Browsing by Author "Means, Anthony R"
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Item Open Access CaMKK2 Contributes to the Regulation of Energy Balance(2011) Lin, FuminThe incidence of obesity and associated diseases such as type 2-diabetes and hypertension has reached epidemic portions worldwide and attracted increased interest to understand the mechanisms that are responsible for these diseases. Obesity can result from excessive energy intake, and increasing evidence has emphasized the role of the central nervous system, especially the hypothalamus, in regulating food intake. White adipose, as a direct target of obesity and an important endocrine organ, also has long been a subject of scientific inquiry. AMPK, a conserved energy sensor, has been shown to play important roles in both the hypothalamus and adipose. Recently, CaMKK2 was shown to function as an AMPK kinase. I used intracerebroventricular cannulation as a means to acutely inhibit hypothalamic CaMKK2 with STO-609 and characterize the appetite change associated with loss of CaMKK2 function. Infusion of STO-609 in wild-type mice, but not CaMKK2-null mice, inhibited appetite and promoted weight loss consistent with reduced NPY and AgRP mRNA. Furthermore, intraperitoneal injection of ghrelin increased food intake in wild-type but not CaMKK2-null mice, and 2-DG increased appetite in both types of mice, indicating that CaMKK2 functions downstream of ghrelin to activate AMPK and upregulate appetite. As CaMKK2-null mice were protected from high-fat diet-induced obesity and diabetes, I performed a pair feeding experiment using a high-fat diet and demonstrated that protection of CaMKK2-null mice did not require reduced food consumption. Analysis of brown adipose tissue and metabolic analysis indicated that CaMKK2-null mice did not expend more energy than WT mice. Interestingly, we were surprised to find that CaMKK2-null mice had more adipose than wild-type mice when fed standard chow (5001). By real-time PCR and immunoblot, I identified CaMKK2 expression in preadipocytes and showed that it decreased during adipogenesis. I used STO-609 or shRNA to block CaMKK2 activity in preadipocytes, which resulted in enhanced adipogenesis and increased mRNA of adipogenic genes. I also identified AMPK as the relevant downstream target of CaMKK2 involved in inhibiting adipogenesis via a pathway that maintained Pref-1 mRNA. Consistent with the in vitro data, we further demonstrated that CaMKK2-null mice have more adipocytes but fewer preadipocytes, which supports our hypothesis that loss of CaMKK2 enhances adipogenesis by depleting the preadipocyte pool. Together the data presented herein contribute to our understanding of distinct mechanisms by which CaMKK2 contributes to feeding behavior and adipogenesis.
Item Open Access Distinct Functions and Regulation of Nonmuscle Myosin II Isoforms a and B in Cell Motility(2008-04-23) Sandquist, Joshua CThe ability of cells to migrate is of fundamental importance to a diverse array of biological processes, both physiological and pathological, such as development, the immune response and cancer cell metastasis, to name a few. The process of cell movement is a complicated cycle of coordinated steps involving dynamic and precise rearrangement of the actin-myosin cytoskeleton. As a critical component of the migration machinery, the molecular motor protein nonmuscle myosin II (myosin II) has long been a subject of scientific inquiry. It is now generally accepted that the contractile forces generated by myosin II contribute directly or indirectly to every step in migration. Interestingly, three isoforms of myosin II (myosin IIA, IIB and IIC) have been identified, and although each isoform performs the same basic molecular functions, recent findings suggest that the different myosin II isoforms make unique contributions to the motile process. In this dissertation work I used RNA interference technology to specifically deplete cells of myosin IIA and IIB in order to characterize the distinct migration phenotypes associated with loss-of-function of each individual isoform. Surprisingly, I found that the two myosin II isoforms perform not only distinct but opposing functions in cell migration, with myosin IIA and IIB normally inhibiting and facilitating proper cell movement, respectively. Furthermore, using pharmacological and microscopy techniques, I investigated the cellular mechanisms allowing for isoform-specific function. My results provide evidence for at least two isoform-specific regulatory mechanisms, namely selectivity in signaling pathways and subcellular distribution. A particularly significant finding is the identification of the different assembly properties of myosin IIA and IIB as the key element responsible for directing isoform-distinct distribution. Together the data presented herein represent a considerable advance in our understanding of the distinct functions and regulation of myosin II in cell motility.
Item Open Access Growth factor erv1-like modulates Drp1 to preserve mitochondrial dynamics and function in mouse embryonic stem cells.(Mol Biol Cell, 2010-04-01) Todd, Lance R; Damin, Matthew N; Gomathinayagam, Rohini; Horn, Sarah R; Means, Anthony R; Sankar, UmaThe relationship of mitochondrial dynamics and function to pluripotency are rather poorly understood aspects of stem cell biology. Here we show that growth factor erv1-like (Gfer) is involved in preserving mouse embryonic stem cell (ESC) mitochondrial morphology and function. Knockdown (KD) of Gfer in ESCs leads to decreased pluripotency marker expression, embryoid body (EB) formation, cell survival, and loss of mitochondrial function. Mitochondria in Gfer-KD ESCs undergo excessive fragmentation and mitophagy, whereas those in ESCs overexpressing Gfer appear elongated. Levels of the mitochondrial fission GTPase dynamin-related protein 1 (Drp1) are highly elevated in Gfer-KD ESCs and decreased in Gfer-overexpressing cells. Treatment with a specific inhibitor of Drp1 rescues mitochondrial function and apoptosis, whereas expression of Drp1-dominant negative resulted in the restoration of pluripotency marker expression in Gfer-KD ESCs. Altogether, our data reveal a novel prosurvival role for Gfer in maintaining mitochondrial fission-fusion dynamics in pluripotent ESCs.Item Open Access Identification and inhibitory properties of a novel Ca(2+)/calmodulin antagonist.(Biochemistry, 2010-05-18) Colomer, Josep; Schmitt, Allison A; Toone, Eric J; Means, Anthony RWe developed a high-throughput yeast-based assay to screen for chemical inhibitors of Ca(2+)/calmodulin-dependent kinase pathways. After screening two small libraries, we identified the novel antagonist 125-C9, a substituted ethyleneamine. In vitro kinase assays confirmed that 125-C9 inhibited several calmodulin-dependent kinases (CaMKs) competitively with Ca(2+)/calmodulin (Ca(2+)/CaM). This suggested that 125-C9 acted as an antagonist for Ca(2+)/CaM rather than for CaMKs. We confirmed this hypothesis by showing that 125-C9 binds directly to Ca(2+)/CaM using isothermal titration calorimetry. We further characterized binding of 125-C9 to Ca(2+)/CaM and compared its properties with those of two well-studied CaM antagonists: trifluoperazine (TFP) and W-13. Isothermal titration calorimetry revealed that binding of 125-C9 to CaM is absolutely Ca(2+)-dependent, likely occurs with a stoichiometry of five 125-C9 molecules to one CaM molecule, and involves an exchange of two protons at pH 7.0. Binding of 125-C9 is driven overall by entropy and appears to be competitive with TFP and W-13, which is consistent with occupation of similar binding sites. To test the effects of 125-C9 in living cells, we evaluated mitogen-stimulated re-entry of quiescent cells into proliferation and found similar, although slightly better, levels of inhibition by 125-C9 than by TFP and W-13. Our results not only define a novel Ca(2+)/CaM inhibitor but also reveal that chemically unique CaM antagonists can bind CaM by distinct mechanisms but similarly inhibit cellular actions of CaM.Item Open Access Identification of Novel Regulators in Hematopoiesis: Roles for Gfer in Hematopoietic Stem Cell Proliferation and CaMKK2 in the Restriction of Granulopoiesis(2011) Teng, Ellen ChaoHematopoiesis is the process in which billions of blood cells are produced on a daily basis, and is vital for sustaining life. This process is tightly regulated by a dynamic balance between hematopoietic stem cell (HSC) self-renewal and differentiation, and maintenance of this balance is of critical importance as dysregulation of HSCs can lead to hematopoietic deficiencies or malignancies such as leukemogenesis. While the signaling mechanisms that regulate HSC homeostasis and function are not well understood, our previous studies have identified a calcium/calmodulin (CaM)-dependent protein kinase, CaMKIV, that is intrinsically required for regulating normal proliferation and survival in HSCs. These findings suggest not only the importance of calcium-initiated pathways including CaMKIV-dependent signaling in hematopoietic cells, but also the potential for other calcium/CaM-dependent effector proteins such as other CaM-kinases to be involved in regulating HSCs and hematopoiesis.
The first major section of this dissertation work presented herein was based on the usage of RNA interference (RNAi) technology to specifically deplete HSCs of growth factor erv1-like (Gfer), a gene whose expression appeared to be absent in CaMKIV null HSCs based on comparative microarray analysis with wild-type HSCs, and seemed a potential target of CaMKIV. We showed that depletion of Gfer in HSCs compromised their in vivo engraftment potential and triggered a hyper-proliferative response that led to their exhaustion. We further assessed Gfer-depleted HSCs by using microscopy techniques and found that these cells possessed significantly reduced levels of the cyclin-dependent kinase inhibitor (CDKI) p27kip1. In contrast, ectopic over-expression of Gfer in HSCs resulted in significantly elevated total and nuclear p27kip1. We next performed immunoprecipitation-immunoblot analyses to determine whether alteration of Gfer levels would affect p27kip1's binding with its inhibitor, the COP9 signalosome subunit jun activation-domain binding protein 1 (Jab1), that would subsequently lead to its ubiquitination, and determined that depletion of Gfer resulted in enhanced binding of p27kip1 to Jab1. Conversely, over-expression of Gfer resulted in its enhanced binding to Jab1 and inhibition of the Jab1-p27kip1 interaction. Furthermore, normalization of p27kip1 in Gfer-KD HSCs rescued their in vitro proliferation deficits. These results provide evidence for a novel Gfer-Jab1-p27kip1 pathway present in HSCs that functions to restrict abnormal proliferation.
The second major section of this dissertation work describes our studies of a CaMKIV kinase, CaMKK2, and its role in HSCs and hematopoietic development. These studies were largely based on the usage of mice genetically ablated for the Camkk2 gene in the germline. Herein, we identified a role for CaMKK2 in the restriction of granulocytic fate commitment and differentiation of myeloid progenitor cells. We performed bone marrow transplantation studies and discovered that engraftment by Camkk2-/- donor cells resulted in the increased production of mature granulocytes in the bone marrow and peripheral blood. Similarly, we used fluorescence activated cell sorting (FACS) to determine that Camkk2-/- mice possessed elevated numbers of common myeloid progenitor cells, and exhibited an accelerated granulopoietic phenotype in the bone marrow. Expression of ectopic CaMKK2 in Camkk2-/- common myeloid progenitors was sufficient to rescue aberrant granulocyte differentiation, and when over-expressed in 32Dcl3 cells was also sufficient to impede granulocyte differentiation in a kinase activity-dependent manner. Collectively, our results reveal a novel role for CaMKK2 as an inhibitor of granulocytic fate commitment and differentiation in early myeloid progenitors.
While our original intent was to identify and link a downstream target and upstream kinase to CaMKIV in HSCs, our results ultimately did not suggest that Gfer or CaMKK2 function in the same pathway in HSCs as discussed in the following chapters. Nonetheless, our findings represent a considerable advance in identifying and characterizing the functions of two novel regulators, Gfer and CaMKK2, that are important for HSC proliferation and the commitment and early differentiation steps of granulopoiesis, respectively.
Item Open Access Pharmacological targeting of the mitochondrial phosphatase PTPMT1.(2009) Doughty-Shenton, DahliaThe dual specificity protein tyrosine phosphatases comprise the largest and most diverse group of protein tyrosine phosphatases and play integral roles in the regulation of cell signaling events. The dual specificity protein tyrosine phosphatases impact multiple cellular processes including mitogenesis, differentiation, adhesion, migration, insulin secretion and programmed cell death. Thus, the dysregulation of these enzymes has been implicated in a myriad of human disease states. While the large volume of genetic data that has become available following genome sequencing efforts over the last decade has led to the rapid identification of many new dual specificity protein tyrosine phosphatases, the elucidation of the cellular function and substrates of these enzymes has been much slower. Hence, there is a need for new tools to study the dual specificity protein tyrosine phosphatases and the identification of inhibitors of these enzymes is regarded as an attractive prospect, potentially affording not only new means of studying these enzymes, but also possible therapeutics for the treatment of diseases caused by their dysregulation. However, the identification of potent, selective inhibitors of the dual specificity protein tyrosine phosphatases has proven somewhat difficult. PTPMT1, Protein Tyrosine Phosphatase Localized to the Mitochondrion 1 is a recently discovered, mitochondrion-localized, dual specificity phosphatase which has been implicated in the regulation of insulin secretion. However, the details of the mechanism by which PTPMT1 impacts insulin secretion, as well as its substrate in the pancreatic β-cell, have yet to be uncovered. Thus, the identification of a potent, selective inhibitor of the enzyme would aid in further study of PTPMT1. This work describes the identification of such an inhibitor of PTPMT1 following an in vitro screen of small molecule, chemical compounds using an artificial substrate. Following the screen, the lead compound emerged as a potent and potentially selective inhibitor of PTPMT1 both in vitro and in cells. Studies using this compound have shown that the compound induces increased secretion of insulin in a dose-dependent manner and thus support the notion that PTPMT1 may serve as a potential target for the treatment of Type II diabetes.Item Open Access Prolyl Isomerase Pin1 Is a Conditional Tumor Suppressor(2011) Teng, Brian LewPin1 specifically binds to and catalyzes the cis-trans isomerization of phosphorylated-Ser/Thr-Pro motifs, which modulate the stability, localization, and function of numerous Pin1 substrates involved in cell cycle and tumorigenesis. During cell cycle progression, the timely synthesis and degradation of key regulatory proteins is required to maintain genomic integrity. Previously, we determined that Pin1 binds to and promotes the degradation of the oncoprotein c-Myc. Pin1 and the SCFCdc4 ubiquitin ligase recognize a “phosphodegron” sequence in c-Myc, which leads to its ubiquitination and degradation by the 26 S proteasome. Since cyclin E is another oncoprotein regulated by SCFCdc4, we hypothesized that Pin1 may also bind to and facilitate cyclin E turnover. Here we show that Pin1 binds to the cyclin E-Cdk2 complex in a manner that requires Ser384 of cyclin E, which is phosphorylated by Cdk2. The absence of Pin1 in mouse embryonic fibroblasts (MEFs) results in stabilization of cyclin E, and impairment of G1-S phase progression. Furthermore, deregulated cyclin E and c-Myc levels correlate with accelerated genomic instability in Pin1-/- MEFs, which results in sensitization of these cells to more aggressive Ras-dependent transformation and tumorigenesis.
However, the role of Pin1 in cancer is controversial as it has been proposed to conditionally promote or suppress tumorigenesis depending on the genetic context. In human cancer, Pin1 protein levels are frequently altered in several cancers. Interestingly, the PIN1 gene is located on chromosome 19p13.2, which is a region subject to loss of heterozygosity in several tumors. Since Pin1 protein is frequently under-expressed in kidney cancer, we tested the hypothesis that it may have a tumor suppressive role in human clear cell renal cell carcinoma (ccRCC). Here we show evidence for PIN1 gene deletion and mRNA under-expression as a mechanism of Pin1 reduction in ccRCC tumors. We demonstrate that restoration of Pin1 in cell lines found to be deficient in Pin1 protein expression can attenuate the growth of ccRCC cells in soft agar and a xenograft tumor model. Moreover, this ability of Pin1 to negatively influence tumor growth in ccRCC cells may be dependent on the presence of functional p53, which is infrequently mutated in ccRCC. These observations suggest Pin1 may function as a conditional tumor suppressor.
Item Open Access Regulation of CaMKKβ Dependent Signaling Pathways(2011) Green, Michelle FrancesCa2+/Calmodulin-dependent protein kinase kinase β(CaMKKβ) is a serine/threonine directed kinase which is activated following increases in intracellular Ca2+. CaMKKβ activates Ca2+/Calmodulin-dependent protein kinase I (CaMKI), Ca2+/Calmodulin-dependent protein kinase IV (CaMKIV), and the AMP-dependent protein kinase (AMPK) in a number of physiological pathways including learning and memory formation, neuronal differentiation, and regulation of energy balance. The purpose of the work presented in this dissertation is to better understand the regulation of CaMKKβ activity and specificity in CaMKKβ-dependent signaling cascades. First, the CaMKKβ-AMPK signaling complex is examined using biochemical assays. In both brain and cell lysates CaMKKβ and AMPK form a stable complex which can be examined by co-immunoprecipitation. This complex lacks the AMPKγ subunit and is not allosterically activated by adenosine 5'-monophohphate (AMP) binding. Using a series of CaMKKβ and AMPK mutants it was determined that the kinase domains of CaMKKβ and AMPK are necessary for their interaction and CaMKKβ must be active and bound to adenosine 5'-triphosphate (ATP) to form a complex with AMPK. However, CaMKKβ need not be active or bound to ATP to bind CaMKIV. This illustrates that the CaMKKβ-AMPK signaling complex differs from the CaMKKβ-CaMKIV signaling complex. These observations indicate that the CaMKKβ-AMPK signaling complex could be specifically targeted without effecting CaMKKβ-CaMKIV signaling.
Second, the regulation of CaMKKβ by multi-site phosphorylation is examined. Three phosphorylation sites in the N-terminus of CaMKKβ were identified by mass spectrometry which regulates its Ca2+/CaM-independent autonomous activity. The kinases responsible for these phosphorylations are identified as CDK5 and GSK3. These phosphorylation events are sequential with CDK5 priming for subsequent GSK3 phosphorylation. In addition to regulation of autonomous activity, phosphorylation of CaMKKβ regulates its half-life as determined in a radioactive pulse-chase assay. Examination of CaMKKβ in a cerebellar granule neuron model system demonstrates that CaMKKβ levels correlate with CDK5 activity and are regulated developmentally. In addition, appropriate phosphorylation of CaMKKβ is critical for its role in neurite development. These results reveal a novel regulatory mechanism for CaMKKβ-dependent signaling cascades.
Overall the work presented in this dissertation illustrates additional levels of regulation of CaMKKβ-dependent signaling pathways. In the future, these novel methods of CaMKKβ regulation will need to be considered when studying CaMKKβ-dependent signaling pathways.
Item Open Access Roles for Pin1 in Modulating Cells of the Innate Immune System(2011) Barberi, TheresaPin1 is a ubiquitously expressed phosphorylation-specific prolyl isomerase that regulates substrate function by catalyzing the cis-trans isomerization of prolyl bonds. Through this modulation, Pin1 has been shown to influence the stability, localization, and/or activity of a diverse set of protein substrates that participate in a variety of cellular responses, such as cell cycle progression, modulation of cell stress, and apoptosis. In addition to extensive studies in non-hematopoietic cells, Pin1 has also been shown to regulate immune cell function. Indeed, Pin1 participates in germinal center B cell development and eosinophil granulocyte survival. It also facilitates cytokine production in T cells, eosinophil granulocytes, and plasmacytoid dendritic cells. Through specific activities such as these, Pin1 has been demonstrated to modulate responses to viral challenge, respiratory allergens, and organ transplantation.
Due to previously described functions of Pin1 in regulating cells of both the innate and adaptive immune system, we predicted that Pin1 would participate in systemic inflammatory responses. Upon inducing systemic inflammation in mice, we observed a profound reduction in circulating cytokine concentrations in Pin1-null mice compared to WT mice. This result prompted further investigations, which are described in chapter 3 and chapter 4 of this dissertation. In chapter 3, we evaluate the potential contribution of macrophages to the defects we observe in LPS-challenged Pin1-null mice. Using primary macrophages, bone marrow-derived macrophages, and MEF, we ultimately exclude a role for Pin1 in modulating LPS-induced production of pro-inflammatory cytokines in these cells. In chapter 4, we uncover a defect in the accumulation of conventional dendritic cells (cDC) in LPS-challenged Pin1-null mice. Upon more careful examination of spleen cDC subsets in Pin1-null mice, we discovered a defect in the CD8+ subset. Experiments described in this chapter collectively indicate a role for Pin1 in preferentially modulating late stages of development of the CD8+ subset of cDC. Consistent with such a defect, the expansion of adoptively transferred WT CD8+ T cells was less robust in Pin1-null mice than WT mice upon infection with the bacterium Listeria monocytogenes . At the end of chapter 4, we provide evidence that Pin1 facilitates the degradation of the hematopoietic transcription factor PU.1, and propose that deregulation of PU.1 expression may be one mechanism by which Pin1 modulates CD8+ cDC development. The work described in this dissertation began by evaluating a potential role for Pin1 in modulating pro-inflammatory cytokine production in macrophages; ultimately, however, we uncovered a novel role for Pin1 in preferentially modulating the development of the CD8+ subset of cDC. The results presented herein expand the current understanding of DC development and further implicate Pin1 as an important modulator of both innate and adaptive immune responses.
Item Open Access SGC-CAMKK2-1: A Chemical Probe for CAMKK2.(Cells, 2023-01) Wells, Carrow; Liang, Yi; Pulliam, Thomas L; Lin, Chenchu; Awad, Dominik; Eduful, Benjamin; O'Byrne, Sean; Hossain, Mohammad Anwar; Catta-Preta, Carolina Moura Costa; Ramos, Priscila Zonzini; Gileadi, Opher; Gileadi, Carina; Couñago, Rafael M; Stork, Brittany; Langendorf, Christopher G; Nay, Kevin; Oakhill, Jonathan S; Mukherjee, Debarati; Racioppi, Luigi; Means, Anthony R; York, Brian; McDonnell, Donald P; Scott, John W; Frigo, Daniel E; Drewry, David HThe serine/threonine protein kinase calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) plays critical roles in a range of biological processes. Despite its importance, only a handful of inhibitors of CAMKK2 have been disclosed. Having a selective small molecule tool to interrogate this kinase will help demonstrate that CAMKK2 inhibition can be therapeutically beneficial. Herein, we disclose SGC-CAMKK2-1, a selective chemical probe that targets CAMKK2.