{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/388655"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/388655","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Characterising the effects of ACK-mediated phosphorylation on the Class IA PI3-Kinase regulatory isoforms","abstract":"Activated Cdc42-associated kinase (ACK), a non-receptor tyrosine (Tyr) kinase, was the first effector discovered for the small GTPase Cdc42. ACK is emerging as an important feature of the cancer landscape and thus, is a promising potential target for the treatment of many malignancies. ACK orchestrates its oncogenic activity through interactions with diverse proteins including the androgen receptor, the oestrogen receptor and WW domain containing oxidoreductase. Despite enormous progress in recent years, much remains to be learnt concerning ACK's role in oncogenic signalling. To gain further insight, the Owen lab conducted a yeast two-hybrid screen to identify novel ACK cellular partners. This screen revealed that ACK interacts with three of the Class IA PI3-kinase regulatory subunits (p85α, p85β, and p55γ). ACK has since been shown to bind to all five p85 isoforms and phosphorylate four (not p55γ) at a Tyr residue (equivalent to p85α Tyr607) in the inter-SH2 (iSH2) domain, a region situated between the N-terminal SH2 (nSH2) and C-terminal SH2 (cSH2) domains. Such phosphorylation was proposed to facilitate the formation of C-terminally mediated p85 isoform dimers driven by interactions between the pTyr residue of one monomer and nSH2 domain of the other monomer. Given the critical role of aberrant PI3-Kinase signalling in cancer, and the emerging role of ACK, it is vital the impact of ACK-mediated phosphorylation of the Class IA PI3-Kinase regulatory subunits is understood. This is particularly important considering that the p85 isoforms serve as key negative regulators of PI3-Kinase signalling, and phosphorylation of the p85 isoforms at the ACK-target site has been shown to promote cell proliferation. Since protein dimerisation is a well-established mechanism for modulating protein function, it is possible that phosphorylation-driven dimerisation of the p85 isoforms could represent a novel mechanism for regulating PI3-Kinase signalling. Accordingly, this project aimed to further characterise the effect of ACK-induced phosphorylation on p85α and p55α assembly in vivo and in vitro. Since phosphomimics (e.g. CMF, Asp, and Glu) proved ineffective at mimicking the role of pTyr in the pTyr–p85 nSH2 interaction, phosphorylated p85 for the in vitro experiments was generated using in vitro kinase assays with recombinant ACK. Interestingly, the SEC-MALS experiments with phosphorylated p85α and p55α showed no evidence for phosphorylation-driven p85 isoform dimerisation. Yet, further analysis suggested that the absence of detected dimerisation was likely attributed to low levels of phosphorylated p85 protein. However, these experiments also suggested that ACK may phosphorylate p85α and p55α at multiple sites, in addition to Tyr607 and Tyr337 respectively. While follow-up mass spectrometry confirmed that ACK phosphorylates p55α at Tyr337 and detected potential phosphorylation at Thr333 and Thr405, these Thr phosphorylation events are likely non-physiological, given ACK has been characterised as a tyrosine kinase. Size exclusion chromatography experiments using cell extracts indicated that p85α phosphorylated at Tyr607 and p55α phosphorylated at Tyr337 eluted in fractions potentially corresponding to dimeric species. However, as these results are obtained from a single independent experiment and the technique offers limited resolution, the data are insufficient to confidently confirm that p85α pTyr607 and p55α pTyr337 exist as dimers. Subsequent co-immunoprecipitation and native PAGE experiments also failed to provide any additional insights into whether ACK-induced phosphorylation facilitates p85 assembly. However, the Native PAGE experiments did confirm that ACK stabilizes the p85 isoforms, which is consistent with previous studies that showed ACK protects p85α from polyubiquitination and degradation. In conclusion, this project has advanced our understanding of the most effective approaches for studying phosphorylation-driven protein assembly in vitro and in vivo. Although the further characterisation of p85 assembly could not be completed in the timeframe of this research project, this work has established the key methods that will enable efficacious exploration of the p85 dimers in future. Ultimately, further research is required to build on these findings to obtain a comprehensive understanding of how ACK-induced phosphorylation affects the structure and function of the p85 isoforms. A thorough understanding of the relationship between ACK and p85 isoforms could be vital for understanding whether p85 or ACK could serve as viable novel anti-cancer therapy targets.","abstract_html":"Activated Cdc42-associated kinase (ACK), a non-receptor tyrosine (Tyr) kinase, was the first effector discovered for the small GTPase Cdc42. ACK is emerging as an important feature of the cancer landscape and thus, is a promising potential target for the treatment of many malignancies. ACK orchestrates its oncogenic activity through interactions with diverse proteins including the androgen receptor, the oestrogen receptor and WW domain containing oxidoreductase. Despite enormous progress in recent years, much remains to be learnt concerning ACK's role in oncogenic signalling. To gain further insight, the Owen lab conducted a yeast two-hybrid screen to identify novel ACK cellular partners. This screen revealed that ACK interacts with three of the Class IA PI3-kinase regulatory subunits (p85α, p85β, and p55γ). ACK has since been shown to bind to all five p85 isoforms and phosphorylate four (not p55γ) at a Tyr residue (equivalent to p85α Tyr607) in the inter-SH2 (iSH2) domain, a region situated between the N-terminal SH2 (nSH2) and C-terminal SH2 (cSH2) domains. Such phosphorylation was proposed to facilitate the formation of C-terminally mediated p85 isoform dimers driven by interactions between the pTyr residue of one monomer and nSH2 domain of the other monomer. Given the critical role of aberrant PI3-Kinase signalling in cancer, and the emerging role of ACK, it is vital the impact of ACK-mediated phosphorylation of the Class IA PI3-Kinase regulatory subunits is understood. This is particularly important considering that the p85 isoforms serve as key negative regulators of PI3-Kinase signalling, and phosphorylation of the p85 isoforms at the ACK-target site has been shown to promote cell proliferation. Since protein dimerisation is a well-established mechanism for modulating protein function, it is possible that phosphorylation-driven dimerisation of the p85 isoforms could represent a novel mechanism for regulating PI3-Kinase signalling. Accordingly, this project aimed to further characterise the effect of ACK-induced phosphorylation on p85α and p55α assembly in vivo and in vitro. Since phosphomimics (e.g. CMF, Asp, and Glu) proved ineffective at mimicking the role of pTyr in the pTyr–p85 nSH2 interaction, phosphorylated p85 for the in vitro experiments was generated using in vitro kinase assays with recombinant ACK. Interestingly, the SEC-MALS experiments with phosphorylated p85α and p55α showed no evidence for phosphorylation-driven p85 isoform dimerisation. Yet, further analysis suggested that the absence of detected dimerisation was likely attributed to low levels of phosphorylated p85 protein. However, these experiments also suggested that ACK may phosphorylate p85α and p55α at multiple sites, in addition to Tyr607 and Tyr337 respectively. While follow-up mass spectrometry confirmed that ACK phosphorylates p55α at Tyr337 and detected potential phosphorylation at Thr333 and Thr405, these Thr phosphorylation events are likely non-physiological, given ACK has been characterised as a tyrosine kinase. Size exclusion chromatography experiments using cell extracts indicated that p85α phosphorylated at Tyr607 and p55α phosphorylated at Tyr337 eluted in fractions potentially corresponding to dimeric species. However, as these results are obtained from a single independent experiment and the technique offers limited resolution, the data are insufficient to confidently confirm that p85α pTyr607 and p55α pTyr337 exist as dimers. Subsequent co-immunoprecipitation and native PAGE experiments also failed to provide any additional insights into whether ACK-induced phosphorylation facilitates p85 assembly. However, the Native PAGE experiments did confirm that ACK stabilizes the p85 isoforms, which is consistent with previous studies that showed ACK protects p85α from polyubiquitination and degradation. In conclusion, this project has advanced our understanding of the most effective approaches for studying phosphorylation-driven protein assembly in vitro and in vivo. Although the further characterisation of p85 assembly could not be completed in the timeframe of this research project, this work has established the key methods that will enable efficacious exploration of the p85 dimers in future. Ultimately, further research is required to build on these findings to obtain a comprehensive understanding of how ACK-induced phosphorylation affects the structure and function of the p85 isoforms. A thorough understanding of the relationship between ACK and p85 isoforms could be vital for understanding whether p85 or ACK could serve as viable novel anti-cancer therapy targets.","abstract_has_math":false,"creators":["Hodder, Samantha"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Broadhurst, Bill","Machesky, Laura"],"committee_chairs":[],"committee_members":[],"year":2025,"date_issued":"2025-01-30","date_published":"2025-01-30","updated_at":"2026-07-22T22:24:23Z","subjects":["ACK","Dimers","p85","PI3-Kinase"],"languages":["eng"],"rights":[],"rights_urls":["https://www.repository.cam.ac.uk/bitstreams/72111700-4bd2-4c96-8053-b38dc08641a7/download","http://purl.org/NET/rdflicense/allrightsreserved"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.120897","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Broadhurst, Bill","Machesky, Laura"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["CRUK Cambridge Cancer Centre"]},{"key":"dc:creator","label":"Author","values":["Hodder, Samantha"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2025-01-30"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/388655"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["ACK","Dimers","p85","PI3-Kinase"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://www.repository.cam.ac.uk/bitstreams/72111700-4bd2-4c96-8053-b38dc08641a7/download","http://purl.org/NET/rdflicense/allrightsreserved"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.120897"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://www.repository.cam.ac.uk/bitstreams/7e1abbe0-a6ab-4101-b6e6-656777acb529/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Activated Cdc42-associated kinase (ACK), a non-receptor tyrosine (Tyr) kinase, was the first effector discovered for the small GTPase Cdc42. 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Such phosphorylation was proposed to facilitate the formation of C-terminally mediated p85 isoform dimers driven by interactions between the pTyr residue of one monomer and nSH2 domain of the other monomer. Given the critical role of aberrant PI3-Kinase signalling in cancer, and the emerging role of ACK, it is vital the impact of ACK-mediated phosphorylation of the Class IA PI3-Kinase regulatory subunits is understood. This is particularly important considering that the p85 isoforms serve as key negative regulators of PI3-Kinase signalling, and phosphorylation of the p85 isoforms at the ACK-target site has been shown to promote cell proliferation. Since protein dimerisation is a well-established mechanism for modulating protein function, it is possible that phosphorylation-driven dimerisation of the p85 isoforms could represent a novel mechanism for regulating PI3-Kinase signalling. Accordingly, this project aimed to further characterise the effect of ACK-induced phosphorylation on p85α and p55α assembly in vivo and in vitro. Since phosphomimics (e.g. CMF, Asp, and Glu) proved ineffective at mimicking the role of pTyr in the pTyr–p85 nSH2 interaction, phosphorylated p85 for the in vitro experiments was generated using in vitro kinase assays with recombinant ACK. Interestingly, the SEC-MALS experiments with phosphorylated p85α and p55α showed no evidence for phosphorylation-driven p85 isoform dimerisation. Yet, further analysis suggested that the absence of detected dimerisation was likely attributed to low levels of phosphorylated p85 protein. However, these experiments also suggested that ACK may phosphorylate p85α and p55α at multiple sites, in addition to Tyr607 and Tyr337 respectively. While follow-up mass spectrometry confirmed that ACK phosphorylates p55α at Tyr337 and detected potential phosphorylation at Thr333 and Thr405, these Thr phosphorylation events are likely non-physiological, given ACK has been characterised as a tyrosine kinase. Size exclusion chromatography experiments using cell extracts indicated that p85α phosphorylated at Tyr607 and p55α phosphorylated at Tyr337 eluted in fractions potentially corresponding to dimeric species. However, as these results are obtained from a single independent experiment and the technique offers limited resolution, the data are insufficient to confidently confirm that p85α pTyr607 and p55α pTyr337 exist as dimers. Subsequent co-immunoprecipitation and native PAGE experiments also failed to provide any additional insights into whether ACK-induced phosphorylation facilitates p85 assembly. However, the Native PAGE experiments did confirm that ACK stabilizes the p85 isoforms, which is consistent with previous studies that showed ACK protects p85α from polyubiquitination and degradation. In conclusion, this project has advanced our understanding of the most effective approaches for studying phosphorylation-driven protein assembly in vitro and in vivo. Although the further characterisation of p85 assembly could not be completed in the timeframe of this research project, this work has established the key methods that will enable efficacious exploration of the p85 dimers in future. Ultimately, further research is required to build on these findings to obtain a comprehensive understanding of how ACK-induced phosphorylation affects the structure and function of the p85 isoforms. 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Such phosphorylation was proposed to facilitate the formation of C-terminally mediated p85 isoform dimers driven by interactions between the pTyr residue of one monomer and nSH2 domain of the other monomer. Given the critical role of aberrant PI3-Kinase signalling in cancer, and the emerging role of ACK, it is vital the impact of ACK-mediated phosphorylation of the Class IA PI3-Kinase regulatory subunits is understood. This is particularly important considering that the p85 isoforms serve as key negative regulators of PI3-Kinase signalling, and phosphorylation of the p85 isoforms at the ACK-target site has been shown to promote cell proliferation. Since protein dimerisation is a well-established mechanism for modulating protein function, it is possible that phosphorylation-driven dimerisation of the p85 isoforms could represent a novel mechanism for regulating PI3-Kinase signalling. Accordingly, this project aimed to further characterise the effect of ACK-induced phosphorylation on p85α and p55α assembly in vivo and in vitro. Since phosphomimics (e.g. CMF, Asp, and Glu) proved ineffective at mimicking the role of pTyr in the pTyr–p85 nSH2 interaction, phosphorylated p85 for the in vitro experiments was generated using in vitro kinase assays with recombinant ACK. Interestingly, the SEC-MALS experiments with phosphorylated p85α and p55α showed no evidence for phosphorylation-driven p85 isoform dimerisation. Yet, further analysis suggested that the absence of detected dimerisation was likely attributed to low levels of phosphorylated p85 protein. However, these experiments also suggested that ACK may phosphorylate p85α and p55α at multiple sites, in addition to Tyr607 and Tyr337 respectively. While follow-up mass spectrometry confirmed that ACK phosphorylates p55α at Tyr337 and detected potential phosphorylation at Thr333 and Thr405, these Thr phosphorylation events are likely non-physiological, given ACK has been characterised as a tyrosine kinase. Size exclusion chromatography experiments using cell extracts indicated that p85α phosphorylated at Tyr607 and p55α phosphorylated at Tyr337 eluted in fractions potentially corresponding to dimeric species. However, as these results are obtained from a single independent experiment and the technique offers limited resolution, the data are insufficient to confidently confirm that p85α pTyr607 and p55α pTyr337 exist as dimers. Subsequent co-immunoprecipitation and native PAGE experiments also failed to provide any additional insights into whether ACK-induced phosphorylation facilitates p85 assembly. However, the Native PAGE experiments did confirm that ACK stabilizes the p85 isoforms, which is consistent with previous studies that showed ACK protects p85α from polyubiquitination and degradation. In conclusion, this project has advanced our understanding of the most effective approaches for studying phosphorylation-driven protein assembly in vitro and in vivo. Although the further characterisation of p85 assembly could not be completed in the timeframe of this research project, this work has established the key methods that will enable efficacious exploration of the p85 dimers in future. Ultimately, further research is required to build on these findings to obtain a comprehensive understanding of how ACK-induced phosphorylation affects the structure and function of the p85 isoforms. A thorough understanding of the relationship between ACK and p85 isoforms could be vital for understanding whether p85 or ACK could serve as viable novel anti-cancer therapy targets."],"dc:format.checksum.md5":["5cc7e6ed5667f3793ae5e67b4fc23063","87eda9de84448d1f82354d60eee3eb5f"],"dc:identifier.doi":["https://doi.org/10.17863/CAM.120897"],"dc:identifier.uri":["https://www.repository.cam.ac.uk/bitstreams/7e1abbe0-a6ab-4101-b6e6-656777acb529/download"],"dc:language":["eng"],"dc:publisher.institution":["University of Cambridge"],"dc:relation.isreferencedby.uri":["https://www.repository.cam.ac.uk/handle/1810/388655"],"dc:rights":["https://www.repository.cam.ac.uk/bitstreams/72111700-4bd2-4c96-8053-b38dc08641a7/download","http://purl.org/NET/rdflicense/allrightsreserved"],"dc:subject":["ACK","Dimers","p85","PI3-Kinase"],"dc:title":["Characterising the effects of ACK-mediated phosphorylation on the Class IA PI3-Kinase regulatory isoforms"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-22T22:24:23Z"}