A mutant gelsolin, [His321]gelsolin, was isolated from R1, a flat revertant of human activated c-Ha-ras oncogene-transformed NIH/3T3 cells (EJ-NIH/3T3) produced by ethylmethanesulfonate treatment. [His321]Gelsolin has a histidine instead of a proline residue at position 321 and suppresses the tumorigenicity of EJ-NIH/3T3 cells when it is constitutively expressed [Müllauer, L., Fujita, H., Ishizaki, A. & Kuzumaki, N. (1993) Oncogene 8, 2531-2536]. To investigate the biochemical consequences of the amino acid substitution of His321, we expressed the [His321]gelsolin and wild-type gelsolin in Escherichia coli, purified them, and analyzed their effects on actin, polyphosphoinositol lipids and phospholipase C. [His321]Gelsolin has decreased actin-filament-severing activity and increased nucleating activity compared with wild-type gelsolin in vitro. Furthermore, compared to wild-type gelsolin both nucleation and severing by [His321]gelsolin are inhibited more strongly by the phosphoinositol lipids phosphatidylinositol 4-phosphate (PtdInsP) and phosphatidylinositol 4,5-bisphosphate (PtdInsP2). In addition, [His321]gelsolin inhibits PtdInsP2 hydrolysis by phospholipase C gamma 1 more strongly than wild-type gelsolin in vitro because of its higher binding capacity for phosphoinositol lipid. Gelsolin has six homologous amino acid repeats called S1-S6. Our results suggest that the segment S3 which contains the mutation is functionally relevant for regulation of gelsolin's activities even though the relevant actin-binding domains are in segments 1, 2, and 4-6, and that the region around the residue 321 may contain a phosphoinositol-lipid-binding site. Altered functions of [His321]gelsolin might be important for the loss of tumorigenicity of the ras-transformed cells.
The activities of Src-family non-receptor tyrosine kinases are regulated by structural changes that alter the orientation of key residues within the catalytic domain. In this study, we investigate the eects of activation loop mutations on regulation of the lymphocyte-speci®c kinase Lck (p56 lck ). Substitution of 5 ± 7 residues amino terminal to the conserved activation loop tyrosine (Y 394 ) increases kinase activity and oncogenic potential regardless of regulatory C-terminal tail phosphorylation levels (Y 505 ), while most mutations in the 13 residues carboxyl to Y 394 decrease kinase activity. Phosphorylation of the Cterminal regulatory tail is carried out by the cytosolic tyrosine kinase Csk and we ®nd that mutations upstream or downstream of Y394 or mutation of Y 394 do not aect the level of Y 505 phosphorylation. In addition, we report that mutations on either side of Y 394 aect substrate speci®city in vivo. We conclude that the high degree of conservation across the entire activation loop of Srcfamily kinases is critical for normal regulation of kinase activity and oncogenicity as well as substrate selection.
The affinity of monomeric actin for several actin-binding proteins, including gelsolin, depends on adenine nucleotides. Gelsolin binds faster and with higher affinity to ADP-actin than to ATP-actin. Here, we show that the C-terminal actin-binding domain of gelsolin, which is required for filament nucleating activity but not for filament severing activity, contains the site that distinguishes between ATP-actin and ADP-actin monomers.In contrast, actin binding to the N-terminal half of gelsolin depends on solution ATP concentrations, but not on the nucleotide (ATP or ADP) tightly bound in the cleft of the actin monomer. Binding is stronger in the absence of free nucleotide or in the presence of 0.5 mM ADP than in solutions containing 0.5 mM ATP. Complexes formed using different nucleotide concentrations differ in their filament-severing activities as well as in their abilities to increase the fluorescence of 4-chloro-7-nitrobenzeno-2-oxa-l,3-diazole-labeled actin monomers. These results suggest that, at physiologic concentrations of nucleotides, both free and actin-bound ATP may affect the binding of actin to its accessory proteins and that gelsolin, actin, or the gelsolin-actin complex, contains a low-affinity nucleotide-binding site.Keywords: gelsolin ; actin ; nucleotides ; ATP.Actin monomers bind ATP with nanomolar affinity and exchange ATP at a low rate (for reviews, see [I -41). Bound ATP is hydrolyzed to ADP and P, after actin monomers assemble into filaments, a reaction which generates both ATP-actin and ADPactin in cells [5, 61. One possible consequence of the ATPase activity of actin is that the actin-bound ATP or actin-bound ADP regulates the binding of proteins to actin monomers (G-actin) and polymers (F-actin Abbreviations. tS1-3,45-kDa thermolysin-generated N-terminal half of gelsolin; 64-6, 47-kDa thermolysin-generated C-terminal half of gelsolin ; F-actin, filamentous actin; G-actin, monomeric actin; NBD: 4-chloro-7-nitrobenzeno-2-oxa-1,3-diazole; rS1-3, recombinant plasma gelsolin containing amino acids 1-406; S1-3, gelsolin N-terminal half fragment containing homologous repeats 1-3 ; S4-6:gelsolin C-terminal half fragment containing homologous repeats 4-6. We recently reported that gelsolin binds ADP-actin preferentially to ATP-actin [8]. In this report, we localize the site on gelsolin that distinguishes between ADP-actin and ATP-actin monomers to its C-terminal half. In contrast, the actin-binding domains located at the N-terminal half of gelsolin do not distinguish between the nucleotides bound to the tight binding cleft of actin, but the rates and affinity of binding depend strongly on sub-millimolar concentrations of nucleoside triphosphates in
Actin exhibits ATPase activity of unknown function that increases when monomers polymerize into filaments. Differences in the kinetics of ATP hydrolysis and the release of the hydrolysis products ADP and inorganic phosphate suggest that phosphate-rich domains exist in newly polymerized filaments. We examined whether the enrichment of phosphate on filamentous ADP-actin might modulate the severing activity of gelsolin, a protein previously shown to bind differently to ATP and ADP actin monomers. Binding of phosphate, or the phosphate analogs aluminum fluoride and beryllium fluoride, to actin filaments reduces their susceptibility to severing by gelsolin. The concentration and pH dependence of inhibition suggest that HPO 4 2؊ binding to actin filaments generates this resistant state. We also provide evidence for two different binding sites for beryllium fluoride on actin. Actin has been postulated to contain two P i binding sites. Our data suggest that they are sequentially occupied following ATP hydrolysis by HPO 4 2؊ which is subsequently titrated to H 2 PO 4 ؊ . We speculate that beryllium fluoride and aluminum fluoride bind to the HPO 4 2؊ binding site. The cellular consequences of this model of phosphate release are discussed.
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