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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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Shank3 interacts with Grb2. (a) Schematic representation of the used Ret9 and Shank3 mutants. The consensus Grb2-binding motif in Shank3 is underlined. (b) Interaction of Shank3 with endogenous Grb2. HEK293 cells were cotransfected with Flag-tagged Shank3–Pro2 or Shank3–Pro2 containing the Y1006F mutant and Ret9, Ret9FA, or Ret9Y1062F. Immunoprecipitation of Shank3 using Flag–M2 Sepharose beads was performed followed by SDS-PAGE and Western blotting. Western blots of immunoprecipitations and total lysates were developed with antibodies against Grb2, Flag, phospho-tyrosine (PY), and Ret9 as indicated.
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fig7: Shank3 interacts with Grb2. (a) Schematic representation of the used Ret9 and Shank3 mutants. The consensus Grb2-binding motif in Shank3 is underlined. (b) Interaction of Shank3 with endogenous Grb2. HEK293 cells were cotransfected with Flag-tagged Shank3–Pro2 or Shank3–Pro2 containing the Y1006F mutant and Ret9, Ret9FA, or Ret9Y1062F. Immunoprecipitation of Shank3 using Flag–M2 Sepharose beads was performed followed by SDS-PAGE and Western blotting. Western blots of immunoprecipitations and total lysates were developed with antibodies against Grb2, Flag, phospho-tyrosine (PY), and Ret9 as indicated.

Mentions: Several adaptor molecules such as Shc, Grb2, FRS2, and dok4/5 are known to be involved in stimulation of the Erk–MAPK pathway downstream of Ret and are recruited to the plasma membrane (Borrello et al., 1996; Arighi et al., 1997; Alberti et al., 1998; Grimm et al., 2001; Kurokawa et al., 2001). Analysis of the Shank3–Pro2 sequence revealed a potential binding site for the Grb2 SH2 domain, with the consensus sequence pYXNX. Indeed, endogenous Grb2 was recruited to this binding site, as shown by coimmunoprecipitation of Shank3–Pro2 upon coexpression of Ret9 (Fig. 7 b). Coexpression of the PDZ-binding motif mutant Ret9 FA did not allow binding of Grb2 to Shank3–Pro2. Moreover, coexpression of Ret9 leads to significantly higher levels of Shank3 phosphorylation than Ret9 FA. We determined that Grb2 is not complexed indirectly with Ret9 through Shc, because Shank3–Pro2 still binds Grb2 when the Shc-binding–deficient Ret9 Y1062F mutant is coexpressed (Fig. 7, a and b). Finally, we mutated tyrosine 1006 to phenylalanine in the Grb2-binding site of Shank3 (Shank3–Pro1 Y1006F), which does not interact with Grb2 (Fig. 7, a and b).


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)

Shank3 interacts with Grb2. (a) Schematic representation of the used Ret9 and Shank3 mutants. The consensus Grb2-binding motif in Shank3 is underlined. (b) Interaction of Shank3 with endogenous Grb2. HEK293 cells were cotransfected with Flag-tagged Shank3–Pro2 or Shank3–Pro2 containing the Y1006F mutant and Ret9, Ret9FA, or Ret9Y1062F. Immunoprecipitation of Shank3 using Flag–M2 Sepharose beads was performed followed by SDS-PAGE and Western blotting. Western blots of immunoprecipitations and total lysates were developed with antibodies against Grb2, Flag, phospho-tyrosine (PY), and Ret9 as indicated.
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Related In: Results  -  Collection

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fig7: Shank3 interacts with Grb2. (a) Schematic representation of the used Ret9 and Shank3 mutants. The consensus Grb2-binding motif in Shank3 is underlined. (b) Interaction of Shank3 with endogenous Grb2. HEK293 cells were cotransfected with Flag-tagged Shank3–Pro2 or Shank3–Pro2 containing the Y1006F mutant and Ret9, Ret9FA, or Ret9Y1062F. Immunoprecipitation of Shank3 using Flag–M2 Sepharose beads was performed followed by SDS-PAGE and Western blotting. Western blots of immunoprecipitations and total lysates were developed with antibodies against Grb2, Flag, phospho-tyrosine (PY), and Ret9 as indicated.
Mentions: Several adaptor molecules such as Shc, Grb2, FRS2, and dok4/5 are known to be involved in stimulation of the Erk–MAPK pathway downstream of Ret and are recruited to the plasma membrane (Borrello et al., 1996; Arighi et al., 1997; Alberti et al., 1998; Grimm et al., 2001; Kurokawa et al., 2001). Analysis of the Shank3–Pro2 sequence revealed a potential binding site for the Grb2 SH2 domain, with the consensus sequence pYXNX. Indeed, endogenous Grb2 was recruited to this binding site, as shown by coimmunoprecipitation of Shank3–Pro2 upon coexpression of Ret9 (Fig. 7 b). Coexpression of the PDZ-binding motif mutant Ret9 FA did not allow binding of Grb2 to Shank3–Pro2. Moreover, coexpression of Ret9 leads to significantly higher levels of Shank3 phosphorylation than Ret9 FA. We determined that Grb2 is not complexed indirectly with Ret9 through Shc, because Shank3–Pro2 still binds Grb2 when the Shc-binding–deficient Ret9 Y1062F mutant is coexpressed (Fig. 7, a and b). Finally, we mutated tyrosine 1006 to phenylalanine in the Grb2-binding site of Shank3 (Shank3–Pro1 Y1006F), which does not interact with Grb2 (Fig. 7, a and b).

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