REEXPRESSION OF THE MAJOR PROTEIN-KINASE-C SUBSTRATE, SSECKS, SUPPRESSES V-SRC-INDUCED MORPHOLOGICAL TRANSFORMATION AND TUMORIGENESIS

SSeCKS (pronounced essex) encodes a major protein kinase C substrate, the expression of which is down-regulated in src- and ras-transformed rodent fibroblasts hut not in raf-transformed rodent fibroblasts (X. L in et al, Mol. Cell, Biol., 15: 2754-2762, 1995), Using a panel of ras -transformed or revertant Rat-6 cells that exhibit selective parameter s of transformation, we show that down-regulation of SSeCKS correlates with anchorage independent growth. Cotransfection of NIH3T3 fibroblas ts with an SSeCKS expression plasmid decreased 6-30-fold the ability o f a v-src expressor plasmid to induce colonies in soft agar. To differ entiate between possible tumor suppressive or growth-inhibitory effect s of SSeCKS, we developed conditionally transformed cell lines (expres sing ts72v-src) with tetracycline-regulated SSeCKS expression, SSeCKS suppressed the ability of v-src to induce increased cellular refractil ity, focus formation, soft agar colony formation, in vitro invasivenes s in Matrigel, and growth in low serum (0.5%) but did not inhibit cell proliferation in high serum (10%) at the permissive (35 degrees C) te mperature for src kinase activity. However, at the nonpermissive (39.5 degrees C) temperature, SSeCKS induced growth arrest, SSeCKS expressi on did not affect: (a) the protein level, in vivo or in vitro kinase a ctivity of ts72src; (b) the activity of jun NH2-terminal kinase; and ( c) the level of mitogen-activated protein kinase (extracellular signal -regulated kinase 2) protein, However, extracellular signal-regulated kinase 2 activity aas induced 5-10-fold by SSeCKS in the presence of a ctive src, SSeCKS reversed the ability of v-src to decrease the format ion of vinculin-associated adhesion plaques, actin-based stress fibers , and filopodia structures, These data suggest a tumor suppressive rol e for SSeCKS via the control of cytoskeletal architecture and cell sig naling.