Tropomodulin 3 binds to actin monomers.
Regulation of the actin cytoskeleton by filament capping proteins is critical to myriad dynamic cellular functions. The ability of these proteins to bind both filaments as well as monomers is often central to their cellular functions. The ubiquitous pointed end capping protein Tmod3 (tropomodulin 3) acts as a negative regulator of cell migration, yet mechanisms behind its cellular functions are not understood. Analysis of Tmod3 effects on kinetics of actin polymerization and steady state monomer levels revealed that Tmod3, unlike previously characterized tropomodulins, sequesters actin monomers with an affinity similar to its affinity for capping pointed ends. Furthermore, Tmod3 is found bound to actin in high speed supernatant cytosolic extracts, suggesting that Tmod3 can bind to monomers in the context of other cytosolic monomer binding proteins. The Tmod3-actin complex can be efficiently cross-linked with 1-ethyl-3-(dimethylaminopropyl)carbodiimide/N-hydroxylsulfosuccinimide in a 1:1 complex. Subsequent tryptic digestion and liquid chromatography/tandem mass spectrometry revealed two binding interfaces on actin, one distinct from other actin monomer binding proteins, and two potential binding sites in Tmod3, which are independent of the previously characterized leucine-rich repeat structure involved in pointed end capping. These data suggest that the Tmod3 isoform may regulate actin dynamics differently in cells than the previously described tropomodulin isoforms.
Fischer
Robert S
RS
Department of Cell Biology, The Scripps Research Institute, La, Jolla, California 92037, USA. bfischer@scripps.edu
Yarmola
Elena G
EG
Weber
Kari L
KL
Speicher
Kaye D
KD
Speicher
David W
DW
Bubb
Michael R
MR
Fowler
Velia M
VM
D000199:Actins / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D002465:Cell Movement; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms; D011817:Rabbits; D051348:Tropomodulin / Q000502:physiology*
D000199:Actins; D003432:Cross-Linking Reagents; D020033:Protein Isoforms; C495836:TMOD3 protein, human; D051348:Tropomodulin