Proteins of the 14-3-3 family have been implicated in various physiological processes, and are thought to function as adaptors in various signal transduction pathways. In addition, 14-3-3 proteins may contribute to the reorganization of the actin cytoskeleton by interacting with as yet unidentified actin-binding proteins. Here we show that the 14-3-3ζ isoform interacts with both the actin-depolymerizing factor cofilin and its regulatory kinase, LIM (Lin-11/Isl-1/Mec-3)-domain-containing protein kinase 1 (LIMK1). In both yeast two-hybrid assays and glutathione S-transferase pull-down experiments, these proteins bound efficiently to 14-3-3ζ. Deletion analysis revealed consensus 14-3-3 binding sites on both cofilin and LIMK1. Furthermore, the C-terminal region of 14-3-3ζ inhibited the binding of cofilin to actin in co-sedimentation experiments. Upon co-transfection into COS-7 cells, 14-3-3ζ-specific immunoreactivity was redistributed into characteristic LIMK1-induced actin aggregations. Our data are consistent with 14-3-3-protein-induced changes to the actin cytoskeleton resulting from interactions with cofilin and/or LIMK1.
- actin-depolymerizing factor
- cytoskeletal dynamics
- testicular protein kinase 1 (TESK1)
Abbreviations used: ADF, actin-depolymerizing factor; GST, glutathione S-transferase; HA, haemagglutinin; HEK, human embryonic kidney; LIM domain, Lin-11/Isl-1/Mec-3 domain; LIMK, LIM-domain-containing protein kinase; PAK, p21-activated protein kinase; PDZ domain, PSD-95/Dlg/ZO-1 domain; ROCK, Rho-associated, coiled-coil-forming protein kinase; TESK, testicular protein kinase; TRITC, tetramethylrhodamine β-isothiocyanate; VSV-G, vesicular stomatitis virus glycoprotein.
- The Biochemical Society, London ©2003