No description available.

Cellular needs are fulfilled by the combined activity of functional proteins. Consequently, cells are equipped with a complex proteostatic network that controls protein production in response to cellular requisites. Thereby, protein synthesis is induced or attenuated depending on the particular cellular conditions. One of the mechanisms to control protein synthesis is the phosphorylation of eIF2, which triggers the so-called Integrated Stress Response (ISR). Kinases that sense stresses induce the phosphorylation of eIF2, which, on the one hand, attenuates global rates of protein synthesis and, on the other hand, activates the expression of specific proteins that help to alleviate the stress. One of the proteins preferentially expressed during the ISR is PPP1R15A, a regulatory subunit of Protein Phosphatase 1 (PP1). The PP1/PPP1R15A holophosphatase dephosphorylates eIF2 and terminates the ISR once the stresses are resolved. Hence, eIF2 kinases and phosphatases work together to control levels of phosphorylated eIF2. Maintaining the right balance between the activity of these kinases and phosphatases is important, as is seen by the correlation between their perturbance and the appearance of certain cellular malfunctions or diseases. However, affecting this balance has been also suggested to have beneficial effects. For example, genetic interference with the PPP1R15A regulatory subunit is proposed to confer protection to mice and cells under ER-stress conditions. This observation led to the search for compounds with the ability to modulate the ISR, in particular, by acting on the eIF2 phosphatases. Three compounds (Salubrinal, Guanabenz and Sephin1) have been proposed as eIF2 phosphatase inhibitors with potential use as therapeutic tools in protein misfolding diseases. However, their precise mechanism of action and their direct effect on the enzyme remains an open question. This thesis focuses on the in vitro reconstitution of the eIF2 phosphatase, which served as a platform for characterizing the enzyme (in terms of its structure, activity and assembly) and for studying the proposed inhibitors. This report details key structural features of the PPP1R15/PP1 holophosphatase, the discovery of a cellular cofactor of the enzyme and the conclusions obtained after analysing the effect of its proposed inhibitors. It also includes the development of several in vitro assays, which could potentially be used to screen libraries of compounds in search for modulators of the enzyme.

In many ways cancer is a disease of cellular signalling disequilibrium. When the equilibrium of key signalling pathways is upset, critical biological functions such as cell growth, survival, motility, proliferation, metabolism and apoptosis are affected, and can lead to the initiation of cancer. Reversible protein phosphorylation is an extremely important mechanism by which the activity of enzymes and proteins in signalling cascades can be regulated. Dual specificity phosphatases (DUSPs) are a unique subgroup of the protein tyrosine phosphatases (PTPs) in that they can dephosphorylate both phospho-tyrosine and phospho-serine/threonine residues within the one substrate. Many DUSPs have been implicated in cancer as critical regulators of key cancer- associated signalling cascades including the mitogen activated protein kinase (MAPK) pathway. Transcript profiling of 51 primary ovarian tumours and four normal ovaries as controls identified an uncharacterised atypical DUSP, DUSP26 as being potentially down-regulated in all histological subtypes of ovarian cancer compared with normal ovaries. DUSP26 is located at 8p12, a chromosomal region previously shown to exhibit allelic imbalance in ovarian cancer. DUSP26 is predominantly expressed in neuro-endocrine tissue, with high expression also in skeletal muscle, prostate and ovary. DUSP26 mRNA expression is reduced in brain cancer, neuroblastoma, and ovarian cancer cell lines compared to normal, consistent with a role for DUSP26 as a tumour suppressor gene. Furthermore, DUSP26 can negatively affect the proliferation of epithelial cells, also consistent with a role as a tumour suppressor gene. Expression of DUSP26 in primary ovarian cancer samples is variable however, and analysis of DUSP26 protein expression is required to reconcile these results. Preliminary results suggest that DUSP26 is epigenetically regulated and that hypermethylation may contribute to its silencing in cancer. In the literature, there is great controversy in regards to the substrate specificity of DUSP26. Results presented in this thesis conclusively demonstrate that DUSP26 is not a MAPK phosphatase, despite reports to the contrary. Instead, using a substrate trapping approach, two novel potential DUSP26 substrates were identified: DNA-dependent protein kinase (DNA-PK) and nuclear mitotic apparatus protein (NuMA), which are often dysregulated in cancer. Consequently, DUSP26 may affect the pathogenesis of cancer via DNA-PK and or NuMA.

Reversible protein phosphorylation is an essential regulatory mechanism used by eukaryotes to coordinate the biochemical processes of the cell. The PP2A phosphatase functions to dephosphorylate specific proteins originally targeted by stimulus-activated protein kinases. The identification of a sub-network surrounding the human PP2A-Striatin holoenzyme, termed STRIPAK, provides insight into novel mechanisms for PP2A function and regulation. I reveal that STRIPAK participates in at least two mutually-exclusive sub-complexes, one of which contains the putative cortactin-binding protein, CTTNBP2NL. I show that CTTNBP2NL is enriched at the actin cytoskeleton, likely in a STRIPAK-independent manner. This study also reveals that STRIPAK interacts with a subunit of the dynein motor, at least partially, through CTTNBP2NL. This work will serve as a platform for the structural and functional characterization of STRIPAK and will ultimately assist in defining novel mechanisms of regulation and function for the human PP2A phosphatase.

Reversible protein phosphorylation is an essential regulatory mechanism used by eukaryotes to coordinate the biochemical processes of the cell. The PP2A phosphatase functions to dephosphorylate specific proteins originally targeted by stimulus-activated protein kinases. The identification of a sub-network surrounding the human PP2A-Striatin holoenzyme, termed STRIPAK, provides insight into novel mechanisms for PP2A function and regulation. I reveal that STRIPAK participates in at least two mutually-exclusive sub-complexes, one of which contains the putative cortactin-binding protein, CTTNBP2NL. I show that CTTNBP2NL is enriched at the actin cytoskeleton, likely in a STRIPAK-independent manner. This study also reveals that STRIPAK interacts with a subunit of the dynein motor, at least partially, through CTTNBP2NL. This work will serve as a platform for the structural and functional characterization of STRIPAK and will ultimately assist in defining novel mechanisms of regulation and function for the human PP2A phosphatase.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / "May 2001." Typescript. Vita. Includes bibliographical references (leaves 143-156). Also available on the Internet.

published_or_final_version / Biological Sciences / Master / Master of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Protein phosphorylation is a central regulatory mechanism in countless cellular processes. Deletion of the PP1 serine/threonine phosphatase gene Ppp1cc in mice results in male infertility due to a severe impairment in spermatogenesis. This disruption in spermatogenesis is hypothesized to arise due to a deficiency of the testis specific Ppp1cc isoform PPP1CC2. To learn more about the function of PPP1CC2 in spermatogenesis, we have employed several proteomic approaches aimed at identifying both regulatory proteins and substrates that interact with PPP1CC2 in the testis. First, we created transgenic mouse embryonic stem cell lines expressing a tandem affinity tagged version of PPP1CC2. Tandem affinity purification using these cell lines identified a number of known PP1 interacting proteins, and one novel interactor DDOST (dolichyl-di-phosphooligosaccharide-protein glycotransferase) which we hypothesize to have a role in spermatogenesis. In a second approach, we conducted GST pull down assays from mouse testis lysates to identify PPP1CC2 interacting proteins. TSSK1 (testis-specific serine kinase 1) was identified as a novel PPP1CC2 interacting protein. We then demonstrated that TSSK1 interacts with PPP1CC2 in an indirect manner via a common interacting protein TSKS (testis-specific serine kinase substrate). Binding of TSKS to PPP1CC2 is regulated via phosphorylation of a PP1 docking motif on the TSKS surface, and localization of TSSK1 and TSKS in the testis is disrupted in Ppp1cc mutants. Finally, to identify candidate substrates of PPP1CC2 in the testis, we conducted a comparative phosphoproteomic analysis and identified 33 different peptides that were hyperphosphorylated in the testis of 3 week old Ppp1cc knockout mice. Amongst these candidate substrates are several proteins essential for mouse spermatogenesis—HMGA1 (high mobility group AT-hook 1), HSPA4 (heat shock protein 4), YBX2 (Y box protein 2) and SYCP2 (synaptonemal complex protein 2). Taken together, our results suggest that PPP1CC2 interacts with a number of different proteins in the testis, and is likely to play a role at several different stages of spermatogenesis, in both meiotic and post-meiotic spermatogenic cells.

Protein phosphorylation is a central regulatory mechanism in countless cellular processes. Deletion of the PP1 serine/threonine phosphatase gene Ppp1cc in mice results in male infertility due to a severe impairment in spermatogenesis. This disruption in spermatogenesis is hypothesized to arise due to a deficiency of the testis specific Ppp1cc isoform PPP1CC2. To learn more about the function of PPP1CC2 in spermatogenesis, we have employed several proteomic approaches aimed at identifying both regulatory proteins and substrates that interact with PPP1CC2 in the testis. First, we created transgenic mouse embryonic stem cell lines expressing a tandem affinity tagged version of PPP1CC2. Tandem affinity purification using these cell lines identified a number of known PP1 interacting proteins, and one novel interactor DDOST (dolichyl-di-phosphooligosaccharide-protein glycotransferase) which we hypothesize to have a role in spermatogenesis. In a second approach, we conducted GST pull down assays from mouse testis lysates to identify PPP1CC2 interacting proteins. TSSK1 (testis-specific serine kinase 1) was identified as a novel PPP1CC2 interacting protein. We then demonstrated that TSSK1 interacts with PPP1CC2 in an indirect manner via a common interacting protein TSKS (testis-specific serine kinase substrate). Binding of TSKS to PPP1CC2 is regulated via phosphorylation of a PP1 docking motif on the TSKS surface, and localization of TSSK1 and TSKS in the testis is disrupted in Ppp1cc mutants. Finally, to identify candidate substrates of PPP1CC2 in the testis, we conducted a comparative phosphoproteomic analysis and identified 33 different peptides that were hyperphosphorylated in the testis of 3 week old Ppp1cc knockout mice. Amongst these candidate substrates are several proteins essential for mouse spermatogenesis—HMGA1 (high mobility group AT-hook 1), HSPA4 (heat shock protein 4), YBX2 (Y box protein 2) and SYCP2 (synaptonemal complex protein 2). Taken together, our results suggest that PPP1CC2 interacts with a number of different proteins in the testis, and is likely to play a role at several different stages of spermatogenesis, in both meiotic and post-meiotic spermatogenic cells.