Expansion of a polyglutamine (polyQ) tract in the Huntingtin (Htt) protein causes Huntington's disease (HD), a fatal inherited neurodegenerative disorder. Loss of the normal function of Htt is thought to be an important pathogenetic component of HD. However, the function of wild-type Htt is not well defined. Htt is thought to be a multifunctional protein that plays distinct roles in several biological processes, including synaptic transmission, intracellular transport and neuronal transcription. Here, we show with biochemical and live cell imaging studies that wild-type Htt stimulates the transport of nuclear factor κ light-chain-enhancer of activated B cells (NF-κB) out of dendritic spines (where NF-κB is activated by excitatory synaptic input) and supports a high level of active NF-κB in neuronal nuclei (where NF-κB stimulates the transcription of target genes). We show that this novel function of Htt is impaired by the polyQ expansion and thus may contribute to the etiology of HD

Baquet

Bates

Bennett

Bhakar

Blahos

Caviston

Chen

Colin

Colomer

Dragatsis

Dunkley

Edoardo Marcora

Engelender

Fagerlund

Fridmacher

Gauthier

Graham

Gray

Gutekunst

Hanz

Harkiolaki

Hayden

Hering

Hickey

Hunt

Imarisio

Jordan

Joshi

Kaltschmidt

Kennedy

Korkin

Kornau

Leavitt

Lipsky

Marcora

Marini

Mary B. Kennedy

Mattson

Meffert

Menalled

Mikenberg

Mosammaparast

Otis

Patterson

Perlson

Shimojo

Simmons

Song

Strand

Suzuki

Takano

The Huntington's Disease Collaborative Research Group

Thompson

Tian

Towbin

Trushina

Velier

Wang

Wellmann

Zhang

Zuccato

English

PubMed

Marcora, Edoardo

Kennedy, Mary B.

Caltech Authors - Main

1 
 
Supplementary Material 
Results 
I-κB can be phosphorylated by CaMKII, a major protein kinase in the PSD 
The fact that NF-κB is present in the PSD and that the phosphorylated form of I-κB is enriched in 
the PSD (Fig. 4) suggests that the PSD may be a site at which NF-κB is preferentially activated in 
the synapse. Others have shown that activation of Ca
2+
/calmodulin-dependent protein kinase II 
(CaMKII), which is enriched in the PSD fraction (1), is necessary for the activation of neuronal 
NF-κB by synaptic activity (2). Although phosphorylation of I-κBα is believed to be carried out 
almost exclusively by the I-κB kinase (IKK), we could not detect IKK in the PSD fraction (data not 
shown). Therefore, we hypothesized that CaMKII may phosphorylate I-κBα and thus directly 
activate NF-κB in the PSD. To test this hypothesis, we performed an in vitro kinase assay using 
recombinant I-κBα and CaMKII purified from adult rat forebrains.  We found that CaMKII is able 
to phosphorylate I-κBα on serines 32 and 36 (Figure S1).  These findings suggest a model in 
which activation of neuronal NF-κB by synaptic activity is directly mediated by CaMKII via 
phosphorylation of I-κB in response to stimulation of glutamate receptors in the PSD and 
subsequent influx of calcium into the dendritic spine. Whether CaMKII phosphorylates I-κB 
in vivo, or acts through IKK (3) to mediate the activation of NF- κB remains an open question. 
2 
 
Materials and Methods 
CaMKII kinase assay 
CaMKII was purified from adult rat forebrains as described in Bennett et al. (4). Phosphorylation 
of recombinant full-length human GST-tagged I-κBα (sc-4094, Santa Cruz Biotechnology) by 
purified CaMKII was carried out in a reaction mix containing 50 mM Tris-HCl (pH 8.0), 10 mM 
magnesium chloride, 0.4 mM EGTA, 10 mM DTT, 30 µM ATP, 0.7 mM calcium chloride, 
10 µg/ml calmodulin, 3 µg of CaMKII, and 3 µg of I-κBα. Phosphorylation was initiated by 
addition of CaMKII and ATP to a 30 µl reaction mix prewarmed to 30°C for 2 min. The negative 
control reaction contained the same reagents, except that calcium chloride and calmodulin 
were omitted. The reaction was carried out for 2 min, stopped by addition of SDS-PAGE sample 
buffer, and processed for immunoblotting as described above. 
References 
1 Kennedy, M.B., Bennett, M.K. and Erondu, N.E. (1983) Biochemical and 
immunochemical evidence that the "major postsynaptic density protein" is a subunit of a 
calmodulin-dependent protein kinase. Proc. Natl. Acad. Sci. U.S.A., 80, 7357-7361. 
2 Meffert, M.K., Chang, J.M., Wiltgen, B.J., Fanselow, M.S. and Baltimore, D. (2003) NF-κ B 
functions in synaptic signaling and behavior. Nat. Neurosci., 6, 1072-1078. 
3 Hughes, K., Edin, S., Antonsson, A. and Grundstrom, T. (2001) Calmodulin-dependent 
kinase II mediates T cell receptor/CD3- and phorbol ester-induced activation of IkappaB kinase. 
J. Biol. Chem., 276, 36008-36013. 
4 Bennett, M.K., Erondu, N.E. and Kennedy, M.B. (1983) Purification and characterization 
of a calmodulin-dependent protein kinase that is highly concentrated in brain. J. Biol. Chem., 
258, 12735-12744. 
 
3 
 
 
Legends to Figures 
Figure S1: NF-κB can be directly activated by CaMKII. Representative immunoblot (n=2) of 
products of in vitro kinase reactions performed with purified CaMKII as kinase and recombinant 
I-κBα as substrate in the presence (+) and absence (-) of calcium/calmodulin (Ca
2+
/CaM). On the 
left; total I-κBα (red) and I-κBα-pS32 (green); on the right; I-κBα doubly phosphorylated on 
serine 32 and 36 (I-κBα-pS32+pS36, red) and I-κBα-pS32 (green). 
Figures 
 
Figure S1 
 


The Huntington's disease mutation impairs Huntingtin's role in the transport of NF-κB from the synapse to the nucleus

Crossref

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https://authors.library.caltech.edu/21194/5/ddq358.pdf

The Huntington's disease mutation impairs Huntingtin's role in the transport of NF-κB from the synapse to the nucleus

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