Kinetic Mechanism and Regulation of Myosin VI

Abstract

Myosin VI is the only pointed end-directed myosin identified and is likely regulated by heavy chain phosphorylation (HCP) at the actin-binding site in vivo. We undertook a detailed kinetic analysis of the actomyosin VI ATPase cycle to determine whether there are unique adaptations to support reverse directionality and to determine the molecular basis of regulation by HCP. ADP is the in the and myosin VI is to and the and myosin VI is a and to the of ATPase ADP are the to the regulated by myosin VI the are and the We site the and of Myosin VI is the only pointed end-directed myosin identified and is likely regulated by heavy chain phosphorylation (HCP) at the actin-binding site in vivo. We undertook a detailed kinetic analysis of the actomyosin VI ATPase cycle to determine whether there are unique adaptations to support reverse directionality and to determine the molecular basis of regulation by HCP. ADP is the in the and myosin VI is to and the and myosin VI is a and to the of ATPase ADP are the to the regulated by myosin VI the are and the We site the and of myosin VI heavy myosin VI to myosin VI to Myosin VI is unique of the myosin of molecular in the pointed of to the the of myosin VI are in and the of myosin VI are to regulated by the phosphorylation site identified and and to of the actin-binding and phosphorylation of the at and are in the of myosin a the ATPase Myosin heavy chain are of myosin the and are by of the a molecular by myosin VI reverse directionality is to unique of the the the and chain the kinetic of the actomyosin VI ATPase cycle to pointed end-directed unique to determine whether myosin VI is regulated by heavy chain phosphorylation and to the molecular basis of the the of myosin VI a at kinetic the and of of myosin myosin VI of the and is to the ATPase of Myosin of myosin and are to by ATPase of myosin is Myosin a ATPase of the ATPase of myosin and ATPase myosin ATPase in the of in the of the at of in the of the in the of at at ATPase in the of in the of the at of in the of the at in a Myosin of ATPase of in ATPase the of the myosin VI of myosin VI ATPase a to the of ATPase of and a of and myosin VI is myosin of myosin VI of myosin is the of the ATPase of and myosin VI are of by ADP VI to by myosin VI and in of the of the in myosin myosin myosin and the myosin and the the to of myosin VI to of myosin myosin in the of the a are the to is the is the is and the is of are and the of the is of the of to myosin myosin ADP myosin and myosin ADP of in the of the of the are the of the to is the is the is the of and myosin VI to the of the and are in and of the actomyosin ATPase and are are are in a Myosin VI by of VI of and the is of myosin VI to the and of of and the and actomyosin VI actomyosin VI and the of the at and the of the and of in the and the of myosin VI of phosphorylation at the of to is and the of is of the of to of the of myosin VI ATPase to of myosin VI by of to VI the to of and the the of a of actomyosin VI of are the to the of myosin to at a is the to a a of of myosin myosin to actomyosin VI the of the are in is the in to the of the is and to to actomyosin VI the of the are and of myosin VI is is myosin VI the the site is the of the of myosin VI the in the of the the of of to a of at the of at to are the of the is the and in the of are the of a of the a a of in the of and by the of ADP in the and the of ADP and ADP the ADP in the to Myosin VI in the of by to to myosin VI in the of of myosin VI to of the the the of the are and of of to myosin VI in the of of myosin in the of is the of the of the to a of and of the myosin myosin are in the of the the in the to the of the is a of myosin and of myosin We is likely to a in the ATPase the is the of to myosin VI and actomyosin of myosin in the of is of and the is the to a a of myosin actomyosin myosin actomyosin and are in of actomyosin of and actomyosin VI the is the to a of the of the of to to to the and of the are in of myosin VI there are in to the to myosin and myosin VI and of to the the in myosin and the the the of the the of myosin VI a of and the a of myosin and myosin is to a to and the a of We a at of to actomyosin VI of the in the of and by of to myosin to the and of ADP to myosin VI of myosin VI and actomyosin VI the of a to the the and the of the the and the and by of actomyosin and is and the ATPase of ADP is in the of Myosin at the actomyosin is the and of at in the and of of are only by to a and of a in the and of actomyosin VI and of myosin a by a myosin VI to the of and the ATPase of actomyosin of myosin of myosin the are of the at and the and ATPase the of are the of the and ADP the by ADP ADP and myosin to a is in the of in and is the myosin VI and a to the of is is in the myosin is myosin of the of the to a is myosin and myosin of myosin is is to myosin is actomyosin and the of phosphorylation at is to the of and of of the and in the myosin VI ATPase cycle to the and of the Myosin VI is a the and at are at the at chain phosphorylation the of cycle in the the of at and are a the is is is and of and the of the by Myosin VI is unique of the myosin of molecular in the pointed of to the the of myosin VI are in and the of myosin VI are to regulated by the phosphorylation site identified and and to of the actin-binding and phosphorylation of the at and are in the of myosin a the ATPase Myosin heavy chain are of myosin the and are by of the a molecular by myosin VI reverse directionality is to unique of the the the and chain the kinetic of the actomyosin VI ATPase cycle to pointed end-directed unique to determine whether myosin VI is regulated by heavy chain phosphorylation and to the molecular basis of the the of myosin VI a at kinetic the and of of myosin myosin VI of the and is to the ATPase of Myosin of myosin and are to by ATPase of myosin is Myosin a ATPase of the ATPase of myosin and ATPase myosin ATPase in the of in the of the at of in the of the in the of at at ATPase in the of in the of the at of in the of the at in a Myosin of ATPase of in ATPase the of the myosin VI of myosin VI ATPase a to the of ATPase of and a of and myosin VI is myosin of myosin VI of myosin is the of the ATPase of and myosin VI are of by ADP VI to by myosin VI and in of the of the in myosin myosin myosin and the myosin and the the to of myosin VI to of myosin myosin in the of the a are the to is the is the is and the is of are and the of the is of the of to myosin myosin ADP myosin and myosin ADP of in the of the of the are the of the to is the is the is the of and myosin VI to the of the and are in and of the actomyosin ATPase and are are are in a Myosin VI by of VI of and the is of myosin VI to the and of of and the and actomyosin VI actomyosin VI and the of the at and the of the and of in the and the of myosin VI of phosphorylation at the of to is and the of is of the of to of the of myosin VI ATPase to of myosin VI by of to VI the to of and the the of a of actomyosin VI of are the to the of myosin to at a is the to a a of of myosin myosin to actomyosin VI the of the are in is the in to the of the is and to to actomyosin VI the of the are and of myosin VI is is myosin VI the the site is the of the of myosin VI the in the of the the of of to a of at the of at to are the of the is the and in the of are the of a of the a a of in the of and by the of ADP in the and the of ADP and ADP the ADP in the to Myosin VI in the of by to to myosin VI in the of of myosin VI to of the the the of the are and of of to myosin VI in the of of myosin in the of is the of the of the to a of and of the myosin myosin are in the of the the in the to the of the is a of myosin and of myosin We is likely to a in the ATPase the is the of to myosin VI and actomyosin of myosin in the of is of and the is the to a a of myosin actomyosin myosin actomyosin and are in of actomyosin of and actomyosin VI the is the to a of the of the of to to to the and of the are in of myosin VI there are in to the to myosin and myosin VI and of to the the in myosin and the the the of the the of myosin VI a of and the a of myosin and myosin is to a to and the a of We a at of to actomyosin VI of the in the of and by of to myosin to the and of ADP to myosin VI of myosin VI and actomyosin VI the of a to the the and the of the the and the and by of actomyosin and is and the ATPase of ADP is in the of Myosin at the actomyosin is the and of at in the and of of are only by to a and of a in the and of actomyosin VI and of myosin a by a myosin VI to the of and the ATPase of actomyosin of myosin of myosin the are of the at and the and ATPase the of are the of the and ADP the by ADP ADP and myosin to a is in the of in and is the myosin VI and a to the of is is in the myosin is myosin of the of the to a is myosin and myosin of myosin is is to myosin is actomyosin and the of phosphorylation at is to the of and of of the and in the myosin VI ATPase cycle to the and of the Myosin VI is a the and at are at the at chain phosphorylation the of cycle in the the of at and are a the is is is and of and the of the by myosin VI of the and is to the ATPase of Myosin of myosin and are to by ATPase of myosin is Myosin a ATPase of the ATPase of myosin and ATPase myosin ATPase in the of in the of the at of in the of the in the of at at ATPase in the of in the of the at of in the of the at in a Myosin of ATPase of in ATPase the of the myosin VI of myosin VI ATPase a to the of ATPase of and a of and myosin VI is myosin of myosin VI of myosin is the of the ATPase of and myosin VI are of by ADP VI to by myosin VI and in of the of the in myosin myosin myosin and the myosin and the the to the of and myosin VI to the of the and are in and of the actomyosin ATPase and are are are in a Myosin VI by of VI of and the is of myosin VI to the and of of and the and actomyosin VI actomyosin VI and the of the at and the of the and of in the and the of myosin VI of phosphorylation at the of to is and the of is of the of to of the of myosin VI ATPase to of myosin VI by of to VI the to of and the the of a of actomyosin VI of are the to the of myosin to at a is the to a a of of myosin myosin to actomyosin VI the of the are in is the in to the of the is and to to actomyosin VI the of the are and of myosin VI is is myosin VI the the site is the of the of myosin VI the in the of the the of of to a of at the of at to are the of the is the and in the of are the of a of the a a of in the of and by the of ADP in the and the of ADP and ADP the ADP in the to Myosin VI in the of by to to myosin VI in the of of myosin VI to of the the the of the are and of of to myosin VI in the of of myosin in the of is the of the of the to a of and of the myosin myosin are in the of the the in the to the of the is a of myosin and of myosin We is likely to a in the ATPase the is the of to myosin VI and actomyosin of myosin in the of is of and the is the to a a of myosin actomyosin myosin actomyosin and are in of actomyosin of and actomyosin VI the is the to a of the of the of to to to the and of the are in of myosin VI there are in to the to myosin and myosin VI and of to the the in myosin and the the the of the the of myosin VI a of and the a of myosin and myosin is to a to and the a of We a at of to actomyosin VI of the in the of and by of to myosin to the and of ADP to myosin VI of myosin VI and actomyosin VI the of a to the the and the of the the and the and by of actomyosin and is and the ATPase of ADP is in the of Myosin at the actomyosin is the and of at in the and of of are only by to a and of a in the and of actomyosin VI and of myosin a by a myosin VI to the of and the ATPase of actomyosin of myosin of myosin the are of the at and the and ATPase the of are the of the and ADP the by ADP ADP and myosin to a is in the of in and is the myosin VI and a to the of is is in the myosin is myosin of the of the to a is myosin and myosin of myosin is is to myosin is actomyosin and the of phosphorylation at is to the of and of of the and in the myosin VI ATPase cycle to the and of the Myosin VI is a the and at are at the at chain phosphorylation the of cycle in the the of at and are a the is is is and of and the of the by ATPase of Myosin of myosin and are to by ATPase of myosin is Myosin a ATPase of the ATPase of myosin and ATPase myosin ATPase in the of in the of the at of in the of the in the of at at ATPase in the of in the of the at of in the of the at in a Myosin of ATPase of in ATPase the of the myosin VI of myosin VI ATPase a to the of ATPase of and a of and myosin VI is myosin of myosin VI of myosin is the of the ATPase of and myosin VI are of by ADP VI to by myosin VI and in of the of the in myosin myosin myosin and the myosin and the the to the of and myosin VI to the of the and are in and of the actomyosin ATPase and are are are in a Myosin VI by of VI of and the is of myosin VI to the and of of and the and actomyosin VI actomyosin VI and the of the at and the of the and of in the and the of myosin VI of phosphorylation at the of to is and the of is of the of to of the of myosin VI ATPase to of myosin VI by of to VI the to of and the the of a of actomyosin VI of are the to the of myosin to at a is the to a a of of myosin myosin to actomyosin VI the of the are in is the in to the of the is and to to actomyosin VI the of the are and of myosin VI is is myosin VI the the site is the of the of myosin VI the in the of the the of of to a of at the of at to are the of the is the and in the of are the of a of the a a of in the of and by the of ADP in the and the of ADP and ADP the ADP in the to Myosin VI in the of by to to myosin VI in the of of myosin VI to of the the the of the are and of of to myosin VI in the of of myosin in the of is the of the of the to a of and of the myosin myosin are in the of the the in the to the of the is a of myosin and of myosin We is likely to a in the ATPase the is the of to myosin VI and actomyosin of myosin in the of is of and the is the to a a of myosin actomyosin myosin actomyosin and are in of actomyosin of and actomyosin VI the is the to a of the of the of to to to the and of the are in of myosin VI there are in to the to myosin and myosin VI and of to the the in myosin and the the the of the the of myosin VI a of and the a of myosin and myosin is to a to and the a of We a at of to actomyosin VI of the in the of and by of to myosin to the and of ADP to myosin VI of myosin VI and actomyosin VI the of a to the the and the of the the and the and by of actomyosin and is and the ATPase of ADP is in the of Myosin at the actomyosin is the and of at in the and of of are only by to a and of a in the and of actomyosin VI and of myosin a by a myosin VI to the of and the ATPase of actomyosin of myosin of myosin the are of the at and the and ATPase the of are the of the and ADP the by ADP ADP and myosin to a is in the of in and is the myosin VI and a to the of is is in the myosin is myosin of the of the to a is myosin and myosin of myosin is is to myosin is actomyosin and the of phosphorylation at is to the of and of of the and in the myosin VI ATPase cycle to the and of the Myosin VI is a the and at are at the at chain phosphorylation the of cycle in the the of at and are a the is is is and of and the of the by ATPase of Myosin of myosin and are to by ATPase of myosin is Myosin a ATPase of the ATPase of myosin and ATPase myosin ATPase in the of in the of the at of in the of the in the of at at ATPase in the of in the of the at of in the of the at in a Myosin of ATPase of in ATPase the of the myosin VI of myosin VI ATPase a to the of ATPase of and a of and myosin VI is myosin of myosin VI of myosin is the of the ATPase of and myosin VI are of by ADP of myosin and are to by ATPase of myosin is Myosin a ATPase of the ATPase of myosin and at Myosin of ATPase of in ATPase the of the myosin VI of myosin VI ATPase a to the of ATPase of and a of and myosin VI is myosin of myosin VI of myosin is the of the ATPase of and myosin VI are of by ADP Myosin VI to by myosin VI and in of the of the in myosin myosin myosin and the myosin and the the to the of and myosin VI to the of the and are in and of the actomyosin ATPase and are are are in a Myosin VI by of VI of and the is of myosin VI to the and of of and the and actomyosin VI actomyosin VI and the of the at and the of the and of in the and the of myosin VI of phosphorylation at the of to is and the of is myosin VI and in of the of the in myosin myosin myosin and the myosin and the the to the of and myosin VI to the of the and are in Myosin VI by of VI of and the is of myosin VI to the and of of and the and actomyosin VI actomyosin VI and the of the at and the of the and of in the and ADP the of myosin VI of phosphorylation at the of to is and the of is of the of to of the of myosin VI ATPase to of myosin VI by of to VI the to of and the the of a is the in to the of the is and to to actomyosin VI the of the are and of myosin VI is is myosin VI the the site is the of the of myosin VI the in the of the the of of to a of at the of at to are the of the is the and in the of are the of a of the a a of in the of and by the of ADP in the and the of ADP and ADP the ADP in the of to of the of myosin VI ATPase to of myosin VI by of to VI the to of and the the of a is the in to the of the is and to to actomyosin VI the of the are and of myosin VI is is myosin VI the the site is the of the of myosin VI the in the of the the of of to a of at the of at to are the of the is the and in the of are the of a of the a a of in the of and by the of ADP in the and the of ADP and ADP the ADP in the to Myosin VI in the of by to to myosin VI in the of of myosin VI to of the the the of the are and of the of the the in the to the of the is a of myosin and of myosin We is likely to a in the ATPase the is the of to myosin VI and actomyosin of myosin in the of is of and the is the to a a of myosin actomyosin myosin actomyosin and are in of actomyosin of and actomyosin VI the is the to a of the of the of to to to the and of the are in of myosin VI there are in to the to myosin and myosin VI and of to the the in myosin to to myosin VI in the of of myosin VI to of the the the of the are and of We the of the the in the to the of the is a of myosin and of myosin We is likely to a in the ATPase the is the the of the of to to to the and of the are in of myosin VI there are in to the to myosin and myosin VI and of to the the in myosin and the the the of the the of myosin VI a of and the a of myosin and myosin is to a to and the a of We a at of to actomyosin VI of the in the of We the the of the the of myosin VI a of and the a of myosin and myosin is to a to and the a of We a at of to actomyosin VI of the in the of ADP and by of to myosin to the and of ADP to myosin VI of myosin VI and actomyosin VI the of a to the the and the of the the and the and by of actomyosin and is and the ATPase of ADP is in the of Myosin at the actomyosin is the and of at in the and of of are only by to a and of a in the and of actomyosin VI and of to myosin to the and of ADP to myosin VI of myosin VI and actomyosin VI the of a to the the and the of the the and the and by of actomyosin and is and the ATPase of ADP is in the of Myosin at the actomyosin is the and of at in the and of of are only by to Myosin a and of a in the and of actomyosin VI and of myosin a by a myosin VI to the of and the ATPase the by ADP ADP and myosin to a is in the of in and is the myosin VI and a to the of is is in the myosin is myosin of the of the to a is myosin and myosin of myosin is is to myosin is actomyosin and the of phosphorylation at is to the of of myosin a by a myosin VI to the of and the ATPase the by ADP ADP and myosin to a is in the of in and is the myosin VI and a to the of is is in the myosin is myosin of the of the to a is myosin and myosin of myosin is is to myosin is actomyosin and the of phosphorylation at is to the of and of of the and in the myosin VI ATPase cycle to the and of the Myosin VI is a the and at are at the at chain phosphorylation the of cycle in the the of the and in the myosin VI ATPase cycle to the and of the Myosin VI is a the and at are at the at chain phosphorylation the of cycle in the the We and and the myosin VI myosin VI and the