BINDING OF PEL98 PROTEIN, AN S100-RELATED CALCIUM-BINDING PROTEIN, TONONMUSCLE TROPOMYOSIN

The cDNA coding for mouse fibroblast tropomyosin isoform 2 (TM2) was p laced into a bacterial expression vector to produce a fusion protein c ontaining glutathione-S-transferase (GST) and TM2 (GST/TM2). Glutathio ne-Sepharose beads bearing GST/TM2 were incubated with [S-35]methionin e-labeled NIH 3T3 cell extracts and the materials bound to the fusion proteins were analyzed to identify proteins that interact with TM2. A protein of 10 kD was found to bind to GST/TM2, but not to GST. The bin ding of the 10-kD protein to GST/TM2 was dependent on the presence of Ca2+ and inhibited by molar excess of free TM2 in a competition assay. The 10-kD protein-binding site was mapped to the region spanning resi dues 39-107 on TM2 by using several COOH-terminal and NH2-terminal tru ncation mutants of TM2. The 10-kD protein was isolated from an extract of NIH 3T3 cells transformed by v-Ha-ras by affinity chromatography o n a GST/TM2 truncation mutant followed by SDS-PAGE and electroelution. Partial amino acid sequence analysis of the purified 10-kD protein, t wo-dimensional polyacrylamide gel analysis and a binding experiment re vealed that the 10-kD protein was identical to a calcium-binding prote in derived from mRNA named pEL98 or 18A2 that is homologous to S100 pr otein. Immunoblot analysis of the distribution of the 10-kD protein in Triton-soluble and -insoluble fractions of NIH 3T3 cells revealed tha t some of the 10-kD protein was associated with the Triton-insoluble c ytoskeletal residue in a Ca2+-dependent manner. Furthermore, immunoflu orescent staining of NIH 3T3 cells showed that some of the 10-kD prote in colocalized with nonmuscle TMs in microfilament bundles. These resu lts suggest that some of the pEL98 protein interacts with microfilamen t-associated nonmuscle TMs in NIH 3T3 cells.