Missing in metastasis gene, or MTSS1, encodes an intracellular protein that is implicated in actin cytoskeleton reorganization and often down-regulated in certain types of tumor cells. In response to platelet-derived growth factor (PDGF), green fluorescent protein (GFP)-tagged murine Mtss1 (Mtss1-GFP) underwent redistribution from the cytoplasm to dorsal membrane ruffles along with phosphorylation at tyrosine residues in a time-dependent manner. Tyrosine phosphorylation of Mtss1-GFP was also elevated in cells where an oncogenic Src was activated but severely impaired in Src knock-out cells or cells treated with Src kinase inhibitor PP2. Mutagenesis analysis has revealed that phosphorylation occurs at multiple sites, including tyrosine residues Tyr-397 and Tyr-398. Mutation at both Tyr-397 and Tyr-398 abolished the PDGF-mediated tyrosine phosphorylation. Furthermore, recombinant Mtss1 protein was phosphorylated by recombinant Src in a manner dependent on Tyr-397 and Tyr-398. Efficient tyrosine phosphorylation of Mtss1 in response to PDGF also involves a coiled-coil domain, which is essential for a proper distribution to the cell leading edge and dorsal ruffles. Interestingly, overexpression of wild type Mtss1-GFP promoted the PDGF-induced dorsal ruffling, whereas overexpression of a mutant deficient in phosphorylation at Tyr-397 and Tyr-398 or a mutant with deletion of the coiled-coil domain impaired the formation of dorsal ruffles. These data indicate that Mtss1 represents a novel signaling pathway from PDGF receptor to the actin cytoskeleton via Srcrelated kinases.Growth factor-mediated cytoskeletal reorganization plays a pivotal role in cell shape changes, locomotion, endocytosis, and homeostasis. The signal transduction elicited by ligand-occupied receptors initially involves activation of several small guanosine triphosphatase proteins, including Rac, Cdc42, and Rho (1). Activated Cdc42 or Rac binds either directly or indirectly to Wiskott-Aldrich Syndrome protein (WASP) 2 or WASP family verprolin homologous protein (WAVE/Scar), resulting in recruitment of these proteins to the plasma membrane wherein they subsequently activate Arp2/3 complex, an actin nucleation factor for the branched actin filaments that constitute lamellipodia and membrane ruffles (2). On the other hand, activation of Rho promotes the assembly of nonbranched actin filaments through the function of the Formin family proteins, which may be responsible for the formation of stress fibers or actin cables (3). In addition to these relatively well established signaling factors, there are a few actin-associated proteins the role of which in the cytoskeletal reorganization remains to be defined. One of these proteins is MIM, which stands for missing in metastasis, or MTSS1 as recently recommended by Human Genome Organization Gene Nomenclature Committee. Previous studies have reported the existence of multiple MIM splicing transcripts, including MIM-A, the prototype of MIM that encodes only 356 amino acids; MIM-B, which encodes a protein product o...