SSeCKS/Gravin/AKAP12 metastasis suppressor inhibits podosome formation via RhoA- and Cdc42-dependent pathways.

Podosomes are poorly understood actin-rich structures notably found in cancer cell lines or in v-Src-transformed cells that are thought to facilitate some of the invasive properties involved in tumor metastasis. The enrichment of the Tks5/Fish protein, a v-Src substrate, is required for formation of podosomes. We showed previously that the tetracycline-regulated reexpression of the Src-suppressed C kinase substrate (SSeCKS, also known as Gravin/AKAP12) inhibited variables of v-Src-induced oncogenic growth in NIH3T3, correlating with the induction of normal actin cytoskeletal structures and cell morphology but not with gross inhibition of Src phosphorylation activity in the cell. Here, we show that SSeCKS reexpression at physiologic levels suppresses podosome formation, correlating with decreases in Matrigel invasiveness, whereas there is no effect on total cellular tyrosine phosphorylation or on the phosphorylation of Tks5/Fish. Activated forms of RhoA and Cdc42 were capable of rescuing podosome formation in v-Src cells reexpressing SSeCKS, and this correlated with the ability of SSeCKS to inhibit RhoA and Cdc42 activity levels by >5-fold. Interestingly, although activated Rac I had little effect on podosome formation, it could partner with activated RhoA to reverse the cell flattening induced by SSeCKS. These data suggest that v-Src-induced Tks5 tyrosine phosphorylation is insufficient for podosome formation in the absence of RhoA- and/or Cdc42-mediated cytoskeletal remodeling. Additionally, they strengthen the notion that SSeCKS suppresses Src-induced oncogenesis by reestablishing actin-based cytoskeletal architecture.