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Functional and direct interaction between the RNA binding protein HuD and active Akt1.

Fujiwara T, Fukao A, Sasano Y, Matsuzaki H, Kikkawa U, Imataka H, Inoue K, Endo S, Sonenberg N, Thoma C, Sakamoto H - Nucleic Acids Res. (2011)

Bottom Line: Here, we further explored the underlying molecular interactions and found that HuD but not the ubiquitously expressed HuR interacts directly with active Akt1.These results suggest the model whereby RNA-bound HuD functions as an adapter to recruit Akt1 to trigger neurite outgrowth.These data might also help to explain how HuD enhances translation of mRNAs that encode proteins involved in neuronal development.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Disease Biology, Institute of Microbial Chemistry, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan. tosinobu@bikaken.or.jp

ABSTRACT
The RNA binding protein HuD plays essential roles in neuronal development and plasticity. We have previously shown that HuD stimulates translation. Key for this enhancer function is the linker region and the poly(A) binding domain of HuD that are also critical for its function in neurite outgrowth. Here, we further explored the underlying molecular interactions and found that HuD but not the ubiquitously expressed HuR interacts directly with active Akt1. We identify that the linker region of HuD is required for this interaction. We also show by using chimeric mutants of HuD and HuR, which contain the reciprocal linker between RNA-binding domain 2 (RBD2) and RBD3, respectively, and by overexpressing a dominant negative mutant of Akt1 that the HuD-Akt1 interaction is functionally important, as it is required for the induction of neurite outgrowth in PC12 cells. These results suggest the model whereby RNA-bound HuD functions as an adapter to recruit Akt1 to trigger neurite outgrowth. These data might also help to explain how HuD enhances translation of mRNAs that encode proteins involved in neuronal development.

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Role of the HuD–Akt1 interaction in neurite outgrowth. (A) Role of the linker region of HuD to induce neurite outgrowth. Shown is the confocal analysis of PC12 cells that have been transfected with constructs coding for the indicated proteins. Cells were costained with anti-FLAG (green) and with anti-α-tubulin antibody (red). Arrow heads point to induced neurites. Scale bar, 20 µm. The same results were obtained in at least three independent experiments. (B) Role of Akt1 in neuronal differentiation in PC12 cells induced by HuD and HuR-DL. Confocal analysis of PC12 cells that were transfected with constructs coding for the indicated proteins. Arrow heads point to induced neurites. Scale bar, 20 µm. The same results were obtained in at least three independent experiments.
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gkr979-F4: Role of the HuD–Akt1 interaction in neurite outgrowth. (A) Role of the linker region of HuD to induce neurite outgrowth. Shown is the confocal analysis of PC12 cells that have been transfected with constructs coding for the indicated proteins. Cells were costained with anti-FLAG (green) and with anti-α-tubulin antibody (red). Arrow heads point to induced neurites. Scale bar, 20 µm. The same results were obtained in at least three independent experiments. (B) Role of Akt1 in neuronal differentiation in PC12 cells induced by HuD and HuR-DL. Confocal analysis of PC12 cells that were transfected with constructs coding for the indicated proteins. Arrow heads point to induced neurites. Scale bar, 20 µm. The same results were obtained in at least three independent experiments.

Mentions: We next wished to test whether the HuD–Akt1 interaction is functionally important. To address this question, we used an established cell-based model system for studying neuronal differentiation, which is based on PC12 cells (13,14). If the HuD–Akt1 interaction is contributing significantly to the function of HuD, then our data predict that the chimeric HuR mutant should gain and the chimeric HuD mutant should loose its ability to induce neurite outgrowth. To test this prediction, we assayed the ability of the different HuD and HuR mutants described above to induce outgrowth in PC12 cells. As shown in Figure 4A and in concert with the data described above, wild-type HuD and the chimeric mutant HuR but not wild-type HuR or the chimeric HuD mutant transfected into PC12 cells can induce neurite outgrowth. These results suggest that binding of HuD to Akt1 is critical for the neurite-inducing activity of HuD. We note that wild-type HuD and the chimeric mutant HuR-DL that promotes neurite outgrowth are cytoplasmic, while those proteins that are not able to do so (wild-type HuR and the chimeric HuD mutant) are nuclear. To confirm that the Akt1 interaction with HuD and not just the cytoplasmic location is the relevant component, we have further mapped linker region of HuD to identify the critical interaction domain. We find that the HuD mutant 277-385 containing a truncated linker region (277-302) and RBD3 is still capable of (i) interacting with Akt1 (Figure 5B) and (ii) inducing neurite outgrowth in PC12 cells (Figure 5C). Strikingly, we find that the truncated HuR mutant 219-327 containing an almost identical linker sequence (see scheme in Figure 5A for comparison) and RBD3 cannot interact with Akt1 (Figure 5B) and cannot induce outgrowth although it localizes to the cytoplasm (Figure 5C). Thus, we are confident to conclude that the interaction of HuD with Akt1 is the relevant component and not just the cytoplasmic localization.Figure 4.


Functional and direct interaction between the RNA binding protein HuD and active Akt1.

Fujiwara T, Fukao A, Sasano Y, Matsuzaki H, Kikkawa U, Imataka H, Inoue K, Endo S, Sonenberg N, Thoma C, Sakamoto H - Nucleic Acids Res. (2011)

Role of the HuD–Akt1 interaction in neurite outgrowth. (A) Role of the linker region of HuD to induce neurite outgrowth. Shown is the confocal analysis of PC12 cells that have been transfected with constructs coding for the indicated proteins. Cells were costained with anti-FLAG (green) and with anti-α-tubulin antibody (red). Arrow heads point to induced neurites. Scale bar, 20 µm. The same results were obtained in at least three independent experiments. (B) Role of Akt1 in neuronal differentiation in PC12 cells induced by HuD and HuR-DL. Confocal analysis of PC12 cells that were transfected with constructs coding for the indicated proteins. Arrow heads point to induced neurites. Scale bar, 20 µm. The same results were obtained in at least three independent experiments.
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Related In: Results  -  Collection

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gkr979-F4: Role of the HuD–Akt1 interaction in neurite outgrowth. (A) Role of the linker region of HuD to induce neurite outgrowth. Shown is the confocal analysis of PC12 cells that have been transfected with constructs coding for the indicated proteins. Cells were costained with anti-FLAG (green) and with anti-α-tubulin antibody (red). Arrow heads point to induced neurites. Scale bar, 20 µm. The same results were obtained in at least three independent experiments. (B) Role of Akt1 in neuronal differentiation in PC12 cells induced by HuD and HuR-DL. Confocal analysis of PC12 cells that were transfected with constructs coding for the indicated proteins. Arrow heads point to induced neurites. Scale bar, 20 µm. The same results were obtained in at least three independent experiments.
Mentions: We next wished to test whether the HuD–Akt1 interaction is functionally important. To address this question, we used an established cell-based model system for studying neuronal differentiation, which is based on PC12 cells (13,14). If the HuD–Akt1 interaction is contributing significantly to the function of HuD, then our data predict that the chimeric HuR mutant should gain and the chimeric HuD mutant should loose its ability to induce neurite outgrowth. To test this prediction, we assayed the ability of the different HuD and HuR mutants described above to induce outgrowth in PC12 cells. As shown in Figure 4A and in concert with the data described above, wild-type HuD and the chimeric mutant HuR but not wild-type HuR or the chimeric HuD mutant transfected into PC12 cells can induce neurite outgrowth. These results suggest that binding of HuD to Akt1 is critical for the neurite-inducing activity of HuD. We note that wild-type HuD and the chimeric mutant HuR-DL that promotes neurite outgrowth are cytoplasmic, while those proteins that are not able to do so (wild-type HuR and the chimeric HuD mutant) are nuclear. To confirm that the Akt1 interaction with HuD and not just the cytoplasmic location is the relevant component, we have further mapped linker region of HuD to identify the critical interaction domain. We find that the HuD mutant 277-385 containing a truncated linker region (277-302) and RBD3 is still capable of (i) interacting with Akt1 (Figure 5B) and (ii) inducing neurite outgrowth in PC12 cells (Figure 5C). Strikingly, we find that the truncated HuR mutant 219-327 containing an almost identical linker sequence (see scheme in Figure 5A for comparison) and RBD3 cannot interact with Akt1 (Figure 5B) and cannot induce outgrowth although it localizes to the cytoplasm (Figure 5C). Thus, we are confident to conclude that the interaction of HuD with Akt1 is the relevant component and not just the cytoplasmic localization.Figure 4.

Bottom Line: Here, we further explored the underlying molecular interactions and found that HuD but not the ubiquitously expressed HuR interacts directly with active Akt1.These results suggest the model whereby RNA-bound HuD functions as an adapter to recruit Akt1 to trigger neurite outgrowth.These data might also help to explain how HuD enhances translation of mRNAs that encode proteins involved in neuronal development.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Disease Biology, Institute of Microbial Chemistry, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan. tosinobu@bikaken.or.jp

ABSTRACT
The RNA binding protein HuD plays essential roles in neuronal development and plasticity. We have previously shown that HuD stimulates translation. Key for this enhancer function is the linker region and the poly(A) binding domain of HuD that are also critical for its function in neurite outgrowth. Here, we further explored the underlying molecular interactions and found that HuD but not the ubiquitously expressed HuR interacts directly with active Akt1. We identify that the linker region of HuD is required for this interaction. We also show by using chimeric mutants of HuD and HuR, which contain the reciprocal linker between RNA-binding domain 2 (RBD2) and RBD3, respectively, and by overexpressing a dominant negative mutant of Akt1 that the HuD-Akt1 interaction is functionally important, as it is required for the induction of neurite outgrowth in PC12 cells. These results suggest the model whereby RNA-bound HuD functions as an adapter to recruit Akt1 to trigger neurite outgrowth. These data might also help to explain how HuD enhances translation of mRNAs that encode proteins involved in neuronal development.

Show MeSH