Role of myosin phosphatase isoforms in cGMP-mediated smooth muscle relaxation

In vitro experiments showing the activation of the myosin phosphatase via heterophilic leucine zipper interactions between its targeting subunit (MYPT1) and cGMP-dependent protein kinase I suggested a pathway for smooth muscle relaxation (Surks, H. K., Mochizuki, N., Kasai, Y., Georgescu, S. P., Tang, K. M., Ito, M., Lincoln, T. M., and Mendelsohn, M. E. (1999) Science 286, 1583-1587). The relationship between MYPT1 isoform expression and smooth muscle responses to cGMP signaling in vivo has not been explored. MYPT1 isoforms that contain or lack a C-terminal leucine zipper are generated in birds and mammals by cassette-type alternative splicing of a 31-nucleotide exon. The avian and mammalian C-terminal isoforms are highly conserved and expressed in a tissue-specific fashion. In the mature chicken the tonic contracting aorta and phasic contracting gizzard exclusively express the leucine zipper positive and negative MYPT1 isoforms, respectively. Expression of the MYPT1 isoforms is also developmentally regulated in the gizzard, which switches from leucine zipper positive to negative isoforms around the time of hatching. This switch coincides with the development in the gizzard of a cGMP-resistant phenotype, i.e. inability to dephosphorylate myosin and relax in response to 8-bromo-cGMP after calcium activation. Furthermore, association of cGMP-dependent protein kinase I with MYPT1 is detected by immunoprecipitation only in the tissue that expresses the leucine zipper positive isoform of MYPT1. These results suggest that the regulated splicing of MYPT1 is an important determinant of smooth muscle phenotypic diversity and the variability in the response of smooth muscles to the calcium desensitizing effect of cGMP signaling.     

Agonist-induced force enhancement: the role of isoforms and phosphorylation of the myosin-targeting subunit of myosin light chain phosphatase.

The magnitude of agonist-induced Ca(2+) sensitization of force is tissue-dependent, but an explanation for this diversity is unknown. Ca(2+) sensitization is thought to involve a G-protein-mediated inhibition of myosin light chain phosphatase activity by phosphorylation of the myosin-targeting subunit (MYPT). The MYPT has two isoforms that differ by a central insert, which lies near this phosphorylation site. Expression of MYPT isoforms is both developmentally regulated and tissue-specific. We hypothesized that the presence or absence of the central insert determines the magnitude of agonist-induced Ca(2+) sensitization. Throughout development, the chicken aorta exclusively expresses the splice-in MYPT isoform, and guanosine 5'-O-(thiotriphosphate) (GTPgammaS) produces a significant force enhancement. Early during development, the chicken gizzard expresses the splice-in MYPT isoform, and GTPgammaS produced a Ca(2+) sensitization. In the gizzard coincident with the shift in expression from the splice-in to splice-out MYPT isoform, GTPgammaS no longer produced force enhancement. In addition, adenosine 5'-O-(thiotriphosphate) (ATPgammaS) phosphorylated only adult gizzard tissue, the only tissue that did not demonstrate a Ca(2+) sensitization. These results suggest that the relative expression of splice-in/splice-out MYPT isoforms determines the magnitude of agonist-induced force enhancement and that MYPT phosphorylation is not required for Ca(2+) sensitization.     

A myosin phosphatase targeting subunit isoform transition defines a smooth muscle developmental phenotypic switch

Smooth muscle myosin phosphatasedephosphorylates the regulatory myosin light chain and thus mediatessmooth muscle relaxation. The activity of this myosin phosphatase isdependent upon its myosin-targeting subunit (MYPT1). Isoforms of MYPT1have been identified, but how they are generated and their relationship to smooth muscle phenotypes is not clear. Cloning of the middle sectionof chicken and rat MYPT1 genes revealed that each gene gave rise toisoforms by cassette-type alternative splicing of exons. In chicken, a123-nucleotide exon was included or excluded from the mature mRNA,whereas in rat two exons immediately downstream were alternative. MYPT1isoforms lacking the alternative exon were only detected in maturechicken smooth muscle tissues that display phasic contractileproperties, but the isoform ratios were variable. The patterns ofexpression of rat MYPT1 mRNA isoforms were more complex, with threemajor and two minor isoforms present in all smooth muscle tissues atvarying stoichiometries. Isoform switching was identified in thedeveloping chicken gizzard, in which the exon-skipped isoform replacedthe exon-included isoform around the time of hatching. This isoformswitch occurred after transitions in myosin heavy chain and myosinlight chain (MLC₁₇) isoforms and correlated with aseveralfold increase in the rate of relaxation. The developmentalswitch of MYPT1 isoforms is a good model for determining the mechanismsand significance of alternative splicing in smooth muscle.

     

The control of protein phosphatase-1 by targetting subunits. The major myosin phosphatase in avian smooth muscle is a novel form of protein phosphatase-1.

The major protein phosphatase that dephosphorylates smooth-muscle myosin was purified from chicken gizzard myofibrils and shown to be composed of three subunits with apparent molecular masses of 130, 37 and 20 kDa, the most likely structure being a heterotrimer. The 37-kDa component was the catalytic subunit, while the 130-kDa and 20-kDa components formed a regulatory complex that enhanced catalytic subunit activity towards heavy meromyosin or the isolated myosin P light chain from smooth muscle and suppressed its activity towards phosphorylase, phosphorylase kinase and glycogen synthase. The catalytic subunit was identified as the beta isoform of protein phosphatase-1 (PP1) and the 130-kDa subunit as the PP1-binding component. The distinctive properties of smooth and skeletal muscle myosin phosphatases are explained by interaction of PP1 beta with different proteins and (in conjunction with earlier analysis of the glycogen-associated phosphatase) establish that the specificity and subcellular location of PP1 is determined by its interaction with a number of specific targetting subunits.     

Novel ZIP kinase isoform lacks leucine zipper

Zipper-interacting protein kinase (ZIP kinase) has been thought to be involved in apoptosis and the C-terminal leucine zipper motif is important for its function. Recent studies have revealed that ZIP kinase also plays a role in regulating myosin phosphorylation. Here, we found novel ZIP kinase isoform in which the C-terminal non-kinase domain containing a leucine zipper is eliminated (hZIPK-S). hZIPK-S binds to myosin phosphatase targeting subunit 1(MYPT1) similar to the long isoform (hZIPK-L). In addition, we found that hZIPK-S as well as hZIPK-L bind to myosin. These results indicate that a leucine zipper is not critical for the binding of ZIP kinase to MYPT1 and myosin. Consistently, hZIPK-S localized with stress-fibers where they co-localized with myosin. The residues 278-311, the C-terminal side of the kinase domain common to the both isoforms, is involved in the binding to MYPT1, while the myosin binding domain is within the kinase domain. These results suggest that the newly found hZIPK-S as well as the long isoform play an important role in the regulation of myosin phosphorylation.     

Determinants of the contractile properties in the embryonic chicken gizzard and aorta

Smooth muscle is generally grouped into two classes of differing contractile properties. Tonic smooth muscles show slow rates of force activation and relaxation and slow speeds of shortening (V(max)) but force maintenance, whereas phasic smooth muscles show poor force maintenance but have fast V(max) and rapid rates of force activation and relaxation. We characterized the development of gizzard and aortic smooth muscle in embryonic chicks to identify the cellular determinants that define phasic (gizzard) and tonic (aortic) contractile properties. Early during development, tonic contractile properties are the default for both tissues. The gizzard develops phasic contractile properties between embryonic days (ED) 12 and 20, characterized primarily by rapid rates of force activation and relaxation compared with the aorta. The rapid rate of force activation correlates with expression of the acidic isoform of the 17-kDa essential myosin light chain (MLC(17a)). Previous data from in vitro motility assays (Rover AS, Frezon Y, and Trybus KM. J Muscle Res Cell Motil 18: 103-110, 1997) have postulated that myosin heavy chain (MHC) isoform expression is a determinant for V(max) in intact tissues. In the current study, differences in V(max) did not correlate with previously published differences in MHC or MLC(17a) isoforms. Rather, V(max) was increased with thiophosphorylation of the 20-kDa regulatory myosin light chain (MLC(20)) in the gizzard, suggesting that a significant internal load exists. Furthermore, V(max) in the gizzard increased during postnatal development without changes in MHC or MLC(17) isoforms. Although the rate of MLC(20) phosphorylation was similar at ED 20, the rate of MLC(20) dephosphorylation was significantly higher in the gizzard versus the aorta, correlating with expression of the M130 isoform of the myosin binding subunit in the myosin light chain phosphatase (MLCP) holoenzyme. These results indicate that unique MLCP and MLC(17) isoform expression marks the phasic contractile phenotype.     

Unzipping the role of myosin light chain phosphatase in smooth muscle cell relaxation

Recently, it has been hypothesized that myosin light chain (MLC) phosphatase is activated by cGMP-dependent protein kinase (PKG) via a leucine zipper-leucine zipper (LZ-LZ) interaction through the C-terminal LZ in the myosin-binding subunit (MBS) of MLC phosphatase and the N-terminal LZ of PKG (Surks, H. K., Mochizuki, N., Kasai, Y., Georgescu, S. P., Tang, K. M., Ito, M., Lincoln, T. M., and Mendelsohn, M. E. (1999) Science 286, 1583-1587). Alternative splicing of a 3'-exon produces a LZ+ or LZ- MBS, and the sensitivity to cGMP-mediated smooth muscle relaxation correlates with the relative expression of LZ+/LZ- MBS isoforms (Khatri, J. J., Joyce, K. M., Brozovich, F. V., and Fisher, S. A. (2001) J. Biol. Chem. 276, 37250 -37257). In the present study, we determined the effect of LZ+/LZ- MBS isoforms on cGMP-induced MLC20 dephosphorylation. Four avian smooth muscle MBS-recombinant adenoviruses were prepared and transfected into cultured embryonic chicken gizzard smooth muscle cells. The expressed exogenous MBS isoforms were shown to replace the endogenous isoform in the MLC phosphatase holoenzyme. The interaction of type I PKG (PKGI) with the MBS did not depend on the presence of cGMP or the MBS LZ. However, direct activation of PKGI by 8-bromo-cGMP produced a dose-dependent decrease in MLC20 phosphorylation (p<0.05) only in smooth muscle cells expressing a LZ+ MBS. These results suggest that the activation of MLC phosphatase by PKGI requires a LZ+ MBS, but the binding of PKGI to the MBS is not mediated by a LZ-LZ interaction. Thus, the relative expression of LZ+/LZ- MBS isoforms could explain differences in tissue sensitivity to NO-mediated vasodilatation.     

Dephosphorylation of myosin by the catalytic subunit of a type-2 phosphatase produces relaxation of chemically skinned uterine smooth muscle

It is now well-established that phosphorylation of the 20,000-dalton light chain of smooth muscle myosin (LC20) is a prerequisite for muscle contraction. However, the relationship between myosin dephosphorylation and muscle relaxation remains controversial. In the present study, we utilized a highly purified catalytic subunit of a type-2, skeletal muscle phosphoprotein phosphatase (protein phosphatase 2A) and a glycerinated smooth muscle preparation to determine if myosin dephosphorylation, in the presence of saturating calcium and calmodulin, would cause relaxation of contracted uterine smooth muscle. Addition of the phosphatase catalytic subunit (0.28 microM) to the muscle bath produced complete relaxation of the muscle. The phosphatase-induced relaxation could be reversed by adding to the muscle bath either purified, thiophosphorylated, chicken gizzard 20,000-dalton myosin light chains or purified, chicken gizzard myosin light chain kinase. Incubation of skinned muscles with adenosine 5'-O-(thiotriphosphate) prior to the addition of phosphatase resulted in the incorporation of 0.93 mol of PO4/mol of LC20 and prevented phosphatase-induced relaxation. Under all of the above conditions, changes in steady-state isometric force were associated with parallel changes in myosin light chain phosphorylation over a range of phosphorylation extending from 0.01 to 0.97 mol of PO4/mol of LC20. We found no evidence that dephosphorylation of contracted uterine smooth muscles, in the presence of calcium and calmodulin, could produce a latch-state where isometric force was maintained in the absence of myosin light chain phosphorylation. These results show that phosphorylation or dephosphorylation of the 20,000-dalton myosin light chain is adequate for the regulation of contraction or relaxation, respectively, in glycerinated uterine smooth muscle.     

Recombinant small subunit of smooth muscle myosin light chain phosphatase. Molecular properties and interactions with the targeting subunit.

We expressed the small subunit of smooth muscle myosin light chain phosphatase (MPs) in Escherichia coli, and have studied its molecular properties as well as its interaction with the targeting subunit (MPt). MPs (M(r) = 18,500) has an anomalously low electrophoretic mobility, running with an apparent M(r) of approximately 21,000 in sodium dodecyl sulfate-gel electrophoresis. CD spectroscopy shows that it is approximately 45% alpha-helix and undergoes a cooperative temperature-induced unfolding with a transition midpoint of 73 degrees C. Limited proteolysis rapidly degrades MPs to a stable C-terminal fragment (M(r) = 10,000) that retains most of the helical content. Rotary shadowing electron microscopy reveals that it is an elongated protein with two domains. Sedimentation velocity measurements show that recombinant MPt (M(r) = 107,000), intact MPs, and the 10-kDa MPs fragment are all dimeric, and that MPs and MPt form a complex with a molar mass consistent with a 1:1 heterodimer. Sequence analysis predicts that regions in the C-terminal portions of both MPs and MPt have high probabilities for coiled coil formation. A synthetic peptide from a region of MPs encompassing residues 77-116 was found to be 100% alpha-helical, dimeric, and formed a complex with MPt with a molecular mass corresponding to a heterodimer. Based on these results, we propose that MPs is an elongated molecule with an N-terminal head and a C-terminal stalk domain. It dimerizes via a coiled coil interaction in the stalk domain, and interacts with MPt via heterodimeric coiled coil formation. Since other proteins with known regulatory function toward MP also have predicted coiled coil regions, our results suggest that these regulatory proteins target MP via the same coiled coil strand exchange mechanism with MPt.     

Tissue-specific and developmentally regulated alternative splicing of a visceral isoform of smooth muscle myosin heavy chain.

Previous work demonstrated that the rabbit smooth muscle myosin heavy chain gene showed sequence divergence at the 25kDa/50kDa junction of the S1 subfragment when compared to chicken gizzard and chicken epithelial nonmuscle myosin. RNase protection analysis with a probe spanning this region detected two partially protected fragments which were not present in RNA from vascular tissue and only found in RNA from visceral tissue. The polymerase chain reaction was used to amplify a 162bp product from primers spanning the putative region of divergence and DNA sequence analysis revealed a seven amino acid insertion not previously detected in other characterised cDNA clones. RNase protection analysis using the PCR product as probe showed that the inserted sequence was expressed exclusively in RNA from visceral tissue. Similar RNA analysis showed that the visceral isoform was not expressed in 20 day fetal rabbit smooth muscle tissues. These results indicated that the new visceral isoform was expressed in a tissue-specific and developmentally regulated manner. Genomic DNA sequencing and mapping of the exon-intron boundaries showed that the visceral isoform was the product of cassette-type alternative splicing. The inclusion of a visceral-specific sequence near the Mg-ATPase domain and at the 25kDa/50kDa junction suggests that the visceral isoform may be important for myosin function in smooth muscle cells.     

Regulation of MYPT1 stability by the E3 ubiquitin ligase SIAH2

Myosin phosphatase target subunit 1 (MYPT1), together with catalytic subunit of type1 δ isoform (PP1cδ) and a small 20-kDa regulatory unit (M20), form a heterotrimeric holoenzyme, myosin phosphatase (MP), which is responsible for regulating the extent of myosin light chain phosphorylation. Here we report the identification and characterization of a molecular interaction between Seven in absentia homolog 2 (SIAH2) and MYPT1 that resulted in the proteasomal degradation of the latter in mammalian cells, including neurons and glia. The interaction involved the substrate binding domain of SIAH2 (aa 116-324) and a central region of MYPT1 (aa 445-632) containing a degenerate consensus Siah-binding motif RLAYVAP (aa 493-499) evolutionally conserved from fish to humans. These findings suggest a novel mechanism whereby the ability of MP to modulate myosin light chain might be regulated by the degradation of its targeting subunit MYPT1 through the SIAH2-ubiquitin-proteasomal pathway. In this manner, the turnover of MYPT1 would serve to limit the duration and/or magnitude of MP activity required to achieve a desired physiological effect.     

Identification in turkey gizzard of an acidic protein related to the C-terminal portion of smooth muscle myosin light chain kinase

The isolation of an acidic protein, pI 4.5, that is abundant in turkey gizzard is described. Its apparent molecular weight measured by electrophoretic procedures is 24,000. This protein is phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and one phosphorylation site is indicated. From sequence determinations of tryptic peptides it is concluded that this protein is closely related to the C-terminal part of smooth muscle myosin light chain kinase. The initiation site for the protein is to the C-terminal side of the calmodulin-binding site. From the sequence data an estimated molecular weight is 18,000. This protein is expressed independently, as indicated by a blocked N terminus, and is probably the translation product of the 2.7-kilobase RNA detected previously in chicken gizzard (Guerriero, V., Jr., Russo, M. A., Olson, N. J., Putkey, J. A., and Means, A. R. (1986) Biochemistry 25, 8372-8381). Because of its putative origin as the C-terminal end of smooth muscle myosin light chain kinase, it is termed "telokin" (from a combination of kinase and the Greek telos, "end").     

Coiled-coil interactions modulate multimerization, mitochondrial binding and kinase activity of myotonic dystrophy protein kinase splice isoforms

The myotonic dystrophy protein kinase polypeptide repertoire in mice and humans consists of six different splice isoforms that vary in the nature of their C-terminal tails and in the presence or absence of an internal Val-Ser-Gly-Gly-Gly motif. Here, we demonstrate that myotonic dystrophy protein kinase isoforms exist in high-molecular-weight complexes controlled by homo- and heteromultimerization. This multimerization is mediated by coiled-coil interactions in the tail-proximal domain and occurs independently of alternatively spliced protein segments or myotonic dystrophy protein kinase activity. Complex formation was impaired in myotonic dystrophy protein kinase mutants in which three leucines at positions a and d in the coiled-coil heptad repeats were mutated to glycines. These coiled-coil mutants were still capable of autophosphorylation and transphosphorylation of peptides, but the rates of their kinase activities were significantly lowered. Moreover, phosphorylation of the natural myotonic dystrophy protein kinase substrate, myosin phosphatase targeting subunit, was preserved, even though binding of the myotonic dystrophy protein kinase to the myosin phosphatase targeting subunit was strongly reduced. Furthermore, the association of myotonic dystrophy protein kinase isoform C to the mitochondrial outer membrane was weakened when the coiled-coil interaction was perturbed. Our findings indicate that the coiled-coil domain modulates myotonic dystrophy protein kinase multimerization, substrate binding, kinase activity and subcellular localization characteristics.     

Purification of chicken gizzard myosin light-chain kinase, and its calcium and strontium sensitivities as compared with those of superprecipitation and ATPase activities of actomyosin

1. A purified preparation of myosin light-chain kinase (MLCK) was obtained from chicken gizzard, and it was shown to consist of two subunits; 130,000 (130 K)-dalton subunit and 17,000 (17 K)-dalton subunit. In amino acid composition the 130 K and 17 K subunits were identical with the 105 K and 17 K subunits of Dabrowska et al. (1977 and 1978), respectively. In disc gel electrophoresis, the 17 K subunit of our MLCK preparation responded to Ca2+ ions in the same way as bovine calmodulin, and differently from skeletal troponin C. There appeared to be one minor difference between 17 K subunit and calmodulin in the primary structure of the C-terminal region. 2. The Ca2+ and Sr2+ concentrations required for the three activities (ATPase and superprecipitation activities and MLCK activity) were measured. Two types of "reconstituted" myosin B were used; one contained 17 K subunit of gizzard MLCK and the other contained bovine brain calmodulin. The two types of "reconstituted" myosin B were practically identical with "natural" myosin B in the Ca2+ and Sr2+ requirements for the three activities measured above. 3. Both the extent and the activity of superprecipitation, and both the limited and steady activities of ATPase were measured. The MLCK activity was estimated in two ways; by urea gel electrophoresis and by measuring 32 P incorporation from [gamma-32P]ATP into myosin. The results thus obtained favor the kinase-phosphatase mechanism of calcium regulation of gizzard muscle contraction.     

Phosphorylation and actin activation of brain myosin

A method is described for obtaining brain myosin that shows significant actin activation, after phosphorylation with chicken gizzard myosin light chain kinase. Myosin with this activity could be obtained only via the initial purification of brain actomyosin. The latter complex, isolated by a method similar to that used for smooth muscle, contained actin, myosin, tropomyosin of the non-muscle type and another actin-binding protein of approximately 100,000 daltons. From the presence of a specific myosin light chain kinase and phosphatase in brain tissue it is suggested that the regulation of actin-myosin interaction operates via phosphorylation and dephosphorylation of myosin.     

Isolation and characterization of a cDNA encoding a novel human transcription factor TFIID subunit containing similarities with histones H2B and H3

Using the yeast two-hybrid system, we isolated a human cDNA that encodes a protein (hp22) interacting with TATA box-binding factor TFIID subunit p80 containing similarity with histone H4. Sequence analysis showed that the open reading frame (ORF) specifies a 161-amino-acid (aa) polypeptide homologous to Drosophila melanogaster TFIID subunit p22 (dp22). Comparison of the aa sequence of human TFIID subunit p22 (hp22) with that of dp22 revealed that p22 is composed of two distinct regions; the less conserved N-terminal (20% identity) and the highly conserved C-terminal (65% identity) regions. Additionally, the C-terminal region was found to contain similarities with histones H2B and H3. Northern blot analysis showed mRNA corresponding to hp22 to be expressed in all tissues examined     

Myosin phosphatase target subunit: Many roles in cell function

Phosphorylation of myosin II is important in many aspects of cell function and involves a myosin kinase, e.g. myosin light chain kinase, and a myosin phosphatase (MP). MP is regulated by the myosin phosphatase target subunit (MYPT1). The domain structure, properties, and genetic analyses of MYPT1 and its isoforms are outlined. MYPT1 binds the catalytic subunit of type 1 phosphatase, delta isoform, and also acts as an interactive platform for many other proteins. A key reaction for MP is with phosphorylated myosin II and the first process shown to be regulated by MP was contractile activity of smooth muscle. In cell division and cell migration myosin II phosphorylation also plays a critical role and these are discussed. However, based on the wide range of partners for MYPT1 it is likely that MP is implicated with substrates other than myosin II. Open questions are whether the diverse functions of MP reflect different cellular locations and/or specific roles for the MYPT1 isoforms.     

Embryonic chicken gizzard: smooth muscle and non-muscle myosin isoforms

Antibodies to smooth muscle and non-muscle myosin allow the development of smooth muscle and its capillary system in the embryonic chicken gizzard to be followed by immunofluorescent techniques. Although smooth muscle development proceeds in a serosal to luminal direction, angiogenetic cell clusters develop independently at the luminal side close to the epithelial layer, and the presumptive capillaries invade the developing muscle in a luminal to serosal direction. The smooth muscle and non-muscle myosin heavy chains in this avian system cannot be separated by SDS polyacrylamide gel electrophoresis and do not show isoform specificity in immunoblotting, unlike the system found in mammals. Only two myosin heavy chains with M_r of 200 and 196 kDa were separable and considerable immunological cross-reactivity was found between the denatured myosin isoform heavy chains.     

Characterization of chicken skeletal muscle myosin light chain kinase. Evidence for muscle-specific isozymes

Myosin light chain kinase purified from chicken white skeletal muscle (Mr = 150,000) was significantly larger than both rabbit skeletal (Mr = 87,000) and chicken gizzard smooth (Mr = 130,000) muscle myosin light chain kinases, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Km and Vmax values with rabbit or chicken skeletal, bovine cardiac, and chicken gizzard smooth muscle myosin P-light chains were very similar for the chicken and rabbit skeletal muscle myosin light chain kinases. In contrast, comparable Km and Vmax data for the chicken gizzard smooth muscle myosin light chain kinase showed that this enzyme was catalytically very different from the two skeletal muscle kinases. Affinity-purified antibodies to rabbit skeletal muscle myosin light chain kinase cross-reacted with chicken skeletal muscle myosin light chain kinase, but the titer of cross-reacting antibodies was approximately 20-fold less than the anti-rabbit skeletal muscle myosin light chain kinase titer. There was no detectable antibody cross-reactivity against chicken gizzard myosin light chain kinase. Proteolytic digestion followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or high performance liquid chromatography showed that these enzymes are structurally very different with few, if any, overlapping peptides. These data suggest that, although chicken skeletal muscle myosin light chain kinase is catalytically very similar to rabbit skeletal muscle myosin light chain kinase, the two enzymes have different primary sequences. The two skeletal muscle myosin light chain kinases appear to be more similar to each other than either is to chicken gizzard smooth muscle myosin light chain kinase.