Year of Publication
Doctor of Philosophy (PhD)
Molecular and Cellular Biochemistry
Dr. Douglas A. Andres
The small GTP-binding protein Rad (RRAD, Ras associated with diabetes) is the founding member of the RGK (Rad, Rem, Rem2, and Gem/Kir) family that regulates voltage-dependent calcium channel function. Given its expression in both excitable and non-excitable cell types, the control mechanisms for Rad regulation and the potential for novel functions for Rad beyond calcium channel modulation are open questions. Here we report a novel interaction between Rad and Enigma, a scaffolding protein that also binds to the E3 ubiquitin ligase Smad ubiquitin regulatory factor 1 (Smurf1). Overexpression of Smurf1, but not of a catalytically inactive mutant enzyme, results in ubiquitination of Rad and down regulation of Rad protein levels. The Smurf1-mediated decrease in Rad levels is sensitive to proteasome inhibition and requires the ubiquitination site Lys204, suggesting that Smurf1 targets Rad for degradation. Rad protein levels, but notably not mRNA levels, are increased in the hearts of Enigma-/- mice, leading to the hypothesis that Enigma may function as a scaffold to enhance Smurf1 regulation of Rad.
In addition to ubiquitination, phosphorylation of RGK proteins represents another potential means of regulation. Indeed, Rem phosphorylation has been shown to abolish calcium channel inhibition. We demonstrate that b-adrenergic signaling promotes Rad phosphorylation at Ser39. Rad Ser39 phosphorylation is correlated with a decrease in the interaction between Rad and the CaVb subunit of the calcium channel and an increase in Rad binding to 14-3-3. Interestingly, Enigma overexpression promotes an increase in Rad Ser39 phosphorylation as well. Despite an interaction between Enigma and the CaV1.2 calcium channel subunit, overexpression of Enigma had no effect on Rad-mediated channel inhibition. Thus, Rad Ser39 phosphorylation alters its association with the calcium channel, but its impact on calcium channel regulation has yet to be determined.
Finally, we report a novel function for Rad in the regulation of bone homeostasis. Rad deletion in mice results in a significant decrease in bone mass. Dynamic histomorphometry in vivo and primary calvarial osteoblast assays in vitro demonstrate that bone formation and osteoblast mineralization rates are depressed in the absence of Rad. Microarray analysis revealed that canonical osteogenic gene expression is not altered in Rad-/- osteoblasts; instead robust up-regulation of matrix Gla protein (MGP, +11-fold), an inhibitor of mineralization and a protein secreted during adipocyte differentiation, was observed. Strikingly, Rad deficiency also resulted in significantly higher bone marrow adipose tissue (BMAT) levels in vivo and promoted spontaneous in vitro adipogenesis of primary calvarial osteoblasts. Adipogenic differentiation of WT osteoblasts resulted in the loss of endogenous Rad protein, further supporting a role for Rad in the control of BMAT levels. These findings reveal a novel in vivo function for Rad signaling in the complex physiological control of skeletal homeostasis and bone marrow adiposity.
In summary, this dissertation expands our understanding of Rad regulation through identification of a novel binding partner and characterization of post-translational regulatory mechanisms for Rad function. This work also defines a new role for Rad that may not depend upon its calcium channel regulatory properties: regulation of the bone-fat balance. These findings suggest that the regulation of Rad GTPase is likely more complex than guanine nucleotide cycling and that functions of Rad in non-excitable tissues warrant further study.
Digital Object Identifier (DOI)
Withers, Catherine Nicole Kaminski, "RAD GTPASE: IDENTIFICATION OF NOVEL REGULATORY MECHANISMS AND A NEW FUNCTION IN MODULATION OF BONE DENSITY AND MARROW ADIPOSITY" (2017). Theses and Dissertations--Molecular and Cellular Biochemistry. 34.
Available for download on Friday, August 03, 2018