PKMζ, But Not PKCλ, Is Rapidly Synthesized and Degraded at the Neuronal Synapse

Palida, Sakina F.; Butko, Margaret T.; Ngo, John T.; Mackey, Mason; Gross, Larry A.; Ellisman, Mark H.; Tsien, Roger Y.

doi:10.1523/jneurosci.0004-15.2015

Cited by 30 publications

(42 citation statements)

References 51 publications

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“…The kinase PKMζ has been implicated in long-term memory maintenance and is upregulated following neuronal activity (Shao, et al, 2012), but the function and precise sub-synaptic localization of these new PKMζcopies is unclear. We fused PKMζcDNA to a TimeSTAMP reporter, TS:YSOG3 (Palida, et al, 2015) that contains both YFP and miniSOG and allows newly synthesized proteins to be labeled in a drug-dependent manner using the small molecule BILN-2061. New copies can be visualized by correlated light and electron microscopy in a manner similar to previous TimeSTAMP reporters incorporating a split YFP and miniSOG (Butko, et al, 2012) (Figure S5).…”

Section: Resultsmentioning

confidence: 99%

“…Cells were treated 48 hours after transfection with 2 μM Arg 10 -IRDye 700DX (prepared in distilled water) and added to the culture media for 2 hours at 37 °C, after which time the media was removed and cells were rinsed once in HBSS. Primary mouse cortical neurons were cultured, transfected with TS:YSOG3-PKMζ, and chemically stimulated with 50 μM forskolin and 0.1 μM rolipram for 10 min, then incubated with 1 μM BILN-2061 as previously described (Palida, et al, 2015). …”

Section: Methodsmentioning

confidence: 99%

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Multicolor Electron Microscopy for Simultaneous Visualization of Multiple Molecular Species

Adams

Mackey

Ramachandra

et al. 2016

Cell Chemical Biology

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Summary Electron microscopy (EM) remains the primary method for imaging cellular and tissue ultrastructure, although simultaneous localization of multiple specific molecules continues to be a challenge for EM. We present a method for obtaining multicolor EM views of multiple subcellular components. The method uses sequential, localized deposition of different lanthanides by photosensitizers, small molecule probes or peroxidases. Detailed view of biological structures is created by overlaying conventional electron micrographs with pseudocolor lanthanide elemental maps derived from distinctive electron energy-loss spectra (EELS) of each lanthanide deposit via energy-filtered transmission electron microscopy (EFTEM). This results in multicolor EM images analogous to multicolor fluorescence but with the benefit of the full spatial resolution of EM. We illustrate the power of this methodology by visualizing hippocampal astrocytes to show that processes from two astrocytes can share a single synapse. We also show that polyarginine-based cell-penetrating peptides enter the cell via endocytosis, and that newly synthesized PKMζ in cultured neurons preferentially localize to the post-synaptic membrane.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Multicolor Electron Microscopy for Simultaneous Visualization of Multiple Molecular Species

Adams

Mackey

Ramachandra

et al. 2016

Cell Chemical Biology

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“…The stability of PKMs has been an area of some controversy as they have been assumed to be quite stable for their role in the maintenance of memory, but some studies have shown relatively short half-lives (Palida et al 2015). In rodents and in Aplysia, PKM is stabilized through binding to the scaffold protein KIBRA, suggesting a model where correctly localized PKMs are stable, but free PKMs are degraded (Vogt-Eisele et al, 2014;Hu et al 2017b).…”

Section: Kibra Classical Calpain and Pkm Stabilitymentioning

confidence: 99%

The zinc fingers of the small optic lobes calpain bind polyubiquitin

Hastings

Qiu

Zha

et al. 2018

Journal of Neurochemistry

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The small optic lobes (SOL) calpain is a highly conserved member of the calpain family expressed in the nervous system. A dominant negative form of the SOL calpain inhibited consolidation of one form of synaptic plasticity, non-associative facilitation, in sensory-motor neuronal cultures in Aplysia, presumably by inhibiting cleavage of protein kinase Cs (PKCs) into constitutively active protein kinase Ms (PKMs) (Hu et al. 2017a). SOL calpains have a conserved set of 5-6 N-terminal zinc fingers. Bioinformatic analysis suggests that these zinc fingers could bind to ubiquitin. In this study, we show that both the Aplysia and mouse SOL calpain (also known as Calpain 15) zinc fingers bind ubiquitinated proteins, and we confirm that Aplysia SOL binds poly- but not mono- or diubiquitin. No specific zinc finger is required for polyubiquitin binding. Neither polyubiquitin nor calcium was sufficient to induce purified Aplysia SOL calpain to autolyse or to cleave the atypical PKC to PKM in vitro. In Aplysia, over-expression of the atypical PKC in sensory neurons leads to an activity-dependent cleavage event and an increase in nuclear ubiquitin staining. Activity-dependent cleavage is partially blocked by a dominant negative SOL calpain, but not by a dominant negative classical calpain. The cleaved PKM was stabilized by the dominant negative classical calpain and destabilized by a dominant negative form of the PKM stabilizing protein KIdney/BRAin protein. These studies provide new insight into SOL calpain's function and regulation. Open Data: Materials are available on https://cos.io/our-services/open-science-badges/ https://osf.io/93n6m/.

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“…The nascent PKMζ is then fully activated through co-translational phosphorylation by PDK1 (14) and stabilized by signaling mechanisms downstream of brain-derived neurotrophic factor (28) and by binding to the protein KIBRA (29). The newly synthesized, autonomously active PKMζ is then thought to be “tagged” to specific activated synapses, as shown by synaptic pathway–tagging experiments in hippocampal slices (30) and the translocation of newly synthesized PKMζ to postsynaptic sites during chemically induced LTP of primary cultured neurons (31). …”

Section: Pkmζ and Late Ltpmentioning

confidence: 99%

The genetics of PKMζ and memory maintenance

Sacktor

Hell

2017

Sci. Signal.

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Elucidating the molecular mechanisms that maintain long-term memory is a fundamental goal of neuroscience. Accumulating evidence suggests that persistent signaling by the atypical protein kinase C (PKC) isoform protein kinase Mζ (PKMζ) might maintain synaptic long-term potentiation (LTP) and long-term memory. However, the role of PKMζ has been challenged by genetic data from PKMζ-knockout mice showing intact LTP and long-term memory. Moreover, the PKMζ inhibitor peptide ζ inhibitory peptide (ZIP) reverses LTP and erases memory in both wild-type and knockout mice. Data from four papers using additional isoform-specific genetic approaches have helped to reconcile these conflicting findings. First, a PKMζ-antisense approach showed that LTP and longterm memory in PKMζ-knockout mice are mediated through a compensatory mechanism that depends on another ZIP-sensitive atypical isoform, PKCι/λ. Second, short hairpin RNAs decreasing the amounts of individual atypical isoforms without inducing compensation disrupted memory in different temporal phases. PKCι/λ knockdown disrupted short-term memory, whereas PKMζ knockdown specifically erased long-term memory. Third, conditional PKCι/λ knockout induced compensation by rapidly activating PKMζ to preserve short-term memory. Fourth, a dominant-negative approach in the model system Aplysia revealed that multiple PKCs form PKMs to sustain different types of long-term synaptic facilitation, with atypical PKM maintaining synaptic plasticity similar to LTP. Thus, under physiological conditions, PKMζ is the principal PKC isoform that maintains LTP and long-term memory. PKCι/λ can compensate for PKMζ, and because other isoforms could also maintain synaptic facilitation, there may be a hierarchy of compensatory mechanisms maintaining memory if PKMζ malfunctions.

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PKMζ, But Not PKCλ, Is Rapidly Synthesized and Degraded at the Neuronal Synapse

Cited by 30 publications

References 51 publications

Multicolor Electron Microscopy for Simultaneous Visualization of Multiple Molecular Species

Multicolor Electron Microscopy for Simultaneous Visualization of Multiple Molecular Species

The zinc fingers of the small optic lobes calpain bind polyubiquitin

The genetics of PKMζ and memory maintenance

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