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The neuronal scaffold protein Shank3 mediates signaling and biological function of the receptor tyrosine kinase Ret in epithelial cells.

Schuetz G, Rosário M, Grimm J, Boeckers TM, Gundelfinger ED, Birchmeier W - J. Cell Biol. (2004)

Bottom Line: The PDZ domain-containing Shank3 protein was found to represent a novel interaction partner of the receptor tyrosine kinase Ret, which binds specifically to a PDZ-binding motif present in the Ret9 but not in the Ret51 isoform.Shank3 protein mediates sustained Erk-MAPK and PI3K signaling, which is crucial for tubule formation, through recruitment of the adaptor protein Grb2.These results demonstrate that the Shank3 adaptor protein can mediate cellular signaling, and provide a molecular mechanism for the biological divergence between the Ret9 and Ret51 isoform.

View Article: PubMed Central - PubMed

Affiliation: MaxDelbrück-Center for Molecular Medicine, Berlin, Germany.

ABSTRACT
Shank proteins, initially also described as ProSAP proteins, are scaffolding adaptors that have been previously shown to integrate neurotransmitter receptors into the cortical cytoskeleton at postsynaptic densities. We show here that Shank proteins are also crucial in receptor tyrosine kinase signaling. The PDZ domain-containing Shank3 protein was found to represent a novel interaction partner of the receptor tyrosine kinase Ret, which binds specifically to a PDZ-binding motif present in the Ret9 but not in the Ret51 isoform. Furthermore, we show that Ret9 but not Ret51 induces epithelial cells to form branched tubular structures in three-dimensional cultures in a Shank3-dependent manner. Ret9 but not Ret51 has been previously shown to be required for kidney development. Shank3 protein mediates sustained Erk-MAPK and PI3K signaling, which is crucial for tubule formation, through recruitment of the adaptor protein Grb2. These results demonstrate that the Shank3 adaptor protein can mediate cellular signaling, and provide a molecular mechanism for the biological divergence between the Ret9 and Ret51 isoform.

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The Ret9 PDZ-binding motif mediates sustained Erk–MAPK and PI3K signaling. MDCK cells, expressing Ret9 (a and e), Ret9 FA (b and f), Ret51 (c and g), or control cells without Ret (d and h) were stimulated with GDNF and sGFRα1 and examined for Erk–MAPK and Akt phosphorylation by SDS-PAGE. Phosphorylated Erk1/2 (P-Erk1/2) or Akt (P-Akt) proteins, total Erk2 and total Akt were detected by Western blotting with specific antibodies. The specific bands for phosphorylated and unphosphorylated proteins are indicated by arrows. Western blots were quantified and activation levels over time are shown. Error bars show standard deviation from three independent experiments.
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fig6: The Ret9 PDZ-binding motif mediates sustained Erk–MAPK and PI3K signaling. MDCK cells, expressing Ret9 (a and e), Ret9 FA (b and f), Ret51 (c and g), or control cells without Ret (d and h) were stimulated with GDNF and sGFRα1 and examined for Erk–MAPK and Akt phosphorylation by SDS-PAGE. Phosphorylated Erk1/2 (P-Erk1/2) or Akt (P-Akt) proteins, total Erk2 and total Akt were detected by Western blotting with specific antibodies. The specific bands for phosphorylated and unphosphorylated proteins are indicated by arrows. Western blots were quantified and activation levels over time are shown. Error bars show standard deviation from three independent experiments.

Mentions: Tubulogenesis by HGF/SF and Met has been shown to require sustained activation of the Erk–MAPK and PI3K pathways (Khwaja et al., 1998; Maroun et al., 2000; Schaeper et al., 2000). Previous investigations have shown that these pathways are also activated by Ret9 and Ret51 (van Puijenbroek et al., 1997; Besset et al., 2000; Hayashi et al., 2000; Melillo et al., 2001; Pelicci et al., 2002). Here we have addressed the question whether activation of the Erk–MAPK and PI3K pathways is modulated by the Ret9 PDZ-binding motif and Shank3 interaction, using the MDCK cell lines described above. Activation of Erk1/2, as measured by phosphorylation, is induced 10 min after GDNF–sGFRα1 stimulation of Ret9-expressing cells, and remains sustained for at least 8 h (Fig. 6 a). Maximal phosphorylation of PKB–Akt, as indicator of activation of the PI3K pathway, in Ret9-expressing cells occurs at 30 min after stimulation, and also remains sustained for at least 8 h (Fig. 6e). However, mutation of the Ret9 PDZ-binding motif (Ret9 FA) prevents sustained but not transient activation of Erk1/2 and activation of PKB–Akt (Fig. 6, b and f). Stimulation of Ret51-expressing cells also fail to induce sustained activation of Erk–MAPK and cannot activate PKB–Akt (Fig. 6, c and g). MDCK cells expressing the proline-rich domain of Shank3 fused to Ret (Ret–Shank3–Pro1), but not other Ret–Shank3 fusion proteins, exhibit sustained Erk–MAPK and PI3K activation similar to Ret9 (unpublished results).


The neuronal scaffold protein Shank3 mediates signaling and biological function of the receptor tyrosine kinase Ret in epithelial cells.

Schuetz G, Rosário M, Grimm J, Boeckers TM, Gundelfinger ED, Birchmeier W - J. Cell Biol. (2004)

The Ret9 PDZ-binding motif mediates sustained Erk–MAPK and PI3K signaling. MDCK cells, expressing Ret9 (a and e), Ret9 FA (b and f), Ret51 (c and g), or control cells without Ret (d and h) were stimulated with GDNF and sGFRα1 and examined for Erk–MAPK and Akt phosphorylation by SDS-PAGE. Phosphorylated Erk1/2 (P-Erk1/2) or Akt (P-Akt) proteins, total Erk2 and total Akt were detected by Western blotting with specific antibodies. The specific bands for phosphorylated and unphosphorylated proteins are indicated by arrows. Western blots were quantified and activation levels over time are shown. Error bars show standard deviation from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172453&req=5

fig6: The Ret9 PDZ-binding motif mediates sustained Erk–MAPK and PI3K signaling. MDCK cells, expressing Ret9 (a and e), Ret9 FA (b and f), Ret51 (c and g), or control cells without Ret (d and h) were stimulated with GDNF and sGFRα1 and examined for Erk–MAPK and Akt phosphorylation by SDS-PAGE. Phosphorylated Erk1/2 (P-Erk1/2) or Akt (P-Akt) proteins, total Erk2 and total Akt were detected by Western blotting with specific antibodies. The specific bands for phosphorylated and unphosphorylated proteins are indicated by arrows. Western blots were quantified and activation levels over time are shown. Error bars show standard deviation from three independent experiments.
Mentions: Tubulogenesis by HGF/SF and Met has been shown to require sustained activation of the Erk–MAPK and PI3K pathways (Khwaja et al., 1998; Maroun et al., 2000; Schaeper et al., 2000). Previous investigations have shown that these pathways are also activated by Ret9 and Ret51 (van Puijenbroek et al., 1997; Besset et al., 2000; Hayashi et al., 2000; Melillo et al., 2001; Pelicci et al., 2002). Here we have addressed the question whether activation of the Erk–MAPK and PI3K pathways is modulated by the Ret9 PDZ-binding motif and Shank3 interaction, using the MDCK cell lines described above. Activation of Erk1/2, as measured by phosphorylation, is induced 10 min after GDNF–sGFRα1 stimulation of Ret9-expressing cells, and remains sustained for at least 8 h (Fig. 6 a). Maximal phosphorylation of PKB–Akt, as indicator of activation of the PI3K pathway, in Ret9-expressing cells occurs at 30 min after stimulation, and also remains sustained for at least 8 h (Fig. 6e). However, mutation of the Ret9 PDZ-binding motif (Ret9 FA) prevents sustained but not transient activation of Erk1/2 and activation of PKB–Akt (Fig. 6, b and f). Stimulation of Ret51-expressing cells also fail to induce sustained activation of Erk–MAPK and cannot activate PKB–Akt (Fig. 6, c and g). MDCK cells expressing the proline-rich domain of Shank3 fused to Ret (Ret–Shank3–Pro1), but not other Ret–Shank3 fusion proteins, exhibit sustained Erk–MAPK and PI3K activation similar to Ret9 (unpublished results).

Bottom Line: The PDZ domain-containing Shank3 protein was found to represent a novel interaction partner of the receptor tyrosine kinase Ret, which binds specifically to a PDZ-binding motif present in the Ret9 but not in the Ret51 isoform.Shank3 protein mediates sustained Erk-MAPK and PI3K signaling, which is crucial for tubule formation, through recruitment of the adaptor protein Grb2.These results demonstrate that the Shank3 adaptor protein can mediate cellular signaling, and provide a molecular mechanism for the biological divergence between the Ret9 and Ret51 isoform.

View Article: PubMed Central - PubMed

Affiliation: MaxDelbrück-Center for Molecular Medicine, Berlin, Germany.

ABSTRACT
Shank proteins, initially also described as ProSAP proteins, are scaffolding adaptors that have been previously shown to integrate neurotransmitter receptors into the cortical cytoskeleton at postsynaptic densities. We show here that Shank proteins are also crucial in receptor tyrosine kinase signaling. The PDZ domain-containing Shank3 protein was found to represent a novel interaction partner of the receptor tyrosine kinase Ret, which binds specifically to a PDZ-binding motif present in the Ret9 but not in the Ret51 isoform. Furthermore, we show that Ret9 but not Ret51 induces epithelial cells to form branched tubular structures in three-dimensional cultures in a Shank3-dependent manner. Ret9 but not Ret51 has been previously shown to be required for kidney development. Shank3 protein mediates sustained Erk-MAPK and PI3K signaling, which is crucial for tubule formation, through recruitment of the adaptor protein Grb2. These results demonstrate that the Shank3 adaptor protein can mediate cellular signaling, and provide a molecular mechanism for the biological divergence between the Ret9 and Ret51 isoform.

Show MeSH
Related in: MedlinePlus