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Vol. 17, Issue 12, 5115-5130, December 2006

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Myristoylated, Alanine-rich C-Kinase Substrate Phosphorylation Regulates Growth Cone Adhesion and Pathfinding

Jesse C. Gatlin^*, Adriana Estrada-Bernal, Staci D. Sanford, and Karl H. Pfenninger

Departments of Pediatrics and of Cell and Developmental Biology, University of Colorado School of Medicine, and University of Colorado Cancer Center, Aurora, CO 80045

Submitted December 30, 2005; Revised August 30, 2006; Accepted September 7, 2006
Monitoring Editor: Marianne Bronner-Fraser

Repellents evoke growth cone turning by eliciting asymmetric, localized loss of actin cytoskeleton together with changes in substratum attachment. We have demonstrated that semaphorin-3A (Sema3A)-induced growth cone detachment and collapse require eicosanoid-mediated activation of protein kinase C (PKC) and that the major PKC target is the myristoylated, alanine-rich C-kinase substrate (MARCKS). Here, we show that PKC activation is necessary for growth cone turning and that MARCKS, while at the membrane, colocalizes with ₃-integrin in a peripheral adhesive zone of the growth cone. Phosphorylation of MARCKS causes its translocation from the membrane to the cytosol. Silencing MARCKS expression dramatically reduces growth cone spread, whereas overexpression of wild-type MARCKS inhibits growth cone collapse triggered by PKC activation. Expression of phosphorylation-deficient, mutant MARCKS greatly expands growth cone adhesion, and this is characterized by extensive colocalization of MARCKS and ₃-integrin, resistance to eicosanoid-triggered detachment and collapse, and reversal of Sema3A-induced repulsion into attraction. We conclude that MARCKS is involved in regulating growth cone adhesion as follows: its nonphosphorylated form stabilizes integrin-mediated adhesions, and its phosphorylation-triggered release from adhesions causes localized growth cone detachment critical for turning and collapse.

These authors contributed equally to this work.

* Present address: Department of Biology, University of North Carolina, Chapel Hill, NC 27599.

Address correspondence to: Karl H. Pfenninger (karl.pfenninger{at}uchsc.edu)

Abbreviations used: Bis, bisindolylmaleimide I; DRG, dorsal root ganglion; ED, effector domain; GCP, growth cone particle; 12(S)-HETE, 12(S)-hydroxyeicosatetraenoic acid; IRM, interference reflection microscopy; MARCKS, myristoylated alanine-rich C-kinase substrate; PAZ, peripheral adhesive zone; P-MARCKS, MARCKS phosphorylated in the ED; Sema3A, semaphorin 3A; TPA, 12-O-tetradecanoyl-phorbol-13-acetate.

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