Post-translational Modification of STEP61 by BDNF Signaling: Implications in Drug Abuse

Jian Xu, Child Study Center, Yale University


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.


Cahill E, Salery M, Vanhoutte P, Caboche J (2014) Convergence of dopamine and glutamate signaling onto striatal ERK activation in response to drugs of abuse. Front Pharmacol 4:172.

Chiodi V, Mallozzi C, Ferrante A, Chen JF, Lombroso PJ, Di Stasi AM, Popoli P, Domenici MR (2014) Cocaine-induced changes of synaptic transmission in the striatum are modulated by adenosine A2A receptors and involve the tyrosine phosphatase STEP. Neuropsychopharmacology 39:569-578.

Lu Y, Christian K, Lu B (2008) BDNF: a key regulator for protein synthesis-dependent LTP and long-term memory? Neurobiol Learn Mem 89:312-323.

McGinty JF, Whitfield TW Jr, Berglind WJ (2010) Brain-derived neurotrophic factor and cocaine addiction. Brain Res 1314:183-193.

McGinty JF, Zelek-Molik A, Sun WL (2014) Cocaine self-administration causes signaling deficits in corticostriatal circuitry that are reversed by BDNF in early withdrawal. Brain Res pii: S0006-8993(14)01299-2.

Nguyen TH, Liu J, Lombroso PJ (2002) Striatal enriched phosphatase 61 dephosphorylates Fyn at phosphotyrosine 420. J Biol Chem 277:24274-24279.

Paul S, Nairn AC, Wang P, Lombroso PJ (2003) NMDA-mediated activation of the tyrosine phosphatase STEP regulates the duration of ERK signaling. Nat Neurosci 6: 34-42.

Puhl MD, Berg AR, Bechtholt AJ, Coyle JT (2015) Availability of N-Methyl-d-Aspartate Receptor Coagonists Affects Cocaine-Induced Conditioned Place Preference and Locomotor Sensitization: Implications for Comorbid Schizophrenia and Substance Abuse. J Pharmacol Exp Ther 353:465-470.

Saavedra A, Puigdellívol M, Tyebji S, Kurup P, Xu J, Ginés S, Alberch J, Lombroso PJ, Pérez-Navarro E (2015) BDNF Induces Striatal-Enriched Protein Tyrosine Phosphatase 61 Degradation Through the Proteasome. Mol Neurobiol doi:10.1007/s12035-015-9335-7. [Epub ahead of print]

Snyder EM, Nong Y, Almeida CG, Paul S, Moran T, Choi EY, Nairn AC, Salter MW, Lombroso PJ, Gouras GK, Greengard P (2005) Regulation of NMDA receptor trafficking by amyloid-beta. Nat Neurosci 8:1051-1058.

Sun WL, Zelek-Molik A, McGinty JF (2013) Short and long access to cocaine self-administration activates tyrosine phosphatase STEP and attenuates GluN expression but differentially regulates GluA expression in the prefrontal cortex. Psychopharmacology (Berl) 229:603-613.

Venkitaramani DV, Paul S, Zhang Y, Kurup P, Ding L, Tressler L, Allen M, Sacca R, Picciotto MR, Lombroso PJ (2009) Knockout of striatal enriched protein tyrosine phosphatase in mice results in increased ERK1/2 phosphorylation. Synapse 63:69-81.

Xu J, Kurup P, Bartos JA, Patriarchi T, Hell JW, Lombroso PJ (2012) Striatal-enriched protein-tyrosine phosphatase (STEP) regulates Pyk2 kinase activity. J Biol Chem 287: 20942-20956.

Xu J, Kurup P, Azkona G, Baguley TD, Saavedra A, Nairn AC, Ellman JA, Pérez-Navarro E, Lombroso PJ (2015) Down-regulation of BDNF in cell and animal models increases striatal-enriched protein tyrosine phosphatase 61 (STEP61 ) levels. J Neurochem doi:10.1111/jnc.13295. [Epub ahead of print]

Xu J, Kurup P, Zhang Y, Goebel-Goody SM, Wu PH, Hawasli AH, Baum ML, Bibb JA, Lombroso PJ (2009) Extrasynaptic NMDA receptors couple preferentially to excitotoxicity via calpain-mediated cleavage of STEP. J Neurosci 29: 9330-9343.

Zhang Y, Kurup P, Xu J, Anderson GM, Greengard P, Nairn AC, Lombroso PJ (2011) Reduced levels of the tyrosine phosphatase STEP block beta amyloid-mediated GluA1/GluA2 receptor internalization. J Neurochem 119:664-672.

Zhang Y, Venkitaramani DV, Gladding CM, Zhang Y, Kurup P, Molnar E, Collingridge GL, Lombroso PJ (2008) The tyrosine phosphatase STEP mediates AMPA receptor endocytosis after metabotropic glutamate receptor stimulation. J Neurosci 28:10561-10566.