Presence of up-stream and downstream components of a mitogen-activated protein kinase pathway in the PSD of the rat forebrain

doi:10.1016/S0006-8993(99)01762-X

Brain Research

Volume 840, Issues 1–2, 4 September 1999, Pages 36-44

https://doi.org/10.1016/S0006-8993(99)01762-X Get rights and content

Abstract

We previously reported the presence of Erk2 type mitogen-activated protein kinase (MAPK) and enrichment of its substrates in the post-synaptic density (PSD) fraction, and suggested a role for MAPK in the synaptic transmission and its modulation [Suzuki, T., Okumura-Noji, K., Nishida, E., ERK2-type mitogen-activated protein kinase (MAPK) and its substrates in post-synaptic density fractions from the rat brain, Neurosci. Res., 22 (1995) 277–285.]. In this paper, synaptic localization of the upstream and downstream components of a MAPK cascade was examined. We found that RSK1, Sos1, N-Shc 66 kDa, N-Shc 52 kDa, and Grb2 were present in the PSD fraction, and cPLA₂ was present in the synaptic plasma membrane fraction. RSK2, Sos2, and N-Shc 46 kDa were not present in the PSD fraction. Post-synaptic localization of RSK1 and Sos1 was confirmed by immunohistochemical examination at the electron microscopic level: the two immunoreactivities were localized in the PSDs, both in the spines and dendrites. These results suggest that all the MAPK cascade components examined were associated with PSD or the synaptic plasma membrane, suggesting the role(s) of the MAPK cascade for synaptic transmission and its regulation at post-synaptic sites.

Introduction

Mitogen-activated protein kinase (MAPK) phosphorylates a number of substrates in vitro and is related to a variety of cellular functions 1, 15. The role of MAPK in a certain cellular compartments may depend on specific substrate protein(s) co-localizing in that compartment. MAPK, as well as protein kinase A (PKA), protein kinase C (PKC), Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), and protein tyrosine kinase (TK), are believed to play a role in the modulation of neuronal signal transduction at synapses, especially at post-synaptic sites 19, 28, 38, 47. Specific MAPK isoform, Erk2, but not Erk1 localizes at the purified post-synaptic density (PSD) and Erk2 substrates are enriched in the preparation [56]. MAPKK (MEK) has also been detected in PSD fractions [56]and Raf, an upstream component of MAPKK, has also been localized in the PSD in vivo, which was confirmed immunohistochemically at the electron microscopic level 39, 40. These findings strongly suggest the roles of this kinase at the post-synaptic site. However, precise molecular events involving Erk2 at the sites have not yet been determined. We speculate that other MAPK pathway components, such as an upstream ras/MAPK pathway and downstream components of MAPK, are also localized at the same sites, forming a functional complex with Erk2 and its substrates.

In this paper, we examined the upstream and downstream components of the ras/MAPK cascade at the post-synaptic sites in the rat central nervous system. Synaptic localization of the components has not been investigated in detail before. Information on upstream and downstream post-synaptic MAPK is essential to fully understand post-synaptic molecular events related to MAPK.

Section snippets

Materials

Mouse monoclonal anti-RSK1 antibody (#R23820), rabbit polyclonal anti-Sos1 (Son of sevenless) and -Sos2 antibody (#S15530), mouse monoclonal anti-Grb2 antibody (#G16720), rabbit polyclonal anti-Shc antibody (#S14630), anti-N-Shc (ShcC) antibody (#SS5720), and mouse monoclonal anti-PSD-95 (#P43520) were purchased from Transduction Laboratories; rabbit polyclonal anti-RSK2 antibody (#06-300) was from Upstate Biotechnology; rabbit polyclonal anti-cPLA₂ antibody (N-216, #sc-438) and rabbit

Identification of various MAPK pathway components in the PSD fraction

We previously showed the presence of Erk2 type MAPK and its substrates in a PSD fraction [56]. In the first place we confirmed the result by using a different anti-MAPK antibody, which detected both Erk1 and Erk2. The PSD fraction contained exclusively Erk2, whereas the synaptosome fraction had both Erk1 and Erk2 (data not shown). We also confirmed the enrichment of Erk2 substrates in the PSD fraction by phosphorylation in the presence of purified Erk2 (data not shown).

We next examined other

Discussion

We showed in this paper the presence of components of the upstream or downstream TK/MAPK signaling cascade in the PSD. This finding, together with the presence of MAPK, MAPKK and Raf in the PSD fraction or PSD in vivo 21, 39, 56, suggest the functional significance of the MAPK system for the synaptic function.

Acknowledgements

This research was supported in part by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science and Culture, Uehara Memorial Foundation, and Toyota Physical and Chemical Research Institute.

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Research reportPresence of up-stream and downstream components of a mitogen-activated protein kinase pathway in the PSD of the rat forebrain

Abstract

Introduction

Section snippets

Materials

Identification of various MAPK pathway components in the PSD fraction

Discussion

Acknowledgements

Brain Res.

Neuron

J. Biol. Chem.

FEBS Lett.

J. Biol. Chem.

Trends Biochem. Sci.

J. Biol. Chem.

J. Biol. Chem.

Cell. Signalling

J. Biol. Chem.

J. Biol. Chem.

Neuroscience

J. Biol. Chem.

Cell

Mol. Brain Res.

Neuron

Neuron

Cell

J. Biol. Chem.

Neurosci. Lett.

Brain Res.

Brain Res.

Mol. Brain Res.

J. Biol. Chem.

J. Biol. Chem.

Trends Neurosci.

Neuron

Exp. Cell Res.

Biochem. Biophys. Res. Commun.

Neurosci. Res.

Neurosci. Res.

Brain Res.

Mol. Brain Res.

Brain Res.

Brain Res.

Trends Biochem. Sci.

Research report
Presence of up-stream and downstream components of a mitogen-activated protein kinase pathway in the PSD of the rat forebrain