Chimeras of Dictyostelium myosin II head and neck domains with Acanthamoeba or chicken smooth muscle myosin II tail domain have greatly increased and unregulated actin-dependent MgATPase activity

Abstract

Phosphorylation of the regulatory light chain of Dictyostelium myosin II increases V(max) of its actin-dependent MgATPase activity about 5-fold under normal assay conditions. Under these assay conditions, unphosphorylated chimeric myosins in which the tail domain of the Dictyostelium myosin II heavy chain is replaced by either the tail domain of chicken gizzard smooth muscle or Acanthamoeba myosin II are 20 times more active because of a 10- to 15-fold increase in V(max) and a 2- to 7-fold decrease in apparent K(ATPase) and are only slightly activated by regulatory light chain phosphorylation. Actin-dependent MgATPase activity of the Dictyostelium/Acanthamoeba chimera is not affected by phosphorylation of serine residues in the tail whose phosphorylation completely inactivates wild-type Acanthamoeba myosin II. These results indicate that the actin-dependent MgATPase activity of these myosins involves specific, tightly coupled, interactions between head and tail domains.

Figure 1

Schematic representation of the structures of the heavy chains of Acanthamoeba, Dictyostelium, and chimeric myosins. The 1,298-residue tail, beginning at Pro-819, of wild-type Dictyostelium myosin II heavy chain was replaced by the 1,131-residue and 663-residue tails of chicken gizzard smooth muscle and Acanthamoeba myosin II in chimera-Sm and chimera-Acwt heavy chains, respectively. The three serines whose phosphorylation inactivates the actin-dependent MgATPase of Acanthamoeba myosin II, and which were replaced by alanines in chimera-Acala, are numbered differently in the chimeras and wild-type heavy chains because the head/neck domain of Dictyostelium myosin II is 28 residues shorter than the head/neck domain of Acanthamoeba myosin II.

Figure 2

SDS/PAGE analysis of purified wild-type Dictyostelium and chimeric myosins. The positions of the heavy chains (HC), regulatory (RLC), and essential (ELC) light chains are indicated.

Figure 3

Rotary-shadowed electron microscopic images of filaments of wild-type Dictyostelium myosin II (two upper rows), chimera-Acwt (middle row), and chimera-Sm (two bottom rows). See Materials and Methods for details. The magnification is the same for all images; bar is 100 nm.

Figure 4

Urea/SDS/glycerol PAGE analysis of phosphorylation of RLC of wild-type Dictyostelium and chimeric myosins by Dictyostelium myosin II light chain kinase. Wild-type and chimeric myosins were incubated with MgATP with (lanes 2, 4, 6, 8) or without (lanes 1, 3, 5, 7) MLCK as described in Materials and Methods. The gel was stained with Coomassie blue. The positions of unphosphorylated (RLC) and phosphorylated (P-RLC) regulatory light chains and essential light chain (ELC) are indicated.

Figure 5

Actin dependence of the MgATPase activities of wild-type Dictyostelium myosin II (Dicty-wt) and chimeric myosins (chimera-Acwt, chimera-Sm). Curves were fit to all of the data points by the Michaelis-Menton equation and the calculated values for V_max and K_ATPase are shown in Table 1. ○, Phosphorylated RLC; ●, unphosphorylated RLC.

Figure 6

SDS/PAGE and autoradiogram of phosphorylation of wild-type Dictyostelium myosin II and chimeric myosins by Acanthamoeba myosin II heavy chain kinase. Myosins were incubated with MHCK and [³²P]ATP as described in Materials and Methods. Only the heavy chains (HC) and RLCs are shown.

Figure 7

Quantitative assay of phosphorylation of chimera-Acwt by Acanthamoeba myosin II heavy chain kinase.

Figure 8

Mg²⁺ dependence of the actin-dependent MgATPase activity of unphosphorylated and RLC-phosphorylated wild-type Dictyostelium myosin II and chimera-Acwt. (Inset) Activity of phosphorylated divided by activity of unphosphorylated (fold activation). ●, Phosphorylated; ○, unphosphorylated.