Regulation of the filament structure and assembly ofAcanthamoebamyosin II by phosphorylation of serines in the heavy-chain nonhelical tailpiece

Abstract

Acanthamoeba myosin II (AMII) has two heavy chains ending in a 27-residue nonhelical tailpiece and two pairs of light chains. In a companion article, we show that five, and only five, serine residues can be phosphorylated both in vitro and in vivo: Ser639 in surface loop 2 of the motor domain and serines 1489, 1494, 1499, and 1504 in the nonhelical tailpiece of the heavy chains. In that paper, we show that phosphorylation of Ser639 down-regulates the actin-activated MgATPase activity of AMII and that phosphorylation of the serines in the nonhelical tailpiece has no effect on enzymatic activity. Here we show that bipolar tetrameric, hexameric, and octameric minifilaments of AMII with the nonhelical tailpiece serines either phosphorylated or mutated to glutamate have longer bare zones and more tightly clustered heads than minifilaments of unphosphorylated AMII, irrespective of the phosphorylation state of Ser639. Although antiparallel dimers of phosphorylated and unphosphorylated myosins are indistinguishable, phosphorylation inhibits dimerization and filament assembly. Therefore, the different structures of tetramers, hexamers, and octamers of phosphorylated and unphosphorylated AMII must be caused by differences in the longitudinal stagger of phosphorylated and unphosphorylated bipolar dimers and tetramers. Thus, although the actin-activated MgATPase activity of AMII is regulated by phosphorylation of Ser639 in loop 2 of the motor domain, the structure of AMII minifilaments is regulated by phosphorylation of one or more of four serines in the nonhelical tailpiece of the heavy chain.A s summarized in the accompanying paper (1), Acanthamoeba myosin II (AMII) is a typical class II myosin with two heavy chains and two pairs of light chains (2, 3). The coiled-coil helical tails of the heavy chains terminate with a 27-residue nonhelical tailpiece, 1483PSSRGGSTRGASARGASVRAGSARAEE1509, which has a pattern of four contiguous repeats of XXSXR (4). Previous work had shown that phosphorylation of two or more of these serines (residues 1489, 1494, 1499, and 1504) correlates with, and was assumed to be responsible for, inactivation of AMII's actin-activated MgATPase activity (5, 6). Also, it had been concluded that only filamentous AMII has actin-activated MgATPase activity (7, 8), and it was inferred that the ATPase activity is regulated by a change in the conformation of the bipolar minifilaments (9-11). However, detailed studies by the Pollard laboratory found no significant differences in either the polymerization properties or electron microscopic images of minifilaments of phosphorylated and dephosphorylated myosins (12-16).The earlier studies were carried out with purified endogenous myosin. To avoid possible complications in enzymatic and structural studies caused by the partial phosphorylation of purified endogenous myosin and incomplete dephosphorylation by phosphatase, we initiated studies on the enzymatic activity and structure of expressed recombinant wild-type, truncated, and mutant myosins before and after phos...

Various Themes of Myosin Regulation

“…Apart from myosin light chain phosphorylation, myosin-2 heavy chain phosphorylation in the tail coiled-coil and the non-helical tailpiece (NHT) is a regulatory targeting mechanism and implicated in the inhibition of filament formation and the interaction with binding partners [44–48]. …”

“…In Acanthamoeba , Ser phosphorylation of the NHT regulates filament assembly by inhibiting the dimerization of myosin monomers and the assembly pathway of dimers into filaments [44]. Recently, phosphorylation of a Ser in an actin-binding surface loop of the motor domain was shown to reduce the steady-state ATPase activity by the allosteric reduction of the rate-limiting P i release ~ 3-fold (Fig.…”

Kinetic Characterization of the ATPase and Actin-activated ATPase Activities of Acanthamoeba castellanii Myosin-2

“…In contrast to eukaryotic muscle and nonmuscle myosin-2s, filament assembly and actin-activated ATPase activity of AM2 are regulated by phosphorylation of heavy chain serines (6). Phosphorylation of four serines within the non-helical tailpiece regulates filament assembly and the structure of the bipolar minifilaments (7), whereas phosphorylation of Ser-639 within the motor domain down-regulates actinactivated ATPase activity of AM2 (6,7). Furthermore, phosphorylation of Ser-639 also inhibits the activity of recombinant single-headed subfragment-1 (S1), and the recombinant phosphomimetic S1 mutant, S639D, has similarly low actinactivated ATPase activity (6).…”

“…The amoeboid myosin-2 assembles via dimeric and tetrameric precursors into antiparallel bipolar minifilaments with octameric structures. Those filaments can further associate laterally to form higher ordered arrays of thick filaments (7,31). Dynamic filament assembly/disassembly transitions from octamers to thick filaments and vice versa have been reported in vitro and in vivo (5,7).…”

Kinetic Monte Carlo simulations of the assembly of filamentous biomacromolecules by the dimer addition mechanism

“…S1). 13–19 It is believed that the dimer addition mechanism produces oligomers of size 2 i (i=1, 2, 3…) since the basic building blocks are dimers. In the past few decades, several attempts have been made to model the kinetics of assembly of proteins through the dimer addition scheme by solving differential equations where reaction-limit was implicitly assumed.…”