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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.

Show MeSH
HuD is not a substrate of Akt1. In vitro kinase reaction using recombinant GST-HuD or MBP-FOXO4 and active Akt1 (see ‘Material and Methods’ section for further details). Phosphorylation levels are shown in the left panel, CBB stain is shown in the right panel. The positions of MBP-FOXO4, Akt1 and GST-HuD are indicated by arrows.
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gkr979-F7: HuD is not a substrate of Akt1. In vitro kinase reaction using recombinant GST-HuD or MBP-FOXO4 and active Akt1 (see ‘Material and Methods’ section for further details). Phosphorylation levels are shown in the left panel, CBB stain is shown in the right panel. The positions of MBP-FOXO4, Akt1 and GST-HuD are indicated by arrows.

Mentions: We finally asked whether HuD is itself substrate for Akt1 phosphorylation or whether HuD recruits the kinase to a different substrate. To address this question, we have performed an in vitro kinase reaction using GST-HuD and active Akt1 (see ‘Materials and Methods’ section for further details). The data shown in Figure 7 reveal that HuD is not phosphorylated by Akt1, whereas the positive control FOXO4 is. On the basis of these results, we conclude that HuD itself is not the target of Akt1. Taken together, with the results described above, our data suggest the model whereby RNA-bound HuD recruits Akt1 to activate neurite outgrowth.Figure 7.


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)

HuD is not a substrate of Akt1. In vitro kinase reaction using recombinant GST-HuD or MBP-FOXO4 and active Akt1 (see ‘Material and Methods’ section for further details). Phosphorylation levels are shown in the left panel, CBB stain is shown in the right panel. The positions of MBP-FOXO4, Akt1 and GST-HuD are indicated by arrows.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr979-F7: HuD is not a substrate of Akt1. In vitro kinase reaction using recombinant GST-HuD or MBP-FOXO4 and active Akt1 (see ‘Material and Methods’ section for further details). Phosphorylation levels are shown in the left panel, CBB stain is shown in the right panel. The positions of MBP-FOXO4, Akt1 and GST-HuD are indicated by arrows.
Mentions: We finally asked whether HuD is itself substrate for Akt1 phosphorylation or whether HuD recruits the kinase to a different substrate. To address this question, we have performed an in vitro kinase reaction using GST-HuD and active Akt1 (see ‘Materials and Methods’ section for further details). The data shown in Figure 7 reveal that HuD is not phosphorylated by Akt1, whereas the positive control FOXO4 is. On the basis of these results, we conclude that HuD itself is not the target of Akt1. Taken together, with the results described above, our data suggest the model whereby RNA-bound HuD recruits Akt1 to activate neurite outgrowth.Figure 7.

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