Structure-function Analysis of the STRIPAK Complex

Reversible phosphorylation events regulate critical aspects of biology. Kinases and phosphatases interact not only with their substrates but also with regulatory subunits and scaffolds. In recent years, affinity purification coupled to mass spectrometry (AP-MS) has proven to be a powerful tool to identify protein-protein interactions (PPIs) involving kinases and phosphatases. Using AP-MS we discovered a novel-signaling complex, which we termed STRIPAK (for STRiatin Interacting Phosphatase and Kinase). The STRIPAK complex contains a protein phosphatase 2A (PP2A) holoenzyme and a family of Ste20 kinases (the GCK-III kinases), as well as the protein MOB4, a member of the MOB family of proteins, which are known kinase regulators. Interestingly the protein CCM3, one of three proteins mutated in familial cases of cerebral cavernous malformations (CCMs), was also found to reside within the STRIPAK complex. My project focused on the structural and functional characterization of STRIPAK. I mapped direct interactions and interacting regions on these proteins and determined that the PP2A substrate binding subunit (striatin) and CCM3 act as adapter molecules to bridge the kinase and phosphatase subunits of STRIPAK. I also characterized the functional role for STRIPAK components in Golgi positioning, and demonstrated that depletion of CCM3 or striatin affects GCK-III kinase and Golgi polarization in an opposite manner. Further AP-MS experiments revealed that CCM3 and the GCK-III kinases reside within at least two distinct protein complexes, either with STRIPAK or with the protein CCM2, which is also mutated in familial cases of CCM disease, and that CCM3 is found in complex with CCM1 and CCM2, thus revealing a possible common pathogenesis for CCM proteins in CCM disease. To gain insight into the role of MOB4 within STRIPAK, I characterized the entire MOB family and their known binding partners, the NDR/LATS kinases, using AP-MS. These studies identified many new MOB and kinase interactors, including a family of guanine nucleotide exchange factors and protein phosphatase 6 (PP6).

Department dc:contributor.department

Molecular and Medical Genetics

Year dc:date.issued

2013

Author dc:creator

• Kean, Michelle

Advisors dc:contributor.advisor

• Gingras, Anne-Claude
• Sicheri, Frank

• STRIPAK
• CCM3
• Cerebral Cavernous Malformations
• PP2A
• MOB proteins
• Affinity Purification-Mass Spectrometry

Language dc:language.iso

en_ca

Handle dc:identifier.uri

http://hdl.handle.net/1807/68949

OAI identifier oai:identifier

oai:utoronto.scholaris.ca:1807/68949