Post-translational Modification of STEP61 by BDNF Signaling: Implications in Drug Abuse
STriatal-Enriched protein tyrosine Phosphatase 61 kDa (STEP61) normally opposes synaptic strengthening by dephosphorylation of regulatory tyrosine residues on its substrates. These substrates include GluN2B, GluA2, Fyn, Pyk2 and ERK1/2 (Snyder et al., 2005; Xu et al., 2009; Zhang et al., 2008, Zhang et al., 2011; Nguyen et al., 2002; Xu et al., 2012; Venkitaramani et al., 2009; Paul et al., 2003). All these molecules have been implicated in the neuroadaption that occurs in response to drugs of abuse (Cahill et al., 2014; Puhl et al., 2015). Recent studies have suggested a role of STEP61 in cocaine-seeking behaviors. Acute or long-term self-administration of cocaine in rodent models results in increased STEP61 protein and decreased Tyr phosphorylation of its substrates, suggesting over-activation of STEP61 upon cocaine treatment (Sun et al., 2013; Chiodi et al., 2014).
Brain-derived neurotrophic factor (BDNF) regulates synaptic strengthening and memory consolidation (Lu et al., 2008). Altered BDNF expression is implicated in drug-induced long-term neuroadaptation, which is often disrupted by psychostimulants such as cocaine (McGinty et al., 2010). Infusion of BDNF into the dorsomedial prefrontal cortex (dmPFC) immediately following a final session of cocaine self-administration blocked cocaine-induced decreases in GluN2B and ERK phosphorylation and attenuated relapse to cocaine seeking for as long as three weeks (McGinty et al., 2014). The recent findings that BDNF signaling leads to degradation of STEP61 suggest that STEP61 may play a role in this process (Saavedra et al., 2015; Xu et al., 2015). Understanding the molecular basis for the cross-talk between BDNF/TrkB signaling and STEP61 function will hopefully lead to better therapeutic strategies in treating drug abuse and addiction.
Specific Aim 1
Determine the residues involved in BDNF-induced phosphorylation of STEP61. We hypothesize that BDNF/TrkB/PLCγ signaling leads to a PKC-mediated phosphorylation of STEP61, which is required for its subsequent ubiquitination and degradation. We will perform PKC phosphorylation of STEP61 in vitro and determine the sites by mass spectrometry. Mutational analyses will confirm these sites in neuronal cultures.
Specific Aim 2
Determine the lysine residues involved in BDNF-induced ubiquitination of STEP61. We will treat rat cortical neurons with BDNF in the presence of the proteasome inhibitor MG-132. All STEP species will be pulled down by immunoprecipitation using anti-STEP antibody. Ubiquitinated STEP61 peptides will be enriched using anti-K-GG antibody after trypsin digestion. Lysine sites that are modified will be determined by mass spectrometry.
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