ACTIN DEPOLYMERIZING FACTOR AND COFILIN PHOSPHORYLATION DYNAMICS - RESPONSE TO SIGNALS THAT REGULATE NEURITE EXTENSION

The actin assembly-regulating activity of actin depolymerizing factor (ADF)/ cofilin is inhibited by phosphorylation. Studies were undertake n to characterize the signaling pathways and phosphatases involved in activating phosphorylated ADF (pADF), emphasizing signals related to n euronal process extension. Western blots using antibodies to ADF and c ofilin, as well as an ADF/cofilin phosphoepitope specific antibody cha racterized in this paper, were used to measure changes in the phosphor ylation state and phosphate turnover of ADF/cofilin in response to inh ibitors and agents known to influence growth cone motility. Increases in both [Ca2+](i) and cAMP levels induced rapid pADF dephosphorylation in HT4 and cortical neurons. Calcium-dependent dephosphorylation depe nded on the activation of protein phosphatase 2B (PP2B), while cAMP-de pendent dephosphorylation was likely through activation of PPI. Growth factors such as NGF and insulin also induced rapid pADF/pcofilin deph osphorylation, with NGF-stimulated dephosphorylation in PC12 cells cor related with the translocation of ADF/cofilin to ruffling membranes. O f special interest was the finding that the rate of phosphate turnover on both pADF and pcofilin could be enhanced by growth factors without changing net pADF levels, demonstrating that growth factors can activ ate bifurcating pathways that promote both phosphorylation and dephosp horylation of ADF/cofilin. All experimental results indicated that dyn amics of phosphorylation on ADF and cofilin are coordinately regulated . Signals that decreased pADF levels are associated with increased pro cess extension, while agents that increased pADF levels, such as lysop hosphatidic acid, inhibit process extension. These data indicate that dephosphorylation/activation of pADF is a significant response to the activation of signal pathways that regulate actin dynamics and alter c ell morphology and neuronal outgrowth. (C) 1998 Wiley-Liss, Inc.