Actin cytoskeletal remodeling is essential for cell morphological changes, motility, and migration. In neurons, actin filament remodeling plays a critical role in the control of neurite outgrowth and guidance (1-3). A number of actin-binding proteins and their upstream signaling molecules have been implicated in the regulation of neurite extension, retraction, and guidance (1-3). Cofilin is an actin-binding protein that plays a key role in controlling actin filament dynamics and reorganization by stimulating the depolymerization and severing of actin filaments (4, 5). These activities of cofilin are negatively regulated by phosphorylation at Ser-3 by LIM kinases (LIMKs) 2 (6, 7), and the inactive Ser-3-phosphorylated cofilin (P-cofilin) is reactivated by dephosphorylation catalyzed by protein phosphatases of the Slingshot (SSH) family (8, 9). LIMK1 is activated by phosphorylation at Thr-508 (in the kinase catalytic domain) by ROCK and PAK, downstream kinases of the Rho family small GTPases Rho, Rac, and Cdc42 (10 -12), or phosphorylation at Ser-323 (outside the catalytic domain) by mitogen-activated protein kinase-activated protein kinase-2, a kinase downstream of p38 mitogen-activated protein kinase (13). Several lines of evidence suggest that cofilin and its upstream regulators, LIMKs and SSHs, play crucial roles in controlling neurite outgrowth and guidance (14 -21). Calcium ion is a critical second messenger that mediates a variety of neuronal functions, including neurite outgrowth, axonal guidance, neuronal differentiation, synaptic plasticity, and memory formation. Numerous Ca 2ϩ -evoked responses are mediated by the Ca 2ϩ