Modulation of Actin Filament Behavior by GAP-43 (Neuromodulin) Is Dependent on the Phosphorylation Status of Serine 41, the Protein Kinase C Site

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Abstract
Synthesis of GAP-43 (also known as neuromodulin) in neurons is induced during axon growth, and high concentrations (estimated between 50 and 100 μm) accumulate in the growth cone. GAP-43 is tightly associated with the growth cone membrane skeleton, the structure that transduces extracellular guidance cues into alterations in morphology by spatially regulating polymerization of actin filaments, thereby causing directional changes in axon growth. GAP-43 cosediments with actin filaments, and its phosphorylation on serine 41 by PKC, too, is spatially regulated so that phosphorylated GAP-43 is found in areas where growth cones make productive, stable contacts with other cells. In contrast, unphosphorylated GAP-43, which binds calmodulin, is always found in parts of the growth cone that are retracting. Here we have used a cell-free assay to investigate how the phosphorylation status of GAP-43 affects its interactions with actin and show that both phosphorylated and unphosphorylated GAP-43 have different, independent effects on actin filament structure. Phosphorylated GAP-43 stabilizes long actin filaments (Kd= 161 nm), and antibodies to phosphorylated GAP-43 inhibit binding of actin to phalloidin, implying a lateral interaction with filaments. In contrast, unphosphorylated GAP-43 reduces filament length distribution (Kd= 1.2 μm) and increases the critical concentration for polymerization. Prebinding calmodulin potentiates this effect. The results show that spatially regulated post-translational modifications of GAP-43 within the growth cone, which can be regulated in response to extracellular signals, have the ability to directly influence the structure of the actin cytoskeleton.