Targeting Protein Phosphatase 1 (PP1) to the Actin Cytoskeleton: the Neurabin I/PP1 Complex Regulates Cell Morphology

Abstract

Neurabin I, a neuronal actin-binding protein, binds protein phosphatase 1 (PP1) and p70 ribosomal S6 protein kinase (p70S6K), both proteins implicated in cytoskeletal dynamics. We expressed wild-type and mutant neurabins fused to green fluorescent protein in Cos7, HEK293, and hippocampal neurons. Biochemical and cellular studies showed that an N-terminal F-actin-binding domain dictated neurabin I localization at actin cytoskeleton and promoted disassembly of stress fibers. Deletion of the C-terminal coiled-coil and sterile alpha motif domains abolished neurabin I dimerization and induced filopodium extension. Immune complex assays showed that neurabin I recruited an active PP1 via a PP1-docking sequence,⁴⁵⁷KIKF⁴⁶⁰. Mutation of the PP1-binding motif or PP1 inhibition by okadaic acid and calyculin A abolished filopodia and restored stress fibers in cells expressing neurabin I. In vitro and in vivo studies suggested that the actin-binding domain attenuated protein kinase A (PKA) phosphorylation of neurabin I. Modification of a major PKA site, serine-461, impaired PP1 binding. Finally, p70S6K was excluded from neurabin I/PP1 complexes and required the displacement of PP1 for recruitment to neurabin I. These studies provided new insights into the assembly and regulation of a neurabin I/PP1 complex that controls actin rearrangement to promote spine development in mammalian neurons.

We thank M. D. Ehlers and T. A. Blanpied (Duke University) for their help in culturing rat hippocampal neurons and Kathy Reaves for technical assistance.

The work was supported by NIH grants DK52054 and NS41063 (to S.S.), NS37508 (to R.J.C.), and CA40042 and GM56362 (to D.L.B.). C.J.O. is supported by a predoctoral fellowship from the Department of Defense Breast Cancer Program (DAMD17-18-1-8075), and R.T.T.-L. is supported by a predoctoral fellowship from the National Science Foundation.