The Regulation of PREX2 by Phosphorylation

Phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3)-dependent RAC exchanger 2 (PREX2) is a guanine nucleotide exchange factor (GEF) for the Ras-related C3 botulinum toxin substrate 1 (RAC1) GTPase. As a GEF, PREX2 facilitates the exchange of GDP for GTP on RAC1. GTP bound RAC1 then activates its downstream effectors, including p21-activated kinases (PAK). PREX2, RAC1, and PAK kinases all have key roles within the insulin signaling pathway. The insulin receptor is a tyrosine kinase that phosphorylates the insulin receptor substrate (IRS) family of adaptor proteins, leading to the activation of phosphatidylinositide 3-kinase (PI3K) and the generation of PI(3,4,5)P3. PI(3,4,5)P3 then activates numerous downstream signaling proteins, including AKT and RAC1, to regulate several important cellular processes, such as glucose metabolism and cell proliferation. In addition to being a RAC1 GEF, PREX2 affects the insulin signaling pathway by inhibiting the lipid phosphatase activity of phosphatase and tensin homolog (PTEN), which dephosphorylates PI(3,4,5)P3 to antagonize PI3K. PREX2 is also important in cancer, which is likely a consequence of both its role as a RAC1 GEF and as a PTEN inhibitor. PREX2 GEF activity is activated by PI(3,4,5)P3 and by Gβγ, which is a heterodimer that is released after GPCR activation. However, PREX2 regulation within specific signaling pathways is poorly understood. This thesis aims to understand the regulation of PREX2 downstream of ligand binding to receptors on the cell surface, with a focus on insulin. This is achieved by studying the phosphorylation of PREX2 after insulin stimulation and by characterizing protein-protein interactions involving PREX2 and key proteins in the insulin signaling pathway. Herein, we identified PI(3,4,5)P3-dependent phosphorylation events on PREX2 that occur downstream of insulin stimulation. Phosphorylation of PREX2 also occurred downstream of Gβγ, suggesting that phosphorylation was associated with the activation of PREX2 GEF activity. Interestingly, phosphorylation of PREX2 reduced GEF activity towards RAC1 and a phospho-mimicking mutation of PREX2 at an insulin-mediated phosphorylation site reduced cancer cell invasion. Phosphorylation of PREX2 also decreased PREX2 binding to the cellular membrane, PI(3,4,5)P3, and Gβγ, providing a mechanism for reduced GEF activity. These data suggested that phosphorylation was part of a negative feedback circuit to decrease the RAC1 signal, which led to the identification of the PAK kinases as mediators of PREX2 phosphorylation. Importantly, insulin-induced phosphorylation of PREX2 was delayed compared to AKT, which is consistent with a model where PREX2 phosphorylation by PAK occurs after activation of PREX2 to attenuate its function. Altogether, we propose that second messengers activate the PREX2-RAC1 signal, which sets in motion a cascade whereby PAK kinases phosphorylate and negatively regulate PREX2 to decrease RAC1 activation. This type of regulation would allow for transient activation of the PREX2-RAC1 signal. We then asked whether PAK phosphorylation of PREX2 was altered in cancer. To do this, we analyzed four recurrent somatic PREX2 tumor mutations, R155W, R297C, R299Q, and R363Q. Interestingly, all four mutants had reduced insulin and PAK1 dependent phosphorylation, and R297C had lower levels of phosphorylation induced by PI3K activating tumor mutants. This suggests that tumors might be mutating PREX2 in order to avoid PAK mediated negative regulation of RAC1. Lastly, we characterized PREX2 interactions with proteins that are critical for insulin signaling, with a focus on the interaction between the PREX2 pleckstrin homology (PH) domain and PTEN. PREX2 inhibition of PTEN is mediated by the PH domain, and we discovered that the β3β4 loop of the PH domain was required for binding of the isolated PH domain to PTEN. We also found that PREX2 co-immunoprecipitates with other insulin related proteins, including the p85 regulatory subunit of PI3K, IRS4, and the insulin receptor. Taken together, the studies in this thesis solidify the role of PREX2 in insulin signaling by showing that PREX2 GEF activity is tightly regulated by insulin and PAK-induced phosphorylation and also by characterizing PREX2 interactions with critical insulin related proteins. Further, this PAK dependent negative regulatory circuit downstream of both PI(3,4,5)P3 and Gβγ activation of PREX2 could have impacts in many aspects of biology given the roles that PREX2 and RAC1 have in critical cellular functions such as cell motility and glucose metabolism, and in diseases such as cancer and diabetes

Akt Regulates TNFα Synthesis Downstream of RIP1 Kinase Activation during Necroptosis

Necroptosis is a regulated form of necrotic cell death that has been implicated in the pathogenesis of various diseases including intestinal inflammation and systemic inflammatory response syndrome (SIRS). In this work, we investigated the signaling mechanisms controlled by the necroptosis mediator receptor interacting protein-1 (RIP1) kinase. We show that Akt kinase activity is critical for necroptosis in L929 cells and plays a key role in TNFα production. During necroptosis, Akt is activated in a RIP1 dependent fashion through its phosphorylation on Thr308. In L929 cells, this activation requires independent signaling inputs from both growth factors and RIP1. Akt controls necroptosis through downstream targeting of mammalian Target of Rapamycin complex 1 (mTORC1). Akt activity, mediated in part through mTORC1, links RIP1 to JNK activation and autocrine production of TNFα. In other cell types, such as mouse lung fibroblasts and macrophages, Akt exhibited control over necroptosis-associated TNFα production without contributing to cell death. Overall, our results provide new insights into the mechanism of necroptosis and the role of Akt kinase in both cell death and inflammatory regulation

The integrated stress response remodels the microtubule-organizing center to clear unfolded proteins following proteotoxic stress

Cells encountering stressful situations activate the integrated stress response (ISR) pathway to limit protein synthesis and redirect translation to better cope. The ISR has also been implicated in cancers, but redundancies in the stress-sensing kinases that trigger the ISR have posed hurdles to dissecting physiological relevance. To overcome this challenge, we targeted the regulatory node of these kinases, namely, the S51 phosphorylation site of eukaryotic translation initiation factor eIF2α and genetically replaced eIF2α with eIF2α-S51A in mouse squamous cell carcinoma (SCC) stem cells of skin. While inconsequential under normal growth conditions, the vulnerability of this ISR-null state was unveiled when SCC stem cells experienced proteotoxic stress. Seeking mechanistic insights into the protective roles of the ISR, we combined ribosome profiling and functional approaches to identify and probe the functional importance of translational differences between ISR-competent and ISR-null SCC stem cells when exposed to proteotoxic stress. In doing so, we learned that the ISR redirects translation to centrosomal proteins that orchestrate the microtubule dynamics needed to efficiently concentrate unfolded proteins at the microtubule-organizing center so that they can be cleared by the perinuclear degradation machinery. Thus, rather than merely maintaining survival during proteotoxic stress, the ISR also functions in promoting cellular recovery once the stress has subsided. Remarkably, this molecular program is unique to transformed skin stem cells, hence exposing a vulnerability in cancer that could be exploited therapeutically

Akt Regulates TNF? Synthesis Downstream of RIP1 Kinase Activation during Necroptosis

Necroptosis is a regulated form of necrotic cell death that has been implicated in the pathogenesis of various diseases including intestinal inflammation and systemic inflammatory response syndrome (SIRS). In this work, we investigated the signaling mechanisms controlled by the necroptosis mediator receptor interacting protein-1 (RIP1) kinase. We show that Akt kinase activity is critical for necroptosis in L929 cells and plays a key role in TNF? production. During necroptosis, Akt is activated in a RIP1 dependent fashion through its phosphorylation on Thr308. In L929 cells, this activation requires independent signaling inputs from both growth factors and RIP1. Akt controls necroptosis through downstream targeting of mammalian Target of Rapamycin complex 1 (mTORC1). Akt activity, mediated in part through mTORC1, links RIP1 to JNK activation and autocrine production of TNF?. In other cell types, such as mouse lung fibroblasts and macrophages, Akt exhibited control over necroptosis-associated TNF? production without contributing to cell death. Overall, our results provide new insights into the mechanism of necroptosis and the role of Akt kinase in both cell death and inflammatory regulation

Regulation of PTEN Inhibition by the Pleckstrin Homology Domain of P-REX2 During Insulin Signaling and Glucose Homeostasis

Insulin activation of phosphoinositide 3-kinase (PI3K) signaling regulates glucose homeostasis through the production of phosphatidylinositol 3,4,5-trisphosphate (PIP3). The dual-specificity phosphatase and tensin homolog deleted on chromosome 10 (PTEN) blocks PI3K signaling by dephosphorylating PIP3, and is inhibited through its interaction with phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 2 (P-REX2). The mechanism of inhibition and its physiological significance are not known. Here, we report that P-REX2 interacts with PTEN via two interfaces. The pleckstrin homology (PH) domain of P-REX2 inhibits PTEN by interacting with the catalytic region of PTEN, and the inositol polyphosphate 4-phosphatase domain of P-REX2 provides high-affinity binding to the postsynaptic density-95/Discs large/zona occludens-1-binding domain of PTEN. P-REX2 inhibition of PTEN requires C-terminal phosphorylation of PTEN to release the P-REX2 PH domain from its neighboring diffuse B-cell lymphoma homology domain. Consistent with its function as a PTEN inhibitor, deletion of Prex2 in fibroblasts and mice results in increased Pten activity and decreased insulin signaling in liver and adipose tissue. Prex2 deletion also leads to reduced glucose uptake and insulin resistance. In human adipose tissue, P-REX2 protein expression is decreased and PTEN activity is increased in insulin-resistant human subjects. Taken together, these results indicate a functional role for P-REX2 PH-domain-mediated inhibition of PTEN in regulating insulin sensitivity and glucose homeostasis and suggest that loss of P-REX2 expression may cause insulin resistance

'Beer used to belong to older men': drink and authority among the Nyakyusa of Tanzania

In her last major work on social change among the Nyakyusa of southwestern Tanzania, Monica Wilson returned to an argument about the changing role of beer drinking which had featured in her earlier writings (1963: 176, 1977: 92-3, 131). She suggested that the selling of locally made grain beer for cash, and its consumption in commercial clubs, had played a major role in altering relationships between young and old, and men and women. Generational separation had broken down: young men had lost respect for elder men and women had acquired a new economic autonomy founded on the cash income they earned from selling beer. Beer thus lay at the heart of a pattern of social change (1977: 186). This article examines changes in authority and the drinking of beer in Nyakyusa society from the 1890s-when written records become available-to the end of the twentieth century. It argues that patterns of deference and authority have suffered less dislocation than Wilson suggested and that-while locally made grain beer has been physically transformed, has disappeared from certain public rituals of well-being and has come increasingly into the cash market-its commoditisation has not necessarily empowered women, nor has it eliminated the role of beer in patterns of behaviour and discourse which sustain elder men's claims to authority

A Molecular Switch between Mammalian MLL Complexes Dictates Response to Menin-MLL Inhibition

Menin interacts with oncogenic MLL1-fusion proteins, and small molecules that disrupt these associations are in clinical trials for leukemia treatment. By integrating chromatin-focused and genome-wide CRISPR screens with genetic, pharmacologic, and biochemical approaches, we discovered a conserved molecular switch between the MLL1-Menin and MLL3/4-UTX chromatin-modifying complexes that dictates response to Menin-MLL inhibitors. MLL1-Menin safeguards leukemia survival by impeding the binding of the MLL3/4-UTX complex at a subset of target gene promoters. Disrupting the Menin-MLL1 interaction triggers UTX-dependent transcriptional activation of a tumor-suppressive program that dictates therapeutic responses in murine and human leukemia. Therapeutic reactivation of this program using CDK4/6 inhibitors mitigates treatment resistance in leukemia cells that are insensitive to Menin inhibitors. These findings shed light on novel functions of evolutionarily conserved epigenetic mediators like MLL1-Menin and MLL3/4-UTX and are relevant to understand and target molecular pathways determining therapeutic responses in ongoing clinical trials

Measurement issues: measures of infant mental health

Background: Emotional and behavioural problems emerging in very young children can represent a challenge to the child and family and warrant early identification and appropriate support or intervention. Diagnostic systems are being developed that allow for specific difficulties to be identified and this review summarises them. The review describes the psychometric properties and potential for use in clinical practice of a range of instruments and methods that are available to identify infant mental health difficulties, and which may be suitable for use in primary care settings, including observations, questionnaires and checklists. Conclusions: While debate continues about whether infant mental health problems can or should be identified, the use of standardised tools may help clinicians to compare observations of infants so that those emerging as atypical can receive additional attention, reflecting a more targeted approach to primary care services (DH 2009; DH 2010)

The impact of immediate breast reconstruction on the time to delivery of adjuvant therapy: the iBRA-2 study

Background: Immediate breast reconstruction (IBR) is routinely offered to improve quality-of-life for women requiring mastectomy, but there are concerns that more complex surgery may delay adjuvant oncological treatments and compromise long-term outcomes. High-quality evidence is lacking. The iBRA-2 study aimed to investigate the impact of IBR on time to adjuvant therapy. Methods: Consecutive women undergoing mastectomy ± IBR for breast cancer July–December, 2016 were included. Patient demographics, operative, oncological and complication data were collected. Time from last definitive cancer surgery to first adjuvant treatment for patients undergoing mastectomy ± IBR were compared and risk factors associated with delays explored. Results: A total of 2540 patients were recruited from 76 centres; 1008 (39.7%) underwent IBR (implant-only [n = 675, 26.6%]; pedicled flaps [n = 105,4.1%] and free-flaps [n = 228, 8.9%]). Complications requiring re-admission or re-operation were significantly more common in patients undergoing IBR than those receiving mastectomy. Adjuvant chemotherapy or radiotherapy was required by 1235 (48.6%) patients. No clinically significant differences were seen in time to adjuvant therapy between patient groups but major complications irrespective of surgery received were significantly associated with treatment delays. Conclusions: IBR does not result in clinically significant delays to adjuvant therapy, but post-operative complications are associated with treatment delays. Strategies to minimise complications, including careful patient selection, are required to improve outcomes for patients