<p dir="ltr">This dissertation delves into two research projects that aim to characterize post-translational modifications in two distinct proteins, each originating from a different species – one from the eukaryotic sea slug Aplysia californica and the other from the bacterial pathogen Bordetella bronchiseptica.</p><p dir="ltr">Aplysia have an unusually large neuron and therefore serve as an excellent model for studying cell signaling regulating neuronal chemotaxis. Cortactin is an actin binding protein that is regulated by post-translational modifications, including acetylation and phosphorylation. Studies have shown that Src2 tyrosine kinase phosphorylates cortactin to regulate lamellipodia protrusion and filopodia formation in Aplysia bag cell neurons. However, these in vivo phenotypes have not been tested mechanistically in vitro. To this end, the goal of my thesis work was to validate in vivo observations. The following work describes the methodology we developed to purify homogenous non-phosphorylated proteins. Our collaborative results show that Src2 phosphorylates cortactin at Y499, although Y505 is the preferred site in vitro.</p><p dir="ltr"> Filamentation induced by cAMP (Fic) proteins constitute a recently characterized family of enzymes that are being recognized to regulate diverse cellular processes in bacteria and metazoans. While Fic proteins predominantly utilize adenosine triphosphate (ATP) to post-translationally modify target proteins via a covalent addition of AMP, two Fic proteins have been reported that utilize uridine triphosphate (UTP) and cytidine diphosphate-choline (CDP-choline) to alter the activity of their target. In this dissertation, we report the discovery of the first guanosine triphosphate (GTP) specific Fic protein – BB0907 (BbFic) from Bordetella bronchiseptica. BbFic displays weak to no binding to ATP; instead has a 10-fold increased preferential usage for GTP. We identify key residues involved in GTP recognition. Additionally, sequence similarity network (SSN) analyses reveal that BbFic represents a distinct clade of Fic proteins, highlighting BbFic as a representative new class of guanylyltransferase. Our discovery adds to the functional diversity of the growing Fic protein family and frames the groundwork for understanding Fic-mediated GMPylation as a novel signaling paradigm. </p><p dir="ltr">Taken together, my thesis work provides novel insights into biological consequences of Fic-mediated GMPylation in bacteria and Src-mediated phosphorylation in filopodia formation.</p><p><br></p>