Heart disease is a complex and heterogeneous disease. Notably, studies have demonstrated gender differences in the expression and types of cardiovascular disease, such as dilated cardiomyopathy (DCM), a major underlying cause of heart failure. Previously we showed that loss of A-Kinase Anchoring Protein 13 (Akap13), a unique proto-oncogene and estrogen receptor modulator, resulted in enlarged embryonic hearts, defective cardiac sarcomere formation, and embryonic lethality in mice. Data have also shown cAMP-dependent Protein Kinase A (PKA) to be involved in DCM pathophysiology. Given the established role of AKAP13 in cell signaling, its ability to bind and modulate ligand-activated nuclear hormone receptors and transcription factors, and its association with actin and other cytoskeletal components, we hypothesized that a functional AKAP13 protein was required for cardiomyocyte function in the adult heart; defective function of AKAP13 could promote DCM. To this end, we established an inducible, cardiac-specific Akap13 conditional knockout (Akap13cKO) mouse model using a Cre-lox recombination strategy with two separate Cre-recombinase expressing mouse models (α-MHC-MerCreMer and Tnnt2-rtTA; TetO-Cre). Cardiac functional examination of Akap13cKO mice revealed significant biventricular dilated cardiomyopathy with compensatory hypertrophic remodeling of the left ventricle and left atrial enlargement, decreased left and right ventricular systolic function, and abnormal left ventricular diastolic function. Of note, female Akap13cKO mice displayed a more pronounced cardiac phenotype and were more likely to die post-recombination.

RasGAP is a GTPase activating protein (GAP) that deactivates Ras by promoting Ras-GTP hydrolysis to Ras-GDP. In Drosophila melanogaster, RasGAP is required for the long-term survival of neurons in the adult brain because mutants in the RasGAP gene (vap) show an age-related neurodegenerative phenotype, with dying neurons showing morphological features of autophagy. RasGAP was shown to have a GAP-independent role within fly neurons that is dependent on its SH2 domains. The aim of this study was to identify proteins that interact with the SH2 domains of RasGAP and to understand the roles of these proteins in neuronal survival. By using tagged RasGAP affinity purification and mass spectrometry of RasGAP protein complexes from S2 cells, Sprint, a Ras effector and putative activator of the endocytic GTPase Rab5, was identified as a novel SH2-dependent RasGAP interacting protein. The interaction between Sprint and RasGAP is phosphotyrosine-dependent, since it requires tyrosine phosphorylation of Sprint. In addition, Sprint and RasGAP interaction requires the SH2 domains of RasGAP but not Sprint or the conserved site of RasGAP tyrosine phosphorylation (pTyr363), indicating an association between these two molecules. RasGAP and Sprint co-localised with Rab5-positive early endosomes and this co-localisation depended on the SH2 domains of both RasGAP and Sprint. This study demonstrates a key role for this interaction in neurodegeneration: mutation of Sprint (or Rab5) suppressed the autophagic neuronal cell death caused by the loss of RasGAP. These results indicate that the long-term survival of adult neurons in Drosophila depends on a critical balance between Ras activation and endocytosis, and that this balance is maintained by the interplay between RasGAP and Sprint.

The molecular weight of the vasoactive intestinal peptide (VIP) receptor was assessed in bovine aorta, and rat liver, lung, and brain by covalent cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The receptor in all four tissues was found to be a single polypeptide of approximate Mr 54,000, contradicting previous claims for substantial heterogeneity in the molecular weight of this receptor. Guanine nucleotides inhibit cross-linking of 125I-VIP to its receptor, and cross-linking with ethylene glycolbis(succinimidylsuccinate) provides further evidence for complex formation between VIP, its receptor and a guanine nucleotide-binding regulatory protein (G-protein). The precise mechanism of receptor-G-protein coupling may differ between the aorta and other tissues.

No description available.

The Rho family of guanosine triphosphatases (GTPases) function as binary molecular switches, which play an important role in the regulation of actin cytoskeleton rearrangement and are involved in several critical cellular processes including cell adhesion, division and migration. Rho GTPases are specifically activated by their associated guanine nucleotide exchange factors (RhoGEFs). Dysregulation of RhoGEFs function through mutation or overexpression has been implicated in oncogenic transformation of cells and linked to several kinds of invasive and metastatic forms of cancer. T-cell lymphoma invasion and metastasis 1 (Tiam1) is a multi-domain Dbl family GEF protein and specifically activates Rho GTPase Rac1 through the catalytic Dbl homology and Pleckstrin homology (DH-PH) bi-domain. Previous works have shown that the nucleotide exchange function of the full-length Tiam1 is auto-inhibited and can be activated by N-terminal truncation, phosphorylation and protein-protein interactions. However, the molecular mechanisms of Tiam1 GEF auto-inhibition and activation have not yet been determined. In this study, the N-terminal PH-CC-Ex domain of Tiam1 is shown to directly inhibit the GEF function of the catalytic DH-PH domain in vitro. Using fluorescencebased kinetics experiments, we demonstrate that the auto-inhibition of Tiam1 GEF function occurs by a competitive inhibition model. In this model, the maximum velocity of catalytic activity remains unchanged, but the Michaelis-Menten constant of the auto-inhibited Tiam1 (the PH-PH fragment) on the substrate Rac1 is increased compared to the activated Tiam1 (the catalytic DH-PH domain alone). Through small angle X-ray scattering (SAXS), the structure of auto-inhibited Tiam1 (the PH-PH fragment) is shown to form a closed conformation in which the catalytic DH-PH domain is blocked by the N-terminal PH-CC-Ex domain. Taken together, these findings demonstrate the molecular mechanism of Tiam1 GEF autoinhibition in which the PH-CC-Ex domain of Tiam1 inhibits its GEF function by preventing the substrate Rho GTPase Rac1 from accessing the catalytic DH-PH bi-domain.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on Feb. 3, 2010). Includes bibliographical references.

The Rho family small GTPases are key regulators of the cytoskeleton, through which they impact and control many vital cellular functions, including growth, vesicle trafficking, intercellular junctions, transepithelial transport, migration, and gene transcription. Activation of Rho GTPases is induced by Guanine Nucleotide Exchange Factors (GEFs). We have previously shown that Tumour Necrosis Factor-α (TNF), plasma membrane depolarization, and immunosuppressive drugs activate RhoA through a specific exchange factor, GEF-H1. However, the question of whether other stimuli, such as hyperosmolarity, that activate RhoA, act through GEF-H1 and whether GEF-H1 activates other RhoGTPases was not known. The overall objective of this research project has been to gain insights into the complex mechanism through which the Rho GTPases, Rac and RhoA, are regulated in tubular cells. Specifically, we wished to explore the role and pathway-specific regulation of GEF-H1 in hyperosmotic stress- and TNF-induced signalling in tubular cells. In order to accomplish our goals, we optimized and used affinity precipitation assays to detect GEF-H1 activation (RhoA(G17A) and Rac(G15A)). We found that 1) GEF-H1 is activated by hyperosmotic stress and mediates the hyperosmolarity-induced RhoA activation, as well as nuclear translocation of the Myocardin-Related Transcription Factor (MRTF); 2) TNF induces activation of both Rac and RhoA through GEF-H1, but via different mechanisms. Epidermal Growth Factor Receptor (EGFR)- and Extracellular signal Regulated Kinase (ERK)-dependent phosphorylation at the Thr678 site of GEF-H1 is a prerequisite for RhoA activation only, while both Rac and RhoA activation require GEF-H1 phosphorylation on Ser885. Interestingly, Rac is required for TNF-induced RhoA activation. Together these findings highlight a role for GEF-H1 as an osmosensitive molecule that regulates cellular reprogramming through MRTF. Importantly, we have also uncovered a novel mechanism explaining hierarchical activation of Rac and RhoA by TNF. Such a mechanism could be key in coordinating GEF function and fine-tuning Rac and RhoA activation.

Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available in electronic form as viewed 2/16/2009.

Doctor of Philosophy / Biochemistry Interdepartmental Program / Qize Wei / This dissertation describes the role of myosin-interacting guanine nucleotide exchange factor (MyoGEF) and centrosome/spindle pole associated protein (CSPP) in mitotic progression and cytokinesis. We have identified three mouse isoforms of CSPP, all of which interact and colocalize with MyoGEF to the central spindle in anaphase cells. The N-terminus of MyoGEF interacts with myosin whereas the C terminus interacts with the N-terminus of CSPP, forming a complex. The N-terminus of CSPP appears to be important for both localization and interaction with MyoGEF. CSPP plays a role in mitotic progression since its depletion by RNAi resulted in metaphase arrest. MyoGEF is required for completion of cytokinesis. Both MyoGEF and CSPP are phosphorylated by mitotic kinases including Plk1 and Aurora. Importantly, MyoGEF is phosphorylated at Thr-574 in mitosis by Polo-like kinase 1, and this phosphorylation is required for activation of RhoA. Thr-543 of MyoGEF is required for Plk1 binding in mitosis and phosphorylation of MyoGEF by Cdk1/cyclinB, possibly at Thr-543 may generate a Plk1 docking site, i.e., Cdk1 can phosphorylate MyoGEF at Thr-543, thereby allowing Plk1 to bind and phosphorylate MyoGEF at Thr-574. Finally, MyoGEF and CSPP are also phosphorylated by Aurora-B kinase in vitro. Taken together, we propose that Aurora-B may phosphorylate and recruit MyoGEF and CSPP to the central spindle, where phosphorylation of MyoGEF at Thr-543 promotes Polo kinase binding and additional phosphorylation of MyoGEF, leading to the activation of RhoA at the cleavage furrow.

We have shown previously [Hosohata, K., Logan, J.K., Varga, E., Burkey, T.H., Vanderah, T.W., Porreca, F., Hruby, V.J., Roeske, W.R., Yamamura, H.I., 2000. The role of the G protein γ2 subunit in opioid antinociception in mice. Eur. J. Pharmacol. 392, R9-R11] that intracerebroventricular (i.c.v.) treatment of mice with a phosphorothioate oligodeoxynucleotide antisense to the γ2 subunit (Gγ2) of the heterotrimeric G proteins (antisense ODN) significantly attenuates antinociception by a δ-opioid receptor agonist. In the present study, we examined the involvement of Gγ2 in antinociception mediated by other (μ- or κ-opioid, cannabinoid, α2-adrenoreceptor) analgesic agents in a warm (55°C) water tail-flick test in mice. Interestingly, i.c.v. treatment with the antisense ODN attenuated antinociception by each analgesic agent. Missense phosphorothioate oligodeoxynucleotide treatment, on the other hand, had no effect on antinociception mediated by these agonists. The antinociceptive response recovered in 6 days after the last antisense ODN injection, indicating a lack of nonspecific tissue damage in the animals. These results suggest a pervasive role for the G protein γ2 subunits in supraspinal antinociception.