Slingshot (SSH) phosphatases and LIM kinases (LIMK)regulate actin dynamics via a reversible phosphorylation (inactivation) of serine 3 in actin-depolymerizing factor (ADF) and cofilin. Here we demonstrate that a multi-protein complex consisting of SSH-1L, LIMK1, actin, and the scaffolding protein, 14-3-3f, is involved, along with the kinase, PAK4, in the regulation of ADF/cofilin activity. Endogenous LIMK1 and SSH-1L interact in vitro and co-localize in vivo, and this interaction results in dephosphorylation and downregulation of LIMK1 activity. We also show that the phosphatase activity of purified SSH-1L is F-actin dependent and is negatively regulated via phosphorylation by PAK4. 14-3-3f binds to phosphorylated slingshot, decreases the amount of slingshot that co-sediments with F-actin, but does not alter slingshot activity. Here we define a novel ADF/cofilin phosphoregulatory complex and suggest a new mechanism for the regulation of ADF/cofilin activity in mediating changes to the actin cytoskeleton.

Neurotrophins activate multiple signaling pathways in neurons. However, the precise roles of these signaling molecules in cell survival are not well understood. In this report, we show that nerve growth factor (NGF) activates the transcription factors NF-B and AP-1 in cultured sympathetic neurons. Activated NF-B complexes were shown to consist of heterodimers of p50 and Rel proteins (RelA, as well as c-Rel), and NF-B activation was found to occur independently of de novo protein synthesis but in a manner that required the action of the proteasome complex. Treatment with the NF-B inhibitory peptide SN50 in the continuous presence of NGF resulted in dosedependent induction of cell death. Under the conditions used, SN50 was shown to selectively inhibit NF-B activation but not the activation of other cellular transcription factors such as AP-1 and cAMP response element-binding protein. Cells treated with SN50 exhibited morphological and biochemical hallmarks of apoptosis, and the kinetics of cell killing were accelerated relative to death induced by NGF withdrawal. Finally, experiments were conducted to test directly whether NF-B could act as a survival factor for NGF-deprived neurons. Microinjection of cells with an expression plasmid encoding NF-B (c-Rel) resulted in enhanced neuronal survival after withdrawal of NGF, whereas cells that were transfected with a vector encoding a mutated derivative of c-Rel lacking the transactivation domain underwent cell death to the same extent as control cells. Together, these findings suggest that the activation of NF-B/Rel transcription factors may contribute to the survival of NGF-dependent sympathetic neurons.

Actin and microtubules are major cytoskeletal elements of most cells including neurons. In order for a cell to move and change shape, its cytoskeleton must undergo rearrangements that involve breaking down and reforming filaments. Many recent reviews have focused on the signaling pathways emanating from receptors that ultimately affect axon growth and growth cone steering. This particular review will address changes in the actin cytoskeleton modulated by the family of actin dynamizing proteins known as actin depolymerizing factor (ADF)/cofilin or AC proteins. Though much is known about inactivation of AC proteins through phosphorylation at ser3 by LIM or TES kinases, new mechanisms of regulation of AC have recently emerged. A novel phosphatase, slingshot (SSH), and the 14-3-3 family of regulatory proteins have also been found to affect AC activity. The potential role of AC proteins in modulating the actin organizational changes that accompany neurite initiation, axonogenesis, growth cone guidance, and dendritic spine formation will be discussed.

The molecular mechanisms by which neurotrophins regulate growth cone motility are not well understood. This study investigated the signaling involved in transducing BDNF-induced increases of filopodial dynamics. Our results indicate that BDNF regulates filopodial length and number through a Rho kinase-dependent mechanism. Additionally, actin depolymerizing factor (ADF)/cofilin activity is necessary and sufficient to transduce the effects of BDNF. Our data indicate that activation of ADF/cofilin mimics the effects of BDNF on filopodial dynamics, whereas ADF/cofilin inactivity blocks the effects of BDNF. Furthermore, BDNF promotes the activation of ADF/ cofilin by reducing the phosphorylation of ADF/cofilin. Although inhibition of myosin II also enhances filopodial length, our results indicate that BDNF signaling is independent of myosin II activity and that the two pathways result in additive effects on filopodial length. Thus, filopodial extension is regulated by at least two independent mechanisms. The BDNF-dependent pathway works via regulation of ADF/cofilin, independently of myosin II activity.

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Background: The Kv2.1 delayed-rectifier K + channel regulates membrane excitability in hippocampal neurons where it targets to dynamic cell surface clusters on the soma and proximal dendrites. In the past, Kv2.1 has been assumed to be absent from the axon initial segment.

Sympathetic neurons undergo apoptosis when deprived of nerve growth factor (NGF). Inhibitors of RNA or protein synthesis block this death, suggesting that gene expression is important for apoptosis in this system. We have identified SM-20 as a new gene that increases in expression in sympathetic neurons after NGF withdrawal. Expression of SM-20 also increases during neuronal death caused by cytosine arabinoside or the phosphatidylinositol 3-kinase inhibitor LY294002. In addition, SM-20 protein synthesis is elevated in NGF-deprived neurons compared with neurons maintained with NGF. Importantly, expression of SM-20 in sympathetic neurons causes cell death in the presence of NGF. These results suggest that SM-20 may function to regulate cell death in neurons. Key Words: Nerve growth factor-Apoptosis-Sympathetic neuron-Gene expression-Cell death. J. Neurochem. 73, 429 -432 (1999).Inhibitors of RNA or protein synthesis block neuronal death after neurotrophic factor deprivation (Martin et al., 1988;Scott and Davies, 1990;Milligan et al., 1994), suggesting that gene expression may be important for regulating this kind of cell death. Consistent with this hypothesis, a small number of genes has been identified that increase in expression in sympathetic neurons after removal of nerve growth factor (NGF) Freeman et al., 1994;Ham et al., 1995). These genes, which include c-jun, JunB, cyclin D1, c-myb, c-fos, FosB, NGFI-A, and Mkp-1, are maximally expressed 10 -20 h after NGF withdrawal, generally preceding the onset of cell death in this system. Several reports suggest the involvement of c-jun and cyclin D1 in neuronal death. Blocking c-Jun function with c-Junneutralizing antibodies or dominant-negative forms of c-Jun protects sympathetic neurons and PC12 cells from death caused by NGF withdrawal and cerebellar granule neurons from withdrawal of insulin-like growth factor-1 Ham et al., 1995;Xia et al., 1995;Watson et al., 1998). Moreover, overexpression of c-Jun is sufficient to induce death in the presence of neurotrophic factors (Ham et al., 1995;Watson et al., 1998). Cyclin D1 protein content and cyclin-dependent protein kinase activity increase during death of serum-deprived N1E-115 neuroblastoma cells, and expression of a cyclindependent protein kinase inhibitor blocks the death (Kranenburg et al., 1996). Cyclin D1-associated kinase activity also increases in cortical neurons undergoing death induced by DNA damage (Park et al., 1998), and cyclin-dependent protein kinase inhibitors can reduce death in neurons deprived of NGF (Park et al., 1997).We now describe an additional gene, called SM-20, that is selectively expressed in sympathetic neurons after NGF withdrawal. SM-20 was originally identified as a growth factorresponsive gene in vascular smooth muscle cells (Wax et al., 1994). SM-20 expression has also been detected after activation of a temperature-sensitive p53 protein in fibroblasts; in these cells p53 activation results in both growth arrest and apoptosis (Madden et al., 1996). Although the biochemic...

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