Properties of Acanthamoeba Myosin I Heavy Chain Kinase Bound to Phospholipid Vesicles

Abstract

The actin-activated Mg 2؉ -ATPase and in vitro motility activities of the three Acanthamoeba myosin I isozymes depend upon phosphorylation of their single heavy chains by myosin I heavy chain kinase. Previously, the kinase had been shown to be activated by autophosphorylation, which is enhanced by acidic phospholipids, or simply by binding to purified plasma membranes in the absence of significant autophosphorylation. In this paper, we show that the rate of phosphorylation of myosin I by unphosphorylated kinase is ϳ20-fold faster when both the myosin I and the kinase are bound to acidic phospholipid vesicles than when both are soluble. This activation is not due to an increase in the local concentrations of vesicle-bound kinase and myosin I. Thus, acidic phospholipids, like membranes, can activate myosin I heavy chain kinase in the absence of significant autophosphorylation, i.e. membrane proteins are not required. Kinetic studies show that both binding of kinase to phospholipid vesicles and autophosphorylation of kinase in the absence of phospholipid increase the V max relative to soluble, unphosphorylated kinase with either an increase in the apparent K m (when myosin I is the substrate) or no significant change in K m (when a synthetic peptide is the substrate). Kinetic data showed that autophosphorylation of phospholipid-bound kinase is both intermolecular and intervesicular, and that phosphorylation of phospholipid-bound myosin I by phospholipid-bound kinase is also intervesicular even when the kinase and myosin are bound to the same vesicles. The relevance of these results to the activation of myosin I heavy chain kinase and phosphorylation of myosin I isozymes in situ are discussed.Each of the three isoforms of Acanthamoeba myosin I has a single ϳ110 -140-kDa heavy chain with an ϳ80-kDa N-terminal domain, that is highly similar in sequence to the subfragment 1 domain of conventional myosin II, and a ϳ50-kDa nonfilamentous C-terminal domain that has no sequence similarity to the C-terminal domain of conventional myosin (1, 2; for reviews, see . The N-terminal head domain contains an ATP-binding site (6) and an ATP-sensitive, F-actinbinding site (7,8), as do all myosins. The C-terminal tail domain contains an ATP-insensitive, F-actin-binding site (8 -10) and a membrane (and acidic phospholipid)-binding site (10, 11) which, thus far, appear to be unique to the myosin I family. Actin-activated Mg 2ϩ -ATPase activity is expressed in vitro only when a single serine (myosin IB and IC) or threonine (myosin IA), situated between the ATP-and actin-binding sites in the globular head (6), is phosphorylated (12, 13). The biological importance of phosphorylation of the myosin I heavy chain is evidenced by the observations (14) that ϳ80% of myosin IA and ϳ20% of myosin IB and IC are phosphorylated in situ, that the fraction of phosphorylated myosin IC associated with the contractile vacuole in situ varies with the stage of the contractile vacuole cycle (14), and that antibodies that specifically inhibit phosphorylation ...

Identification by mass spectrometry of the phosphorylated residue responsible for activation of the catalytic domain of myosin I heavy chain kinase, a member of the PAK/STE20 family

“…Although Ser-627 is the only specific residue that must be phosphorylated for full activation of the expressed catalytic domain of Acanthamoeba MIHCK, additional phosphorylations may be necessary for activation of full-length, native kinase (13,14). It is likely that the native Acanthamoeba MIHCK, as proposed for other PAKs (2), is folded such that its N-terminal region blocks the catalytic domain.…”

“…It is likely that the native Acanthamoeba MIHCK, as proposed for other PAKs (2), is folded such that its N-terminal region blocks the catalytic domain. The catalytic domain may then be opened to substrate either by the kinase binding to membranes or phospholipid and͞or by phospholipid͞membrane-stimulated autophosphorylation (13,14,25). Consistent with this proposal, autophosphorylation of the 54-kDa C-terminal fragment of native kinase (19) and of the expressed catalytic domain (data not shown) are much faster than autophosphorylation of native kinase.…”

“…The activity of MIHCK increases about 50-fold coincident with autophosphorylation of up to 8-10 mol of P per mol (13,14). About 2-3 mol of P per mol are retained in the fully active, Ϸ35 kDa, C-terminal fragment produced by trypsin digestion of phosphorylated Acanthamoeba MIHCK (15).…”

The Catalytic Domain of Acanthamoeba Myosin I Heavy Chain Kinase

“…Highly purified MIHC kinase autophosphorylates (intermolecularly) slowly; incorporation of 7-8 mol of phosphate/ mol of kinase enhances its activity over 50-fold (18), and phosphorylation can reach 16 mol/mol. Association of MIHC kinase with acidic phospholipids (18,19) or isolated plasma membranes (20) enhances the rate of phosphorylation of myosin I and of autophosphorylation of kinase (ϳ20-fold). These stimulatory effects of phospholipid are Ca 2ϩ -independent but are inhibited by Ca 2ϩ -calmodulin (21), probably because Ca 2ϩ -calmodulin and acidic phospholipids compete for the same binding site on the kinase.…”

Regulation of Class I and Class II Myosins by Heavy Chain Phosphorylation

“…Heavy Chain Kinases-Acanthamoeba myosin I heavy chain kinase (MIHCK) is a 97-kDa monomer that is 50-fold activated by intermolecular autophosphorylation of 8 sites (45,46). Association of Acanthamoeba MIHCK with acidic phospholipids (or, to a lesser extent, isolated plasma membranes) enhances the rates of phosphorylation of myosin I and of kinase autophosphorylation ϳ20-fold (45,47).…”