Phosphorylation of a threonine unique to the short C-terminal isoform of betaII-spectrin links regulation of alpha-beta spectrin interaction to neuritogenesis

Spectrin tetramers are cytoskeletal proteins required in the formation of complex animal tissues. Mammalian II- and II-spectrin subunits form dimers that associate head-to-head with high affinity to form tetramers, but it is not known if this interaction is regulated. We show here that the short C-terminal splice variant of II-spectrin (IIsigma2) is a substrate for phosphorylation. In vitro, protein kinase CK2 phosphorylates S2110 and T2159; PKA phosphorylates T2159. Anti-phospho-T2159 peptide antibody detected phosphorylated IIsigma2 in Cos-1 cells. Immunoreactivity was increased in Cos-1 cells by treatment with forskolin, indicating that phosphorylation is promoted by elevated cAMP. The effect of forskolin was counteracted by the cAMP-dependent kinase inhibitor, H89. In vitro, PKA phosphorylation of an active fragment of IIsigma2 greatly reduced its interaction with II-spectrin at the tetramerization site. Mutation of T2159 to alanine eliminated inhibition by phosphorylation. Among the processes that require spectrin in mammals is the formation of neurites (incipient nerve axons). We tested the relationship of spectrin phosphorylation to neuritogenesis by transfecting the neuronal cell line, PC12, with enhanced green fluorescent protein-coupled fragments of IIsigma2-spectrin predicted to act as inhibitors of spectrin tetramer formation. Both wild type and T2159E mutant fragments allowed neuritogenesis in PC12 cells in response to nerve growth factor. The mutant T2159A inhibited neuritogenesis. Since the T2159A mutant represents a high affinity inhibitor of tetramer formation, we conclude that tetramers are requisite for neuritogenesis. Furthermore, since both the T2159E mutant and the wild-type allow neuritogenesis, we conclude that the short C-terminal II-spectrin is phosphorylated during this process.