The specificity of PTPs towards their substrates arises through amino acid modules that target PTPs to cellular compartments, while additional motifs lead to their interactions with substrate proteins. As mentioned above, STEP, along with its closest relatives HePTP and PTP-SL, contain a KIM domain that is necessary for binding to MAPK family members ERK, p38α, and JNK (33
). All three of these PTPs dephosphorylate the regulatory tyrosine in the activation loop of MAPKs and thereby inactivate them (30
The ability of STEP to regulate ERK () has been shown in a number of studies. In corticostriatal cultures, ERK is rapidly activated (within 2 minutes) in response to glutamate stimulation, followed by a delayed inactivation of ERK to baseline phosphorylation levels by 20–30 minutes. This delayed inactivation of ERK is mediated by STEP through its delayed dephosphorylation within the KIM domain in response to NMDA receptor-dependent activation of calcineurin (30
) (). Thus, STEP acts to regulate the temporal profile of ERK activity, and consequently helps to control its translocation to the nucleus, and subsequent downstream nuclear signaling.
STEP dephosphorylates ERK, Fyn and the NMDA receptor complex
In a second study, STEP was found to play an important role in a signal transduction cascade that mediates the effects of psychostimulant drugs on ERK activation (32
). Psychostimulant drugs of abuse exert their addictive effects by increasing extracellular dopamine in the nucleus accumbens, where they likely alter the plasticity of corticostriatal glutamatergic transmission. Cocaine and amphetamine activate ERK in a subset of medium spiny neurons of the dorsal striatum and nucleus accumbens, through the combined action of NMDA and D1-dopaminergic receptors. The activation of ERK involves D1-dopamine receptor-dependent regulation of PKA, phosphorylation of the regulatory protein DARPP-32, inhibition of the serine/threonine phosphatase, PP-1, and inhibition of STEP. Thus, activation of ERK, by a protein phosphatase cascade, functions as a detector of coincidence of dopamine and glutamate signals converging on accumbens medium spiny neurons and is critical for long-lasting effects of drugs of abuse.
Recently, a series of in vivo
investigations directly tested the hypothesis that STEP might be involved in regulating synaptic plasticity (37
). ERK activation is required for the consolidation of many forms of long-term memory, including fear conditioning (38
). Mutations of PTPs in their catalytic domain create inactive variants that may be used as substrate-trapping proteins to identify potential substrates. Inactive PTPs bind to their substrates but do not release them, as release requires dephosphorylation of the target protein (39
). A substrate-trapping mutant of STEP46
was made by mutating a required cysteine in the catalytic domain to a serine. This STEP variant was made cell permeable by attaching a TAT-peptide to the N-terminus. It was infused into the lateral amygdala of rats to determine whether it would bind to ERK, disrupt ERK signaling, and thereby block consolidation of long-term memories after fear conditioning. Animals were trained on a standard protocol where a shock is paired with an acoustic cue. Short-term memory was not affected in these animals, implying that the substrate trapping TAT-STEP protein did not block the acquisition of this form of memory. However, 24 hours after fear conditioning, long-term memory was disrupted, indicating an effect on the consolidation of fear memories.
There were two striking observations in that study. The first was the rapidity of ERK activation after fear conditioning. Phosphorylated ERK (pERK) was detected in lateral amygdala neurons within five minutes of training, returned to baseline levels by 15 minutes, and then increased again by one hour. The initial activation of ERK is thought to occur through the convergence onto lateral amygdala neurons of auditory thalamic inputs in response to the conditioning stimulus (tone) and somatosensory thalamic inputs in response to the unconditioned stimulus (electrical foot shock). Both inputs are required for the establishment of LTP in the lateral amygdala and the consolidation of fear conditioning (40
Activation of ERK was followed within an additional few minutes by the de novo
translation of STEP (37
). The translation of STEP was blocked by anisomysin, not affected by actinomycin D, and blocked by inhibitors of MAPK. Importantly, neither shock alone nor tone alone led to ERK activation or STEP translation. Within minutes after the de novo
synthesis of STEP, pERK levels returned to baseline levels. These results support a feedback model by which STEP regulates the duration that ERK is active. Additional modulatory inputs are likely to be involved. For example, if a dopaminergic input arrives to these same neurons, then STEP will be phosphorylated and no longer interact with ERK, leading to a more persistent pERK signal. Additional studies are needed to determine whether the infused TAT-STEP that prevented the consolidation of fear conditioning did so through its ability to block ERK signaling only, or whether it also disrupts other components of synaptic plasticity, through the regulation of STEP substrates such as Fyn or NMDA receptors.
As was mentioned above, mutations of PTPs in their catalytic domain create inactive variants that may be used as substrate-trapping proteins. This type of inactive STEP protein was used to identify a second STEP substrate, the non-receptor tyrosine kinase Fyn () (22
). STEP interacts with Fyn through its KIM domain, although the first polyproline sequence present in STEP61
is also involved in Fyn binding (22
). Interestingly, the related tyrosine kinases, Src, Lyn and Pyk2, which are also present within the postsynaptic density, did not interact directly with STEP under the conditions used in this study (22
). Two tyrosine residues are phosphorylated in the Src family of non-receptor kinases, and the enzymatic activity of these proteins depends upon which tyrosine is phosphorylated. STEP specifically catalyzes the dephosphorylation of Tyr420
, leading to the inactivation of Fyn. Conversely, a second PTP (PTPα) dephosphorylates Tyr531
, and dephosphorylation of this residue activates Fyn (42
The NMDA receptor is a third potential STEP substrate. The NR1 subunit was initially shown to associate with STEP through co-immunoprecipitation experiments using hippocampal tissue (10
) and more recently it has been shown that NMDA receptor subunits and STEP interact directly (44
). STEP regulates NMDA receptor trafficking by controlling the level of tyrosine phosphorylation of the NR2B subunit (45
). Tyrosine phosphorylation of NR2B at Tyr1472
by Src-family members, including Fyn, is required for the movement of NMDA receptors into membranes (46
). Dephosphorylation of the NR2B subunit at that same residue leads to endocytosis of NMDA receptors through a clathrin- and adaptor protein-2-mediated mechanism (48
). Current studies are determining whether this is through the direct dephosphorylation of the NMDA receptor by STEP, an indirect effect through its ability to reduce Fyn activity and thus decrease NMDA Tyr1472
phosphorylation levels, or whether both mechanisms work together in a cooperative fashion ().
STEP activation may lead to abnormal NMDA receptor endocytosis in Alzheimer’s disease
An initial electrophysiological study looked at the ability of STEP to regulate NMDA receptor channel properties (10
). STEP affects the function of synaptic NMDA receptors in both spinal cord cultures and hippocampal CA1 pyramidal neurons. Exogenously applied STEP decreased the open probability and mean channel open time of NMDA receptors in single channel recording from excised patches of spinal cord neurons (10
). Furthermore, infusing a functionally inhibitory STEP antibody increased the NMDA receptor-mediated component of synaptic responses. Because NMDA receptors are critically important for the induction of LTP, it was important to examine the role of STEP in this form of synaptic plasticity (10
). Microinfusion of active STEP protein into the postsynaptic neuron blocked LTP induction at hippocampal Schaffer collateral CA1 synapses. Conversely, infusion of the functionally inhibitory antibody caused an increase in basal synaptic transmission, thereby occluding LTP induction. Thus, STEP appears to directly affect the conductance properties of NMDA receptors as well as regulating NMDA receptor trafficking, and together, these mechanisms oppose the development of synaptic plasticity.