Regulation of Dictyostelium myosin I and II.

Dictyostelium expresses 12 different myosins, including seven single-headed myosins I and one conventional two-headed myosin II. In this review we focus on the signaling pathways that regulate Dictyostelium myosin I and myosin II. Activation of myosin I is catalyzed by a Cdc42/Rac-stimulated myosin I heavy chain kinase that is a member of the p21-activated kinase (PAK) family. Evidence that myosin I is linked to the Arp2/3 complex suggests that pathways that regulate myosin I may also influence actin filament assembly. Myosin II activity is stimulated by a cGMP-activated myosin light chain kinase and inhibited by myosin heavy chain kinases (MHCKs) that block bipolar filament assembly. Known MHCKs include MHCK A and MHCK B, which have a novel type of kinase catalytic domain joined to a WD repeat domain, and MHC-protein kinase C (PKC), which contains both diacylglycerol kinase and PKC-related protein kinase catalytic domains. A Dictyostelium PAK (PAKa) acts indirectly to promote myosin II filament formation, suggesting that the MHCKs may be indirectly regulated by Rac GTPases.

Hydrodynamic properties of Vibrio harveyi acyl carrier protein and its fatty-acylated derivatives.

The amino acid sequence of Vibrio harveyi acyl carrier protein (ACP) is 86% identical to that of Escherichia coli ACP, although five nonconservative amino acid differences are concentrated in the loop region between helices I and II (residues 18-25). We have investigated the influence of these sequence differences on the hydrodynamic properties of the two ACPs and their fatty acylated derivatives. Hydropathy analysis suggests that V. harveyi ACP is more hydrophobic than E. coli ACP in the loop region, a prediction supported by stronger binding of V. harveyi acyl-ACPs (C12 to C16) to octyl-Sepharose. Gel filtration experiments indicated that both ACPs undergo a similar conformational expansion when pH was elevated from 7.5 (R(s) = 24 A) to 9.0 (R(s) = 30 A). Fatty acylation reversed this expansion: R(s) for 16:0-ACP was 12 A, independent of ACP source and pH. By contrast, V. harveyi and E. coli ACPs exhibited distinct gel electrophoretic properties. Fatty acylation of V. harveyi ACP produced a greater increase in mobility on a conformationally sensitive native gel system. Moreover, while both V. harveyi and E. coli ACPs migrated anomalously at 20 kDa on SDS-polyacrylamide gel electrophoresis, they exhibited strikingly different behavior on SDS gels upon acylation with longer chain fatty acids. These results indicate that E. coli and V. harveyi ACPs exhibit similar overall pH- and fatty acid-dependent conformational changes, but gel electrophoresis is more sensitive to structural differences due to variations of hydrophobicity and charge.