Investigation of the effects of the Small Linear Peptide KiR-SLiM in the assembly and function of Protein Phosphatase 1 (PP1) holoenzyme complexes

Abstract

Protein phosphatase 1 (PP1) is an essential phosphatase responsible for many de-phosphorylating events in cells throughout the cell cycle. PP1 functions as holoenzyme complex consisting of its catalytic subunit and regulatory subunits determining its substrate specificity by recruiting PP1 to de-phosphorylation targets. The holoenzyme complex formation is mediated by several small linear motifs (SLiMs) within the regulatory subunit. A specific SLiM termed KiR-SLiM (Ki-67-Repo-Man SLiM) was discovered which is only used by Cdca2 and Ki-67 to bind to PP1. In some cancers, protein levels of holoenzyme complex proteins are dysregulated leading to aberrant dephosphorylation and uncontrolled cell proliferation. Despite this, the development of controlled PP1 inhibition has proven challenging. A molecular understanding of PP1 holoenzyme formation, recruitment and substrate specificity is still quite patchy needing further investigation to discover specific PP1 holoenzyme inhibitors for treating specifically dysfunctional PP1 holoenzyme complexes in cancer. We have shown that a KiR-SLiM peptidomimetic is sufficient to recruit PP1cγ to an artificially created location in the chromatin. We discovered that KiR-SLiM localises to the nucleoli which can be impaired by removing PP1, depleting RRP1B or Ki-67 or increasing the size of KiR-SLiM. We have shown the stable binding of PP1cγ and PP1cβ to KiR-SLiM by pull-down in vivo and identified the interactome of KiR-SLiM. Furthermore, we have shown that the overexpression of KiR-SLiM leads to a G1-phase arrest in HeLa cells, however the mechanism is still elusive. We established a reliable gene editing method to endogenously label PP1cβ generating a stable HCT116:TETONOSTR1 cell line; these ground-breaking tools allow the in vivo investigation of endogenous PP1cβ. This work gives an important insight into the molecular effects of KiR-SLiM as binding motif; the ability of KiR-SLiM to bind PP1 in vivo leading to cell cycle arrest could pave the way to use interfering peptides to modulate dysfunctional PP1 activity in cells.