Abstract
We have isolated a protein from Dictyostelium with a molecular mass of 110 kDa as judged by SDS-gel electrophoresis that can stimulate the actin-activated MgATPase activity of Dictyostelium myosin ID (MyoD). In the presence of MgATP the 110-kDa protein incorporated phosphate into itself and into the heavy chain, but not the light chain, of MyoD. Phosphorylation to 0.5 mol of Pi/mol increased the MyoD actin-activated MgATPase rate from 0.2 to 3 mumol/min/mg. Renaturation following SDS-gel electrophoresis demonstrated that the 110-kDa protein contained intrinsic protein kinase and autophosphorylation activity. Autophosphorylation to 1 mol of Pi/mol enhanced the rate at which the 110-kDa protein kinase phosphorylated MyoD by 40-fold. The rate of autophosphorylation was strongly dependent on the 110-kDa protein kinase concentration, indicating an intermolecular reaction. Synthetic peptides of 9-11 residues corresponding to the heavy chain phosphorylation site of Acanthamoeba myosin IC and the homologous sites in Dictyostelium myosin IB (MyoB) and MyoD were poor substrates for the 110-kDa protein kinase. The 110-kDa protein kinase was unable to phosphorylate the MyoB isozyme suggesting that it may be specific for MyoD.
Highlights
Heavy chains of -125 kDa in size [7, 8, 11], while MyoA and MyoE are smaller with heavy chains of -115 kDa [6, 9]
The tail regions of myosin IB (MyoB), MyoC, and myosin ID (MyoD) contain a basic domain (TH1) at the head/tail junction that is implicated in membrane binding [12], a domain consisting of a repetitive GPX motif (TH2) that is responsible for ATP-independent actin binding [11, 13] and a domain with homology to SH3 domains (TH3) [14, 15]
Purification ofa 110-kDa Protein That Stimulates the Actinactivated MgATPase Activity of MyoD-As isolated, MyoD displays an actin-activated MgATPase activity of less than 0.2 1-LmolJmin/mg, which is considerably below the 2-8 1-LmolJ min/mg actin-activated MgATPase rates reported for other Acanthamoeba and Dictyostelium myosin I isozymes [23, 26, 27, 30, 38]
Summary
Heavy chains of -125 kDa in size [7, 8, 11], while MyoA and MyoE are smaller with heavy chains of -115 kDa [6, 9]. Though, to suggest that heavy chain phosphorylation, which stimulates the actinactivated MgATPase activity of the Acanthamoeba myosin I isozymes [22, 23], and is required for these enzymes to support movement in in vitro motility assays [24, 25], may play a role in regulating the properties of some of the Dictyostelium myosin I isozymes. The strongest direct evidence is derived from a study showing that the purified Acanthamoeba myosin I heavy chain kinase [26] can stimulate the actin-activated MgATPase activity of one of the Dictyostelium myosin I isozymes [27] (later identified as MyoB [8]). By assaying fractions for their ability to stimulate the actinactivated MgATPase activity of MyoD we have purified a protein with a molecular mass of 110 kDa that displays intrinsic
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