Chemotaxis of bacteria requires regulated methylation of chemoreceptors. However, despite considerable effort in the 1980s, transmethylation has never been established as a component of eukaryotic cell chemotaxis. S-adenosylhomocysteine (SAH), the product formed when the methyl group of the universal donor S-adenosylmethionine (SAM) is transferred to an acceptor molecule, is a potent inhibitor of all transmethylation reactions. In eukaryotic cells, this inhibition is relieved by hydrolysis of SAH to adenosine and homocysteine catalyzed by S-adenosylhomocysteine hydrolase (SAHH), which is an important component of the methylation machinery. We now report that SAHH, which is diffuse in the cytoplasm of non-motile Dictyostelium amoebae and human neutrophils, concentrates with F-actin in pseudopods at the front of motile, chemotaxing cells, but is not present in filopodia or at the very leading edge. Tubercidin, an inhibitor of SAHH, substantially inhibits chemotaxis in both Dictyostelium and neutrophils at concentrations that have little effect on cell viability. Tubercidin does not inhibit starvation-induced expression of the cAMP receptor, cAR1, nor does it inhibit the G protein-mediated stimulation of adenylyl cyclase activity and actin polymerization in Dictyostelium. Tubercidin has no effect on either capping of concanavalin A receptors or phagocytosis in Dictyostelium. These results add SAHH to the list of proteins that redistribute in response to chemotactic signals in Dictyostelium and neutrophils, and strongly suggest a role for transmethylation in chemotaxis of eukaryotic cells.? ? Dictyostelium actin was previously shown to become phosphorylated on Tyr53 late in the developmental cycle and when cells in the amoeboid stage are subjected to stress, but the phosphorylated actin had not been purified and characterized. We have separated phosphorylated and unphosphorylated actin and shown that Tyr53-phosphorylation substantially reduces actin's ability to inactivate DNase I, increases actin's critical concentration, and greatly reduces its rate of polymerization. Tyr53-phosphorylation substantially, if not completely, inhibits nucleation and elongation from the pointed-end of actin filaments, and reduces the rate of elongation from the barbed-end. Negatively stained electron microscopic images of polymerized Tyr53-phosphorylated actin show a variable mixture of small oligomers and filaments, which are converted to more typical, long filaments upon addition of myosin subfragment 1. Tyr53-phosphorylated and unphosphorylated actin co-polymerize in vitro, and phosphorylated and unphosphorylated actin co-localize in amoebae. Tyr53-phosphorylation does not affect the ability of filamentous actin to activate myosin ATPase.? ? The structure of the tail of the heavy chain of Acanthamoeba myosin IC (AMIC), a 466 amino acid residue protein, was investigated to determine if there is any interaction between its N-terminal and C-terminal regions of the protein, as was suggested by cryo-electron microscopy (Ishikawa, T. et al., 2004). The tail comprises an N-terminal 220-residue basic region (BR) followed by a 56-residue Gly/Pro/Ala-rich region (GPA1), a 55-residue SH3 domain and a C-terminal 135-residue region (GPA2). Comparison of NMR spectra of the full-length tail and the individually expressed BR and GPA1-SH3-GPA2 regions showed some specific differences, implying an interaction between the N-terminal and C-terminal regions of the full-length tail. From the results of the NMR experiments, we assigned many of the BR and GPA1-SH3-GPA2 amino acid residues that are critical for the interaction. By combining homology modeling with the NMR data, we suggest how the two regions might interact in the full length AMIC tail. Further, we suggest that the N-terminal basic region contains a PH (pleckstrin homology) domain, consistent with the predominant association of AMIC with plasma membranes and large vacuole and contractile vacuole membranes.
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