhibited state (Fig. 1). In each case, both single charge reversal and alanine substitution mutations of residues Lys, Lys, Lys, and Lys resulted in kinases that were more easily activated by Ca/calmodulin (decreased [Ca] and K values relative to wild-type). The results with Lys are most interesting since this residue is close to the junction between the catalytic core and the connecting region. These findings support the hypothesis that an intrasteric, autoinhibitory mechanism for myosin light chain kinase involves structures that extend beyond the proposed pseudosubstrate sequence. They also support the idea that the connecting region between the catalytic core and calmodulin binding domain may be bound to the surface of the catalytic core similar to the binding of the carboxyl-terminal tail of twitchin kinase to its catalytic core (Hu et al., 1994). The multiple intramolecular contacts made by this connecting region may be involved in autoinhibition and, because of the extensive contacts, single mutations would not be expected to produce profound effects on Ca/calmodulin activation properties. However, Shoemaker et al.(1990) showed that multiple charge reversal mutations in a portion of this autoinhibitory sequence could result in a constitutively active myosin light chain kinase.

Many acidic residues in the catalytic core appear to have specific interactions with the autoinhibitory sequence, but not light chain. The conservative neutral mutation, E858Q, created a kinase with wild-type catalytic properties but a 7-fold lower K value. This result is consistent with the suggestion that Glu is involved in maintenance of autoinhibition but not in binding to the regulatory light chain. As predicted, D898K, D913K, and D914K each displayed similar [Ca] and K values as wild-type myosin light chain kinase, suggesting that these acidic residues do not bind to or near the autoinhibitory sequence. However, evidence was not obtained that other predicted acidic residues bind to the autoinhibitory sequence (Asp, Glu, and Asp). Thus, the acidic residues on the surface of the catalytic core, which have been implicated from this and previous mutational analyses (Gallagher et al., 1993) in binding to or near the autoinhibitory sequence, are predicted to be mainly in the D -helix (Fig. 3). The placement of these residues defines a binding pathway distinct from that of PKI binding to the G -helix of cAMP-dependent protein kinase. By inference then, this pathway is also distinct from the purported substrate-binding groove that predicts pseudosubstrate as well as substrate binding contacts with residues in the G -helix (Asp, Glu, and Asp) (Knighton et al., 1992; Kemp et al., 1994). In fact, none of these acidic residues nor any of those in the vicinity of the G -helix (Asp, Asp and Asp) can be implicated by our mutational analyses in binding to or near the autoinhibitory domain. Additional proposed contacts include Glu, which is found in a loop spatially situated between the D -helix and the G -helix, and Glu, which is found in the active site cleft on the D -helix (Knighton et al., 1992). However, Glu also does not appear to bind the light chain. These results suggest that the autoinhibitory sequence may bind on the surface that comprises the substrate binding pocket immediately surrounding the catalytic site, but it is likely to have more contacts with the D -helix than with the G -helix.


Figure 3: Ribbon diagram of a model of the catalytic core of smooth/nonmuscle muscle myosin light chain kinase. The ribbon was drawn using the program MOLSCRIPT (Kraulis, 1991). The model is derived from the chicken smooth muscle myosin light chain kinase catalytic core model (Knighton et al., 1992). The letters displayed correspond to the respective -helices as defined for the cAMP-dependent protein kinase structure (Knighton et al., 1991). The D and G -helices are highlighted in red and yellow, respectively, for clarification. Some amino acid residues are displayed in ball-and-stick. The 2 acidic residues, Glu and Glu (highlighted in blue), are proposed to bind to basic residues in both the autoinhibitory domain and the arginine at P-3 in the regulatory light chain. The 7 acidic residues, Glu, Glu, Glu, Glu, Asp, Glu, and Glu (highlighted in red), that display a distinct pathway projecting from the catalytic site to the end of the catalytic core have been implicated, from this and previous mutational analyses (Gallagher et al., 1993), in binding to or near the inhibitory domain, but not to the light chain. Charge reversal mutations of several other acidic residues (highlighted in yellow), including Asp, Glu, and Asp, which had been predicted by the pseudosubstrate theory to bind to both the autoinhibitory domain and the regulatory light chain, had little or no effect on the catalytic or activation properties of the kinase. These and other residues (Asp and Asp) are predicted not to play important roles in binding either the regulatory light chain or the autoinhibitory domain.



These results also raise a question regarding the identification of acidic residues in the catalytic core that bind to the 3 basic residues in the consensus phosphorylation sequence of the light chain at P-6 to P-8 positions (Fig. 1). The V/K ratios obtained with the intact light chain substrate for the respective charge reversal mutations at Glu, Asp, Asp, Asp, and Asp are only slightly decreased (2-5-fold) relative to the V/K ratio of the wild-type kinase and are minor compared with the 50-fold effects of alanine substitutions for basic residues in synthetic peptide substrates (Kemp et al., 1983; Kemp and Pearson, 1985). The decreases in V/K ratios for regulatory light chain in the charge reversal mutant kinases are still much smaller in magnitude compared with the 340-fold decrease in the V/K ratio reported for the charge reversal mutation of arginine at the P-3 position in the regulatory light chain (Zhi et al., 1994). Even with an alanine substitution at P-3 in the light chain, the V/K ratio decreased 35-fold. The decreases in the V/K ratio values were modest with individual alanine substitutions of the basic residues in the consensus phosphorylation sequence at P-6 to P-8 in the intact light chain. It is unlikely then that acidic residues Glu, Asp, Asp, Asp, and Asp are important in substrate binding and phosphorylation. This conclusion is consistent with the proposal that it is other interactions, involving the arginine at P-3 and the hydrophobic residues at P+1 to P+3, that are most important for substrate recognition in addition to subdomains I and II of the regulatory light chain (Zhi et al., 1994). Thus, only a portion (Arg at P-3 and hydrophobic residues at P+2 and P+3) of the previously described consensus phosphorylation sequence is important for substrate recognition of the intact light chain. As the importance of the basic residues found amino-terminal to the phosphorylatable serine at P-6 to P-8 positions is diminished in the protein substrate (Zhi et al., 1994), the definition of the pseudosubstrate must also be modified. This hypothesis may explain why only 2 of the 20 acidic residues predicted to be on the substrate-binding surface of the catalytic core appear to be important in substrate recognition. These 2 residues, Glu and Glu, are located near the catalytic site in the cleft between the two lobes of the kinase and are implicated in binding to arginine at P-3 in the regulatory light chain. When these 2 residues are mutated, the V/Kratio decreases 138- and 72-fold, respectively (Herring et al., 1992). These 2 residues also appear to bind to or near the autoinhibitory domain (Gallagher et al., 1993).

The recently reported crystal structure of a related kinase, twitchin, may provide further insights into the structural basis for intrasteric inhibition of myosin light chain kinase (Hu et al., 1994). The portion of the kinase carboxyl-terminal to the catalytic core extends across the surface of the large lobe of the catalytic core similar to the distinct pathway proposed for the autoinhibitory sequence of myosin light chain kinase (Fig. 3). It then extends through the active site between the large and small lobes. Acidic residues in the twitchin kinase, Glu and Glu, are equivalent to Glu and Glu, respectively, in rabbit smooth/nonmuscle myosin light chain kinase (Fig. 4). The twitchin structure shows that these 2 acidic residues make potential contacts (3.5 Å) with Lys and Lys, respectively, in -helix 11. From sequence alignment, Lys in the calmodulin binding domain of myosin light chain kinase and Lys of twitchin are conserved. By inference, it is possible that Lys binds to Glu and/or Glu. The crystal structure of calmodulin bound to a peptide analog of the calmodulin-binding region of chicken smooth muscle myosin light chain kinase reveals that Lys binds to calmodulin (Meador et al., 1992). Consistent with this finding is the observation that charge reversal substitution (K979E) decreased Ca/calmodulin-binding affinity (Fitzsimons et al., 1992). However, additional investigations will be necessary to determine if Lys and other residues in the calmodulin binding domain bind both calmodulin and residues in the catalytic core of myosin light chain kinase.


Figure 4: Amino acid sequence alignments for rabbit smooth/nonmuscle myosin light chain kinase twitchin. Alignment was obtained using the GCG software BESTFIT program (Genetics Computer Group, Inc., 1992). Bestfit allows for optimal alignment by the insertion of gaps to maximize the number of matches utilizing the local homology algorithm of Smith and Waterman (1981). The dots between mismatched sequences represent evolutionarily related amino acids (Gribskov and Burgess, 1986). Boxedsequence indicates conserved catalytic core. The asterisk indicates Lys in myosin light chain kinase, which is equivalent to twitchin residue Lys. The Lys binds to Glu and Glu (Hu et al., 1994). By homology, the indicates the acidic residues in the catalytic core (Glu and Glu) for myosin light chain kinase proposed to bind to basic residues in the autoinhibitory domain (Lys) and light chain substrate (Arg), respectively.



Only 2 acidic residues (Glu and Glu) located near the catalytic site of myosin light chain kinase have been implicated in binding to both the regulatory light chain and the autoinhibitory sequence (herein and Gallagher et al. (1993)). Assuming binding homology between twitchin and myosin light chain kinase, Lys may bind to Glu and Glu. However, basic residues Lys, Lys, Lys, Lys, and Arg amino-terminal to both the calmodulin-binding domain and the proposed pseudosubstrate sequence also appear to be involved in inhibition of myosin light chain kinase activity. In addition, 7 acidic residues following a distinct path across the surface of the catalytic core are implicated in binding to or near the inhibitory sequence but not to basic residues in the regulatory light chain. Since only 2 acidic residues (Glu and Glu) near the catalytic core may bind both to the autoinhibitory domain and the arginine at P-3 in the light chain substrate, exposure of these 2 residues upon calmodulin binding may be necessary and sufficient for light chain phosphorylation. We conclude that the autoinhibitory sequence operates primarily through an intrasteric rather than a simple pseudosubstrate mechanism in inhibiting myosin light chain kinase activity.

  
Table: Kinetic properties of myosin light chain kinase containing point mutations of basic residues within the regulatory domain

Recombinant and mutant myosin light chain kinases were expressed in COS cells as described under ``Experimental Procedures.'' Values are means ± S.E. for at least three experiments with lysates from transfections representing at least two independent mutations. [Ca] values represent the Ca concentration required for half-maximal activation at 1 µM calmodulin. The relative K values were calculated for at least three Ca concentrations. V and K (light chain) values were determined from Lineweaver-Burke plots. KLC refers to K values for the regulatory light chain. ND, values not determined.


  
Table: Kinetic properties of myosin light chain kinases containing catalytic core point mutations

Mutagenesis, expression, and measurements of kinetic properties of myosin light chain kinases were performed as described under ``Experimental Procedures.'' Values represent means ± S.E. for at least three experiments with lysates from transfections representing at least two independent mutants. The relative K values were calculated for at least three Ca concentrations. V and K values were determined from Lineweaver-Burke plots. KLC refers to K values for the regulatory light chain.



FOOTNOTES

*
This work was supported in part by Grant HL26043 and Fellowships T32-HL07360 and F32 HL08874 from the National Institutes of Health and by the Bashour Research Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9040. Tel.: 214-648-6849; Fax: 214-648-2974.

The abbreviations used are: K, concentration of Ca/calmodulin required for half-maximal activation of myosin light chain kinase; [Ca], concentration of Ca required for half-maximal activation of myosin light chain kinase; PKI, inhibitor peptide for cAMP-dependent protein kinase.


ACKNOWLEDGEMENTS

We thank Phyllis Foley for the preparation of this manuscript, Patricia J. Gallagher for antibodies to myosin light chain kinase, and James M. Mottonen for technical assistance with the MOLSCRIPT software.


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