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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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Activation of cAMP-dependent PKA in neurons treated with NT-3. Cells were cultured for 4 d and then incubated in MEM for 3 h. NT-3 (25 ng/ml) was added to the medium for the indicated time. Cells were extracted and the PKA activities were estimated by 32P-ATP incorporation assay (Signa TECT cAMP-dependent protein kinase assay system; Promega).
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Figure 9: Activation of cAMP-dependent PKA in neurons treated with NT-3. Cells were cultured for 4 d and then incubated in MEM for 3 h. NT-3 (25 ng/ml) was added to the medium for the indicated time. Cells were extracted and the PKA activities were estimated by 32P-ATP incorporation assay (Signa TECT cAMP-dependent protein kinase assay system; Promega).

Mentions: To determine whether NT-3 signaling can specifically activate a cAMP signaling pathway, we measured protein kinase A (PKA) activity in cell lysates after cultures were treated with NT-3 at various time points. This assay used a biotinylated Kemptide substrate that is highly specific for cAMP-dependent PKA (see Materials and Methods). PKA activity was observed to peak at 2 min (345% increase over control levels) and then declined to baseline levels (Fig. 9). We conclude from this assay that there is a rapid (within 2 min) increase in PKA activity that precedes our observations of β-actin mRNA localization.


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

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

Activation of cAMP-dependent PKA in neurons treated with NT-3. Cells were cultured for 4 d and then incubated in MEM for 3 h. NT-3 (25 ng/ml) was added to the medium for the indicated time. Cells were extracted and the PKA activities were estimated by 32P-ATP incorporation assay (Signa TECT cAMP-dependent protein kinase assay system; Promega).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2164987&req=5

Figure 9: Activation of cAMP-dependent PKA in neurons treated with NT-3. Cells were cultured for 4 d and then incubated in MEM for 3 h. NT-3 (25 ng/ml) was added to the medium for the indicated time. Cells were extracted and the PKA activities were estimated by 32P-ATP incorporation assay (Signa TECT cAMP-dependent protein kinase assay system; Promega).
Mentions: To determine whether NT-3 signaling can specifically activate a cAMP signaling pathway, we measured protein kinase A (PKA) activity in cell lysates after cultures were treated with NT-3 at various time points. This assay used a biotinylated Kemptide substrate that is highly specific for cAMP-dependent PKA (see Materials and Methods). PKA activity was observed to peak at 2 min (345% increase over control levels) and then declined to baseline levels (Fig. 9). We conclude from this assay that there is a rapid (within 2 min) increase in PKA activity that precedes our observations of β-actin mRNA localization.

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