CONTROL OF CYTOSKELETAL ARCHITECTURE BY THE SRC-SUPPRESSED C-KINASE SUBSTRATE, SSECKS

Activation of protein kinase C (PKC) in many cell types results in cyt oskeletal reorganization associated with cell proliferation. We previo usly described a new cell cycle-regulated myristylated PKC substrate, SSeCKS (pronounced essex), that interacts with the actin cytoskeleton [Lin et al., 1995, 1996]. SSeCKS shares significant homology with Grav in, which encodes kinase scaffolding functions for PKC and PKA [Nauert et al., 1997]. This article describes the cellular effects of ectopic ally expressing SSeCKS in untransformed NIH3T3 fibroblasts. Because th e constitutive overexpression of SSeCKS is toxic [Lin et al., 1995], w e developed cell lines with tetracycline (tet)-regulated SSeCKS expres sion. The induction of SSeCKS (removal of tet) caused significant cell flattening and the elaboration of an SSeCKS-associated cortical cytos keletal matrix resistant to Triton X-100 extraction. Flattened cells w ere growth-arrested and marked by the formation of cellular projection s and the temporary loss of actin stress fibers and vinculin-associate d adhesion plaques. SSeCKS overexpression did not affect steady-state levels of actin, vinculin, or focal adhesion kinase (FAK) but did incr ease integrin-independent FAK tyrosine phosphorylation. Stress fiber l ass was coincident with induced SSeCKS expression, strongly suggesting a direct effect. Cytochalasin, and to a lesser extent nocodazole, inh ibited SSeCKS-induced cell flattening, however, only cytochalasin affe cted the shape of pre-flattened cells, suggesting a greater dependence on microfilaments, rather than microtubules. By contrast, only nocoda zole caused retraction of the filopodia-like processes. These data ind icate a role for SSeCKS in modulating both cytoskeletal and signaling pathways. Thus, we propose to expand SSeCKS scaffolding functions to i nclude the ability to control actin-based cytoskeletal architecture, a s well as mitogenic signal pathways. (C) 1998 Wiley-Liss, Inc.