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PICK1 links AMPA receptor stimulation to Cdc42.

Rocca DL, Hanley JG - Neurosci. Lett. (2014)

Bottom Line: The Rho-family member Cdc42 regulates dendritic spine morphology via its effector N-WASP, which activates the actin-nucleating Arp2/3 complex.Furthermore, AMPAR stimulation deactivates Cdc42 and alters its detergent solubility in neurons via a PICK1-dependent process.This work suggests a novel role for PICK1 in transducing AMPAR stimulation to Cdc42 function in neurons.

View Article: PubMed Central - PubMed

Affiliation: School of Biochemistry, Medical Sciences Building,University of Bristol, University Walk, Bristol BS8 1TD, UK.

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PICK1 interacts with Cdc42 and Rac1 but not RhoA.(A) PICK1 binds Rac1 and Cdc42. GST-pulldowns were carried out from lysates prepared from COS7 cells expressing myc-tagged Rac1(V12), Rac1(N17), Cdc42(V12) or Cdc42(N17) using GST–PICK1, GST–PAK–CRIB or GST alone. Bound proteins were detected by western blotting using anti-myc.(B) PICK1 does not bind RhoA. GST-pulldowns were carried out from lysates prepared from COS7 cells expressing myc-tagged RhoA(V14) or RhoA(N17) using GST–PICK1, GST-Rhotekin or GST alone. Bound proteins were detected by western blotting using anti-myc.(C) Cdc42 and Rac1 interact with PICK1 in neurons. Lysates prepared from cultured cortical neurons were immunoprecipitated with anti-PICK1 antibody or non-immune IgG as a control, and bound proteins were detected by western blotting using specific antibodies as shown.
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fig0005: PICK1 interacts with Cdc42 and Rac1 but not RhoA.(A) PICK1 binds Rac1 and Cdc42. GST-pulldowns were carried out from lysates prepared from COS7 cells expressing myc-tagged Rac1(V12), Rac1(N17), Cdc42(V12) or Cdc42(N17) using GST–PICK1, GST–PAK–CRIB or GST alone. Bound proteins were detected by western blotting using anti-myc.(B) PICK1 does not bind RhoA. GST-pulldowns were carried out from lysates prepared from COS7 cells expressing myc-tagged RhoA(V14) or RhoA(N17) using GST–PICK1, GST-Rhotekin or GST alone. Bound proteins were detected by western blotting using anti-myc.(C) Cdc42 and Rac1 interact with PICK1 in neurons. Lysates prepared from cultured cortical neurons were immunoprecipitated with anti-PICK1 antibody or non-immune IgG as a control, and bound proteins were detected by western blotting using specific antibodies as shown.

Mentions: To investigate the interaction of PICK1 with Rho-family GTPases, we carried out pulldown assays using GST–PICK1 and lysates prepared from COS cells expressing epitope-tagged Cdc42, Rac1 or RhoA. Since an important functional feature of Rho-family GTPases is that they bind downstream effector proteins preferentially in their active, GTP-bound state [4], we tested constitutively active (CA, V12) and dominant negative (DN, N17) mutant GTPases. p21 activated kinase (PAK) is a known effector for Cdc42 and Rac and binds CA but not DN mutants of both GTPases ([35] and Fig. 1A). GST–PICK1 binds CA and DN mutants equally well for both Rac1 and Cdc42 (Fig. 1A), suggesting that PICK1 is not a Rac1/Cdc42 effector, but perhaps plays a scaffolding role to localise the GTPases to specific subcellular locations. We carried out equivalent experiments for RhoA, using the known effector protein Rhotekin as a positive control [22]. GST–PICK1 does not interact with either RhoA mutant, demonstrating specificity for the interaction with Rac1 and Cdc42 (Fig. 1B). To confirm that Cdc42 and Rac1 interact with PICK1 in neurons, we carried out co-immunoprecipitations (co-IPs) from lysates prepared from cultured cortical neurons using anti-PICK1 antibodies. Both Rac1 and Cdc42 show a robust interaction with PICK1 (Fig. 1C), demonstrating that both GTPases interact with PICK1 in neurons.


PICK1 links AMPA receptor stimulation to Cdc42.

Rocca DL, Hanley JG - Neurosci. Lett. (2014)

PICK1 interacts with Cdc42 and Rac1 but not RhoA.(A) PICK1 binds Rac1 and Cdc42. GST-pulldowns were carried out from lysates prepared from COS7 cells expressing myc-tagged Rac1(V12), Rac1(N17), Cdc42(V12) or Cdc42(N17) using GST–PICK1, GST–PAK–CRIB or GST alone. Bound proteins were detected by western blotting using anti-myc.(B) PICK1 does not bind RhoA. GST-pulldowns were carried out from lysates prepared from COS7 cells expressing myc-tagged RhoA(V14) or RhoA(N17) using GST–PICK1, GST-Rhotekin or GST alone. Bound proteins were detected by western blotting using anti-myc.(C) Cdc42 and Rac1 interact with PICK1 in neurons. Lysates prepared from cultured cortical neurons were immunoprecipitated with anti-PICK1 antibody or non-immune IgG as a control, and bound proteins were detected by western blotting using specific antibodies as shown.
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fig0005: PICK1 interacts with Cdc42 and Rac1 but not RhoA.(A) PICK1 binds Rac1 and Cdc42. GST-pulldowns were carried out from lysates prepared from COS7 cells expressing myc-tagged Rac1(V12), Rac1(N17), Cdc42(V12) or Cdc42(N17) using GST–PICK1, GST–PAK–CRIB or GST alone. Bound proteins were detected by western blotting using anti-myc.(B) PICK1 does not bind RhoA. GST-pulldowns were carried out from lysates prepared from COS7 cells expressing myc-tagged RhoA(V14) or RhoA(N17) using GST–PICK1, GST-Rhotekin or GST alone. Bound proteins were detected by western blotting using anti-myc.(C) Cdc42 and Rac1 interact with PICK1 in neurons. Lysates prepared from cultured cortical neurons were immunoprecipitated with anti-PICK1 antibody or non-immune IgG as a control, and bound proteins were detected by western blotting using specific antibodies as shown.
Mentions: To investigate the interaction of PICK1 with Rho-family GTPases, we carried out pulldown assays using GST–PICK1 and lysates prepared from COS cells expressing epitope-tagged Cdc42, Rac1 or RhoA. Since an important functional feature of Rho-family GTPases is that they bind downstream effector proteins preferentially in their active, GTP-bound state [4], we tested constitutively active (CA, V12) and dominant negative (DN, N17) mutant GTPases. p21 activated kinase (PAK) is a known effector for Cdc42 and Rac and binds CA but not DN mutants of both GTPases ([35] and Fig. 1A). GST–PICK1 binds CA and DN mutants equally well for both Rac1 and Cdc42 (Fig. 1A), suggesting that PICK1 is not a Rac1/Cdc42 effector, but perhaps plays a scaffolding role to localise the GTPases to specific subcellular locations. We carried out equivalent experiments for RhoA, using the known effector protein Rhotekin as a positive control [22]. GST–PICK1 does not interact with either RhoA mutant, demonstrating specificity for the interaction with Rac1 and Cdc42 (Fig. 1B). To confirm that Cdc42 and Rac1 interact with PICK1 in neurons, we carried out co-immunoprecipitations (co-IPs) from lysates prepared from cultured cortical neurons using anti-PICK1 antibodies. Both Rac1 and Cdc42 show a robust interaction with PICK1 (Fig. 1C), demonstrating that both GTPases interact with PICK1 in neurons.

Bottom Line: The Rho-family member Cdc42 regulates dendritic spine morphology via its effector N-WASP, which activates the actin-nucleating Arp2/3 complex.Furthermore, AMPAR stimulation deactivates Cdc42 and alters its detergent solubility in neurons via a PICK1-dependent process.This work suggests a novel role for PICK1 in transducing AMPAR stimulation to Cdc42 function in neurons.

View Article: PubMed Central - PubMed

Affiliation: School of Biochemistry, Medical Sciences Building,University of Bristol, University Walk, Bristol BS8 1TD, UK.

Show MeSH
Related in: MedlinePlus