Browsing by Subject "phosphorylation"
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Item Dopamine Signaling in Substantia Nigra and Its Impact on Locomotor Function-Not a New Concept, but Neglected Reality(MDPI, 2024-01-23) Salvatore, Michael F.The mechanistic influences of dopamine (DA) signaling and impact on motor function are nearly always interpreted from changes in nigrostriatal neuron terminals in striatum. This is a standard practice in studies of human Parkinson's disease (PD) and aging and related animal models of PD and aging-related parkinsonism. However, despite dozens of studies indicating an ambiguous relationship between changes in striatal DA signaling and motor phenotype, this perseverating focus on striatum continues. Although DA release in substantia nigra (SN) was first reported almost 50 years ago, assessment of nigral DA signaling changes in relation to motor function is rarely considered. Whereas DA signaling has been well-characterized in striatum at all five steps of neurotransmission (biosynthesis and turnover, storage, release, reuptake, and post-synaptic binding) in the nigrostriatal pathway, the depth of such interrogations in the SN, outside of cell counts, is sparse. However, there is sufficient evidence that these steps in DA neurotransmission in the SN are operational and regulated autonomously from striatum and are present in human PD and aging and related animal models. To complete our understanding of how nigrostriatal DA signaling affects motor function, it is past time to include interrogation of nigral DA signaling. This brief review highlights evidence that changes in nigral DA signaling at each step in DA neurotransmission are autonomous from those in striatum and changes in the SN alone can influence locomotor function. Accordingly, for full characterization of how nigrostriatal DA signaling affects locomotor activity, interrogation of DA signaling in SN is essential.Item Endothelin-1-Induced Signaling Involved in Extracellular Matrix Remodeling(2006-12-01) He, Shaoqing; Thomas Yorio; Neeraj Agarwal; Peter KoulenET-1-Induced Signaling in ECM Remodeling in Astrocytes. Shaoqing He, Department of Pharmacology & Neuroscience, University of North Texas Health Science Center, Fort Worth, TX 76107. ET-1 levels are elevated under pathophysiological conditions, including glaucoma, however, ET-1’s ocular functions are not fully documented. Therefore, ET-1-induced signaling and ECM remodeling in astrocytes and at the optic nerve head were determined in this study. Three signaling pathways, including ERK1/2, PKC, and P13 kinase, were involved in ET-1-medicated cell proliferation of U373MG astrocytoma cells. Blocking one of these pathways completely abolished cell proliferation. It appeared that ERK1/2 activation was involved, but was independent of PKC and P13 kinase activation by ET-1. It was also determined that the ETB receptor was the dominant receptor involved in ERK1/2 phosphorylation and cell proliferation. In addition, ERK1/2 phosphorylation was not transactivated by the EGF receptor by ET-1. The studies also indicated that there was no activation of c/nPKC, although PKC was involved in cell proliferation. In U373MG astrocytoma cells, MAPK-ERK, PKC and P13K pathways appear to exert their roles in parallel without a direct, apparent “cross-talk”. Based on the signaling pathways obtained from U373MG astrocytoma cells, the regulation of MMPs/TIMPs and fibronectin in ET-1-activated human optic nerve head astroctyes (hONAs) was also determined. ET-1 not only induced rapid phosphorylation of ERK1/2 and PKC βI/ βII/δ but also increased the activity of MMP-2 and the expression of TIMP=1 and 2. The activity of MMP-2 was enhanced in the presence of inhibitors of MAPK or PKC in hONAs, whereas the expression of TIMP-1 and 2 was abolished. ET-1 increased the soluble fibronectin (FN) expression as well as FN matrix formation, however, the expression and deposition of FN were MAPK- and PKC-independent, whereas expression and activity of MMps and TIMPs were MAPK- and PKC-dependent. Therefore, ET-1 shifted the balance of MMPs/TIMPs and substrates that altered the ECM composition and subsequently let to ECM remodeling in activated hONA cells. ET-1’s effects on ECM remodeling at the optic nerve head were also examined following intravitreal administration of ET-1 in rats. The increased expression of MMP-9 and collagen VI was detected in both ETB deficient rats and wildtype Wistar rats post ET-1 intravitreal injection for 2 and 14 days, whereas the deposition of FN and collagen IV was unchanged. There was no significant difference in staining of MMP-9 and collagen VI between ETB deficient rats and wildtype Wistar rats. In this study, ECM remodeling was demonstrated in rats injected with ET-1 into the vitreous. Such changes in the ECM seen in the current study provide additional insight into the mechanisms that might explain the glaucomatous changes observed in ET-1-injection or perfusion models. In summary, ET-1 not only activated several signaling pathways in cell proliferation of astrocytes, but also modulated the expression of ECM molecules in vitro and in vivo, indicating that ET-1 plays a regulatory role in ECM remodeling. These effects coupled with observations that ET-1 levels are elevated in glaucoma patients, suggests that ET-1 may be involved in glaucomatous optic neuropathy.Item Involvement of p70S6K in Cisplatin-Induced Cell Death(2008-05-01) Dhar, Rohini; Basu, Alakananda; Singh, Meharvan; Vishwanatha, JamboorRohini Dhar, Involvement of p70S6K in cisplatin-induced cell death. Doctor of Philosophy (Biochemistry and Molecular Biology) May 2008, 151 pp., 23 illustration, 3 tables, 153 references. Cisplatin is used for the treatment of solid tumors; however its success is often compromised due to relapse and chemoresistance. The purpose of this dissertation is to delineate the role of p70S6K in cisplatin-induced apoptosis. A comparison of p70S6K levels in H69 cells that acquired resistance to cisplatin (H69/CP) compared to parental H69 cells revealed that levels of phosphorylated p70S6K and S6 were elevated. Cisplatin treatment resulted in the activation of p70S6K and downregulation of total p70S6K. Inhibition of the phosphoinositide 3-kinase (P13K) pathway by itself augmented cisplatin-induced PARP cleavage and also blocked the phosphorylation of p70S6K. Inhibition of extracellular signal-regulated kinase (ERK) pathway however attenuated cisplatin-induced PARP cleavage. These results reveal that phosphorylation of p70S6K is associated with increased cisplatin resistance, and inhibition of P13K/ p70S6K pathway could reverse cisplatin resistance. We have found that cisplatin caused a time- and concentration-dependent downregulation of p70S6K. While the calpain and proteasome inhibitors had no effect on the downregulation of p70S6K, the broad specificity caspase inhibitor z-VAD-fmk (z-VAD) reversed p70S6K downregulation by cisplatin. Furthermore, the caspase-3 inhibitor and knockdown of caspase-3 prevented cisplatin-induced proteolytic cleavage of p70S6K. While, cisplatin failed to induce cleavage of p70S6K in MCF-7 cells that lack functional caspase-3, overexpression of caspase-3 in these cells resulted in cisplatin-induced cleavage of p70S6K. Thus, these results demonstrate that p70S6K is a novel substrate for caspase-3. Examination of the role of p70S6K in cisplatin-induced death shows that rapamycin in a pharmacological inhibitor of p70S6K, enhanced cisplatin-induced apoptosis in A549 cells. However, knockdown of p70S6K by siRNA resulted in a decrease in cisplatin-iduced apoptosis. In addition, caspase-3 mediated cleavage of p70S6K at the aspartic acid residue at the 393 position and site-directed mutagenesis of Asp393 to Ala resulted in protection against cisplating-mediated apoptosis. Interestingly, introduction of the N-terminal cleaved fragment [∆(394-525)] resulted in potentiation of cisplatin-induced apoptosis. These results suggest that the proteolytic cleavage of p70S6K by caspase-3 is important for cisplatin-induced apoptosis.Item Phosphorlyation of Annexin A2 is essential for its association with exosomes and for migration, invasion and proliferation in triple negative breast cancer(2018-12) Desai, Priyanka P.; Vishwanatha, Jamboor K.; Basha, Riyaz; Chaudhary, PankajExosomes are membrane enclosed small vesicles that range from 40-120 nm in size and participate in cell-cell communication by transferring proteins to other cells. Annexin A2 (AnxA2), a calcium-dependent phospholipid binding protein, is present on the surface of the exosomes. AnxA2 phosphorylation plays an essential role in many physiological conditions by forming a heterotetrameric complex with p11 or S100A10 on the cell surface. We demonstrate here that the phosphorylation at Tyrosine (Tyr)-23 in the N-terminal region of AnxA2 is consequential for its association with the cell surface. This association increases the migratory, invasive and proliferative capacity of MDA-MB-231 triple negative breast cancer (TNBC) cells. An increase in cell surface AnxA2 further leads to a stronger association of AnxA2 with the exosomal surface. We also demonstrate that AnxA2 enriched exosomes promote proliferative and invasive characteristics of a different recipient cell [CAL (Centre Antoine Lacassagne) - 148]. These results demonstrate that Tyr23 phosphorylation of AnxA2 is pivotal for its association with exosomes and for imparting more malignant characteristics to the other breast cancer cells. Thus, AnxA2 could be used as a targeting approach for developing a treatment of TNBC.Item Studies of Checkpoint Responses Caused by Endogenous Oxidative DNA Damage in DNA Repair Deficient Saccharomyces Cerevisiae(2008-12-01) Pawar, Vaibhav; Alvarez, Rafael; Jiang, Yiwei; Krishnamoorthy, Raghu R.Pawar, Vaibhav., Studies of checkpoint responses caused by endogenous oxidative DNA damage in DNA repair deficient Saccharomyces cerevisiae. Doctor of Philosophy (Biomedical Sciences), December 2008, 163 pp., 21 illustrations, 189 references. In this dissertation project, I aimed to study checkpoint response of stationary phase yeast to DNA damage caused by basal oxidative stress. My study was focused on the regulation of Rad53 phosphorylation in different repair deficient strains to yeast. Rad53 plays decisive roles in cell cycle progression, cell death and transcriptional regulation of repair proteins to a plethora of DNA insults, including oxidative DNA damage. Rad53 activity is upregulated by phosphorylation, generating Rad53 species of various degrees of phosphorylation. I have measured steady state levels of Rad53 phosphorlyation by western blotting following SDS-polyacrylamide gel electrophoresis at different intervals in stationary phase, in various mutant backgrounds. To address the possible contribution of different repair pathways to endogenous DNA damage, I utilized two different sets of DNA repair deficient strains such as those deficient in Base excision repair (BER) and nucleotide excision repair (NER), and other set was deficient in Ku protein and NER. Interestingly, in both BERNER and Yku70rad4 strains, Rad53 phosphorylation was evident in stationary phase that is after 2 days, 4 days and 6 days but not in logarithmic phase. This covalent modification disappears after phosphatase treatment. This Rad53 modification was absent in their respective rho0 mutants, which lack mitochondrial DNA, indicating involvement of mitochondrial ROS in this checkpoint response. We analyzed mutants of different checkpoint proteins for Rad53 phosphorylation. Exclusive involvement of Rad17, Rad50 and Mec1 kinase in Rad53 phosphorylation strongly suggests processed DNA double strand breaks as critical lesions in BERNER cells. Analysis of Yku70 and NER deficient strain showed involvement of ssDNA, which is most likely at telomeres. This study consents with the model of unrepaired oxidative base damage, which can accelerate the appearance of single stranded DNA in the vicinity of double strand breaks (DSBs) or at telomeres.Item The Effects of a 14-3-3 inhibitor peptide on cardiomyocyte hypertrophic gene expression(2006-05-01) Ellis, Joel James; Dillon, Glenn; Mallet, Robert T.; Shepard, AllanJoel James Ellis, The Effects of a 14-3-3 inhibitor peptide on cardiomyocyte hypertrophic gene expression. Doctor of Philosophy (Biomedical Sciences), May 2006, 164 pp, 2 tables, 29 illustrations, references, 117 titles. The myocyte enhancer factor-2 (MEF2) family of transcription factors regulates transcription of muscle-dependent genes in skeletal, smooth and cardiac muscle types. MEF2 is activated by calcium/calmodulin (CaM)-dependent protein kinases I and IV and silenced by CaM kinase IlδC. MEF2 is held inactive in the nucleus by class I histone deacetylases (HDAC4&5) until phosphorylated by either CaM kinase I or IV. This phosphorylation results in HDAC transport out of the nucleus via a 14-3-3-dependent mechanism, thereby freeing MEF2 to drive transcription. 14-3-3 proteins exist as homodimers, which are modulated by the phosphorylation of serines 60 and 65 in the dimerization region. In this study, a HIV TAT protein transduction domain (PTD) fused 14-3-3 peptide inhibitor was generated that is designed to prevent the dimerization of 14-3-3 proteins. The data presented demonstrates that the 14-3-3 inhibitor peptide freely enters cardiomyocytes and is not cytotoxic under culture conditions. The presence of this 14-3-3 inhibitor promotes nuclear localization of class II HDACs in the presence of hypertrophic stimuli. Moreover, the 14-3-3 inhibitor prevented dimerization of wild type 14-3-3β in ventricular cardiomyocytes. Finally, increased MEF2-dependent transcriptional activity, due to CaMKI, CaMKIV and PE, was effectively silenced by this 14-3-3 inhibitor in cardiomyocytes. Atrial natriuretic peptide (ANP) transcriptional activity was also pressed in the presence of the 14-3-3 inhibitor under these same conditions. Taken together, these data suggest that the 14-3-3 inhibitor peptide is able to affect dimerization of 14-3-3, revealing a key regulatory point in the signaling of cardiac hypertrophy. Information from these results may provide a promising point of therapeutic intervention in the progression of heart disease due to cardiomyocyte hypertrophy.