Bimolecular complementation affinity purification (BiCAP) reveals dimer-specific protein interactions for ERBB2 dimers
The dynamic assembly of multiprotein complexes is a central mechanism of many cell signaling pathways. This process is key to maintaining the spatiotemporal specificity required for an accurate, yet adaptive, response to rapidly changing cellular conditions. We describe a technique for the specific...
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| Published in: | Science signaling Vol. 9; no. 436; p. ra69 |
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| Main Authors: | , , , , , , , , , , |
| Format: | Journal Article |
| Language: | English |
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United States
12.07.2016
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| ISSN: | 1937-9145, 1937-9145 |
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| Abstract | The dynamic assembly of multiprotein complexes is a central mechanism of many cell signaling pathways. This process is key to maintaining the spatiotemporal specificity required for an accurate, yet adaptive, response to rapidly changing cellular conditions. We describe a technique for the specific isolation and downstream proteomic characterization of any two interacting proteins, to the exclusion of their individual moieties and competing binding partners. We termed the approach bimolecular complementation affinity purification (BiCAP) because it combines the use of conformation-specific nanobodies with a protein-fragment complementation assay with affinity purification. Using BiCAP, we characterized the specific interactome of the epidermal growth factor receptor (EGFR) family member ERBB2 when in the form of a homodimer or when in the form of a heterodimer with either EGFR or ERBB3. We identified dimer-specific interaction patterns for key adaptor proteins and identified a number of previously unknown interacting partners. Functional analysis for one of these newly identified partners revealed a noncanonical mechanism of extracellular signal-regulated kinase (ERK) activation that is specific to the ERBB2:ERBB3 heterodimer and acts through the adaptor protein FAM59A in breast cancer cells. |
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| AbstractList | The dynamic assembly of multiprotein complexes is a central mechanism of many cell signaling pathways. This process is key to maintaining the spatiotemporal specificity required for an accurate, yet adaptive, response to rapidly changing cellular conditions. We describe a technique for the specific isolation and downstream proteomic characterization of any two interacting proteins, to the exclusion of their individual moieties and competing binding partners. We termed the approach bimolecular complementation affinity purification (BiCAP) because it combines the use of conformation-specific nanobodies with a protein-fragment complementation assay with affinity purification. Using BiCAP, we characterized the specific interactome of the epidermal growth factor receptor (EGFR) family member ERBB2 when in the form of a homodimer or when in the form of a heterodimer with either EGFR or ERBB3. We identified dimer-specific interaction patterns for key adaptor proteins and identified a number of previously unknown interacting partners. Functional analysis for one of these newly identified partners revealed a noncanonical mechanism of extracellular signal-regulated kinase (ERK) activation that is specific to the ERBB2:ERBB3 heterodimer and acts through the adaptor protein FAM59A in breast cancer cells. The dynamic assembly of multiprotein complexes is a central mechanism of many cell signaling pathways. This process is key to maintaining the spatiotemporal specificity required for an accurate, yet adaptive, response to rapidly changing cellular conditions. We describe a technique for the specific isolation and downstream proteomic characterization of any two interacting proteins, to the exclusion of their individual moieties and competing binding partners. We termed the approach bimolecular complementation affinity purification (BiCAP) because it combines the use of conformation-specific nanobodies with a protein-fragment complementation assay with affinity purification. Using BiCAP, we characterized the specific interactome of the epidermal growth factor receptor (EGFR) family member ERBB2 when in the form of a homodimer or when in the form of a heterodimer with either EGFR or ERBB3. We identified dimer-specific interaction patterns for key adaptor proteins and identified a number of previously unknown interacting partners. Functional analysis for one of these newly identified partners revealed a noncanonical mechanism of extracellular signal-regulated kinase (ERK) activation that is specific to the ERBB2:ERBB3 heterodimer and acts through the adaptor protein FAM59A in breast cancer cells.The dynamic assembly of multiprotein complexes is a central mechanism of many cell signaling pathways. This process is key to maintaining the spatiotemporal specificity required for an accurate, yet adaptive, response to rapidly changing cellular conditions. We describe a technique for the specific isolation and downstream proteomic characterization of any two interacting proteins, to the exclusion of their individual moieties and competing binding partners. We termed the approach bimolecular complementation affinity purification (BiCAP) because it combines the use of conformation-specific nanobodies with a protein-fragment complementation assay with affinity purification. Using BiCAP, we characterized the specific interactome of the epidermal growth factor receptor (EGFR) family member ERBB2 when in the form of a homodimer or when in the form of a heterodimer with either EGFR or ERBB3. We identified dimer-specific interaction patterns for key adaptor proteins and identified a number of previously unknown interacting partners. Functional analysis for one of these newly identified partners revealed a noncanonical mechanism of extracellular signal-regulated kinase (ERK) activation that is specific to the ERBB2:ERBB3 heterodimer and acts through the adaptor protein FAM59A in breast cancer cells. |
| Author | Lau, Joseph Iconomou, Mary Han, Jeremy Z R Hastings, Jordan F McKenna, Jessie Aparicio, Samuel Kennedy, Sean P Croucher, David R Shearer, Robert F Wan, Adrian Saunders, Darren N |
| Author_xml | – sequence: 1 givenname: David R surname: Croucher fullname: Croucher, David R email: d.croucher@garvan.org.au, d.saunders@unsw.edu.au organization: The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia. St. Vincent's Hospital Clinical School, University of New South Wales, Sydney, New South Wales 2052, Australia. School of Medicine, University College Dublin, Belfield, Dublin D4, Ireland. d.croucher@garvan.org.au d.saunders@unsw.edu.au – sequence: 2 givenname: Mary surname: Iconomou fullname: Iconomou, Mary organization: The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia – sequence: 3 givenname: Jordan F surname: Hastings fullname: Hastings, Jordan F organization: The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia – sequence: 4 givenname: Sean P surname: Kennedy fullname: Kennedy, Sean P organization: The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia. Systems Biology Ireland, University College Dublin, Belfield, Dublin D4, Ireland – sequence: 5 givenname: Jeremy Z R surname: Han fullname: Han, Jeremy Z R organization: The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia – sequence: 6 givenname: Robert F surname: Shearer fullname: Shearer, Robert F organization: The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia – sequence: 7 givenname: Jessie surname: McKenna fullname: McKenna, Jessie organization: The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia – sequence: 8 givenname: Adrian surname: Wan fullname: Wan, Adrian organization: Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, British Columbia V5Z 1L3, Canada – sequence: 9 givenname: Joseph surname: Lau fullname: Lau, Joseph organization: Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, British Columbia V5Z 1L3, Canada – sequence: 10 givenname: Samuel surname: Aparicio fullname: Aparicio, Samuel organization: Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, British Columbia V5Z 1L3, Canada. Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada – sequence: 11 givenname: Darren N surname: Saunders fullname: Saunders, Darren N email: d.croucher@garvan.org.au, d.saunders@unsw.edu.au organization: The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia. School of Medical Sciences, University of New South Wales, Sydney, New South Wales 2052, Australia. d.croucher@garvan.org.au d.saunders@unsw.edu.au |
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| SubjectTerms | Breast Neoplasms - metabolism Breast Neoplasms - pathology Female HEK293 Cells Humans MAP Kinase Signaling System MCF-7 Cells Protein Multimerization Receptor, Epidermal Growth Factor - isolation & purification Receptor, Epidermal Growth Factor - metabolism Receptor, ErbB-2 - isolation & purification Receptor, ErbB-2 - metabolism Receptor, ErbB-3 - isolation & purification Receptor, ErbB-3 - metabolism |
| Title | Bimolecular complementation affinity purification (BiCAP) reveals dimer-specific protein interactions for ERBB2 dimers |
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