Taking STEPs forward to understand fragile X syndrome

Abstract

A priority of fragile X syndrome (FXS) research is to determine the molecular mechanisms underlying the functional, behavioral, and structural deficits in humans and in the FXS mouse model. Given that metabotropic glutamate receptor (mGluR) long-term depression (LTD) is exaggerated in FXS mice, considerable effort has focused on proteins that regulate this form of synaptic plasticity. STriatal-Enriched protein tyrosine Phosphatase (STEP) is a brain-specific phosphatase implicated as an "LTD protein" because it mediates AMPA receptor internalization during mGluR LTD. STEP also promotes NMDA receptor endocytosis and inactivates ERK1/2 and Fyn, thereby opposing synaptic strengthening. We hypothesized that dysregulation of STEP may contribute to the pathophysiology of FXS. We review how STEP's expression and activity are regulated by dendritic protein synthesis, ubiquitination, proteolysis, and phosphorylation. We also discuss implications for STEP in FXS and other disorders, including Alzheimer's disease. As highlighted here, pharmacological interventions targeting STEP may prove successful for FXS.

Figure 1. Schematic of STEP

To date, four alternatively-spliced variants of STEP (STEP₆₁, STEP₄₆, STEP₃₈, and STEP₂₀) and one calpain cleavage product (STEP₃₃) have been identified. The kinase-interacting motif (KIM) domain is essential for substrate binding, and the consensus protein tyrosine phosphatase (PTP) sequence, [I/V]HCxAGxxR[S/T]G, is required for phosphatase activity. Since STEP₆₁ and STEP₄₆ are the only two that contain both the KIM and PTP sequence, they are the only active forms of STEP. STEP₃₈ and STEP₂₀ do not contain the PTP sequence and are inactive variants of STEP with unknown function. It is possible that these two inactive isoforms function as dominant-negative variants that compete with active STEP variants for substrate binding, or they possess other functions yet to be discovered. A unique ten amino acid sequence at the C-terminus of STEP₃₈ and STEP₂₀ is introduced during splicing. A calpain cleavage site resides within the KIM domain between Ser²²⁴ and Leu²²⁵ which is utilized to generate STEP₃₃. Cleavage at this site disrupts the ability of STEP₃₃ to interact with its substrates. STEP₆₁ also has an additional 172 amino acids in its N-terminus which contains two transmembrane (TM) domains, two polyproline-rich (PP) regions, and an adjacent PEST sequence (not labeled). The TM regions target STEP₆₁ to the endoplasmic reticulum, as well as the post-synaptic density. Without these TM regions, STEP₄₆ is restricted to the cytosol. The PP regions impart substrate binding specificity. PKA phosphorylates STEP within the KIM domain (Ser²²¹ and Ser⁴⁹ on STEP₆₁ and STEP₄₆, respectively), as well as in the region adjacent to the PP regions (Ser¹⁶⁰ on STEP₆₁). Although the function of the additional phosphorylation site on STEP₆₁ remains unclear, current investigations are aimed at determining if phosphorylation at this or other sites is a signal for calpain-mediated cleavage and/or ubiquitination.

Figure 2. Regulation of STEP and its substrates by phosphorylation

In response to dopamine D1 receptor activation, PKA phosphorylation of STEP₆₁ at Ser²²¹ sterically prevents binding of STEP₆₁ to its substrates. In contrast, stimulation of NMDARs initiates calcium influx and activation of PP2B (calcineurin) and PP1 to dephosphorylate and activate STEP₆₁. When active, STEP dephosphorylates ERK1/2 and Fyn at their regulatory tyrosine residues, Tyr²⁰⁴ and Tyr⁴²⁰ (respectively), and inactivates them. STEP₆₁ regulates the phosphorylation of NR2B-containing NMDARs by two parallel mechanisms. First, when Fyn is inactivated by STEP₆₁, Fyn is unable to phosphorylate NR2B Tyr¹⁴⁷². Second, STEP₆₁ dephosphorylates NR2B Tyr¹⁴⁷² directly. Dephosphorylation of Tyr¹⁴⁷² promotes the interaction of NR2B with clathrin adaptor proteins and leads to endocytosis of these receptors. STEP₆₁ is also required for the internalization of GluR1/GluR2-containing AMPARs following mGluR stimulation. While the molecular mechanisms are still incompletely understood, STEP₆₁ appears to promote the endocytosis of AMPARs in a similar manner to NMDARs.

Figure 3. Mechanisms governing STEP protein expression and implications for Fragile X Syndrome

(A) In normal (or wild-type) neurons, brief stimulation of mGluR5 receptors with DHPG triggers translation of STEP mRNA, as well as translation of other mRNAs including APP and FMRP. STEP dephosphorylates a tyrosine residue on GluR2 and initiates endocytosis of AMPARs following DHPG treatment. FMRP associates with both STEP and APP mRNA and likely acts as a translation suppressor to prevent excessive translation of these mRNAs. Ubiquitination and degradation by the proteasome regulate STEP and FMRP protein levels. Upon stimulation of mGluRs with DHPG, FMRP is rapidly degraded by the proteasome, presumably to permit translation of FMRP targets and allow the expression of LTD. (B) In the absence of FMRP, STEP protein expression might be inappropriately elevated by two parallel pathways. First, without the suppression of STEP mRNA translation by FMRP, the steady-state translation rate of STEP would be upregulated. Similarly, translation of APP is increased in Fmr1 KO mice. Elevated levels of APP provide more targets for β- and γ-secretase-mediated cleavage and result in exacerbated Aβ production in aged Fmr1 KO mice. Given that Aβ inhibits the ubiquitin proteasome system (UPS) in Alzheimer’s disease, it is possible that the UPS is blocked in FXS later in life. Consequently, inhibition of UPS by Aβ could lead to reduced degradation of STEP. Elevated STEP levels in FXS could therefore maintain the persistent internalization of AMPARs and exaggerated mGluR-dependent LTD. Of note, for simplicity, NR2B-containing NMDARs, ERK1/2, and Fyn were removed from this figure; however, it is possible that these proteins would also be more dephosphorylated and inactivated in the presence of elevated STEP levels.