The effects of caldesmon on the ATPase activities of rabbit skeletal-muscle myosin

Lim, Megan S.; Walsh, Michael P.

doi:10.1042/bj2380523

Cited by 21 publications

(4 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As shown in Table I, the ATPase activity of the myosin was inhibited by 6 pM caldesmon to 65% of the control value, confirming the result of Lim and Walsh [1986] and Chalovich et al [ 19871 with skeletal muscle myosin. However, the effect of the myosin-binding fragment at 6 pM was stimulatory (Table I).…”

Section: Confirmatory Studies With Skeletal Muscle Myosinsupporting

confidence: 93%

“…Measurements of ATPase activity of skeletal muscle myosin in the presence of actin filament confirmed the data from the motility assay although micromolar levels of caldesmon and the myosin-binding fragment were required for measurements. As shown in Table I, the ATPase activity of the myosin was inhibited by 6 pM caldesmon to 65% of the control value, confirming the result of Lim and Walsh [1986] and Chalovich et al Fig. 5.…”

Section: Confirmatory Studies With Skeletal Muscle Myosinsupporting

confidence: 84%

See 1 more Smart Citation

Stimulation of the ATP‐dependent interaction between actin and myosin by a myosin‐binding fragment of smooth muscle caldesmon

Lin

Ishikawa

Okagaki

et al. 1994

Cell Motil. Cytoskeleton

View full text Add to dashboard Cite

We reported previously that smooth muscle caldesmon stimulates the ATP-dependent interaction between actin and phosphorylated smooth muscle myosin, as monitored by ATPase measurement and in vitro motility assay. Furthermore, this effect changes from stimulatory to inhibitory with increasing concentrations of caldesmon [Ishikawa et al., 1991: J. Biol. Chem. 266:21784-21790]. The N-terminal (myosin-binding) fragment and the C-terminal (actin-binding) fragment were purified from digests of caldesmon. The effects of the myosin-binding fragment and the actin-binding fragment on the interaction were stimulatory and inhibitory, respectively, indicating that stimulatory and inhibitory domains are localized in the myosin-binding domain and actin-binding domain of caldesmon, respectively. The effect of the myosin-binding fragment on the interaction was exclusively stimulatory when the interaction was challenged by caldesmon, both at lower and higher concentrations. However, the actin-binding fragment had no effect on the interaction at lower concentrations and inhibited the interaction at higher concentrations. Thus, the stimulatory effect of caldesmon that is observed at lower concentrations can be explained by the hypothesis that the stimulatory effect of the myosin-binding domain predominates over the inhibitory effect of the actin-binding domain when the concentration of caldesmon is low. With uncleaved caldesmon, we also emphasized the role of the myosin-binding domain in the stimulation as follows; the stimulatory effect of caldesmon became obscured when binding of caldesmon to myosin was competed by the exogenous caldesmon-binding fragment of myosin.

show abstract

Section: Confirmatory Studies With Skeletal Muscle Myosinsupporting

confidence: 93%

Section: Confirmatory Studies With Skeletal Muscle Myosinsupporting

confidence: 84%

Stimulation of the ATP‐dependent interaction between actin and myosin by a myosin‐binding fragment of smooth muscle caldesmon

Lin

Ishikawa

Okagaki

et al. 1994

Cell Motil. Cytoskeleton

View full text Add to dashboard Cite

show abstract

“…Caldesmon inhibits the actin-activated myosin Mg2+-ATPase in a contractile system reconstituted in vitro from purified actin, myosin, tropomyosin, calmodulin and myosin light-chain kinase, without affecting myosin phosphorylation , suggesting that caldesmon may play a role in the regulation of smooth-muscle contraction. These observations have been confirmed by several investigators using caldesmon and myosin from a variety of sources (Dabrowska et al, 1985;Marston & Lehman, 1985;Sobue et al, 1985;Clark et al, 1986;Lash et al, 1986;Lim & Walsh, 1986;Chalovich et al, 1987).…”

Section: Introductionmentioning

confidence: 63%

Autophosphorylation of smooth-muscle caldesmon

Scott-Woo

Walsh

1988

Biochemical Journal

Self Cite

View full text Add to dashboard Cite

Caldesmon, a major actin- and calmodulin-binding protein of smooth muscle, has been implicated in regulation of the contractile state of smooth muscle. The isolated protein can be phosphorylated by a co-purifying Ca2+/calmodulin-dependent protein kinase, and phosphorylation blocks inhibition of the actomyosin ATPase by caldesmon [Ngai & Walsh (1987) Biochem. J. 244, 417-425]. We have examined the phosphorylation of caldesmon in more detail. Several lines of evidence indicate that caldesmon itself is a kinase and the reaction is an intermolecular autophosphorylation: (1) caldesmon (141 kDa) and a 93 kDa proteolytic fragment of caldesmon can be separated by ion-exchange chromatography: both retain caldesmon kinase activity, which is Ca2+/calmodulin-dependent; (2) chymotryptic digestion of caldesmon generates a Ca2+/calmodulin-independent form of caldesmon kinase; (3) caldesmon purified to electrophoretic homogeneity retains caldesmon kinase activity, and elution of enzymic activity from a fast-performance-liquid-chromatography ion-exchange column correlates with caldesmon of Mr 141,000; (4) caldesmon is photoaffinity-labelled with 8-azido-[alpha-32P]ATP; labelling is inhibited by ATP, GTP and CTP, indicating a lack of nucleotide specificity; (5) caldesmon binds tightly to Affi-Gel Blue resin, which recognizes proteins having a dinucleotide fold. Autophosphorylation of caldesmon occurs predominantly on serine residues (83.3%), with some threonine (16.7%) and no tyrosine phosphorylation. Autophosphorylation is site-specific: 98% of the phosphate incorporated is recovered in a 26 kDa chymotryptic peptide. Complete tryptic/chymotryptic digestion of this phosphopeptide followed by h.p.l.c. indicates three major phosphorylation sites. Caldesmon exhibits a high degree of substrate specificity: apart from autophosphorylation, brain synapsin I is the only good substrate among many potential substrates examined. These observations indicate that caldesmon may regulate its own function (inhibition of the actomyosin ATPase) by Ca2+/calmodulin-dependent autophosphorylation. Furthermore, caldesmon may regulate other cellular processes, e.g. neurotransmitter release, through the Ca2+/calmodulin-dependent phosphorylation of other proteins such as synapsin I.

show abstract

“…In vitro, caldesmon inhibits actin-activated myosin ATPase (20), and caldesmon together with tropomyosin protects actin filaments from severing activities of gelsolin and anneals gelsolin-severed actin filaments (21,22). These two activities are likely to be involved in regulating the actomyosin contractility and stability of stress fibers, respectively (23)(24)(25)(26)(27)(28).…”

mentioning

confidence: 99%