Protein interactome of the Cancerous Inhibitor of protein phosphatase 2A (CIP2A) in Th17 cells

• Published in: Current research in immunology 2020-12, Vol.1, p.10-22
• Main Authors: Khan, Mohd Moin, Välikangas, Tommi, Khan, Meraj Hasan, Moulder, Robert, Ullah, Ubaid, Bhosale, Santosh Dilip, Komsi, Elina, Butt, Umar, Qiao, Xi, Westermarck, Jukka, Elo, Laura L., Lahesmaa, Riitta
• Format: Article
• Language: English
• Subjects: CIP2A; Confocal microscopy; Interactome; Mass Spectrometry (MS); Pathway analysis; Selected Reaction Monitoring (SRM) targeted mass spectrometry
• ISSN: 2590-2555; 2590-2555
• DOI: 10.1016/j.crimmu.2020.02.001

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is involved in immune response, cancer progression, and Alzheimer's disease. However, an understanding of the mechanistic basis of its function in this wide spectrum of physiological and pathological processes is limited due to its poorly characterized interaction networks. Here we present the first systematic characterization of the CIP2A interactome by affinity-purification mass spectrometry combined with validation by selected reaction monitoring targeted mass spectrometry (SRM-MS) analysis in T helper (Th) 17 (Th17) cells. In addition to the known regulatory subunits of protein phosphatase 2A (PP2A), the catalytic subunits of protein PP2A were found to be interacting with CIP2A. Furthermore, the regulatory (PPP1R18, and PPP1R12A) and catalytic (PPP1CA) subunits of phosphatase PP1 were identified among the top novel CIP2A interactors. Evaluation of the ontologies associated with the proteins in this interactome revealed that they were linked with RNA metabolic processing and splicing, protein traffic, cytoskeleton regulation and ubiquitin-mediated protein degradation processes. Taken together, this network of protein-protein interactions will be important for understanding and further exploring the biological processes and mechanisms regulated by CIP2A both in physiological and pathological conditions. A schematic representation of the CIP2A interactome study: CIP2A immunoprecipitation (IP) was performed after 72 ​h of polarization of naïve T cells towards the Th17 lineage. Two CIP2A specific antibodies (Ab1 and Ab2), targeting the N-terminal structured region and unstructured C-terminus, were used together with their respective IgG (IgG1 and IgG2) control antibodies. Following LC-MS/MS analysis to identify the CIP2A associated proteins, statistical analysis using the Significance Analysis of INTeractome (SAINT) algorithm was used to distinguish non-specific interactions. [Display omitted] •The first characterisation of the CIP2A interactome in Th17 ​cells.•Key interactions validated by targeted SRM-MS proteomics, western blot and confocal microscopy.•Pathway analysis of the interactome revealed interrelationships with proteins across a broad range of cellular processes.•The study identifies for the first time the interaction of phosphatase PP1 with CIP2A.