published_or_final_version / abstract / toc / Zoology / Doctoral / Doctor of Philosophy

Ca2+ functions as a second messenger in all eukaryotes. Such Ca2+ signaling is used to coordinate plant responses to numerous stimuli, both developmental and environmental. Ca2+ signals are often transduced via the action of the ubiquitous Ca2+ sensor calmodulin (CaM). CaM-dependent protein phosphorylation forms an important component of such signal transduction pathways, including that regulating the initiation and development of symbiotic rhizobial nodules in legumes such as soybean (Glycine max). To further understand the role of Ca2+/CaM during nodule organogenesis, a nodule cDNA expression library was screened using radiolabeled CaM as a probe to identify novel CaM-binding proteins (CaMBPs). This screen resulted in the identification of a previously uncharacterized receptor-like kinase, termed GmCaMK1. The CaM-binding domain (CaMBD) of GmCaMK1 is located in a 24 residue region of GmCaMK1, which overlaps with the subdomain XI of a conserved Ser/Thr kinase domain. This CaMBD bound CaM in a Ca2+-dependent manner, and with high affinity (Kd = 1.4 nM). Furthermore, two hydrophobic residues (V372 and L375) were identified as critical for GmCaMK1-CaM interaction. Recombinant GmCaMK1 exhibited protein kinase activity in vitro, with autophosphorylation activity unaffected by the presence or absence of Ca2+/CaM. GmCaMK1 expression is enriched in developing nodules and main roots, and highest expression level was observed in lateral roots. While the function of CaM-binding to GmCaMK1 remains unclear, the affinity and Ca2+-dependence of the GmCaMK1-CaM interaction strongly suggests that GmCaMK1 is a physiologically relevant CaM target. The Arabidopsis ortholog of GmCaMK1, AtCaMK1 also bound CaM when expressed as a recombinant protein. GmCaMK1 is part of a multi-member family in soybean, as are putative homologs across taxa, suggesting that this is a novel, conserved family of CaMBPs. / Thesis (Master, Biology) -- Queen's University, 2010-01-28 16:00:48.69

Thesis (Ph. D.)--University of Hawaii at Manoa, 1995. / Includes bibliographical references (leaves 118-133). / Microfiche. / xiii, 133 leaves, bound ill. 29 cm

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [69]-76).

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [67]-75).

Thesis (Ph. D.)--University of Chicago, Committee on Neurobiology, March 2000. / Includes bibliographical references. Also available on the Internet.

Universiẗat, Diss., 2005--Würzburg.

As the genetic causes of Hypertrophic Cardiomyopathy (HCM) have become widely recognized, considerable lag in the development of targeted therapeutics has limited interventions to symptom palliation. This is in part due to an oft-noted finding that similar point mutations within myofilament proteins are known to cause differential disease severity, highlighting the need to understand disease progression at the molecular level. One commonly described pathway in HCM progression is calcium homeostasis dysregulation, albeit little is understood about disruption of the pathway. This dissertation investigated the calcium homeostasis of two clinically relevant murine models of HCM expressing similar point mutations within myofilament proteins. A mutation-specific alteration in the calcium dissociation rate from the cardiac myofilament is proposed to as a primary mechanism of down-stream calcium disruption. Two modes of intervention in down-stream calcium homeostasis were tested to as a means of improving directed therapies in HCM progression. The clinically-utilized diltiazem hydrochloride, an L-type calcium channel blocker, revealed mutation-specific symptom palliation but an inability to target within the disease mechanism itself. Due to this insufficient response to diltiazem, we investigated the role of the calcium-dependent kinase, CaMKII, and its persistent (autonomous) activation resulting from calcium dysregulation. Partial inhibition of the autonomous activation of the kinase was shown to improve functional and morphological indices of failure in calcium-dependent HCM progression. Thus, we conclude a myofilament-linked derangement in calcium homeostasis that potentiates aberrant activation of CaMKII. Moreover, we position the kinase as a nodal point in disease progression and a potential therapeutic target for early, robust management of HCM in the clinical population.

The kisspeptin receptor (KISS1R), functioning as a metastasis suppressor and gatekeeper of GnRH neurons, is a potent activator of intracellular Ca2+. The surge in cytoplasmic Ca2+ mediates the exocytosis of GnRH from GnRH neurons. However, the regulatory processes which enable KISS1R to sense increasing intracellular Ca2+ and avoid Ca2+ excitotoxicity via a signalling off-switch mechanism remain unclear. This thesis provides evidence for the interaction between KISS1R and the Ca2+ regulated proteins of calmodulin (CaM), and αCa2+/CaM-dependent-protein kinase II (α-CaMKII). Binding of CaM to KISS1R was shown with three independent approaches. Firstly, cell-free spectrofluorimeter assays showed that CaM selectively binds to intracellular loop (IL) 2 and IL3 of the KISS1R. Secondly, KISS1R co-immunoprecipitation experiments identified ligand/Ca2+-dependent binding of KISS1R to HEK-293 endogenous CaM. Thirdly, confocal experiments showed CFPCaM co-localises with YFP-KISS1R. The functional relevance of CaM binding was examined with alanine substitution of critical residues of the CaM binding motifs in IL2 and IL3 of KISS1R. This approach revealed that the receptor activity (relative maximum responsiveness) was increased in the mutated residues of the juxtamembrane regions of IL3 and the N-terminus of IL2 relative to wild-type KISS1R. The Ca2+/CaM regulated αCaMKII was also found to interact with KISS1R by selectively phosphorylating T77 of IL1. Phosphomimetic mutations of T77 into E or D created a receptor that was unable to elicit inositol phosphate production upon ligand stimulation. Finally, in vivo studies using ovariectomised rats that were intracerebroventricularly administered with a cell-permeable αCaMKII inhibitor augmented the effects of kisspeptin ligand stimulation of plasma luteinizing hormone levels. Taken together, this thesis demonstrates that the KISS1R-G protein coupling is regulated by Ca2+-dependent CaM binding and αCaMKII-mediated KISS1R phosphorylation.

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