This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E16-08-0594) on July 12, 2017.
Abstract
Extracellular
netrin-1 and its receptor deleted in colorectal cancer (DCC) promote
axon branching in developing cortical neurons. Netrin-dependent
morphogenesis is preceded by multimerization of DCC, activation of FAK
and Src family kinases, and increases in exocytic vesicle fusion, yet
how these occurrences are linked is unknown. Here we demonstrate that tripartite motif
protein 9 (TRIM9)-dependent ubiquitination of DCC blocks the
interaction with and phosphorylation of FAK. Upon netrin-1 stimulation
TRIM9 promotes DCC multimerization, but TRIM9-dependent ubiquitination
of DCC is reduced, which promotes an interaction with FAK and subsequent
FAK activation. We found that inhibition of FAK activity blocks
elevated frequencies of exocytosis in vitro and elevated axon branching
in vitro and in vivo. Although FAK inhibition decreased soluble N-ethylmaleimide attachment protein receptor
(SNARE)-mediated exocytosis, assembled SNARE complexes and vesicles
adjacent to the plasma membrane increased, suggesting a novel role for
FAK in the progression from assembled SNARE complexes to vesicle fusion
in developing murine neurons.
Introduction
Introduction
During
development, extracellular axon guidance cues direct the extension and
branching of axons, essential for appropriate anatomy and function in
the adult brain. Disruption of axon extension and branching may lead to
the defective connectivity implicated in neurodevelopmental and
neuropsychiatric disorders (Engle, 2010 
; Grant et al., 2012 ).
In the mammalian neocortex, the secreted axon guidance cue netrin-1
promotes attractive guidance and axon branching through the receptor
deleted in colorectal cancer (DCC) (Kennedy and Tessier-Lavigne, 1995 ; Keino-Masu et al., 1996 ). Gene trap–mediated disruption or deletion of either Dcc or the gene encoding netrin-1 (Ntn1)
in mice leads to defects in fiber tracts in the forebrain, including
the corpus callosum and hippocampal commissure, and perinatal or
embryonic lethality (Serafini et al., 1996 ; Fazeli et al., 1997 ; Bin et al., 2015 ; Yung et al., 2015 ).
Netrin-dependent neuronal morphogenesis is preceded by several
subcellular events. In the presence of netrin-1, DCC is recruited to
clusters within the plasma membrane, where it homomultimerizes (Mille et al., 2009 ; Matsumoto and Nagashima, 2010 ; Wang et al., 2014 ; Gopal et al., 2016 ).
The noncatalytic cytoplasmic tail of DCC interacts with nonreceptor
tyrosine kinases, leading to their activation. These targets include FAK
and Src family kinases (SFKs), which are involved in cell adhesion,
migration survival, neuritogenesis, and axon outgrowth (Li et al., 2004 ; Liu et al., 2004 ; Meriane et al., 2004 ; Ren et al., 2004 ).
The molecular regulators that control and coordinate DCC relocalization
and downstream kinase activation are not fully elucidated.
; Grant et al., 2012 ).
In the mammalian neocortex, the secreted axon guidance cue netrin-1
promotes attractive guidance and axon branching through the receptor
deleted in colorectal cancer (DCC) (Kennedy and Tessier-Lavigne, 1995 ; Keino-Masu et al., 1996 ). Gene trap–mediated disruption or deletion of either Dcc or the gene encoding netrin-1 (Ntn1)
in mice leads to defects in fiber tracts in the forebrain, including
the corpus callosum and hippocampal commissure, and perinatal or
embryonic lethality (Serafini et al., 1996 ; Fazeli et al., 1997 ; Bin et al., 2015 ; Yung et al., 2015 ).
Netrin-dependent neuronal morphogenesis is preceded by several
subcellular events. In the presence of netrin-1, DCC is recruited to
clusters within the plasma membrane, where it homomultimerizes (Mille et al., 2009 ; Matsumoto and Nagashima, 2010 ; Wang et al., 2014 ; Gopal et al., 2016 ).
The noncatalytic cytoplasmic tail of DCC interacts with nonreceptor
tyrosine kinases, leading to their activation. These targets include FAK
and Src family kinases (SFKs), which are involved in cell adhesion,
migration survival, neuritogenesis, and axon outgrowth (Li et al., 2004 ; Liu et al., 2004 ; Meriane et al., 2004 ; Ren et al., 2004 ).
The molecular regulators that control and coordinate DCC relocalization
and downstream kinase activation are not fully elucidated.
We identified vertebrate TRIM9, an evolutionarily conserved class I tripartite motif (TRIM) protein (Berti et al., 2002 ; Tanji et al., 2010 ), as a key regulator of netrin-dependent morphogenesis in cortical and hippocampal neurons (Winkle et al., 2014 ; Winkle, Olsen, et al., 2016 ; Menon, Boyer, et al., 2015 ). The single invertebrate orthologues of Ntn1 and of Dcc regulate axon development through the single class I TRIM orthologue (Hao et al., 2010 ; Morikawa et al., 2011 ).
Mammalian TRIM9 directly interacts with the cytoplasmic tail of DCC,
the neuronal exocytic target (t)-SNARE SNAP25, and the filopodial actin
polymerase VASP (Li et al., 2001 ; Winkle et al., 2014 ; Menon, Boyer, et al., 2015 ). Genetic deletion of murine Trim9
in cortical neurons is associated with a loss of netrin-1
responsiveness, elevated exocytosis, enhanced growth cone filopodial
stability in vitro, and defects in axon branching and axon projections
in vitro and in vivo (Winkle et al., 2014 ; Winkle, Olsen, et al., 2016 ; Menon, Boyer, et al., 2015 ).
However, whether TRIM9 plays a role in DCC localization or
FAK-dependent intracellular signal transduction downstream of
DCC/netrin-1 remains unknown.
; Tanji et al., 2010 ), as a key regulator of netrin-dependent morphogenesis in cortical and hippocampal neurons (Winkle et al., 2014 ; Winkle, Olsen, et al., 2016 ; Menon, Boyer, et al., 2015 ). The single invertebrate orthologues of Ntn1 and of Dcc regulate axon development through the single class I TRIM orthologue (Hao et al., 2010 ; Morikawa et al., 2011 ).
Mammalian TRIM9 directly interacts with the cytoplasmic tail of DCC,
the neuronal exocytic target (t)-SNARE SNAP25, and the filopodial actin
polymerase VASP (Li et al., 2001 ; Winkle et al., 2014 ; Menon, Boyer, et al., 2015 ). Genetic deletion of murine Trim9
in cortical neurons is associated with a loss of netrin-1
responsiveness, elevated exocytosis, enhanced growth cone filopodial
stability in vitro, and defects in axon branching and axon projections
in vitro and in vivo (Winkle et al., 2014 ; Winkle, Olsen, et al., 2016 ; Menon, Boyer, et al., 2015 ).
However, whether TRIM9 plays a role in DCC localization or
FAK-dependent intracellular signal transduction downstream of
DCC/netrin-1 remains unknown.
Here we find that DCC is ubiquitinated in a TRIM9-dependent manner. Our
data are consistent with the hypothesis that DCC ubiquitination blocks
the activation of FAK and SFK in the absence of netrin-1. Following
netrin-1 stimulation, DCC ubiquitination is reduced, and TRIM9-dependent
clustering and multimerization of DCC occurs. FAK also becomes
phosphorylated and activated, and SNARE-mediated exocytosis and axon
branching increase. Inhibition of FAK activity blocks netrin-dependent
exocytosis and axon branching but, surprisingly, increases the number of
assembled SNARE complexes and the density of vesicles found immediately
adjacent to the plasma membrane. This suggests a novel requirement for
FAK activity in the progression from an assembled SNARE complex to
SNARE-mediated fusion, which is necessary for plasma membrane expansion
during axon branching.
..... etc.
..... etc.
TRIM9 sai nimen SPRING
J Biol Chem. 2001 Nov 2;276(44):40824-33. Epub 2001 Aug 27.
Spring, a novel RING finger protein that regulates synaptic vesicle exocytosis.
The
synaptosome-associated protein of 25 kDa (SNAP-25) interacts with
syntaxin 1 and vesicle-associated membrane protein 2 (VAMP2) to form a
ternary soluble N-ethylmaleimide-sensitive fusion protein attachment
protein receptor (SNARE) complex that is essential for synaptic vesicle
exocytosis. We report a novel RING finger protein, Spring, that
specifically interacts with SNAP-25. Spring is exclusively expressed in
brain and is concentrated at synapses. The association of Spring with
SNAP-25 abolishes the ability of SNAP-25 to interact with syntaxin 1 and
VAMP2 and prevents the assembly of the SNARE complex. Overexpression of
Spring or its SNAP-25-interacting domain reduces Ca(2+)-dependent
exocytosis from PC12 cells. These results indicate that Spring may act
as a regulator of synaptic vesicle exocytosis by controlling the
availability of SNAP-25 for the SNARE complex formation.
TRIM9 (Tripartite Motif Containing 9) is a Protein Coding gene.
Among its related pathways are Class I MHC mediated antigen processing and presentation and Innate Immune System.
GO annotations related to this gene include protein homodimerization activity and ubiquitin-protein transferase activity.
An important paralog of this gene is TRIM67.
- Geenitieto:
Entrez Gene Summary for TRIM9 Gene
-
The protein encoded by this gene is a member of
the tripartite motif (TRIM) family. The TRIM motif includes three
zinc-binding domains, a RING, a B-box type 1 and a B-box type 2, and a
coiled-coil region. The protein localizes to cytoplasmic bodies. Its
function has not been identified. Alternate splicing of this gene
generates two transcript variants encoding different isoforms. [provided
by RefSeq, Jul 2008]
GeneCards Summary for TRIM9 Gene
UniProtKB/Swiss-Prot for TRIM9 Gene
- E3 ubiquitin-protein ligase which ubiquitinates itself in cooperation with an E2 enzyme UBE2D2/UBC4 and serves as a targeting signal for proteasomal degradation. May play a role in regulation of neuronal functions and may also participate in the formation or breakdown of abnormal inclusions in neurodegenerative disorders. May act as a regulator of synaptic vesicle exocytosis by controlling the availability of SNAP25 for the SNARE complex formation.
- ( Tästä aiheesta on päivitys 25.4. 2018 ja enemmän suomesnosta)
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