Actin depolymerizing factor and cofilin phosphorylation dynamics: Response to signals that regulate neurite extension

Abstract

The actin assembly‐regulating activity of actin depolymerizing factor (ADF)/cofilin is inhibited by phosphorylation. Studies were undertaken to characterize the signaling pathways and phosphatases involved in activating phosphorylated ADF (pADF), emphasizing signals related to neuronal process extension. Western blots using antibodies to ADF and cofilin, as well as an ADF/cofilin phosphoepitope‐specific antibody characterized in this paper, were used to measure changes in the phosphorylation state and phosphate turnover of ADF/cofilin in response to inhibitors 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 depended on the activation of protein phosphatase 2B (PP2B), while cAMP‐dependent dephosphorylation was likely through activation of PP1. Growth factors such as NGF and insulin also induced rapid pADF/pcofilin dephosphorylation, with NGF‐stimulated dephosphorylation in PC12 cells correlated with the translocation of ADF/cofilin to ruffling membranes. Of 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 activate bifurcating pathways that promote both phosphorylation and dephosphorylation of ADF/cofilin. All experimental results indicated that dynamics of phosphorylation on ADF and cofilin are coordinately regulated. Signals that decreased pADF levels are associated with increased process extension, while agents that increased pADF levels, such as lysophosphatidic 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 cell morphology and neuronal outgrowth. Cell Motil. Cytoskeleton 39:172–190, 1998. © 1998 Wiley‐Liss, Inc.

Regulation of Growth Cone Actin Dynamics by ADF/Cofilin

“…Therfore, a net change in phosphorylation of AC may not be as critical to actin dynamics as is the turnover rate of the pAC pools (i.e., phosphocycling). Evidence for AC phosphocycling in non-neuronal cells has been presented (Meberg et al 1998). In this way, ACs possess the properties to enhance actin turnover at the leading edge of migrating cells, including growth cones.…”

How does an axon grow?

“…At the transitional zone between actin tails and microtubules, the actin filament-severing protein gelsolin and the filament depolymerizing family of ADF/ cofilin proteins increase actin turnover. Growth cones from gelsolin knockout mice fail to retract their filipodia (Lu et al 1997), and ADF/cofilin activation increases neurite outgrowth (Meberg et al 1998). Another actin monomer-binding protein, ␤-thymosin, is enriched in growth cones and is up-regulated in regenerating zebrafish RGCs (Roth et al 1999).…”

Signaling mechanisms that regulate actin‐based motility processes in the nervous system

“…There is evidence to suggest this occurs in response to growth factors (see below). Several potential activating phosphatases have been identified, including PP1, PP2A, and PP2B (Meberg et al 1998;Ambach et al 2000). Slingshot (SSH) is a recently discovered family of phosphatases that have apparent specificity for ADF/cofilin (Niwa et al 2002).…”

“…ADF is expressed in the dendrites of Purkinje cells, but the axons of mossy and climbing fibers (Lena et al 1991). ADF phosphorylation state is regulated in neurons by second messenger systems and growth factors (Meberg et al 1998). Increases in intracellular calcium and cAMP levels cause a rapid dephosphorylation of ADF in cortical neurons.…”