Protein production in three different expression vectors from a single polymerase chain reaction product.
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Base Sequence↗
Biomedical subjects
J Kendrick-Jones
Publications and source records attributed to J Kendrick-Jones.
Dystrophin and utrophin: the missing links!
There is considerable sequence homology between dystrophin and utrophin, both at the protein and DNA level, and consequently it was assumed that their domain structures and functions would be similar. As more of the detailed biochemical and cell biological properties of these two proteins become known, so it becomes clear that there are subtle if not significant differences between them. We review recent findings and present new hypotheses into the structural and functional properties of the actin-binding domain, central coiled-coil region and regulatory/membrane protein-binding regions of dystrophin and utrophin.
Actins↗
Tilting of the light-chain region of myosin during step length changes and active force generation in skeletal muscle.
Force generation and relative sliding between the myosin and actin filaments in muscle are thought to be caused by tilting of the head region of the myosin crossbridges between the filaments. Structural and spectroscopic experiments have demonstrated segmental flexibility of myosin in muscle, but have not shown a direct linkage between tilting of the myosin heads and either force generation or filament sliding. Here we use fluorescence polarization to detect changes in the orientation of the light-chain region of the head, the part most likely to tilt, and synchronized head movements by imposing rapid length steps. We found that the light-chain region of the myosin head tilts both during the imposed filament sliding and during the subsequent quick force recovery that is thought to signal the elementary force-generating event.
Animals↗
Determinants of ion specificity on EF-hands sites. Conversion of the Ca2+/Mg2+ site of smooth muscle myosin regulatory light chain into a Ca(2+)-specific site.
Calcium binding proteins mediate a large number of cellular processes. These processes respond to micromolar fluctuations of cytosolic calcium in the presence of a large excess of magnesium. The metal binding sites present in these proteins are either calcium-specific (regulatory sites) or capable of binding both calcium and magnesium (structural sites). Using site-directed mutagenesis we were able to convert the single Ca2+/Mg2+ site present in chicken smooth muscle myosin regulatory light chain (RLC) into a Ca(2+)-specific site. The replacement of the aspartic acid present in the 12th position (-Z coordinating position) of the metal binding loop with a glutamic acid increases calcium affinity and abolishes magnesium binding, rendering the site calcium-specific. To explain this observation, we hypothesize that restrictions on the ability of side chains to change conformation, contributing one (for Mg2+ binding) or two (for Ca2+ binding) coordinations could alter the metal specificity in EF-hands. Other mutations which decrease or abolish calcium binding have also been characterized. When used to substitute the endogenous scallop myosin RLC, these mutants were capable of restoring the Ca2+ regulation to the actin-activated myosin ATPase demonstrating that in these hybrid myosins, the regulatory function of the Ca(2+)-specific site (present on the essential light chain) does not depend on the occupancy of the Ca2+/Mg2+ site (present on the regulatory light chain).
Adenosine Triphosphatases↗
Full-length and short forms of utrophin, the dystrophin-related protein.
All previous studies of the localization of utrophin (the dystrophin-related protein) in muscle and other tissues have been performed only with antibodies against the C-terminal region of the protein. Since several short forms of dystrophin, the apo-dystrophins, are produced from the 3' end of the dystrophin gene, there is a possibility that similar short forms of utrophin exist and that these could be responsible for some of the many different localizations of 'utrophin' in muscle. We have produced a new panel of 15 mAbs against the N-terminal region of utrophin and we have used it together with mAbs against the C-terminal region to show that full-length utrophin is present at neuromuscular junctions, in nerves, blood vessels and capillaries in normal muscle and in the sarcolemma of patients with muscular dystrophy and dermatomyositis. However, two of the 15 mAbs also recognised rat/mouse utrophin and both of these detected an additional 62 kDa protein on Western blots of rat C6 glioma cells. This potential 62 kDa 'apo-utrophin' was not detected in human cerebral cortex, in rat Schwannoma cells nor in any of the non-nerve cells and tissues tested.
Animals↗
Calcium/calmodulin-dependent regulation of the NH2-terminal F-actin binding domain of utrophin.
The cytoskeletal proteins utrophin, dystrophin and alpha-actinin are predicted to form antiparallel dimers thus potentially bringing their NH2-terminal F-actin binding domains in close proximity to their EF-hand containing COOH-terminal domains. This arrangement would allow for calcium-dependent regulation of F-actin binding. We tested this hypothesis by determining the effect of the ubiquitous calcium binding protein calmodulin on their F-actin binding capabilities. Binding of the NH2-terminal F-actin binding domain of utrophin to F-actin was inhibited by increasing concentrations of calmodulin in a calcium-dependent manner. The homologous F-actin binding domains from dystrophin and alpha-actinin were not regulated by calmodulin in the presence or absence of calcium. These findings have implications for the structural organisation of utrophin dimers and also for the replacement of dystrophin by over-expression of utrophin in dystrophic muscle.
Actinin↗
Coiled-coil regions in the carboxy-terminal domains of dystrophin and related proteins: potentials for protein-protein interactions.
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Amino Acid Sequence↗
Calmodulin regulation of utrophin actin binding.
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Actinin↗
Intracellular mechanisms involved in the regulation of vascular smooth muscle tone.
Vascular smooth muscle contraction is thought to occur by a mechanism similar to that described for striated muscles, i.e., via a cross-bridge cycling--sliding filament mechanism. This symposium focused on Ca2+ signalling and the role of intracellular free Ca2+ concentration, [Ca2+]i, in regulating vascular tone: how contractile stimuli leading to an increase in [Ca2+]i trigger vasoconstriction and how relaxant signals reduce [Ca2+]i causing vasodilation. M.P. Walsh opened the symposium with an overview emphasizing the central role of myosin phosphorylation-dephosphorylation in the regulation of vascular tone and identifying recent developments concerning regulation of [Ca2+]i, Ca2+ sensitization and desensitization of the contractile response, Ca(2+)-independent protein kinase C induced contraction, and direct regulation of cross-bridge cycling by the thin filament associated proteins caldesmon and calponin. The remainder of the symposium focused on three specific areas related to the regulation of vascular tone: Ca2+ signalling in relation to smooth muscle structure, structure-function relations of myosin, and the role of cyclic GMP (cGMP) dependent protein kinase. G.J. Kargacin described how smooth muscle cells are structured and how second messenger signals such as Ca2+ might be modified or influenced by this structure. J. Kendrick-Jones then discussed the results of mutagenesis studies aimed at understanding how the myosin light chains, particularly the phosphorylatable (Ca(2+)-calmodulin dependent) regulatory light chains, control myosin. The vasorelaxant effects of signalling molecules such as beta-adrenergic agents and nitrovasodilators are mediated by cyclic nucleotide dependent protein kinases, leading principally to a reduction in [Ca2+]i. T.M. Lincoln described the roles of cyclic nucleotide dependent protein kinases, in particular cyclic GMP dependent protein kinase, in vasodilation.
Animals↗
Utrophin actin binding domain: analysis of actin binding and cellular targeting.
Utrophin, or dystrophin-related protein, is an autosomal homologue of dystrophin. The protein is apparently ubiquitously expressed and in muscle tissues the expression is developmentally regulated. Since utrophin has a similar domain structure to dystrophin it has been suggested that it could substitute for dystrophin in dystrophic muscle. Like dystrophin, utrophin has been shown to be associated with a membrane-bound glycoprotein complex. Here we demonstrate that expressed regions of the predicted actin binding domain in the NH2 terminus of utrophin are able to bind to F-actin in vitro, but do not interact with G-actin. The utrophin actin binding domain was also able to associate with actin-containing structures, stress fibres and focal contacts, when microinjected into chick embryo fibroblasts. The expressed NH2-terminal 261 amino acid domain of utrophin has an affinity for skeletal F-action (Kd 19 +/- 2.8 microM), midway between that of the corresponding domains of alpha-actinin (Kd 4 microM) and dystrophin (Kd 44 microM). Moreover, this utrophin domain binds to non-muscle actin with a approximately 4-fold higher affinity than to skeletal muscle actin. These data (together with those of Matsumura et al. (1992) Nature, 360, 588-591) demonstrate for the first time that utrophin is capable of performing a functionally equivalent role to that of dystrophin. The NH2 terminus of utrophin binds to actin and the COOH terminus binds to the membrane associated glycoprotein complex, thus in non-muscle and developing muscle utrophin performs the same predicted 'spacer' or 'shock absorber' role as dystrophin in mature muscle tissues. These data suggest that utrophin could replace dystrophin functionally in dystrophic muscle.
Actins↗
Single-molecule mechanics of heavy meromyosin and S1 interacting with rabbit or Drosophila actins using optical tweezers.
Single-molecule mechanical interactions between rabbit heavy meromyosin (HMM) or subfragment 1 (S1) and rabbit actin were measured with an optical tweezers piconewton, nanometer transducer. Similar intermittent interactions were observed with HMM and S1. The mean magnitude of the single interaction isotonic displacements was 20 nm for HMM and 15 nm with S1. The mean value of the force of single-molecule interactions was 1.8 pN for HMM and 1.7 pN with S1. The stiffness of myosin S1 was determined by applying a sinusoidal length change to the thin filament and measuring the corresponding force; the mean stiffness was 0.13 pN nm-1. By moving an actin filament over a long distance past an isolated S1 head, we found that cross-bridge attachment occurred preferentially at a periodicity of about 40 nm, similar to that of the actin helical repeat. Rate constants for the probability of detachment of HMM from actin were determined from histograms of the lifetime of the attached state. This gave a value of 8 s-1 or 0.8 x 10(6) M-1 s-1 for binding of ATP to the rigor complex. We conclude (1) that our HMM-actin interactions involve just one head, (2) that compliance of the cross-bridge is not in myosin subfragment 2, although we cannot say to what extent contributions arise from myosin S1 or actin, and (3) that the elemental movement can be caused by a change of shape of the S1 head, but that this would have to be much greater than the movements suggested from structural studies of S1 (Rayment et al., 1993).
Actins↗
A myosin-like protein from a higher plant.
As part of a study of the diversity of myosins, we have cloned a cDNA encoding a myosin-like protein from Arabidopsis thaliana. This is the first molecular motor of any kind to be cloned from a higher plant. The predicted polypeptide (molecular weight 131 kDa) has a motor domain (head) very similar to those of other myosins, but the remainder of the sequence is unusual. The tail contains four potential calmodulin binding sites ("IQ-motifs"), but no sequence motifs suggestive of actin or phospholipid binding, like those found in other myosins. There is also a small region of probable alpha-helical coiled-coil, which suggests that the molecule could be dimeric, though unlikely to form filaments. The N-terminal and C-terminal regions of the molecule are unique. We present a phylogenetic analysis of myosin head sequences, which suggests that this is a new type of myosin.
Amino Acid Sequence↗
Sequences of sea urchin kinesin light chain isoforms.
We have deduced the amino acid sequences of four sea urchin (Strongylocentrotus purpuratus; SP) kinesin light chain (KLC) isoforms (SPKLC 1-4) and compared them to rat brain light chain sequences. Examination of the SPKLC open reading frames (SPKLC1, 649; SPKLC2, 677; SPKLC3, 686; and SPKLC4, 451 amino acid residues) reveals that the first 500 or so residues of the KLCs are highly conserved but the C-terminal ends of rat and sea urchin light chains are divergent; SPKLCs 1, 2 and 3 share a highly basic, 86 residue C-terminal segment that is missing from the shorter rat light chains and SPKLC4. The insertion of 28 and 37 residue segments at residue 563 of SPKLCs 2 and 3, respectively, gives rise to sequence heterogeneity at the C-terminal ends of the sea urchin KLCs. C-terminal sequence differences between light chains may provide inter- and intraspecies differences in the functional properties of the presumptive cargo attachment elements of kinesin.
Amino Acid Sequence↗
The C-terminal helix in subdomain 4 of the regulatory light chain is essential for myosin regulation.
In vertebrate smooth/non-muscle myosins, phosphorylation of the regulatory light chains by a specific calmodulin-activated kinase controls both myosin head interaction with actin and assembly of the myosin into filaments. Previous studies have shown that the C-terminal domain of the regulatory light chain is crucial for the regulation of these myosin functions. To further dissect the role of this region of the light chain in myosin regulation, a series of chicken smooth muscle myosin regulatory light chain mutants has been constructed with successive C-terminal deletions. These mutants were synthesized in Escherichia coli and analysed by their ability to restore Ca2+ regulation to scallop myosin that had been stripped of its native regulatory light chains ('desensitized'). The results show that regulatory light chain mutants with deletions in the C-terminal helix in subdomain 4 were able to reform the regulatory Ca2+ binding site on the scallop myosin head, but had lost the ability to suppress scallop myosin filament assembly and interaction with actin in the absence of Ca2+. Further deletions in the C-terminal domain led to a gradual loss of ability to restore the regulatory Ca2+ binding site. Thus, the regions in the C-terminal half of the regulatory light chain responsible for myosin regulation can be identified.
Actins↗
Structural studies on the conformations of myosin.
Myosin is the major motor protein found in vertebrate striated and smooth muscle and in non-muscle cells where, in association with actin, its main role is to convert chemical energy into mechanical work. Smooth muscle and non-muscle myosin adopts a number of different conformations: for example an unfolded (6S) form which is capable of forming filaments and generating force and a 'folded' (10S) form, which is most probably a storage form incapable of forming filaments. In the 10S form the products of ATP cleavage are trapped by the folded tails and the ATPase activity of myosin is greatly reduced. It is believed that a transition to the unfolded 6S form is necessary prior to filament formation. We report here on two relatively low resolution structural techniques for studying hydrated myosin. We have used a relatively recent development in microscopy, the scanning tunneling microscope, to image a series of biologically interesting specimen, mainly to evaluate the potential of the technique. There are significant potential advantages for imaging biological specimen with the STM as the imaging is done in air and the specimen can be imaged without a metal coating. Our experience with imaging myosin suggests that good images of hydrated myosin can be obtained but with poor reproducibility. We have also carried out small angle solution x-ray scattering studies on the two myosin conformations to explore the possibilities of doing kinetic measurements on the transition between the two states. Small angle scattering from the S1 fragment are re-constituted parts of the rod have also been carried out and the data is compared with expected scattering from model structures.
Animals↗
Dystrophin and related proteins.
During the past year significant progress has been made in understanding how dystrophin deficiency leads to muscle cell necrosis in Duchenne muscular dystrophy and Becker muscular dystrophy. Dystrophin interacts with a glycoprotein complex spanning the muscle sarcolemma, effectively linking the actin cytoskeleton to the extracellular matrix. The carboxyl terminus of dystrophin is required for glycoprotein binding. Interestingly, at least three mRNAs transcribed from the distal end of the DMD gene in tissues other than muscle have been shown to encode this domain. Deficiency of a second component of the dystrophin-associated glycoprotein complex has been shown to occur in another muscle-wasting disorder, severe childhood autosomal recessive muscular dystrophy. Sequence analysis of the entire cDNA for the autosomal dystrophin-related protein utrophin has shown that dystrophin and utrophin are closely related. Furthermore, both of these proteins have been shown to bind to the same or a similar glycoprotein complex in muscle.
Animals↗
Primary structure of dystrophin-related protein.
Dystrophin-related protein (DRP or 'utrophin') is localized in normal adult muscle primarily at the neuromuscular junction. In the absence of dystrophin in Duchenne muscular dystrophy (DMD) patients, DRP is also present in the sarcolemma. DRP is expressed in fetal and regenerating muscle and may play a similar role to dystrophin in early development, although it remains to be determined whether DRP can functionally replace dystrophin in adult tissue. Previously we described a 3.5-kilobase complementary DNA clone that exhibits 80 per cent homology to the C-terminal domain of dystrophin. This sequence identifies a 13-kilobase transcript that maps to human chromosome 6 (refs 2, 11). Antibodies raised against the gene product identify a polypeptide with a relative molecular mass of about 400K in all tissues examined. To investigate the relationship between DRP and dystrophin in more detail, we have cloned and sequenced the whole DRP cDNA. Homology between DRP and dystrophin extends over their entire length, suggesting that they derive from a common ancestral gene. Comparative analysis of primary sequences highlights regions of functional importance, including those that may mediate the localization of DRP and dystrophin in the muscle cell.
Actinin↗
Uncoupling of actin-activated myosin ATPase activity from actin binding by a monoclonal antibody directed against the N-terminus of myosin light chain 1.
The role of the N-terminal region of myosin light chain 1 (LC1) in actomyosin interaction was investigated using an IgG monoclonal antibody (2H2) directed against the N-terminal region of LC1. We defined the binding site of 2H2 by examining its cross-reactivity with myosin light chains from a variety of species and with synthetic oligopeptides. Our findings suggest that 2H2 is directed against the N-terminal region of LC1 which includes the trimethylated alanine residue at the N-terminus. In the presence of 2H2, the rate of actomyosin superprecipitation was reduced, although the extent was not. 2H2 caused a reduction in the Vmax of both myosin and chymotryptic S1(A1) actin-activated ATPase activity, while the Km appeared to be unaltered. The Mg(2+)-ATPase activity of myosin alone was also unaffected. Binding studies revealed that 2H2 did not prevent the formation of acto-S1 complex, either in the presence or in the absence of ATP, nor did it affect the ability of ATP to dissociate S1 from F-actin. Our findings suggest that the N-terminal region of LC1 is not essential for actin binding but is involved in modulating actin-activated ATPase activity of myosin.
Actins↗