SPECs, small binding proteins for Cdc42

The Rho GTPase, Cdc42, regulates a wide variety of cellular activities incl uding actin polymerization, focal complex assembly, and kinase signaling. W e have identified a new family of very small Cdc42-binding proteins, design ated SPECs (for Small Protein Effector of Cdc42), that modulates these regu latory activities. The two human members, SPEC1 and SPECS, encode proteins of 79 and 84 amino acids, respectively. Both contain a conserved N-terminal region and a centrally located CRIB (Cdc42/Rac Interactive Binding) domain . Using a yeast two-hybrid system, we found that both SPECs interact strong ly with Cdc42, weakly with Rad, and not at all with RhoA. Transfection anal ysis revealed that SPEC1 inhibited Cdc42-induced c-Jun N-terminal kinase (J NK) activation in COS1 cells in a manner that required an intact CRIB domai n. Immunofluorescence experiments in MH-3T3 fibroblasts demonstrated that b oth SPEC1 and SPECS showed a cortical localization and induced the formatio n of cell surface membrane blebs, which was not dependent on Cdc42 activity . Cotransfection experiments demonstrated that SPEC1 altered Cdc42-induced cell shape changes both in COS1 cells and in NIH-3T3 fibroblasts and that t his alteration required an intact CRIB domain. These results suggest that S PECs act as novel scaffold molecules to coordinate and/or mediate Cdc42 sig naling activities.