Reversible protein phosphorylation, the best studied post-translational modification, regulates most cellular processes, including signaling, migration, cell cycle progression, DNA damage repair, stress response and modulaton of the activities of metabolic enzymes. Therefore, it has emerged as a key therapeutic target in diseases in which these processes are deregulated. Unlike kinases, protein phosphatase 1 (PP1) is a promiscuous enzyme that gains its substrate specificity from a large group of “regulatory subunits” with which it associates to form a range of holoenzyme complexes targeted to specific subcellular localizations and substrates. Inhibition of a specific dephosphorylation event therefore relies on targeting the regulatory rather than the catalytic subunit. The present study uses GFP as a molecular reporter to assess the localization of PP1c and identify the underlying binding events that govern it via a combination of fluorescence imaging, cellular fractionation, affinity purification and quantitative mass spectrometry. While there is some overlap in their targeting and intracellular roles, the three PP1 isoforms show distinct localizations based on relative preferences for particular regulatory subunits. In this study we assembled a comprehensive map of isoform- and compartment- specific phosphatase complexes in three different cultured human cell lines, using the data to extrapolate, with confidence, the distribution of each PP1 isoform between a large pool of known/predicted and novel regulatory subunits. Network analysis also highlighted key multiprotein complexes to which PP1 is targeted by these regulatory subunits, and identified a novel regulatory subunit that links phosphatase activity to regulation of protein degradation. Our work confirmed that Mypt1, the regulatory subunit that targets PP1 activity to the myosin light chain, preferentially associates with the beta isoform of PP1c. We further demonstrated that they are in complex in both the cytoplasm and nucleus, and represent ~30% of the total PP1β holoenzyme complexes in both interaphase and mitotic cells. Further investigation of these complexes led to the discovery of Specc1 and Specc1L, which associate with Mypt1/PP1β via direct binding to Mypt1. Specc1/1L are microtubule binding proteins that can also associate with actin filaments, and we demonstrated that they mediate the distribution of Mypt1/PP1β complexes between these two cytoskeletal networks. Given that disruption of this balance has been implicated in disease states including cancer and hypertension, the Specc1/L family represents a novel therapeutic target for the regulation of Mypt1/PP1 activity. With PP1 now emerging as a promising therapeutic target and the first PP1-targeted therapeutic drug, Sephin 1, in clinical trials, a better understanding of PP1’s in vivo distribution between holoenzyme complexes is essential. Our work establishes an initial “snapshot” of this distribution against which changes can be assessed, as we demonstrated here by showing its re-distribution in mitotic cells. Dynamic redistributions during specific cell processes such as differentiation or in response to perturbations or disease states can be assessed in a similar fashion in future, facilitating both identification of the relevant complexes and the design of specific strategies to target them therapeutically.

Optineurine ("Optic neuropathy-inducing", Optn) est une protéine exprimée de façon ubiquitaire chez les vertébrés et impliquée dans de nombreux processus cellulaires tels que la régulation du trafic vésiculaire associée à l'appareil de Golgi, la réponse immunitaire innée ou l'autophagie des bactéries. Mon travail de thèse a permis de caractériser une nouvelle fonction d'Optn dans la régulation du cycle cellulaire. Plus précisément, j'ai pu montrer qu'Optn était un régulateur négatif de Polo-like kinase 1 (Plk1), une kinase qui joue un rôle clef dans chacune des étapes de la mitose : de la prophase à la cytokinèse. Les résultats présentés dans cette thèse montrent qu'Optn est phosphorylée par Plk1 sur la sérine 177 en début de mitose provoquant le détachement d'Optn de l'appareil de Golgi et son accumulation dans le noyau. Nous avons montré que la phosphorylation et la translocation nucléaire d'Optn étaient requises pour permettre la régulation négative de Plk1 par le complexe phosphatase MYPT1-PP1 au cours de la mitose. Les conséquences fonctionnelles de la déplétion d'Optn et donc de l'hyperactivité de Plk1 sont des défauts de cytokinèse et de ségrégation des chromosomes, aboutissant à l'apparition de cellules plurinucléées. En conclusion, nos résultats mettent en évidence un mécanisme de rétrocontrôle négatif par lequel Plk1 module la localisation d'Optn pendant la mitose pour réguler sa propre activité

Regulation of smooth muscle contraction depends on the phosphorylated state of myosin light chain (MLC). Although there are many kinases responsible for phosphorylating MLC, the myosin phosphatase complex is solely accountable for its dephosphorylation. Myosin phosphatase, in turn, is tightly regulated by many proteins. One of them being the CPI-17 gene family, which inhibits myosin phosphatase. This family of proteins is composed of CPI-17 itself, PHI-1, KEPI, and GBPI. Zebrafish have two genes each of CPI-17 and PHI-1, which are expressed during early embryonic development. This study sets out to investigate whether the two isoforms of CPI-17 and PHI-1 have diverged in function or expression using zebrafish as a model organism. Through a series of biochemical tests and assays, we have determined that the two isoforms have diverged in their expression pattern from each other, however they have similar function.

Alternative splicing of the zebrafish Myosin Phosphatase Targeting Subunit, MYPT1, produces a novel isoform (TV202). TV202 and the truncated TV202Δ ere shown to form an active complex with Protein Phosphatase 1 β (PP1β) via stress fiber assay. TV202 was also shown to be localized in the cytoplasm, enriched in a paranuclear manner. TV202Δ was found the be localized inside the nucleus. It was also found that TV202 was zygotically, but not maternally, expressed during early zebrafish development via RT-PCR.

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Zipper-Interacting Protein Kinase (ZIPK) is a known modulator of actin-myosin contractility in vertebrate species. Interestingly, rodent and mouse ZIPK has undergone a divergence in regulation in comparison to other vertebrate orthologs including human. Whereas the human ortholog of ZIPK requires phosphorylation of residues TT299/300 for nuclear exit, rodents and mouse require interaction with another protein termed PAR-4. In this project we completed several experiments to examine zebrafish ZIPK in development and its effect on acto-myosin contractility. It was found that zebrafish ZIPK was expressed ubiquitously in maternal stages. In zygotic stages, ZIPK expression dropped dramatically and localized to the anterior portions of the embryo. Zebrafish and human ZIPK, but not rodent ZIPK were able to increase stress fiber formation and myosin light chain-2 (MLC-2) phosphorylation in vitro. Human and zebrafish ZIPK underwent nucleocytoplasmic shuttling without PAR-4 interaction, unlike rodent ZIPK, which required PAR-4 for nuclear exit. Unlike human ZIPK, zebrafish ZIPK TT299/300AA mutants were able to undergo shuttling. Similar to human ZIPK, catalytic mutations to zebrafish ZIPK abolished or dramatically reduced activity. Through these experiments we were able to show human and zebrafish ZIPK homologs function and are regulated similarly, while the rodent ZIPK was much more unique. Although the exhibited phenotypes were similar between human and zebrafish ZIPK orthologs, the mechanism of regulation is not completely conserved.