B‐50/GAP‐43 Binds to Actin Filaments Without Affecting Actin Polymerization and Filament Organization

Abstract

To investigate a possible function of the nervous tissue-specific protein kinase C substrate B-50/GAP-43 in regulation of the dynamics of the submembranous cytoskeleton, we studied the interaction between purified B-50 and actin. Both the phosphorylated and dephosphorylated forms of B-50 cosedimented with filamentous actin (F-actin) in a Ca(2+)-independent manner. Neither B-50 nor phospho-B-50 had any effect on the kinetics of actin polymerization and on the critical concentration at steady state, as measured using pyrenylated actin. Light scattering of F-actin samples was not increased in the presence of B-50, suggesting that B-50 does not bundle actin filaments. The number of actin filaments, determined by [3H]cytochalasin B binding, was not affected by either phospho- or dephospho-B-50, indicating that B-50 has neither a severing nor a capping effect. These observations were confirmed by electron microscopic evaluation of negatively stained F-actin samples, which did not reveal any structural changes in the actin meshwork on addition of B-50. We conclude that B-50 is an actin-binding protein that does not directly affect actin dynamics.

B-50/GAP-43-induced Formation of Filopodia Depends on Rho-GTPase

“…Inclusion of the actin-depolymerizing drug cytochalasin B (0.05 g/ml) during spreading completely prevented the B-50 -induced cortical cytoskeletal changes (our unpublished results), arguing for the dependence of these changes on new actin filament formation. In vitro, B-50 has been shown to cosediment with f-actin (Hens et al, 1993;He et al, 1997). Moreover, phosphorylated B-50 was shown to stabilize long actin filaments, whereas unphosphorylated B-50 reduced filament length and increased the critical concentration for actin polymerization, an effect that could be potentiated by calmodulin binding (He et al, 1997).…”

“…The protein colocalizes to a great extent with the cortical actin cytoskeleton Widmer and Caroni, 1993; and has been shown to cosediment with f-actin in vitro (Hens et al, 1993;He et al, 1997). Modulation of B-50 expression levels has been reported to have drastic effects on cell and growth cone morphology.…”

“…The same elements would be involved in the B-50 -induced extrusion of filopodia and blebs (Wiederkehr et al, 1997). The C-terminal segment of B-50 harbors a highly conserved casein kinase II domain (Pisano et al, 1988;Apel et al, 1991) that might be involved in binding f-actin (Hens et al, 1993;He et al, 1997). In the present study, we did not observe apparent morphological differences or changes in f-actin levels between cells expressing [1-176]B-50, missing the casein kinase II domain, or the wild-type construct (Figure 4, d and e), arguing against a critical role for the distal C-terminal segment (177-226) in B-50 -induced cytoskeletal changes in spreading fibroblasts.…”

“…B-50 is primarily located at the inner leaflet of axonal and growth cone membranes of developing and regenerating neurons as well as in plastic regions of the adult brain . The protein colocalizes to a great extent with the cortical actin cytoskeleton Widmer and Caroni, 1993; and has been shown to cosediment with f-actin in vitro (Hens et al, 1993;He et al, 1997).…”

Expression of Neuronal Kinesin Heavy Chain Is Developmentally Regulated in the Central Nervous System of the Rat

“…In our study, the growth-associated expression and heterogeneous distribution of nKHC appear to be strikingly similar to the ones of GAP 43 and SCGIO, suggesting that nKHC may indeed play a specific role in microtubule-based transport during axon growth and refinement of synaptic connections. In this respect, it is interesting that both GAP 43 and SCG1O have been shown to interact with and regulate the dynamics of cytoskeletal components, such as actin filaments and microtubules, respectively (Hens et al, 1993;Riederer et al, 1997).…”