Critical role of Ena/VASP proteins for filopodia formation in neurons and in function downstream of netrin-1

Ena/VASP proteins play important roles in axon outgrowth and guidance. Ena/VASP activity regulates the assembly and geometry of actin networks within fibroblast lamellipodia. In growth cones, Ena/VASP proteins are concentrated at filopodia tips, yet their role in growth cone responses to guidance signals has not been established. We found that Ena/VASP proteins play a pivotal role in formation and elongation of filopodia along neurite shafts and growth cone. Netrin-1-induced filopodia formation was dependent upon Ena/VASP function and directly correlated with Ena/VASP phosphorylation at a regulatory PKA site. Accordingly, Ena/VASP function was required for filopodial formation from the growth cone in response to global PKA activation. We propose that Ena/VASP proteins control filopodial dynamics in neurons by remodeling the actin network in response to guidance cues.     

A CaMKII/calcineurin switch controls the direction of Ca(2+)-dependent growth cone guidance

Axon pathfinding depends on attractive and repulsive turning of growth cones to extracellular cues. Localized cytosolic Ca2+ signals are known to mediate the bidirectional responses, but downstream mechanisms remain elusive. Here, we report that calcium-calmodulin-dependent protein kinase II (CaMKII) and calcineurin (CaN) phosphatase provide a switch-like mechanism to control the direction of Ca(2+)-dependent growth cone turning. A relatively large local Ca2+ elevation preferentially activates CaMKII to induce attraction, while a modest local Ca2+ signal predominantly acts through CaN and phosphatase-1 (PP1) to produce repulsion. The resting level of intracellular Ca2+ concentrations also affects CaMKII/CaN operation: a normal baseline allows distinct turning responses to different local Ca2+ signals, while a low baseline favors CaN-PP1 activation for repulsion. Moreover, the cAMP pathway negatively regulates CaN-PP1 signaling to inhibit repulsion. Finally, CaMKII/CaN-PP1 also mediates netrin-1 guidance. Together, these findings establish a complex Ca2+ mechanism that targets the balance of CaMKII/CaN-PP1 activation to control distinct growth cone responses.     

Anchoring of Protein Kinase A by ERM (Ezrin-Radixin-Moesin) Proteins Is Required for Proper Netrin Signaling through DCC (Deleted in Colorectal Cancer)

Netrin-1, acting through its principal receptor DCC (deleted in colorectal cancer), serves as an axon guidance cue during neural development and also contributes to vascular morphogenesis, epithelial migration, and the pathogenesis of some tumors. Several lines of evidence suggest that netrin-DCC signaling can regulate and be regulated by the cAMP-dependent protein kinase, PKA, although the molecular details of this relationship are poorly understood. Specificity in PKA signaling is often achieved through differential subcellular localization of the enzyme by interaction with protein kinase A anchoring proteins (AKAPs). Here, we show that AKAP function is required for DCC-mediated activation of PKA and phosphorylation of cytoskeletal regulatory proteins of the Mena/VASP (vasodilator-stimulated phosphoprotein) family. Moreover, we show that DCC and PKA physically interact and that this association is mediated by the ezrin-radixin-moesin (ERM) family of plasma membrane-actin cytoskeleton cross-linking proteins. Silencing of ERM protein expression inhibits DCC-PKA interaction, DCC-mediated PKA activation, and phosphorylation of Mena/VASP proteins as well as growth cone morphology and neurite outgrowth. Finally, although expression of wild-type radixin partially rescued growth cone morphology and tropism toward netrin in ERM-knockdown cells, expression of an AKAP-deficient mutant of radixin did not fully rescue growth cone morphology and switched netrin tropism from attraction to repulsion. These data support a model in which ERM-mediated anchoring of PKA activity to DCC is required for proper netrin/DCC-mediated signaling.     

Growth arrest and DNA damage-inducible protein GADD34 assembles a novel signaling complex containing protein phosphatase 1 and inhibitor 1

The growth arrest and DNA damage-inducible protein, GADD34, was identified by its interaction with human inhibitor 1 (I-1), a protein kinase A (PKA)-activated inhibitor of type 1 protein serine/threonine phosphatase (PP1), in a yeast two-hybrid screen of a human brain cDNA library. Recombinant GADD34 (amino acids 233 to 674) bound both PKA-phosphorylated and unphosphorylated I-1(1-171). Serial truncations mapped the C terminus of I-1 (amino acids 142 to 171) as essential for GADD34 binding. In contrast, PKA phosphorylation was required for PP1 binding and inhibition by the N-terminal I-1(1-80) fragment. Pulldowns of GADD34 proteins expressed in HEK293T cells showed that I-1 bound the central domain of GADD34 (amino acids 180 to 483). By comparison, affinity isolation of cellular GADD34/PP1 complexes showed that PP1 bound near the C terminus of GADD34 (amino acids 483 to 619), a region that shows sequence homology with the virulence factors ICP34.5 of herpes simplex virus and NL-S of avian sarcoma virus. While GADD34 inhibited PP1-catalyzed dephosphorylation of phosphorylase a, the GADD34-bound PP1 was an active eIF-2alpha phosphatase. In brain extracts from active ground squirrels, GADD34 bound both I-1 and PP1 and eIF-2alpha was largely dephosphorylated. In contrast, the I-1/GADD34 and PP1/GADD34 interactions were disrupted in brain from hibernating animals, in which eIF-2alpha was highly phosphorylated at serine-51 and protein synthesis was inhibited. These studies suggested that modification of the I-1/GADD34/PP1 signaling complex regulates the initiation of protein translation in mammalian tissues.     

Lipid rafts mediate chemotropic guidance of nerve growth cones

Axon guidance requires signal transduction of extracellular cues through the plasma membrane for directional motility. Here we present evidence that cholesterol- and sphingolipid-enriched membrane microdomains (lipid rafts) mediate specific guidance responses of nerve growth cones. Disruption of lipid rafts by various approaches targeting cholesterol or gangliosides selectively abolished growth cone attraction and repulsion in BDNF and netrin-1 gradients, respectively, without affecting glutamate-induced attraction. Interestingly, local raft disruption on one side of the growth cone in bath BDNF or netrin-1 produced opposite turning responses to that induced by the gradients. Raft manipulation also blocked Semaphorin 3A-induced growth cone repulsion, inhibition, and collapse. Finally, guidance responses appeared to involve raft-dependent activation of p42/p44 MAPK and ligand-induced receptor recruitment to lipid rafts. Together with the observation of asymmetric receptor-raft associations at the growth cone in guidance gradients, our findings indicate that localized signaling through membrane rafts plays a role in mediating guidance actions of extracellular cues on developing axons.     

Calcium mediates bidirectional growth cone turning induced by myelin-associated glycoprotein

Cytoplasmic second messengers, Ca2+ and cAMP, regulate nerve growth cone turning responses induced by many guidance cues, but the causal relationship between these signaling pathways has been unclear. We here report that, for growth cone turning induced by a gradient of myelin-associated glycoprotein (MAG), cAMP acts by modulating MAG-induced Ca2+ signaling. Growth cone repulsion induced by MAG was accompanied by localized Ca2+ signals on the side of the growth cone facing the MAG source, due to Ca2+ release from intracellular stores. Elevating cAMP signaling activity or membrane depolarization enhanced MAG-induced Ca2+ signals and converted growth cone repulsion to attraction. Directly imposing high- or low-amplitude Ca2+ signals with an extracellular gradient of Ca2+ ionophore was sufficient to trigger either attractive or repulsive turning, respectively. Thus, distinct Ca2+ signaling, which can be modulated by cAMP, mediates the bidirectional turning responses induced by MAG.     

UNC-6/netrin and its receptors UNC-5 and UNC-40/DCC modulate growth cone protrusion in vivo in C. elegans

The UNC-6/netrin guidance cue functions in axon guidance in vertebrates and invertebrates, mediating attraction via UNC-40/DCC family receptors and repulsion via by UNC-5 family receptors. The growth cone reads guidance cues and extends lamellipodia and filopodia, actin-based structures that sense the extracellular environment and power the forward motion of the growth cone. We show that UNC-6/netrin, UNC-5 and UNC-40/DCC modulated the extent of growth cone protrusion that correlated with attraction versus repulsion. Loss-of-function unc-5 mutants displayed increased protrusion in repelled growth cones, whereas loss-of-function unc-6 or unc-40 mutants caused decreased protrusion. In contrast to previous studies, our work suggests that the severe guidance defects in unc-5 mutants may be due to latent UNC-40 attractive signaling that steers the growth cone back towards the ventral source of UNC-6. UNC-6/Netrin signaling also controlled polarity of growth cone protrusion and F-actin accumulation that correlated with attraction versus repulsion. However, filopodial dynamics were affected independently of polarity of protrusion, indicating that the extent versus polarity of protrusion are at least in part separate mechanisms. In summary, we show here that growth cone guidance in response to UNC-6/netrin involves a combination of polarized growth cone protrusion as well as a balance between stimulation and inhibition of growth cone (e.g. filopodial) protrusion.     

Phosphorylation of TIMAP by glycogen synthase kinase-3beta activates its associated protein phosphatase 1

TIMAP (TGF-beta1 inhibited, membrane-associated protein) is a prenylated, endothelial cell-predominant protein phosphatase 1 (PP1c) regulatory subunit that localizes to the plasma membrane of filopodia. Here, we determined whether phosphorylation regulates TIMAP-associated PP1c function. Phosphorylation of TIMAP was observed in cells metabolically labeled with [32P]orthophosphate and was reduced by inhibitors of protein kinase A (PKA) and glycogen synthase kinase-3 (GSK-3). In cell-free assays, immunopurified TIMAP was phosphorylated by PKA and, after PKA priming, by GSK-3beta. Site-specific Ser to Ala substitution identified amino acid residues Ser333/Ser337 as the likely PKA/GSK-3beta phosphorylation site. Substitution of Ala for Val and Phe in the KVSF motif of TIMAP (TIMAPV64A/F66A) abolished PP1c binding and TIMAP-associated PP1c activity. TIMAPV64A/F66A was hyper-phosphorylated in cells, indicating that TIMAP-associated PP1c auto-dephosphorylates TIMAP. Constitutively active GSK-3beta stimulated phosphorylation of TIMAPV64A/F66A, but not wild-type TIMAP, suggesting that the PKA/GSK-3beta site may be subject to dephosphorylation by TIMAP-associated PP1c. Substitution of Asp or Glu for Ser at amino acid residues 333 and 337 to mimic phosphorylation reduced the PP1c association with TIMAP. Conversely, GSK-3 inhibitors augmented PP1c association with TIMAP-PP1c in cells. The 333/337 phosphomimic mutations also increased TIMAP-associated PP1c activity in vitro and against the non-integrin laminin receptor 1 in cells. Finally, TIMAP mutants with reduced PP1c activity strongly stimulated endothelial cell filopodia formation, an effect mimicked by the GSK-3 inhibitor LiCl. We conclude that TIMAP is a target for PKA-primed GSK-3beta-mediated phosphorylation. This phosphorylation controls TIMAP association and activity of PP1c, in turn regulating extension of filopodia in endothelial cells.     

Ena/VASP function in retinal axons is required for terminal arborization but not pathway navigation

The Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family of proteins is required for filopodia formation in growth cones and plays a crucial role in guidance cue-induced remodeling of the actin cytoskeleton. In vivo studies with pharmacological inhibitors of actin polymerization have previously provided evidence for the view that filopodia are needed for growth cone navigation in the developing visual pathway. Here we have re-examined this issue using an alternative strategy to generate growth cones without filopodia in vivo by artificially targeting Xena/XVASP (Xenopus homologs of Ena/VASP) proteins to mitochondria in retinal ganglion cells (RGCs). We used the specific binding of the EVH1 domain of the Ena/VASP family of proteins with the ligand motif FP4 to sequester the protein at the mitochondria surface. RGCs with reduced function of Xena/XVASP proteins extended fewer axons out of the eye and possessed dynamic lamellipodial growth cones missing filopodia that advanced slowly in the optic tract. Surprisingly, despite lacking filopodia, the axons navigated along the optic pathway without obvious guidance errors, indicating that the Xena/XVASP family of proteins and filopodial protrusions are non-essential for pathfinding in retinal axons. However, depletion of Xena/XVASP proteins severely impaired the ability of growth cones to form branches within the optic tectum, suggesting that this protein family, and probably filopodia, plays a key role in establishing terminal arborizations.     

Netrin-1 and axonal guidance: signaling and asymmetrical translation

More than 10 years after its initial discovery, netrin-1 - the first described chimioattractive molecule controlling the guidance of the commissural axons - has recently known a unsuspected wave of interest because of its implication in the development of the nervous system but also, more recently, fot its role in angiogenesis and tumorigenesis. Because, of a series of recent publications on netrin-1 signaling, we propose here to describe the recent insight in netrin-1 signaling via its main receptor DCC (deleted in colorectal cancer), and the recent discovery that netrin controls the assymetric distribution of beta-actin in the growth cone. Thus, it seems that netrin-1, but also the neurotrophic factor BDNF, controls acute growth cone responses such as collapse and turning by the regulation of localized protein translation, such as beta-actin. This process involves both transport of beta-actin mRNA, bound to Vg1RBP, to specific locations, and mRNA translation upon stimulation by local activation of the translation initiation regulator eIF-4E-binding protein 1. Indeed, Netrin-1 induces the movement of Vg1RBP granules into filopodia, and triggers a polarized increase in beta-actin translation on the near side of the growth cone before growth cone turning. The binding of BDNF to its receptor Trk has a similar effect for growth cone attraction, althought it is differentially regulated. Thus, this asymetrically synthesized beta-actin may direct actin polymerization and consequently the migration of the growth cone toward the cue.     

Geranylgeraniol enhances testosterone production via the cAMP/protein kinase A pathway in testis-derived I-10 tumor cells

Testosterone levels in men decrease with age; this decline has been linked to various diseases and can shorten life expectancy. Geranylgeraniol (GGOH) is an isoprenoid found in plants that plays an important role in several biological processes; however, its role in steroidogenesis is unknown. Here, we report that GGOH enhances the production of testosterone and its precursor progesterone in testis-derived I-10 tumor cells. GGOH induced protein kinase A (PKA) activity and increased cAMP levels and was found to regulate cAMP/PKA signaling by activating adenylate cyclase without altering phosphodiesterase activity. GGOH also stimulated mRNA and protein levels of steroidogenic acute regulatory protein, a downstream effector in the cAMP/PKA pathway. These results demonstrate that GGOH enhances steroidogenesis in testis-derived cells by modulating cAMP/PKA signaling. Our findings have potential applications for the development of therapeutics that increase testosterone levels in aging men.     

Dynamic anchoring of PKA is essential during oocyte maturation

In the final stages of ovarian follicular development, the mouse oocyte remains arrested in the first meiotic prophase, and cAMP-stimulated PKA plays an essential role in this arrest. After the LH surge, a decrease in cAMP and PKA activity in the oocyte initiates an irreversible maturation process that culminates in a second arrest at metaphase II prior to fertilization. A-kinase anchoring proteins (AKAPs) mediate the intracellular localization of PKA and control the specificity and kinetics of substrate phosphorylation. Several AKAPs have been identified in oocytes including one at 140 kDa that we now identify as a product of the Akap1 gene. We show that PKA interaction with AKAPs is essential for two sequential steps in the maturation process: the initial maintenance of meiotic arrest and the subsequent irreversible progression to the polar body extruded stage. A peptide inhibitor (HT31) that disrupts AKAP/PKA interactions stimulates oocyte maturation in the continued presence of high cAMP. However, during the early minutes of maturation, type II PKA moves from cytoplasmic sites to the mitochondria, where it associates with AKAP1, and this is shown to be essential for maturation to continue irreversibly.     

Cyclic AMP-dependent protein kinase isoenzymes in human myeloid leukemia (HL60) and breast tumor (MCF-7) cells

Combinations of retinoic acid (RA) and cAMP mediate many biological responses in a large variety of cell types. While the basis for the apparent synergistic effects of RA and cAMP are not clearly defined, it is likely that activation of PKA by cAMP is involved. However, literature reports concerning the identity of PKA isoforms in HL60 and MCF-7 cells are conflicting. The purpose of the present investigation is to identify PKA isoforms in HL60 and MCF-7 cells. Utilization of high-performance anion-exchange liquid chromatography, immunoblotting, and 8-azido-cAMP photoaffinity binding resulted in the finding that HL60 cells contain PKA types I alpha and II alpha, while MCF-7 cells contain PKA types I alpha, II alpha, and II beta. PKA type I alpha in both HL60 and MCF-7 cells eluted from columns as two well-separated peaks. One peak eluted at a low salt concentration in agreement with previous reports. The second HL60 PKA type I alpha peak eluted at a salt concentration intermediate between that eluting the first peak and that eluting PKA type II alpha and contained approximately 62% of the total RI alpha protein. However, the second MCF-7 PKA type I alpha peak contained approximately 66% of the total RI alpha protein and co-eluted with PKA types II alpha and II beta. This "contamination" of PKA type II fractions with PKA type I has led, in some cases, to interpretations that may need reevaluation.     

Targeting protein phosphatase 1 (PP1) to the actin cytoskeleton: the neurabin I/PP1 complex regulates cell morphology

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,(457)KIKF(460). 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.     

Calcium and cAMP levels interact to determine attraction versus repulsion in axon guidance

Correct guidance of axons to their targets depends on an intricate network of signaling molecules in the growth cone. Calcium and cAMP are two key regulators of whether axons are attracted or repelled by molecular gradients, but how these molecules interact to determine guidance responses remains unclear. Here, we constructed a mathematical model for the relevant signaling network, which explained a large range of previous biological data and made predictions for when axons will be attracted or repelled. We then confirmed these predictions experimentally, in particular showing that while small increases in cAMP levels promote attraction large increases do not, and that under some circumstances reducing cAMP levels promotes attraction. Together, these results show that a relatively simple mathematical model can quantitatively predict guidance decisions across a wide range of conditions, and that calcium and cAMP levels play a more complex role in these decisions than previously determined.     

The localization and activity of cAMP-dependent protein kinase affect cell cycle progression in thyroid cells

cAMP signals are received and transmitted by multiple isoforms of cAMP-dependent protein kinases (PKAs), typically determined by their specific regulatory subunits. We describe changes in the cAMP signal transduction pathway during cell cycle progression in synchronized rat thyroid cells. Both PKA type II (PKAII) localization and nuclear cAMP signaling are significantly modified during G(0) and G(1)-S transitions. G(1) is characterized by PKA activation and amplified cAMP signal transduction. This is associated with a decrease in the concentration of RI and RII regulatory subunits and enhanced anchoring of PKAII to the Golgi-centrosome region. Just prior to S, the cAMP pathway is depressed. Up-regulation of the pathway by exogenous cAMP in G(1) inhibited the subsequent decay of the Cdk inhibitor p27 and delayed the onset of S phase. Forced translocation of endogenous PKAII to the cytosol down-regulated cAMP signaling, advancing the timing of p27 decay and inducing premature exit from G(1). These data indicate that membrane-bound PKA amplifies the transduction of cAMP signals in G(1) and that the length of G(1) is influenced by cAMP-PKA.     

Association of the type 1 protein phosphatase PP1 with the A-kinase anchoring protein AKAP220

The cyclic AMP (cAMP)-dependent protein kinase (PKA) and the type 1 protein phosphatase (PP1) are broad-specificity signaling enzymes with opposing actions that catalyze changes in the phosphorylation state of cellular proteins. Subcellular targeting to the vicinity of preferred substrates is a means of restricting the specificity of each enzyme [1] [2]. Compartmentalization of the PKA holoenzyme is mediated through association of the regulatory subunits with A-kinase anchoring proteins (AKAPs), whereas a diverse family of phosphatase-targeting subunits directs the location of the PP1 catalytic subunit (PP1c) [3] [4]. Here, we demonstrate that the PKA-anchoring protein, AKAP220, binds PP1c with a dissociation constant (KD) of 12.1 +/- 4 nM in vitro. Immunoprecipitation of PP1 from cell extracts resulted in a 10.4 +/- 3.8-fold enrichment of PKA activity. AKAP220 co-purified with PP1c by affinity chromatography on microcystin sepharos Immunocytochemical analysis demonstrated that the kinase, the phosphatase and the anchoring protein had distinct but overlapping staining patterns in rat hippocampal neurons. Collectively, these results provide the first evidence that AKAP220 is a multivalent anchoring protein that maintains a signaling scaffold of PP1 and the PKA holoenzyme.     

Substrate enhances the sensitivity of type I protein kinase a to cAMP

The functional significance of the presence of two major (types I and II) isoforms of the cAMP-dependent protein kinase (PKA) is still enigmatic. The present study showed that peptide substrate enhanced the activation of PKA type I at low, physiologically relevant concentrations of cAMP through competitive displacement of the regulatory RI subunit. The effect was similar whether the substrate was a short peptide or the physiological 60-kDa protein tyrosine hydroxylase. In contrast, substrate failed to affect the cAMP-sensitivity of PKA type II. Size exclusion chromatography confirmed that substrate acted to physically enhance the dissociation of the RIalpha and Calpha subunits of PKA type I, but not the RIIalpha and Calpha subunits of PKA type II. Substrate availability can therefore fine-tune the activation of PKA type I by cAMP, but not PKA type II. The cAMP-dissociated RII and C subunits of PKA type II reassociated much faster than the PKA type I subunits in the presence of substrate peptide. This suggests that only PKA type II is able to rapidly reverse its activation after a burst of cAMP when exposed to high substrate concentration. We propose this as a possible reason why PKA type II is preferentially found in complexes with substrates undergoing rapid phosphorylation cycles.