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Neurotrophin regulation of beta-actin mRNA and protein localization within growth cones.

Zhang HL, Singer RH, Bassell GJ - J. Cell Biol. (1999)

Bottom Line: NT-3 treatment resulted in a rapid and transient stimulation of PKA activity that preceded the localization of beta-actin mRNA.Depolymerization of microtubules, but not microfilaments, inhibited the NT-3-induced localization of beta-actin mRNA.These results suggest that NT-3 activates a cAMP-dependent signaling mechanism to promote the microtubule-dependent localization of beta-actin mRNA within growth cones.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461, USA.

ABSTRACT
Neurotrophins play an essential role in the regulation of actin-dependent changes in growth cone shape and motility. We have studied whether neurotrophin signaling can promote the localization of beta-actin mRNA and protein within growth cones. The regulated localization of specific mRNAs within neuronal processes and growth cones could provide a mechanism to modulate cytoskeletal composition and growth cone dynamics during neuronal development. We have previously shown that beta-actin mRNA is localized in granules that were distributed throughout processes and growth cones of cultured neurons. In this study, we demonstrate that the localization of beta-actin mRNA and protein to growth cones of forebrain neurons is stimulated by neurotrophin-3 (NT-3). A similar response was observed when neurons were exposed to forskolin or db-cAMP, suggesting an involvement of a cAMP signaling pathway. NT-3 treatment resulted in a rapid and transient stimulation of PKA activity that preceded the localization of beta-actin mRNA. Localization of beta-actin mRNA was blocked by prior treatment of cells with Rp-cAMP, an inhibitor of cAMP-dependent protein kinase A. Depolymerization of microtubules, but not microfilaments, inhibited the NT-3-induced localization of beta-actin mRNA. These results suggest that NT-3 activates a cAMP-dependent signaling mechanism to promote the microtubule-dependent localization of beta-actin mRNA within growth cones.

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NT-3 signaling of β-actin mRNA localization through cAMP. NT-3 stimulation of β-actin mRNA localization was reduced by kinase inhibitors. K252a (200 nM) or Rp-cAMP (50 μM) was added to the MEM for 1 h after a 5-h starvation. Forskolin and db-cAMP stimulated β-actin mRNA localization in a similar fashion to NT-3. *, P < 0.01 when NT-3/K252a at 10min and NT-3/Rp-cAMP at 10min were compared with NT-3 at 10min. #, P < 0.01 when NT-3/K252a at 2h and NT-3/Rp-cAMP at 2h were compared with NT-3 at 2h.
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Figure 8: NT-3 signaling of β-actin mRNA localization through cAMP. NT-3 stimulation of β-actin mRNA localization was reduced by kinase inhibitors. K252a (200 nM) or Rp-cAMP (50 μM) was added to the MEM for 1 h after a 5-h starvation. Forskolin and db-cAMP stimulated β-actin mRNA localization in a similar fashion to NT-3. *, P < 0.01 when NT-3/K252a at 10min and NT-3/Rp-cAMP at 10min were compared with NT-3 at 10min. #, P < 0.01 when NT-3/K252a at 2h and NT-3/Rp-cAMP at 2h were compared with NT-3 at 2h.

Mentions: SignaTECT cAMP-Dependent Protein Kinase Assay (Promega) was used to monitor neurotrophin-3 stimulation of PKA activity in primary neuronal cultures (Cobb et al., 1992; Goueli et al. 1995; Walsh and Van Patten 1994). This assay uses a biotinylated Kemptide substrate, derived from pyruvate kinase (Piklis et al. 1980), which is highly specific for cAMP-dependent PKA (Km = 5–10 μM). The biotinylated peptide substrate is phosphorylated by cellular PKA using γ-[32P]ATP. The 32P-labeled biotin substrate is then recovered from the reaction mix and captured by a streptavidin-linked matrix (SAMTM Membrane). In brief, cultured neurons were treated with NT-3 as described, washed with HBSS, and then lysed in 0.3 ml cold extraction buffer (25 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 10 mM mercaptoethanol, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 0.5 mM PMSF). Extracts were centrifuged for 5 min at 4°C at 14,000 g and 5 μl of the supernatant was incubated with 20 μl of the reaction mixture containing biotinylated peptide substrate and γ-[32P]ATP for 5 min at 30°C. The reaction was terminated and 10 μl was spotted onto a biotin capture membrane. After washing and drying of the membrane, radioactivity was measured using a liquid scintillation counter. This assay detects 0.012 casein units (2.5 Kemptide units) or less of purified cAMP-dependent PKA. A linear regression analysis, using calibrated amounts of casein units, produced an R2 value >0.95. Triplicate samples were used for each time point and the PKA activities and standard deviation is shown in Fig. 8 (expressed relative to starved cells not exposed to NT-3).


Neurotrophin regulation of beta-actin mRNA and protein localization within growth cones.

Zhang HL, Singer RH, Bassell GJ - J. Cell Biol. (1999)

NT-3 signaling of β-actin mRNA localization through cAMP. NT-3 stimulation of β-actin mRNA localization was reduced by kinase inhibitors. K252a (200 nM) or Rp-cAMP (50 μM) was added to the MEM for 1 h after a 5-h starvation. Forskolin and db-cAMP stimulated β-actin mRNA localization in a similar fashion to NT-3. *, P < 0.01 when NT-3/K252a at 10min and NT-3/Rp-cAMP at 10min were compared with NT-3 at 10min. #, P < 0.01 when NT-3/K252a at 2h and NT-3/Rp-cAMP at 2h were compared with NT-3 at 2h.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2164987&req=5

Figure 8: NT-3 signaling of β-actin mRNA localization through cAMP. NT-3 stimulation of β-actin mRNA localization was reduced by kinase inhibitors. K252a (200 nM) or Rp-cAMP (50 μM) was added to the MEM for 1 h after a 5-h starvation. Forskolin and db-cAMP stimulated β-actin mRNA localization in a similar fashion to NT-3. *, P < 0.01 when NT-3/K252a at 10min and NT-3/Rp-cAMP at 10min were compared with NT-3 at 10min. #, P < 0.01 when NT-3/K252a at 2h and NT-3/Rp-cAMP at 2h were compared with NT-3 at 2h.
Mentions: SignaTECT cAMP-Dependent Protein Kinase Assay (Promega) was used to monitor neurotrophin-3 stimulation of PKA activity in primary neuronal cultures (Cobb et al., 1992; Goueli et al. 1995; Walsh and Van Patten 1994). This assay uses a biotinylated Kemptide substrate, derived from pyruvate kinase (Piklis et al. 1980), which is highly specific for cAMP-dependent PKA (Km = 5–10 μM). The biotinylated peptide substrate is phosphorylated by cellular PKA using γ-[32P]ATP. The 32P-labeled biotin substrate is then recovered from the reaction mix and captured by a streptavidin-linked matrix (SAMTM Membrane). In brief, cultured neurons were treated with NT-3 as described, washed with HBSS, and then lysed in 0.3 ml cold extraction buffer (25 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 10 mM mercaptoethanol, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 0.5 mM PMSF). Extracts were centrifuged for 5 min at 4°C at 14,000 g and 5 μl of the supernatant was incubated with 20 μl of the reaction mixture containing biotinylated peptide substrate and γ-[32P]ATP for 5 min at 30°C. The reaction was terminated and 10 μl was spotted onto a biotin capture membrane. After washing and drying of the membrane, radioactivity was measured using a liquid scintillation counter. This assay detects 0.012 casein units (2.5 Kemptide units) or less of purified cAMP-dependent PKA. A linear regression analysis, using calibrated amounts of casein units, produced an R2 value >0.95. Triplicate samples were used for each time point and the PKA activities and standard deviation is shown in Fig. 8 (expressed relative to starved cells not exposed to NT-3).

Bottom Line: NT-3 treatment resulted in a rapid and transient stimulation of PKA activity that preceded the localization of beta-actin mRNA.Depolymerization of microtubules, but not microfilaments, inhibited the NT-3-induced localization of beta-actin mRNA.These results suggest that NT-3 activates a cAMP-dependent signaling mechanism to promote the microtubule-dependent localization of beta-actin mRNA within growth cones.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461, USA.

ABSTRACT
Neurotrophins play an essential role in the regulation of actin-dependent changes in growth cone shape and motility. We have studied whether neurotrophin signaling can promote the localization of beta-actin mRNA and protein within growth cones. The regulated localization of specific mRNAs within neuronal processes and growth cones could provide a mechanism to modulate cytoskeletal composition and growth cone dynamics during neuronal development. We have previously shown that beta-actin mRNA is localized in granules that were distributed throughout processes and growth cones of cultured neurons. In this study, we demonstrate that the localization of beta-actin mRNA and protein to growth cones of forebrain neurons is stimulated by neurotrophin-3 (NT-3). A similar response was observed when neurons were exposed to forskolin or db-cAMP, suggesting an involvement of a cAMP signaling pathway. NT-3 treatment resulted in a rapid and transient stimulation of PKA activity that preceded the localization of beta-actin mRNA. Localization of beta-actin mRNA was blocked by prior treatment of cells with Rp-cAMP, an inhibitor of cAMP-dependent protein kinase A. Depolymerization of microtubules, but not microfilaments, inhibited the NT-3-induced localization of beta-actin mRNA. These results suggest that NT-3 activates a cAMP-dependent signaling mechanism to promote the microtubule-dependent localization of beta-actin mRNA within growth cones.

Show MeSH
Related in: MedlinePlus