Novel Mechanisms Underlying the Regulation of Src Kinase in the NMDA Receptor Src Signaling Complex

As medicine progresses and the population ages neurodegenerative diseases, such as Parkinson's, Huntington's, and Alzheimer's disease, begin to become more and more prevalent. In order to treat these devastating disorders it is important to understand the mechanisms by which they occur. N-methyl-D-aspartate (NMDA) receptors are implicated in the cellular functions responsible for learning and memory and have surfaced as key players underlying various neurological disorders. The focus of this dissertation is to uncover novel mechanisms of NMDA receptor regulation through the regulation of the Src signaling complex. Previous studies have implicated C-terminal Src Kinase (Csk) in the regulation of a variety of NMDA receptor associated functions through Src family kinases (SFKs) and the Src signaling complex. Increases in Src activity are known to up-regulate NMDA functions and play important roles in many physiological and pathophysiological processes. Csk acts to constrain SFK activity by phosphorylating a regulatory tyrosine in the C-terminal tail of the kinase. Our group has previously shown that Csk can associate with the NR2A and NR2B subunits of NMDA receptors in a Src activity-dependent manner to serve as an intrinsic mechanism to provide a "brake" on the induction of long-term synaptic potentiation (LTP) mediated by NMDA receptors. In contrast to the NR2A and NR2B subunits no apparent tyrosine phosphorylation is found in the NR1 subunit of NMDA receptors. In this work, however, we report that Csk can also associate with the NR1 subunit in a Src activity-dependent manner. Furthermore, we found that truncation of the NR1 C-tail, or mutation of its single C-tail tyrosine (Y837), decreased the association of CSK with the NMDA receptor complex and blocked the inhibition of NMDA receptors observed during the intracellular application of Csk. These findings indicate that both the NR1 and NR2 subunits are required for the regulation of NMDA receptor activity by Csk. Src kinase has been shown to play a critical role as the primary regulator of NMDA receptor activity in the Src signaling complex. To further elucidate how Src is regulated within this complex we explored a stability-associated regulation of Src activity. Using a bacterial expression system we were able to purify a neuronal isoform of Src (n-Src). We additionally expressed and purified a constitutively active and an inactive form of n-Src. Characterization of these enzymes revealed that altering n-Src enzyme activity either pharmacologically (e.g., application of ATP or a Src inhibitor) or genetically (mutation of Y535 or K303) was consistently associated with changes in n-Src stability: an increase in n-Src activity was coupled with a decrease in n-Src stability and vice versa. Furthermore, alterations in n-Src stability (e.g., application of urea or arginine) were again coupled with the same changes in n-Src activity. These findings, therefore, indicate that n-Src activity and stability are interdependent. In vivo n-Src activity is modulated through internal interactions by its regulatory SH2 and SH3 domains. Previous studies have demonstrated that these domains act to stabilize the inactive kinase and help maintain the inactive state. In this work we report that the up-regulation of NMDA receptors induced by expression of constitutively active n-Src is significantly reduced by dysfunctions of the SH2 and SH3 domains of the protein. Furthermore, we found that dysfunctions of SH2 and SH3 domains reduce auto-phosphorylation of the kinase activation loop, depress kinase activity, and decrease NMDA receptor phosphorylation. The SH2 domain plays a greater role than the SH3 domain in the regulation of the active kinase. We additionally found that n-Src binds directly to the C-terminus of the NMDA receptors NR2A subunit in vitro, with a KD of 108.2 ± 13.3 nM. This binding is not Src kinase activity-dependent and dysfunctions of the SH2 and SH3 domains do not significantly affect the binding. These data indicate that the SH2 and SH3 domains may function to promote the catalytic activity of active n-Src in addition to constraining the activity of the inactive kinase. These regulatory functions are important in the regulation of activity-dependent neuroplasticity by NMDA receptors. The importance of NMDA receptors and the necessity for their proper regulation is demonstrated by the wide variety of disorders with which NMDA receptors are associated. In this work we have explored novel mechanisms of NMDA receptor regulation by the Src signaling complex, which is tightly controlled by the regulation of Src kinase itself both extrinsically and intrinsically. Exploration into the regulation of SFKs and the association of SFKs with NMDA receptors will bring with it a better understanding of synaptic plasticity as well as yield potential therapeutic strategies to treat the growing number of NMDA receptor associated disorders.