High Affinity Ca2+ Binding Sites of Calmodulin Are Critical for the Regulation of Myosin Iβ Motor Function

Zhu, Tao; Beckingham, Kathy; Ikebe, Mitsuo

doi:10.1074/jbc.273.32.20481

Cited by 63 publications

(75 citation statements)

References 37 publications

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“…This indicates that interhead interaction is not critical for the Ca 2ϩ -dependent regulation of C442Y myosin VI. Ca 2ϩ -induced activation of the actin-activated ATPase activity has been reported for other unconventional myosins (28,(53)(54)(55)(56)(57)(58). Because bound calmodulin can be dissociated from the heavy chain at high Ca 2ϩ , it was proposed originally that the activation of the ATPase activity is because of the dissociation of calmodulin (53,56).…”

Section: Discussionmentioning

confidence: 99%

Human Deafness Mutation of Myosin VI (C442Y) Accelerates the ADP Dissociation Rate

Satō

White

Inoue

et al. 2004

Journal of Biological Chemistry

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The missense mutation of Cys 442 to Tyr of myosin VI causes progressive postlingual sensorineural deafness. Here we report the affects of the C442Y mutation on the kinetics of the actomyosin ATP hydrolysis mechanism and motor function of myosin VI. The largest changes in the kinetic mechanism of ATP hydrolysis produced by the C442Y mutation are about 10-fold increases in the rate of ADP dissociation from both myosin VI and actomyosin VI. The rates of ADP dissociation from acto-C442Y myosin VI-ADP and C442Y myosin VI-ADP are 20 -40 times more rapid than the steady state rates and cannot be the rate-limiting steps of the hydrolysis mechanism in the presence or absence of actin. The 2-fold increase in the actin gliding velocity of C442Y compared with wild type (WT) may be explained at least in part by the more rapid rate of ADP dissociation. The C442Y myosin VI has a significant increase (ϳ10-fold) in the steady state ATPase rate in the absence of actin relative to WT myosin VI. The steady state rate of actin-activated ATP hydrolysis is unchanged by the C442Y mutation at low (<10 ؊7 M) calcium but is calcium-sensitive with a 1.6-fold increase at high (ϳ10 ؊4 M) calcium that does not occur with WT. The actin gliding velocity of the C442Y mutant decreases significantly at low surface density of myosin VI, suggesting that the mutation hampers the processive movement of myosin VI.Myosin is a motor protein that interacts with actin filaments and converts energy from ATP hydrolysis into mechanical activity. Among diverse myosin superfamily proteins, class VI myosins are identified in various organisms in the animal kingdom (1-4). In mammals, myosin VI is found in various tissues, but its physiological significance is most recognized in the auditory system (5). The mouse myosin VI gene was discovered to be responsible for deafness in the Snell's waltzer mice, which show the characteristic waltzing and circling behavior (6).Myosin VI is expressed in both the inner and outer hair cells of the inner ear and is localized in the pericuticular necklace, present between the cuticular plate and circumferential actin structures. The location suggests that myosin VI may be responsible for the maintenance of sensory hair cells. In Snell's walzer mice, the cochlea shows progressive loss of hair cells soon after birth (6, 7), suggesting that myosin VI function is involved in the maintenance of sensory hair cells. These findings suggest that myosin VI may function to maintain the association of the cuticular plate with the stereocilia rootlets, which in turn stabilize the hair cells (8). Although the functional deficiency of myosin VI mutation is most pronounced in auditory function, myosin VI is expressed in a variety of cell types.The location of class VI myosin in Drosophilia (Drosophila 95F myosin) in the cytoplasmic particles that move along microfilaments suggests that myosin VI is involved in the transport processes (9). In polarized cells from the intestinal brush border, myosin VI is concentrated in the terminal web with a sma...

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Section: Discussionmentioning

confidence: 99%

Human Deafness Mutation of Myosin VI (C442Y) Accelerates the ADP Dissociation Rate

Satō

White

Inoue

et al. 2004

Journal of Biological Chemistry

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“…But recent studies have revealed that the inhibition does not require the dissociation of calmodulin but that Ca 2ϩ binding at the high affinity sites of calmodulin triggers the inhibition presumably because of a large conformational change of calmodulin (13,14,18). Addition of exogenous calmodulin did not rescue the inhibition of the motility activity of myosin X unless Ca 2ϩ concentration was decreased.…”

Section: Fig 13mentioning

confidence: 99%

“…The molecular mass markers used were smooth muscle myosin heavy chain (204 kDa), ␤-galactosidase (116 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), myosin regulatory light chain (20 kDa), and ␣-lactalbumin (14.2 kDa). The amount of the myosin X heavy chain and calmodulin was determined by densitometry as described previously (14). The steady state ATPase activity was determined by measuring liberated 32 P i at 25°C as described previously (22).…”

Section: Methodsmentioning

confidence: 99%

“…The sample was ultracentrifuged at 100,000 ϫ g for 30 min, and the pellets were analyzed by SDS-polyacrylamide gel electrophoresis. The amount of the co-sedimented M10CC heavy chain and calmodulin were determined by densitometry as described previously (14).…”

Section: Methodsmentioning

confidence: 99%

“…The role of the IQ motif in unconventional myosins is best studied in myosin I (13)(14)(15)(16)(17) and V (18). These myosins contain calmodulin as their light chain that associates with the heavy chain at the IQ region.…”

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Motor Function and Regulation of Myosin X

Homma

Saito

Ikebe

et al. 2001

Journal of Biological Chemistry

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Myosin X is a member of the diverse myosin superfamily that is ubiquitously expressed in various mammalian tissues. Although its association with actin in cells has been shown, little is known about its biochemical and mechanoenzymatic function at the molecular level. We expressed bovine myosin X containing the entire head, neck, and coiled-coil domain and purified bovine myosin X in Sf9 cells. The Mg 2؉ -ATPase activity of myosin X was significantly activated by actin with low K ATP . The actin-activated ATPase activity was reduced at Ca 2؉ concentrations above pCa 5 in which 1 mol of calmodulin light chain dissociates from the heavy chain. Myosin X translocates F-actin filaments with the velocity of 0.3 m/s with the direction toward the barbed end. The actin translocating activity was inhibited at concentrations of Ca 2؉ at pCa 6 in which no calmodulin dissociation takes place, suggesting that the calmodulin dissociation is not required for the inhibition of the motility. Unlike class V myosin, which shows a high affinity for F-actin in the presence of ATP, the K actin of the myosin X ATPase was much higher than that of myosin V. Consistently nearly all actin dissociated from myosin X in the presence of ATP. ADP did not significantly inhibit the actin-activated ATPase activity of myosin X, suggesting that the ADP release step is not rate-limiting. These results suggest that myosin X is a nonprocessive motor. Consistently myosin X failed to support the actin translocation at low density in an in vitro motility assay where myosin V, a processive motor, supports the actin filament movement.Myosins are actin-based motor proteins that play a role in diverse cellular movement. Myosin is composed of a motor domain containing an ATP and actin binding region, a neck domain that interacts with specific light chains or calmodulin, and a tail domain that serves to anchor myosin to a specific cellular target. In the last decade, a number of different types of myosins have been discovered by molecular cloning, and based upon their primary structure of the motor domain they are classified in at least 18 classes (1-6).Myosin X is a newly found myosin from bovine, human (7), and mouse (8), and it is expressed ubiquitously in various mammalian tissues. Based upon the deduced amino acid sequence, it is predicted that myosin X consists of a motor domain, three IQ motifs that function as light chain binding sites, a coiled-coil domain, and a tail domain. Because of the presence of a coiled-coil domain, it has been thought that myosin X is a two-headed myosin. There are three pleckstrin homology domains, one myosin tail homology domain, and one FERM (4.1/ Ezrin/Radixin/Moesin) domain in each heavy chain. Although the actual function of these domains is not known, they could play a role in interactions with membrane phospholipids or other proteins that create an anchoring structure.By immunocytochemistry, it was found (7) that myosin X is present at the edge of lamellipodia, membrane ruffles, and the tip of filopodial actin bundles ...

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Calmodulin

Yap¹,

Ikura²

2004

Handbook of Metalloproteins

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CaM is a ubiquitous eukaryotic calcium binding protein that interacts with hundreds of proteins including kinases and phosphatases, transmembrane ion channels and pumps, and cytoskeletal regulatory proteins. In response to an increase in intracellular calcium, CaM undergoes a major conformational change enabling target binding and activation. In some cases, CaM is able to bind target proteins in the absence of, or independently of, Ca 2+ ; in others, CaM binding may induce inactivation. The structural plasticity of calmodulin is demonstrated by the diversity observed in its interaction with various targets.

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High Affinity Ca2+ Binding Sites of Calmodulin Are Critical for the Regulation of Myosin Iβ Motor Function

Cited by 63 publications

References 37 publications

Human Deafness Mutation of Myosin VI (C442Y) Accelerates the ADP Dissociation Rate

Human Deafness Mutation of Myosin VI (C442Y) Accelerates the ADP Dissociation Rate

Motor Function and Regulation of Myosin X

Calmodulin

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