Calcium functionally uncouples the heads of myosin VI.
This study examines the steady state activity and in vitro motility of single-headed (S1) and double-headed (HMM) myosin VI constructs within the context of two putative modes of regulation. Phosphorylation of threonine 406 does not alter either the rate of actin filament sliding or the maximal actin-activated ATPase rate of S1 or HMM constructs. Thus, we do not observe any regulation of myosin VI by phosphorylation within the motor domain. Interestingly, in the absence of calcium, the myosin VI HMM construct moves in an in vitro motility assay at a velocity that is twice that of S1 constructs, which may be indicative of movement that is not based on a "lever arm" mechanism. Increasing calcium above 10 microm slows both the rate of ADP release from S1 and HMM actomyosin VI and the rates of in vitro motility. Furthermore, high calcium concentrations appear to uncouple the two heads of myosin VI. Thus, phosphorylation and calcium are not on/off switches for myosin VI enzymatic activity, although calcium may alter the degree of processive movement for myosin VI-mediated cargo transport. Lastly, calmodulin mutants reveal that the calcium effect is dependent on calcium binding to the N-terminal lobe of calmodulin.
Morris
Carl A
CA
Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6085, USA.
Wells
Amber L
AL
Yang
Zhaohui
Z
Chen
Li-Qiong
LQ
Baldacchino
Corradina V
CV
Sweeney
H Lee
HL
D000199:Actins / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D020409:Molecular Motor Proteins; D009038:Motion; D018995:Myosin Heavy Chains / Q000737:chemistry / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D013552:Swine
D000199:Actins; D020409:Molecular Motor Proteins; C089785:myosin VI; D010744:Phosphoric Monoester Hydrolases; D018995:Myosin Heavy Chains; D002118:Calcium