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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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Protein–protein interaction by HuD and Akt1. (A) Specific coimmunoprecipitation of Akt1 with HuD. HeLa cells were transfected with T7-HuD or T7-GFP and FLAG-Akt1-coding plasmids. HuD was immunoprecipitated with anti-T7 antibody (right panel). Akt1 was immunoprecipitated with anti-FLAG antibody (left panel). Coimmunoprecipitation was monitored by IB. GFP is a negative control. (B) Specific coimmunoprecipitation of endogenous Akt1 with HuD. PC12 cells were transfected with T7-HuD- or T7-GFP-coding plasmids. HuD was immunoprecipitated with anti-T7 antibody. Coimmunoprecipitation of endogenous Akt1 was monitored by IB using anti-Akt1 antibodies.
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gkr979-F1: Protein–protein interaction by HuD and Akt1. (A) Specific coimmunoprecipitation of Akt1 with HuD. HeLa cells were transfected with T7-HuD or T7-GFP and FLAG-Akt1-coding plasmids. HuD was immunoprecipitated with anti-T7 antibody (right panel). Akt1 was immunoprecipitated with anti-FLAG antibody (left panel). Coimmunoprecipitation was monitored by IB. GFP is a negative control. (B) Specific coimmunoprecipitation of endogenous Akt1 with HuD. PC12 cells were transfected with T7-HuD- or T7-GFP-coding plasmids. HuD was immunoprecipitated with anti-T7 antibody. Coimmunoprecipitation of endogenous Akt1 was monitored by IB using anti-Akt1 antibodies.

Mentions: Activation of the mTOR pathway by PI3K-Akt signaling is key for stimulating translation. Akt signaling promotes growth and proliferation including NGF-mediated neurite outgrowth induction (6). We hypothesized that (i) Akt1 function might be critical for the neurite-inducing activity of HuD and (ii) Akt1 might directly interact with HuD to fulfill this function. To examine this possibility, we performed first immunoprecipitation assays using HeLa cell lysates expressing FLAG-tagged Akt1 and T7-tagged HuD or GFP (Figure 1). Indeed, HuD, but not the negative control GFP, copurifies with FLAG-Akt1 (Figure 1A, left panel) and vice versa Akt1 copurifies with T7-HuD, but not with the control T7-GFP (Figure 1A, right panel).Figure 1.


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)

Protein–protein interaction by HuD and Akt1. (A) Specific coimmunoprecipitation of Akt1 with HuD. HeLa cells were transfected with T7-HuD or T7-GFP and FLAG-Akt1-coding plasmids. HuD was immunoprecipitated with anti-T7 antibody (right panel). Akt1 was immunoprecipitated with anti-FLAG antibody (left panel). Coimmunoprecipitation was monitored by IB. GFP is a negative control. (B) Specific coimmunoprecipitation of endogenous Akt1 with HuD. PC12 cells were transfected with T7-HuD- or T7-GFP-coding plasmids. HuD was immunoprecipitated with anti-T7 antibody. Coimmunoprecipitation of endogenous Akt1 was monitored by IB using anti-Akt1 antibodies.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3300026&req=5

gkr979-F1: Protein–protein interaction by HuD and Akt1. (A) Specific coimmunoprecipitation of Akt1 with HuD. HeLa cells were transfected with T7-HuD or T7-GFP and FLAG-Akt1-coding plasmids. HuD was immunoprecipitated with anti-T7 antibody (right panel). Akt1 was immunoprecipitated with anti-FLAG antibody (left panel). Coimmunoprecipitation was monitored by IB. GFP is a negative control. (B) Specific coimmunoprecipitation of endogenous Akt1 with HuD. PC12 cells were transfected with T7-HuD- or T7-GFP-coding plasmids. HuD was immunoprecipitated with anti-T7 antibody. Coimmunoprecipitation of endogenous Akt1 was monitored by IB using anti-Akt1 antibodies.
Mentions: Activation of the mTOR pathway by PI3K-Akt signaling is key for stimulating translation. Akt signaling promotes growth and proliferation including NGF-mediated neurite outgrowth induction (6). We hypothesized that (i) Akt1 function might be critical for the neurite-inducing activity of HuD and (ii) Akt1 might directly interact with HuD to fulfill this function. To examine this possibility, we performed first immunoprecipitation assays using HeLa cell lysates expressing FLAG-tagged Akt1 and T7-tagged HuD or GFP (Figure 1). Indeed, HuD, but not the negative control GFP, copurifies with FLAG-Akt1 (Figure 1A, left panel) and vice versa Akt1 copurifies with T7-HuD, but not with the control T7-GFP (Figure 1A, right panel).Figure 1.

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