Ca2+-dependent Regulation of the Motor Activity of Myosin V

Abstract

Mouse myosin V constructs were produced that consisted of the myosin motor domain plus either one IQ motif (M5IQ1), two IQ motifs (M5IQ2), a complete set of six IQ motifs (SHM5), or the complete IQ motifs plus the coiled-coil domain (thus permitting formation of a double-headed structure, DHM5) and expressed in Sf9 cells. The actin-activated ATPase activity of all constructs except M5IQ1 was inhibited above pCa 5, but this inhibition was completely reversed by addition of exogenous calmodulin. At the same Ca 2؉ concentration, 2 mol of calmodulin from SHM5 and DHM5 or 1 mol of calmodulin from M5IQ2 were dissociated, suggesting that the inhibition of the ATPase activity is due to dissociation of calmodulin from the heavy chain. However, the motility activity of DHM5 and M5IQ2 was completely inhibited at pCa 6, where no dissociation of calmodulin was detected. Inhibition of the motility activity was not reversed by the addition of exogenous calmodulin. These results indicate that inhibition of the motility is due to conformational changes of calmodulin upon the Ca 2؉ binding to the high affinity site but is not due to dissociation of calmodulin from the heavy chain.Myosins are motor proteins that translocate actin filaments upon hydrolysis of ATP, and thus they play a critical role in diverse forms of cell contractility and motility. During the last decade a number of myosin-like proteins have been found, and the myosins are currently organized into 15 classes based upon phylogenetic sequence comparisons of the motor domains (1-5). Class V myosin was originally identified in brain as a calmodulin-binding protein that had actin-dependent ATPase activity (6). Myosin V is a member of the myosin superfamily that is expressed in variety of cell types and is involved in a variety of membrane trafficking and organelle transport functions (1-5). Myosin V has two heads that are connected with a long coiledcoil domain; however, in contrast to conventional myosin, it contains a globular C-terminal domain and does not form thick filaments (7). The head domain is composed of a globular motor domain and an elongated neck domain that is associated with a number of light chains. The sequence at the neck region contains six IQ motifs that have been implicated as calmodulin or myosin light chain binding consensus motifs as found in a variety of calmodulin-binding proteins and myosins (7). Since light chains play a critical role in the regulation of various conventional myosins, it has been proposed that the IQ domain serves as a regulatory component of myosin V. The role of the IQ motif and bound calmodulin serving as a regulatory component of unconventional myosins is best studied for mammalian myosin Is. For both brush border myosin I (8 -10) and myosin I␤ (11-13), high Ca 2ϩ inhibits motor activity due to Ca 2ϩ binding to the calmodulin light chain. Since 1 mol of bound calmodulin dissociates from myosin I at high Ca 2ϩ , it was originally thought that this dissociation of calmodulin was responsible for the inhibition of myosin I...

Mechanoenzymatic Characterization of Human Myosin Vb

“…The slower cycling rate is also consistent with the actin gliding velocity; myosin Vb exhibited approximately twothirds of the velocity of myosin Va (43,58).…”

“…However, there are the regions in the motor domain of myosin Vb whose sequences are quite different from that of myosin Va. Among them, of interest is the loop 2 region that has been suggested to influence the actin binding property of myosin (43)(44)(45)(46)(47) since the K actin of myosin Vb is significantly higher than that of myosin Va. Figure 12 shows the amino acid sequence comparison between human myosin Va and myosin Vb in the motor domain. The loop 2 sequence of myosin Vb is unique and quite different from that of myosin Va.…”

Myosin-Va Regulates Exocytosis through the Submicromolar Ca2+-dependent Binding of Syntaxin-1A

“…Further studies in a bacterial two-hybrid assay, which is a modification of the yeast two-hybrid method, indicated that the six IQ-motifs of the neck bind to syntaxin-1A as well as CaM, although the first IQ alone does not mediate binding ( Figure 5E). Collectively, these results demonstrate that the binding site for syntaxin-1A is in the neck of myosin-Va.CaM is known to be released from the neck of myosin-Va in the presence of micromolar Ca 2ϩ (Cameron et al, 1998;Homma et al, 2000). We confirmed that preincubation of myosin-Va with Ca 2ϩ released the bound CaM ( Figure 5F).…”

“…Recombinant myosin-V (DHM5; ) was produced in Sf9 cells as described previously (Homma et al, 2000) or by in vitro translation (Promega, Madison, WI) using the mouse dilute cDNA (gift of N. A. Jenkins, University of Sao Paolo, Ribeirao Preto, Brazil; Mercer et al, 1991). DHM5 was detected with anti-myosin-Va head antibody (gift of R. E. Larson, National Cancer Institute, Frederick, MD; Nascimento et al, 1996;Evans et al, 1998).…”

The Phosphorylation of Myosin II at the Ser1 and Ser2 Is Critical for Normal Platelet-derived Growth Factor–induced Reorganization of Myosin Filaments

“…MLC 20 was phosphorylated by MLCK and PKC as described (Ikebe and Hartshorne, 1985a;Ikebe et al, 1987). To analyze the fraction of the expressed MLC 20 incorporated into myosin II, we have used an ATP-dependent actin-binding activity of myosin II as described previously (Homma et al, 2000;Wei and Adelstein, 2000) with slight modifications. MEF/3T3 cells expressing myc-tagged MLC 20 s were lysed in buffer I (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 5 mM MgCl 2 , 2 mM EGTA, 1 mM dithiothreitol, 0.2 mM N-␣-p-tosyl-l-lysine chloromethl ketone [TLCK], 0.2 mM tosyl-phenylalanyl chloromethyl ketone [TPCK], 10 g/ml leupeptin, 2 mM phenylmethylsulfonyl fluoride, and 0.01% IGEPAL CA-630) with 2 mM ATP by sonication.…”