1 Department of Pediatric Oncology, Dana-Farber Cancer Institute and Harvard
Medical School, Boston, MA 02115, USA
2 Divison of Newborn Medicine, Children's Hospital, Boston, MA 02115, USA
* Author for correspondence (e-mail: david_rowitch{at}dfci.harvard.edu)
Accepted 30 September 2003
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SUMMARY |
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Key words: Cerebellum, Sonic hedgehog, Proliferation, GSK3, Medulloblastoma, Myc, Neural precursor, Mouse, Nmyc1 (N-myc)
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Introduction |
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Many well-characterized mitogens [e.g. insulin-like growth factor (IGF),
platelet-derived growth factor] signal through receptor tyrosine kinases
(RTKs) that lead to activation of the Ras-MAPK and
phosphatidylinositol-3-kinase (PI3K) pathways
(Marshall, 1995). By contrast,
Shh binding to its receptor Patched (Ptc) relieves inhibition of the signaling
pathway activated by the transmembrane protein Smoothened (Smo)
(Ho and Scott, 2002
),
resulting in activation of Smo target genes including Ptc and the
Gli family of transcription factors. However, several lines of
evidence suggest that RTK signaling might be synergistic with Shh in its
capacity to regulate proliferation of neuronal precursor cells. For example,
although treatment of with CXCL12 (also called SDF1; the cognate ligand of
CXCR4) is not mitogenic in primary cerebellar granule neuron precursor (CGNP)
cultures, it causes marked enhancement of Shh proliferative effects
(Klein et al., 2001
).
Additionally, essential roles for IGF2 signaling have been described during
Hedgehog-pathway-associated cerebellar tumorigenesis
(Hahn et al., 2000
).
The precursors for cerebellar granule neurons are generated in rhombomere 1
of the embryonic hindbrain and migrate dorsally to form the outer layer of the
cerebellum, or external granule layer (EGL). Proliferation of granule cells in
the EGL is largely postnatal in mammals
(Altman and Bayer, 1997).
Previous work has shown that Shh is required for granule cell precursor
proliferation (Ho and Scott,
2002
) and that Shh induces D-type cyclin expression
(Kenney and Rowitch, 2000
). In
previous work, we surveyed a range of known immediate early factors and
determined that proto-oncogene Nmyc1 was a direct target of Shh signaling in
proliferating CGNPs (Kenney et al.,
2003
). Nmyc1 (previously known as N-myc) is a member of the Myc
proto-oncogene family, which includes Myc and L-Myc. As heterodimeric
complexes with the Max protein, Myc family members behave as transcriptional
activators (Henriksson and Luscher,
1996
) and also have gene repression capabilities
(Wanzel et al., 2003
). Nmyc1
activity is necessary for CGNP proliferation
(Kenney et al., 2003
;
Knoepfler et al., 2002
) and
regulation of cyclinD1 and cyclinD2 expression during cerebellar development
(Ciemerych et al., 2002
). Gene
activation by Myc family members involves interactions with chromatin
remodeling machinery (Cole and McMahon,
1999
) and this aspect of Nmyc1 activity that is absolutely
required for CGNP proliferation (Kenney et
al., 2003
; Oliver et al.,
2003
). These studies indicate that Nmyc1 is a crucial determinant
of proliferation and growth arrest during central nervous system (CNS)
development.
Identification of the intracellular events that integrate effects of divergent signaling pathways is crucial for a comprehensive understanding of growth control in both normal and neoplastic neuronal progenitors. To better understand interactions between the Shh and PI3K pathways during neuronal precursor proliferation, we have focused on determinants of Nmyc1 protein turnover and cell cycle progression in primary CGNP cultures. Here, we report that phosphorylation promotes Nmyc1 protein turnover and timely cell cycle exit in CGNP primary cultures. Nmyc1 phosphorylation in CGNPs requires GSK3 activity and is antagonized by PI3K signaling but is regulated independent of Shh activity per se. Our findings provide a mechanism by which complex regulation of a single intracellular target can integrate the effects divergent signaling pathways active in the developing brain.
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Materials and methods |
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Mass spectrometry
HEK 293 cells were transiently transfected with Flag-tagged Nmyc1 in the
pWZL retroviral vector using Fugene 6 transfection reagent (Roche). 36 hours
after transfection, cells were lysed and Flag-Nmyc1 was immunoprecipitated
with anti-Flag-antibody-bound agarose beads (Sigma, M2) at 4°C overnight.
The protein was then eluted with 3x flag peptide (Sigma). The eluate was
separated by SDS-PAGE, and the Coomassie-stained band corresponding to Nmyc1
was excised, trypsinized and subjected to matrix-assisted laser
desorption/ionization time-of-flight (MALDI-TOF) analysis using Applied
Biosystems DE-STR mass analysis instrumentation. Further fragmentation of the
identified phosphorylated peptide at 1741.8544 Da was performed using tandem
electrospray mass spectrometry with ion-trap technology (Thermal Finnegan
LCQ-DECA).
Retroviral constructs
Flag-tagged wild-type Nmyc1 was cloned into the pWZL retroviral vector as
described (Kenney et al.,
2003). Nmyc1T50A, Nmyc1S54A, Nmyc1 T50E and Nmyc1 S54E were
prepared using the QuikChange site-directed mutagenesis kit (Stratagene), with
pWZL-Flag-Nmyc1 as template. Retroviral stock preparation and CGNP infection
were carried out as previously described
(Kenney et al., 2003
).
Western blotting and immunohistochemistry
To detect proteins by immunoblotting, non-denaturing lysates were prepared
from CGNPs as described (Kenney and
Rowitch, 2000). Immunoblots were incubated overnight at 4°C in
primary antibodies (Nmyc1, Santa Cruz sc-791; phospho-T58 Myc, CST 9401;
Cyclin D1, Neomarkers Ab-3; Cyclin D2, Santa-Cruz M-20; ß-tubulin, Sigma
T4026; phospho-GSK3ß, CST 9331; phospho-S473 Akt, CST 9271), then
developed using horseradish-peroxidase (HRP)conjugated anti-rabbit (Pierce) or
anti-mouse (Jackson) secondary antibodies and ECL reagents (Amersham).
Immunohistochemical analysis of cryosections of PN 7, 15 and 21 mouse
cerebella with polyclonal phospho-T58 Myc and monoclonal antibodies against
Calbindin (Sigma) and PCNA (Dako) was carried out using standard protocols
except that antigen retrieval was used. Immunofluorescence of anti-rabbit Cy3
and anti-mouse Cy2-conjugated secondary antibodies was visualized with a Nikon
E600 microscope and documented with a SPOT digital camera.
Northern blotting
Northern blotting was carried out as described
(Kenney et al., 2003).
Prime-It II kits (Stratagene) were used to label cDNA probes with
P32-dCTP. Nmyc1 cDNA probe was a gift from R. DePinho
(Dana-Farber Cancer Institute).
Transactivation assays
HeLa cells were seeded in 24-well plates at a density of
2x104 cells per well in 24-well format and grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum.
The following day, the cells were transfected with a mixture containing 20%
pGL3-hCDK4 promoter, 10% renilla luciferase construct and 70% effector
(pWZL-Nmyc/mutant constructs). Approximately 28 hours after transfection, the
cells were harvested and analysed by the dual-luciferase assay system. The
results are calculated as average fold activation relative to control pGL3-TK
and using renilla luciferase activity values to correct for transfection
efficiency. The experiments shown are averages from at least three independent
experiments with standard deviation of the mean indicated.
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Results |
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Myc, L-Myc and Nmyc1 all feature residues in the highly conserved
N-terminal Myc box 1 (MB1) domain, which could function as phosphate acceptor
sites (Fig. 1A). We first asked
whether Nmyc1 was phosphorylated in the N-terminal transactivation domain. We
overexpressed Flag-tagged Nmyc1 in HEK 293 cells, then affinity purified Nmyc1
and subjected the protein to analysis by mass spectrometry, a precise and
established means of determining peptide post-translational modifications
(Littlepage et al., 2002).
Analysis of peptides derived from Flag-tagged Nmyc1
(Fig. 1B) demonstrated
phosphorylation of the T50 and S54 residues of Nmyc1 Myc box 1.
|
Phosphorylation of Nmyc1 is required for CGNP cell cycle exit
To assess the impact of Nmyc1 phosphorylation on neuronal precursor
proliferation, we infected Shh-treated CGNP cultures with retroviruses
expressing wild-type Nmyc1, Nmyc1T50A or Nmyc1S54A, and then compared the
effects of these on cell cycle progression to green fluorescent protein
(GFP)-virus-infected controls. To determine whether, like Nmyc1, Nmyc1T50A and
Nmyc1S54A are sufficient for CGNP proliferation in the absence of Shh
signaling, we treated infected CGNPs with the protein kinase A activator
forskolin, an effective way of inhibiting Shh-induced CGNP proliferation
(Kenney et al., 2003;
Kenney and Rowitch, 2000
;
Wechsler-Reya and Scott,
1999
). As previously shown
(Kenney et al., 2003
), ectopic
expression of wild-type Nmyc1 maintains proliferation in CGNP cultures
independent of Shh signaling (Fig.
2A). We found that Nmyc1 phosphorylation site mutants were
similarly capable of maintaining proliferation in CGNP cultures despite
treatment with forskolin (Fig.
2A). Indeed, levels of BrdU incorporation in Nmyc1T50A- and
Nmyc1S54A-infected, but not wild-type Nmyc1-infected, cultures surpassed those
with Shh treatment alone. These results suggest that prevention of Nmyc1
phosphorylation at either site does not result in defective Nmyc1 protein and
might even enhance Nmyc1 function in CGNPs.
|
One possible explanation for these findings is that wild-type Nmyc1
but not the phosphorylation mutants undergoes progressive attenuation
of its ability to transactivate target genes necessary for CGNP maintenance in
the cell cycle. Recent biochemical and genetic evidence has implicated D-type
cyclins as likely Nmyc1 targets in CGNPs in vitro and in vivo
(Ciemerych et al., 2002;
Kenney et al., 2003
). We
therefore assessed levels of cyclins D1 and D2 in Shh-treated CGNPs infected
with viruses carrying wild-type Nmyc1 and the Nmyc1 mutants. As shown
(Fig. 2C), at 96 hours after
infection, all three Nmyc1 proteins tested promoted increased levels of cyclin
D1 and D2 relative to untreated, uninfected controls. Notably, wild-type Nmyc1
retains its capacity to upregulate cyclin D1 and cyclin D2 4 days after
infection despite the fact that levels of proliferation are not significantly
elevated. Oliver et al. (Oliver et al.,
2003
) similarly found that ectopic expression of D-type cyclins is
insufficient for CGNP proliferation. We conclude that the enhanced
proliferative effects of Nmyc1 mutant T50A and S54A proteins reflect
regulation of additional components of the cell cycle machinery beyond the
D-type cyclins.
Phosphorylation accelerates Nmyc1 turnover in CGNPs
The mechanism that promotes CGNP proliferation by nonphosphorylatable Nmyc1
mutants could involve activation of additional gene targets. It is also
possible that elevation of intracellular Nmyc1 protein levels per se and
consequent protein-protein interactions could regulate cell cycle progression.
To assess these possibilities further, we first asked whether mutation of
N-terminal phosphorylation sites affected the ability of Nmyc1 to
transactivate target genes. Myc family members activate target genes by
recognizing an E-box motif in the promoter. To determine effects of
phosphorylation site mutation on Nmyc1's transactivation capacity, we
transfected HeLa cells with a luciferase reporter under control of an
E-box-containing promoter, then compared the ability of Nmyc1, Nmyc1T50A and
Nmyc1S54A to transactivate this reporter. As shown in
Fig. 3A, we did not observe
differences between wild-type Nmyc1 and Nmyc1 phosphorylation mutants in terms
of their ability to activate transcription of a luciferase reporter,
suggesting that phosphorylation does not regulate Nmyc1 transactivation
capacity.
|
To determine whether the proliferative effects of Nmyc1 phosphorylation site mutations are restricted to CGNPs or might be observed in other cells of nervous system origin, we assessed the effects of Nmyc1, Nmyc1T50A and Nmyc1S54A expression on proliferation of human SK-N-SH neuroblastoma cells. As shown (Fig. 3C), the proportion of cells in S-phase in SK-N-SH cells infected with Nmyc1T50A or Nmyc1S54A was greater than that of cells infected with wild-type Nmyc1. Increased proliferation in cells infected with Nmyc1 phosphorylation mutants were also observed in Daoy cells, a poorly characterized medulloblastoma cell line (data not shown). Thus, inhibition of Nmyc1 phosphorylation at T50 or S54 increases the proliferative effect of Nmyc1 on several types of nervous-system-originating cells.
Transfer of a phosphate group to an amino acid residue results in the
placement of a negative charge at that site, as well as affecting higher-order
polypeptide structure. To investigate whether the addition of a negatively
charged amino acid would suffice to mimic Nmyc1 phosphorylation, we generated
retroviruses expressing non-phosphorylatable Nmyc1 mutants with aspartic acid
substitutions at positions 50 and 54 (Nmyc1T50E and Nmyc1S54E). We then
infected CGNP cultures with retroviruses expressing these mutants and compared
their stability with wild-type Nmyc1 using the cycloheximide pulse assay. We
found that replacement of S54 with E resulted in a protein with limited
cycloheximide resistance, similar to wild-type Nmyc1
(Fig. 3D), suggesting that the
mechanistic consequences of phosphorylation at this site occur principally as
a result of negative charge transfer. By contrast, Nmyc1T50E showed evidence
of greater cycloheximide resistance than wild-type Nmyc1
(Fig. 3D). We conclude that the
destabilizing effects of phosphorylation at Nmyc1 T50 occur in response to
information provided by the phosphate group in addition to the negative charge
it carries. Indeed, this has been previously reported for proteins whose
half-life is regulated by phosphorylation
(Lo et al., 2001).
Phosphorylation of Nmyc1 on T50 depends on GSK3
Several extracellular signals have been proposed to modulate proliferative
effects of Shh in CGNP cultures, including those that activate RTK, Notch and
chemokine receptors (Klein et al.,
2001; Solecki et al.,
2001
; Wechsler-Reya and Scott,
1999
). However, the precise intracellular mechanisms underlying
such interactions are unclear. Because Nmyc1 phosphorylation and degradation
are associated with CGNP cell cycle exit, we hypothesized that other signaling
pathways might synergize with mitogenic effects of Shh via inhibition of Nmyc1
N-terminal phosphorylation. We first asked whether Shh signaling itself might,
in addition to its roles in promoting Nmyc1 expression
(Kenney et al., 2003
),
regulate Nmyc1 phosphorylation and turnover. We assayed for effects of Shh
signaling on Nmyc1 phosphorylation on T50 by comparing the ratio of
T50-phosphorylated Nmyc1 to total Nmyc1 in Shh-treated CGNPs with that of
CGNPs when Shh signaling was blocked. To inhibit Shh signaling, we treated
CGNP cultures with cyclopamine and submitted protein lysates to western blot
analysis to determine the relative levels of wild-type and T50-phosphorylated
Nmyc1 (Fig. 4A). Because Nmyc1
mRNA and protein levels diminish when Shh signaling is inhibited
(Kenney et al., 2003
), we used
lactacystin [a proteosome inhibitor that blocks Nmyc1 degradation in
neuroblastoma cells (Bonvini et al.,
1998
)] to preserve detectable levels of endogenous Nmyc1 protein.
As shown in Fig. 4A, we
observed that the level of T50-phosphorylated Nmyc1 relative to total Nmyc1
was similar in the presence or absence of Shh signaling. Because T50
phosphorylation requires phosphorylation of S54
(Fig. 1C), Shh activity at that
site would be reflected in increased or decreased levels of T50
phosphorylation relative to total Nmyc1. Together, our results argue against a
role for Shh in the regulation of Nmyc1 phosphorylation in vitro.
|
Phosphorylation of Nmyc1 is regulated by PI3K signaling
If GSK3-mediated phosphorylation of Nmyc1 at T50 triggers its degradation,
it follows that increased activity of GSK would destabilize wild-type Nmyc1.
By contrast, based on our earlier observation
(Fig. 3), we predicted that the
stability of the nonphosphorylatable Nmyc1T50A and Nmyc1S54A would be
unaffected by increasing GSK3 activity. Phosphorylation of GSK3 by PKB/Akt
kinase causes GSK3 inactivation and PKB/Akt activity in CGNP cultures is
positively regulated by PI3K (Dudek et
al., 1997). We asked whether PI3K might stabilize Nmyc1 by
modulating GSK3. We used wortmannin, a PI3K inhibitor, to promote increased
GSK3 activity in CGNP cultures infected with vector carrying GFP, wild-type
Nmyc1, Nmyc1T50A or Nmyc1S54A, and then used western blotting to assay the
relative stability of the ectopically expressed Nmyc1 proteins. The
effectiveness of wortmannin was demonstrated by reduced levels of
phosphorylated (inactive) GSK3 in all of the treated samples
(Fig. 4C). After 3 hours of
wortmannin exposure, we found that levels of Shh-induced, endogenous Nmyc1
were substantially reduced (Fig.
4C, left). Likewise, retrovirally expressed wild-type Nmyc1
protein dropped in the presence of wortmannin
(Fig. 4C, right). These
findings suggest that increased GSK3 activity results in more rapid turnover
of wild-type Nmyc1. Although it is possible that decreased levels of wild-type
Nmyc1 in wortmannin-treated CGNP cultures could reflect reduced Nmyc1 protein
synthesis (Brown and Schreiber,
1996
), this is unlikely because wortmannin treatment had no effect
on levels of retrovirally expressed Nmyc1T50A and Nmyc1S54A proteins
(Fig. 4C, right). Indeed, these
results show that the ability to be phosphorylated at T50 (and S54) is a
requirement for Nmyc1 to be destabilized by PI3K pathway inhibition.
Previous work indicates that the PI3K pathway is required for granule cell
survival and proliferation (Dudek et al.,
1997). Activity of PI3K could regulate Nmyc1 turnover in response
to multiple factors found in the developmental milieu. Indeed, several
PI3K-activating pathways have been reported to enhance granule cell
proliferation in vivo and in vitro. These include signaling through SDF-CXCR4,
integrin receptors (which mediate CGNP cell-cell contacts) and IGF1
(Gao et al., 1991
;
Klein et al., 2001
;
Ye et al., 1996
). In cultured
CGNPs, signaling through the IGF receptor (IGFR) potently activates PI3K
(Dudek et al., 1997
).
CGNPs are normally cultured in N2 medium, which contains insulin at levels
high enough to activate the IGFR (Dudek et
al., 1997). To determine whether IGFR signaling stabilizes
endogenous Nmyc1, we assessed the effects of insulin (N2) withdrawal on
endogenous Nmyc1 protein levels. We cultured cells in N2 in the absence
(Fig. 4D, lane 1) or presence
(Fig. 4D, lanes 2-5) of Shh for
24 hours, at which time the medium was replaced as indicated
(Fig. 4D). We observed a
striking reduction in levels of Nmyc1 protein in CGNP cultures treated for 3
hours with Shh-supplemented medium lacking any N2 or IGF1
(Fig. 4D, lane 3). Reduced IGFR
stimulation resulted in decreased phosphorylation of Akt, a well-established
downstream target of PI3K. We also observed decreased GSK3 phosphorylation,
indicating increased GSK activity. We found similar reductions in Nmyc1
protein levels and evidence of decreased PI3K activity in cells treated with
LY294002, a PI3K inhibitor (Fig.
4D, lane 5). Addition of IGF1 for 3 hours was sufficient to
maintain Nmyc1 protein levels (Fig.
4D, lane 4), and IGF1- and N2-supplemented media were equally able
to sustain phosphorylation of Akt and GSK3. These results indicate that, in
the context of CGNP primary cultures, IGF1 stabilizes Nmyc1 proteins via
activation of the PI3K pathway. They do not rule out the possibility that
there are additional mechanisms to activate PI3K signaling in CGNPs in vivo or
in vitro.
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Discussion |
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In the present study, we have investigated how post-translational
modification of Nmyc1 affects its activity in neuronal precursors using the
extensively characterized Shh-treated CGNP primary culture system. We found
that Nmyc1 is phosphorylated on two highly conserved residues within Myc box
1. Our research has led to the implication of Nmyc1 phosphorylation in an
important biological process, the regulation of cell cycle exit during CNS
development. Indeed, our studies are the first to demonstrate that
phosphorylation is an important mechanism for regulation of Myc family protein
levels during normal development. We found that phosphorylation of endogenous
Nmyc1 can be prevented by inhibition of GSK3, and that endogenous Nmyc1 is
stabilized by PI3K activation. These data, together with previous work
(Kenney et al., 2003;
Oliver et al., 2003
), suggest
that Shh mitogenic effects in the developing cerebellum result primarily from
promoting increased levels of Nmyc1 expression and that, in turn, PI3K
signaling inhibits GSK3-dependent phosphorylation and turnover of Nmyc1
protein. The cumulative result of Shh and PI3K signaling in CGNP cultures is
to promote increased Nmyc1 levels and ongoing proliferation
(Fig. 5).
|
A requirement for Nmyc1 function in CNS precursors was shown by Knoepfler
et al. (Knoepfler et al.,
2002), who followed a CNS-specific conditional gene ablation
strategy. Their animals exhibited marked defects, including overall decreased
brain size and evidence of premature neuronal differentiation. Additional
abnormalities were found in the cerebellum, where granule neuron progenitor
proliferation is known to depend on Shh signaling
(Dahmane and Ruiz-i-Altaba,
1999
; Wallace,
1999
; Wechsler-Reya and Scott,
1999
). In vitro, Shh treatment of CGNPs results in Nmyc1
upregulation but not induction of Myc or L-Myc in the presence or
absence of new protein synthesis (Kenney
et al., 2003
). Thus, specificity for Nmyc1 function in CGNPs is
probably due to precise upstream transcriptional regulation by the Hedgehog
signaling pathway, a mechanism likely to be conserved in vivo, because Nmyc1
is the only Myc family member expressed in the proliferating EGL
(Kenney et al., 2003
).
IGF1-mediated activation of the PI3K pathway is crucial for the long-term
survival of cultured CGNPs (Dudek et al.,
1997) and, under our culture conditions, insulin is present at
levels sufficient to activate the IGF receptor. We found that short-term
withdrawal of insulin substantially destabilized Nmyc1 in cultured CGNPs, and
that this destabilization could be prevented by substitution of insulin with
IGF. IGF signaling is important for CNS development, and increased IGF
activity results in cerebellar hyperplasia
(de Pablo and de la Rosa,
1995
; Ye et al.,
1996
). In addition to enhancing growth by promoting survival,
IGF-mediated activation of PI3K could have positive effects on the cell cycle
regulatory apparatus. Indeed, PI3K negatively regulates Forkhead transcription
factors, which can promote cell cycle exit by repressing cyclin D
(Schmidt et al., 2002
). The
finding that IGF-stimulated PI3K activity can stabilize Nmyc1 provides
additional insight as to the molecular mechanisms underlying the
pro-proliferative effects of PI3K signaling in neuronal precursors.
Our data indicate that Nmyc1 T50 phosphorylation in CGNP cultures depends
on GSK3, suggesting that Nmyc1 turnover is regulated by PI3K signaling. In
addition to IGF, PI3K can be activated by other factors present in the milieu
of the developing cerebellum, including CXCL12-CXCR4 and integrin signaling.
Additionally, Wnt signaling is a powerful antagonist of GSK3 activity,
although Wnt-mediated GSK3 regulation is distinct from the PI3K pathway
(Cohen and Frame, 2001). We
observed that inhibition of Shh signaling did not affect Nmyc1 phosphorylation
in CGNP cultures. However, an indirect role for Shh in regulating Nmyc1
phosphorylation cannot be ruled out. Indeed, several lines of evidence suggest
that IGF pathway components are transcriptional targets of Shh in
medulloblastomas (Hahn et al.,
2000
), CGNPs (Oliver et al.,
2003
; Zhao et al.,
2002
) and a Shh-responsive cell line (Ingram et al., 2002). These
findings suggest that Hedgehog and PI3K signaling interactions could be
regulated during cerebellar development in vivo by local Shh induction of
upstream PI3K activators (e.g. IGF2). We have focused on granule neuron
precursors of the cerebellum but the ability of Shh and IGF to promote
proliferation in neural stem cells
(Arsenijevic et al., 2001
;
Lai et al., 2003
) suggests
that a synergy between PI3K and Shh signaling might apply to additional CNS
precursor populations, including those of the adult brain. In keeping with
this, we note that Nmyc1 is expressed in the post-natal (P10) mouse
hippocampus (A.M.K. and D.H.R., unpublished). Further work is required to
establish precise roles for Nmyc1 phosphorylation in neural progenitors in
vivo.
We observed that increasing GSK3 activity resulted in enhanced Nmyc1
turnover, probably through T50 phosphorylation. Conversely, preventing the
phosphorylation by mutating T50 or S54 to alanine resulted in Nmyc1
accumulation. GSK3 requires a priming phosphorylation event in order to
recognize its target site (Cohen and
Frame, 2001). A probable Nmyc1 GSK3 priming site is S54, which is
analogous to Myc S62. Our findings indicate that S54 phosphorylation is
required for phosphorylation of Nmyc1 T50, consistent with regulation by
GSK3-dependent kinase activity. Further, it is clear that the inability to be
phosphorylated at Nmyc1 S54 stabilizes the protein. In contrast, some studies
have suggested that MAPK activity at S62 transiently stabilizes Myc before T58
phosphorylation by GSK3 in vitro (Sears et
al., 2000
). Others indicate that MAPK might not phosphorylate Myc
(Lutterbach and Hann, 1999
),
and we have shown that MAPK is dispensable for Shh proliferative effects in
CGNP cultures (Kenney and Rowitch,
2000
). We conclude that MAPK is unlikely to prime S54
phosphorylation of Nmyc1 in CGNPs and that the identity of the priming kinase
remains to be determined.
Our observation of Nmyc1S54A mutant protein stabilization and enhanced
proliferative effects is in contrast to some Myc studies, which have suggested
that mutation of S62 might cripple Myc function in fibroblasts or cell lines
(Chang et al., 2000;
Sears et al., 2000
). Others
have shown that mutation of Myc S62 enhances its ability to transform REFs
(Henriksson et al., 1993
).
Although these findings might highlight general differences in the regulation
of Myc and Nmyc1 activity, they might also reflect features of
cell-type-specific regulation in neuronal cells, as opposed to the nonneuronal
cells used in the Myc studies. In any case, our studies demonstrate that
N-terminal phosphorylation of Nmyc1 in CGNPs promotes protein turnover and
cell cycle exit rather than affecting the ability of Nmyc1 to regulate target
gene transcription. The mechanism through which stabilized Nmyc1 enhances CGNP
proliferation remains to be determined and might involve other Myc functions
such as transcriptional repression (Wanzel
et al., 2003
) or direct antagonistic interactions with proteins
that promote cellular differentiation
(Wechsler-Reya et al.,
1998
).
Although we were unable to establish culture conditions of sufficient
duration to determine the effects of GSK3 inhibition on endogenous Nmyc1
accumulation, it has been reported that inhibition of GSK activity enhances
CGNP proliferation (Cui et al., 1998). We observed that accumulation of
phosphorylation-mutant proteins took place over a relatively long time course
(48-96 hours), whereas GSK3-mediated degradation could be measured after only
a few hours. These findings suggest that additional mechanisms for Nmyc1
degradation exist in CGNPs. For example, ubiquitination of Myc via association
Myc box 2 domain with ubiquitin ligases results in protein turnover
(Kim et al., 2003;
Lehr et al., 2003
). In
Drosophila, GSK3 phosphorylation of the canonical Hedgehog signaling
target cubitus interruptus leads to its proteolysis
(Jia et al., 2002
;
Price and Kalderon, 2002
),
raising the intriguing possibility that GSK3-mediated antagonism might be a
general feature of Hedgehog signaling.
Abnormally prolonged or increased activity of the Myc family of basic
helix-loop-helix leucine zipper transcription factors is associated with many
types of cancer (Nesbit et al.,
1999). Nmyc1 function, in particular, is implicated in the
generation and/or maintenance of neuroblastomas, gliomas and
Hedgehog-associated cases of medulloblastoma in humans and mice
(Herms et al., 1999
;
Kenney et al., 2003
;
Nesbit et al., 1999
;
Oliver et al., 2003
;
Pomeroy et al., 2002
). We
observed that Nmyc1 phosphorylation takes place in the developing cerebellum
in vivo, and that preventing Nmyc1 phosphorylation enhances CGNP
proliferation. Furthermore, we show that IGF-mediated PI3K activation is an
important regulator of Nmyc1 stability in primary cerebellar cultures.
Interestingly, evidence of increased IGF pathway activity and IGF2 expression
has been found in human and animal models of medulloblastomas
(Del Valle et al., 2002
;
Pomeroy et al., 2002
). Our
data suggest that increased Nmyc1 stabilization caused by PI3K pathway
activation could occur as a result of enhanced IGF pathway activity in tumors,
thereby contributing to tumor growth. Manipulation of signaling pathways to
promote GSK3-dependent phosphorylation of Nmyc1 protein might therefore prove
an effective adjuvant anti-tumor strategy in such cases of cancer in
humans.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Albert, T., Urlbauer, B., Kohlhuber, F., Hammersen, B. and Eick, D. (1994). Ongoing mutations in the N-terminal domain of c-Myc affect transactivation in Burkitt's lymphoma cell lines. Oncogene 9,759 -763.[Medline]
Altman J. and Bayer, S. A. (1997). Development of the Cerebellar System in Relation to its Evolution, Structure, and Functions. Boca Raton, FL: CRC Press.
Arsenijevic, Y., Weiss, S., Schneider, B. and Aebischer, P.
(2001). Insulin-like growth factor-1 is necessary for neural stem
cell proliferation and demonstrates distinct actions of epidermal growth
factor and fibroblast growth factor-2. J. Neurosci.
21,7194
-7202.
Bonvini, P., Nguyen, P., Trepel, J. and Neckers, L. M. (1998). In vivo degradation of N-Myc in neuroblastoma cells is mediated by the 26S proteasome. Oncogene 16,1131 -1139.[CrossRef][Medline]
Brown, E. J. and Schreiber, S. L. (1996). A signaling pathway to translational control. Cell 86,517 -520.[Medline]
Chang, D. W., Claassen, G. F., Hann, S. R. and Cole, M. D.
(2000). The c-Myc transactivation domain is a direct modulator of
apoptotic versus proliferative signals. Mol. Cell.
Biol. 20,4309
-4319.
Charron, J., Malynn, B. A., Fisher, P., Stewart, V., Jeannotte, L., Goff, S. P., Robertson, E. J. and Alt, F. W. (1992). Embryonic lethality in mice homozygous for a targeted disruption of the N-Myc gene. Genes Dev. 6,2248 -2257.[Abstract]
Chin, L., Schreiber-Agus, N., Pellicer, I., Chen, K., Lee, H. W., Dudast, M., Cordon-Cardo, C. and DePinho, R. A. (1995). Contrasting roles for Myc and Mad proteins in cellular growth and differentiation. Proc. Natl. Acad. Sci. USA 92,8488 -8492.[Abstract]
Ciemerych, M. A., Kenney, A. M., Sicinska, E., Kalaszczynska,
I., Bronson, R. T., Rowitch, D. H., Gardner, H. and Sicinski, P.
(2002). Development of mice expressing a single D-type cyclin.
Genes Dev. 16,3277
-3289.
Cohen, P. and Frame, S. (2001). The renaissance of GSK3. Nat. Rev. Mol. Cell Biol. 2, 769-776.[CrossRef][Medline]
Cole, M. D. and McMahon, S. B. (1999). The Myc oncoprotein: a critical evaluation of transactivation and target gene regulation. Oncogene 18,2916 -2924.[CrossRef][Medline]
Dahmane, N. and Ruiz-i-Altaba, A. (1999). Sonic
hedgehog regulates the growth and patterning of the cerebellum.
Development 126,3089
-3100.
de Pablo, F. and de la Rosa, E. J. (1995). The developing CNS: a scenario for the action of proinsulin, insulin and insulin-like growth factors. Trends Neurosci. 18,143 -150.[CrossRef][Medline]
Del Valle, L., Enam, S., Lassak, A., Wang, J. Y., Croul, S.,
Khalili, K. and Reiss, K. (2002). Insulin-like growth factor
I receptor activity in human medulloblastomas. Clin. Cancer
Res. 8,1822
-1830.
Dildrop, R., Zimmerman, K., DePinho, R. A., Yancopoulos, G. D., Tesfaye, A. and Alt, F. W. (1988). Differential expression of Myc-family genes during development: normal and deregulated N-Myc expression in transgenic mice. Curr. Top. Microbiol. Immunol. 141,100 -109.[Medline]
Dudek, H., Datta, S. R., Franke, T. F., Birnbaum, M. J., Yao,
R., Cooper, G. M., Segal, R. A., Kaplan, D. R. and Greenberg, M. E.
(1997). Regulation of neuronal survival by the serine-threonine
protein kinase Akt. Science
275,661
-665.
Duman-Scheel, M., Weng, L., Xin, S. and Du, W. (2002). Hedgehog regulates cell growth and proliferation by inducing cyclin D and cyclin E. Nature 417,299 -304.[CrossRef][Medline]
Gao, W. O., Heintz, N. and Hatten, M. E. (1991). Cerebellar granule cell neurogenesis is regulated by cell-cell interactions in vitro. Neuron 6, 705-715.[Medline]
Hahn, H., Wojnowski, L., Specht, K., Kappler, R., Calzada-Wack,
J., Potter, D., Zimmer, A., Muller, U., Samson, E. and Quintanilla-Martinez,
L. (2000). Patched target IGF2 is indispensable for the
formation of medulloblastoma and rhabdomyosarcoma. J. Biol.
Chem. 275,28341
-28344.
Hatten, M. E. et al. (1998). The cerebellum: purification and co-culture of identified cell populations. In Culturing Nerve Cells (ed. K. Goslin and G. Banker), pp. 419-459. Cambridge, MA: MIT Press.
Henriksson, M. and Luscher, B. (1996). Proteins of the Myc network: essential regulators of cell growth and differentiation. Adv. Cancer Res. 68,109 -182.[Medline]
Henriksson, M., Bakardjiev, A., Klein, G. and Luscher, B. (1993). Phosphorylation sites mapping in the N-terminal domain of c-Myc modulate its transforming potential. Oncogene 8,3199 -3209.[Medline]
Herms, J. W., von Loewenich, F. D., Behnke, J., Markakis, E. and Kretzschmar, H. A. (1999). c-Myc oncogene family expression in glioblastoma and survival. Surg. Neurol. 51,536 -542.[CrossRef][Medline]
Hirvonen, H., Makela, T. P., Sandberg, M., Kalimo, H., Vuorio, E. and Alitalo, K. (1990). Expression of the Myc proto-oncogenes in developing human fetal brain. Oncogene 5,1787 -1797.[Medline]
Ho, K. S. and Scott, M. P. (2002). Sonic hedgehog in the nervous system: functions, modifications and mechanisms. Curr. Opin. Neurobiol. 12, 57-63.[CrossRef][Medline]
Hurlin, P. J., Queva, C., Koskinen, P. J., Steingrimsson, E., Ayer, D. E., Copeland, N. G., Jenkins, N. A. and Eisenman, R. N. (1995). Mad3 and Mad4: novel Max-interacting transcriptional repressors that suppress c-Myc dependent transformation and are expressed during neural and epidermal differentiation. EMBO J. 14,5646 -5659.[Abstract]
Jia, J., Amanai, K., Wang, G., Tang, J., Wang, B. and Jiang, J. (2002). Shaggy/GSK3 antagonizes Hedgehog signalling by regulating Cubitus interruptus. Nature 416,548 -552.[CrossRef][Medline]
Kenney, A. M. and Rowitch, D. H. (2000). Sonic
hedgehog promotes G1 cyclin expression and sustained cell cycle
progression in mammalian neuronal precursors. Mol. Cell.
Biol. 20,9055
-9067.
Kenney, A. M., Cole, M. D. and Rowitch, D. H.
(2003). Nmyc upregulation by sonic hedgehog signaling promotes
proliferation in developing cerebellar granule neuron precursors.
Development 130,15
-28.
Kim, S. Y., Herbst, A., Tworkowski, K. A., Salghetti, S. E. and Tansey, W. P. (2003). Skp2 regulates Myc protein stability and activity. Mol. Cell 11,1177 -1188.[Medline]
Klein, R. S., Rubin, J. B., Gibson, H. D., DeHaan, E. N.,
Alvarez-Hernandez, X., Segal, R. A. and Luster, A. D. (2001).
SDF-1 induces chemotaxis and enhances Sonic hedgehog-induced
proliferation of cerebellar granule cells. Development
128,1971
-1981.
Knoepfler, P. S., Cheng, P. F. and Eisenman, R. N.
(2002). N-Myc is essential during neurogenesis for the rapid
expansion of progenitor cell populations and the inhibition of neuronal
differentiation. Genes Dev.
16,2699
-2712.
Lai, K., Kaspar, B. K., Gage, F. H. and Schaffer, D. V. (2003). Sonic hedgehog regulates adult neural progenitor proliferation in vitro and in vivo. Nat. Neurosci. 6, 21-27.[CrossRef][Medline]
Lehr, N. v. d., Johansson, S., Wu, S., Bahram, F., Castell, A., Cetinkaya, C., Hydbring, P., Weidung, I., Nakayama, K., Nakayama, K. et al. (2003). The F-box protein Skp2 participates in c-Myc proteosomal degradation and acts as a co-factor for c-Myc regulated transcription. Mol. Cell 11,1189 -1200.[Medline]
Littlepage, L. E., Wu, H., Andresson, T., Deanehan, J. K.,
Amundadottir, L. T. and Ruderman, J. V. (2002).
Identification of phosphorylated residues that affect the activity of the
mitotic kinase Aurora-A. Proc. Natl. Acad. Sci. USA
99,15440
-15445.
Lo, R. S., Wotton, D. and Massague, J. (2001).
Epidermal growth factor signaling via Ras controls the Smad transcriptional
co-repressor TGIF. EMBO J.
20,128
-136.
Lutterbach, B. and Hann, S. R. (1999). c-Myc transactivation domain-associated kinases: questionable role for Map kinases in c-Myc phosphorylation. J. Cell. Biochem. 72,483 -491.[CrossRef][Medline]
Marshall, C. J. (1995). Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 80,179 -185.[Medline]
Miyazawa, K., Himi, T., Garcia, V., Yamagishi, H., Sato, S. and
Ishizaki, Y. (2000). A role for p27/Kip1 in the control of
cerebellar granule cell precursor proliferation. J.
Neurosci. 20,5756
-5763.
Monjaraz, E., Navarrete, A., Lopez-Santiago, L. F., Vega, A. V.,
Arias-Montano, J. A. and Cota, G. (2000). L-type calcium
channel activity regulates sodium channel levels in rat pituitary GH3 cells.
J. Physiol. 523,45
-55.
Nesbit, C. E., Tersak, J. M. and Prochownik, E. V. (1999). MYC oncogenes and human neoplastic disease. Oncogene 18,3004 -3016.[CrossRef][Medline]
Niklinski, J., Claassen, G., Meyers, C., Gregory, M. A.,
Allegra, C. J., Kaye, F. J., Hann, S. R. and Zajac-Kaye, M.
(2000). Disruption of Myctubulin interaction by
hyperphosphorylation of c-Myc during mitosis or by constitutive
hyperphosphorylation of mutant c-Myc in Burkitt's lymphoma. Mol.
Cell. Biol. 20,5276
-5284.
Oliver, T. G., Grasfeder, L. L., Carroll, A. L., Kaiser, C.,
Gillingham, C. L., Lin, S. M., Wickramasinghe, R., Scott, M. P. and
Wechsler-Reya, R. J. (2003). Transcriptional profiling of the
Sonic hedgehog response: a critical role for N-Myc in proliferation of
neuronal precursors. Proc. Natl. Acad. Sci. USA
100,7331
-7336.
Pomeroy, S. L., Tamayo, P., Gaasenbeek, M., Sturla, L. M., Angelo, M., McLaughlin, M. E., Kim, J. Y., Goumnerova, L. C., Black, P. M., Lau, C. et al. (2002). Prediction of central nervous system embryonal tumour outcome based on gene expression. Nature 415,436 -442.[CrossRef][Medline]
Price, M. A. and Kalderon, D. (2002). Proteolysis of the Hedgehog signaling effector Cubitus interruptus requires phosphorylation by glycogen synthase kinase 3 and casein kinase 1. Cell 108,823 -835.[Medline]
Queva, C., Hurlin, P. J., Foley, K. P. and Eisenman, R. N. (1998). Sequential expression of the MAD family of transcriptional repressors during differentiation and development. Oncogene 16,967 -977.[CrossRef][Medline]
Salghetti, S. E., Kim, S. Y. and Tansey, W. P.
(1999). Destruction of Myc by ubiquitin-mediated proteolysis:
cancer-associated and transforming mutations stabilize Myc. EMBO
J. 18,717
-726.
Sawai, S., Shimono, A., Hanaoka, K. and Kondoh, H. (1991). Embryonic lethality resulting from disruption of both N-Myc alleles in mouse zygotes. New Biol. 3, 861-869.[Medline]
Schmidt, M., Fernandez de Mattos, S., van der Horst, A.,
Klompmaker, R., Kops, G. J., Lam, E. W., Burgering, B. M. and Medema, R.
H. (2002). Cell cycle inhibition by FoxO forkhead
transcription factors involves downregulation of cyclin D. Mol.
Cell. Biol. 22,7842
-7852.
Sears, R., Nuckolls, F., Haura, E., Taya, Y., Tamai, K. and
Nevins, J. R. (2000). Multiple Ras-dependent phosphorylation
pathways regulate Myc protein stability. Genes Dev.
14,2501
-2514.
Solecki, D. J., Liu, X. L., Tomoda, T., Fang, Y. and Hatten, M. E. (2001). Activated Notch2 signaling inhibits differentiation of cerebellar granule neuron precursors by maintaining proliferation. Neuron 31,557 -568.[Medline]
Stanton, B. R., Perkins, A. S., Tessarollo, L., Sassoon, D. A. and Parada, L. F. (1992). Loss of N-Myc function results in embryonic lethality and failure of the epithelial component of the embryo to develop. Genes Dev. 6,2235 -2247.[Abstract]
Strieder, V. and Lutz, W. (2002). Regulation of N-Myc expression in development and disease. Cancer Lett. 180,107 -119.[CrossRef][Medline]
Wallace, V. A. (1999). Purkinje-cell-derived Sonic hedgehog regulates granule neuron precursor cell proliferation in the developing mouse cerebellum. Curr. Biol. 9, 445-448.[CrossRef][Medline]
Wanzel, M., Herold, S. and Eilers, M. (2003). Transcriptional repression by Myc. Trends Cell Biol. 13,146 -150.[CrossRef][Medline]
Wechsler-Reya, R. J. and Scott, M. P. (1999). Control of neuronal precursor proliferation in the cerebellum by Sonic Hedgehog. Neuron 22,103 -114.[Medline]
Wechsler-Reya, R. J., Elliott, K. J. and Prendergast, G. C.
(1998). A role for the putative tumor suppressor Bin1 in muscle
cell differentiation. Mol. Cell. Biol.
18,566
-575.
Ye, P., Xing, Y., Dai, Z. and D'Ercole, A. J. (1996). In vivo actions of insulin-like growth factor-1 (IGF-1) on cerebellum development in transgenic mice: evidence that IGF-1 increases proliferation of granule cell progenitors. Brain Res. Dev. Brain Res. 95,44 -54.[Medline]
Zhao, Q., Kho, A., Kenney, A. M., Yuk, D. I., Kohane, I. and
Rowitch, D. H. (2002). Identification of genes expressed with
temporal-spatial restriction to developing cerebellar neuron precursors by a
functional genomic approach. Proc. Natl. Acad. Sci.
USA 99,5704
-5709.