Rapid growth of filopodia is observed in dendrites during the development of the nervous system and following increased synaptic activity. While some filopodia subsequently retract, others proceed to form new spines and synapses. In adult neurons, even mature spines undergo dynamic changes in shape that are mediated by actin rearrangement, and pharmacological agents that promote actin depolymerization result in serious deficits in synaptic efficacy (15
). We have analyzed NrbI-bound PP1 as a potential convergence point for signals that regulate both synaptic plasticity and spine morphology. As in vitro studies of NrbI are limited by the inability to express full-length NrbI (4
), a key goal of our studies was to establish a cell-based assay that exploited in vitro structure-function analyses of NrbI to yield new insights into the physiological role of the NrbI/PP1 complex in the mammalian nervous system.
WT GFP-NrbI (1-1095) bound polymerized F-actin both in vitro and in vivo and modified the morphology of HEK293 and Cos7 cells to reorganize the stress fiber network. GFP-NrbI (286-1095), which lacked actin binding, was cytosolic and had little effect on cell morphology, thereby establishing that actin binding was critical for cytoskeletal reorganization by NrbI. Previous studies showed that NrbI promoted in vitro bundling or cross-linking of actin fibers (20
). As ectopically expressed NrbI and NrbII formed homodimers and heterodimers (16
), this raised the possibility that the presentation of two adjacent actin-binding domains in a dimeric NrbI may promote actin bundling. Thus, we deleted the C-terminal coiled-coil and SAM domains, shown elsewhere for other proteins to self-associate (14
), and by using pull-downs of WT NrbI and NrbII from rat brain extracts established that homodimerization and heterodimerization of neurabins are mediated via their C termini. The monomeric GFP-NrbI (1-552) induced dramatic changes in cell morphology, disrupting the stress fiber network and promoting highly extended filopodia. Live video microscopy showed the rapid growth of filopodia in the GFP-NrbI (1-552)-expressing cells that extended significantly further than those in cells expressing WT GFP-NrbI (1-1095). The NrbI C terminus (amino acids 553 to 1095), while sufficient to bind NrbI (and NrbII), did not localize with the actin cytoskeleton or modify cell morphology. Cell fractionation and immunoprecipitation studies suggested that GFP-NrbI (1-552) bound both F-actin and PP1 more effectively than did WT GFP-NrbI (1-1095), hinting that NrbI dimerization may attenuate its actin- and PP1-binding properties.
The importance of PP1 recruitment for NrbI-induced morphology was established by mutating the core PP1-binding sequence, KIKF, in NrbI to alanines. This not only abolished PP1 binding in vitro (data not shown) and in vivo, but unlike GFP-NrbI (1-552), GFP-NrbI (1-552, 4A) failed to induce filopodia in HEK293 and Cos7 cells or disrupt stress fibers in Cos7 cells. Pharmacological inhibition of PP1 activity by okadaic acid or calyculin A rapidly collapsed filopodia induced by GFP-NrbI (1-552), showing that, in addition to binding F-actin, NrbI must recruit an active PP1 to modify cell morphology. Immune complex assays confirmed that the NrbI-bound PP1 was an active protein phosphatase. This was unexpected, as in vitro studies (26
) (Fig. ) showed that PP1-binding NrbI peptides inhibit its activity against phosphorylase a
. The molecular basis for the difference in the NrbI/PP1 complexes assembled in vitro and in vivo is unclear, but the difference may reflect the presence of additional proteins or covalent modifications of NrbI in cells. In any case, our data supported an active role for the NrbI/PP1 complex in cytoskeletal reorganization.
PP1 binds both substrates (8
) and regulators (22
) that undergo reversible phosphorylation. Covalent modification of PP1 regulators modulates PP1 binding (7
) and activity (12
). Analysis of metabolically labeled HEK293 cells established for the first time that GFP-NrbI (1-1095) was a phosphoprotein and that its phosphorylation in vivo was stimulated by the PKA agonist Sp-cAMPS. Surprisingly, GFP-NrbI (1-552) was essentially unphosphorylated in HEK293 cells. This was not due to elimination of PKA sites, as GFP-NrbI (553-1095) also showed low basal phosphorylation that was not further stimulated by Sp-cAMPS. Deletion of PP1 binding or inhibition of phosphatase activity failed to enhance GFP-NrbI (1-552) phosphorylation, suggesting that PP1 binding did not dictate the phosphorylation state of NrbI. Compared to WT GFP-NrbI (1-1095), soluble GFP-NrbI (286-1095) incorporated much higher levels of [32
P]phosphate that was greatly enhanced by PKA activation. This raised the possibility that the actin-binding domain and/or association with the cytoskeleton inhibited NrbI phosphorylation by PKA. This was confirmed by in vitro studies that showed that NrbI (103-615) lacking N-terminal actin-binding sequences incorporated twofold-higher levels of phosphate than did NrbI (1-615). This did not, however, fully explain the absence of phosphate in GFP-NrbI (1-552), leading us to speculate that F-actin association may further impair NrbI phosphorylation in cells. Mutation of serine-461 to alanine in GFP-NrbI (286-1095) abolished PKA-stimulated phosphorylation and identified the serine immediately adjacent to the PP1-binding motif as a major in vivo PKA phosphorylation site. Analysis of recombinant NrbI (374-516, S461E), in which glutamic acid was introduced to mimic serine-461 phosphorylation, confirmed an earlier observation (17
) that serine-461 modification diminished PP1 association with NrbI and suggested that assembly-disassembly of the NrbI/PP1 complex may be regulated by hormones that elevate intracellular cyclic AMP.
The PDZ domain adjacent to the core PP1-binding site recruits p70S6K, which has effects opposite of those of PP1 on actin dynamics (23
). Affinity isolation of NrbI/PP1 complexes from rat brain or NrbI immunoprecipitation from HEK293 cells overexpressing p70S6K failed to show significant association of p70S6K with NrbI. This demonstrated that NrbI had a significant preference for PP1 over p70S6K and predicted that disruption of PP1 binding would facilitate p70S6K recruitment. Indeed, GFP-NrbI (1-552, 4A) that failed to bind PP1 showed enhanced p70S6K binding. These data can be summarized in the following model for the potential role of NrbI in transducing signals that regulate actin cytoskeleton (Fig. ). Our data suggest that the majority of cellular NrbI is bound to PP1 and is associated with the actin cytoskeleton. This promotes the disassembly of stress fibers. While both dimeric and monomeric forms of NrbI induce filopodia in cultured cells, the enhanced ability by cells expressing monomeric NrbI to extend surface projections leads us to speculate that the dynamics of NrbI dimerization may also play a role in dictating the cell morphology. Physiological signals that activate Rac1 GTPase (29
) promote NrbI shuttling to the cytoskeleton and may target PP1 to the actin cytoskeleton. In contrast, the cytosolic NrbI, by virtue of being an improved substrate for PKA (at serine-461), may display decreased PP1 binding and recruit p70S6K and other targets to the NrbI PDZ domain. This mechanism may also account for the antagonism between PP1 and p70S6K that regulates neuronal morphology. As neither p70S6K overexpression nor inhibition of p70S6K activity by rapamycin had any effect on HEK293 cell morphology, we were unable to establish a role for the NrbI-bound p70S6K in heterologous cells.
FIG. 7. Model for the regulation of cell morphology by the NrbI/PP1 complex. Our experiments propose the following model for the regulation of the neuronal cytoskeleton by the NrbI/PP1 complex. NrbI function may be regulated at many steps including reversible (more ...)
NrbI is expressed exclusively in neurons, while NrbII is more widely expressed. Both neurabins bind an overlapping set of targets that include PP1. Thus, disruption of the mouse NrbII/spinophilin gene that results in defective PP1 signaling and increases spine formation in the hippocampus of young NrbII-null mice (9
) may represent not only the loss of NrbII/PP1 complexes but also the uncontested activity of NrbI/PP1. By overexpressing WT GFP-NrbI (1-1095), we artificially tipped the balance in favor of NrbI/PP1 complexes in cultured hippocampal neurons and induced filopodia, which subsequently matured into spines with GFP-NrbI (1-1095) localized within these structures. This suggested an active role of NrbI/PP1 in spine formation in developing neurons and may explain the abundance of spines seen in the NrbII-null mouse (9
). In conclusion, our work has highlighted several key structural determinants and protein interactions required for the regulation of actin cytoskeleton by a NrbI/PP1 complex and thus set the stage for future studies that will elucidate the physiological role of the NrbI/PP1 complex in neurons.