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First published online 3 December 2003
doi: 10.1242/dev.00891

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Hedgehog and PI-3 kinase signaling converge on Nmyc1 to promote cell cycle progression in cerebellar neuronal precursors

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

View larger version (28K): [in a new window]   Fig. 1. Nmyc1 is phosphorylated on conserved MB1 sites and phosphorylated Nmyc1 can be detected in the developing cerebellum. (A) Known phosphorylation sites in Myc and L-Myc. Conserved putative sites for phosphorylation of Nmyc1 within the MBI (I) region at theronine-50 (T-50) and serine-54 (S-54) are indicated. (B) Chromatographic results of mass spectrometric analysis of Nmyc1 protein overexpressed in HEK293 cells. The tryptic fragment identified as Nmyc1 K44-R57 contained two phosphorylated residues (black arrow). (Inset) The fragment Nmyc1 K44-R57 was subjected to further fragmentation and the specific amino acid residues were identified. Of these, T50 and S54 were phosphorylated. (C) Anti-phosphorylated-T58-Myc antibody recognizes Nmyc1 phosphorylated on T50. CGNP cultures, which do not express Myc (Kenney et al., 2003), were infected with the indicated retroviruses for 24 hours and protein lysates were analysed by western blot. Anti-phosphorylated-T58-Myc antibody recognized wild-type Nmyc1, but not Nmyc1 mutated at T50 or S54. Nmyc1 S54 phosphorylation is evidently required for phosphorylation of T50, consistent with previous findings for Myc (Sears et al., 2000). (D) Phosphorylated Nmyc1 proteins are present in proliferating cells of the developing mouse external granule layer (EGL). (Left) Sections of PN 7 mouse cerebella were immunostained with anti-T58-Myc antibody (red) and Calbindin (green), a marker of Purkinje neurons (Pur) underlying the EGL. Nuclei are labeled with DAPI. (Right) Staining with anti-T58-Myc antibody (red) and PCNA (green), a marker of proliferating cells, confirms co-expression (indicated by merged staining, yellow).

View larger version (29K): [in a new window]   Fig. 2. Alanine replacement of Nmyc1 T50 or S54 residues results in enhanced proliferative effects and delayed CGNP cell cycle exit. (A) After 24 hours of Shh treatment alone (no serum), CGNP cultures were infected with the indicated viruses and forskolin (10 µM) was added 24 hours later. After a further 24 hour incubation period, BrdU was added (2 hour pulse) and proliferation was assessed by BrdU immunostaining. The histogram shows levels of BrdU incorporation normalized to Shh-treated, control CGNP cultures as previously described (Kenney et al., 2003; Kenney and Rowitch, 2000). Wild-type Nmyc1 is sufficient for sustained proliferation in the presence of forskolin (fsk). Nmyc1T50A and Nmyc1S54A supported proliferation to a greater extent than wild-type Nmyc1, despite treatment with fsk. The western blot shows relative Nmyc1 protein levels. (B) Nmyc1T50A and Nmyc1S54A extend the phase of CGNP proliferation relative to Shh treatment alone. CGNP cultures were infected as indicated. Cell cycle phase distribution was assessed by propidium iodide flow cytometry at 48 hours (left) and 96 hours (right) after infection. The average percent of cells in S phase±s.e.m. derived from three independent experiments is shown in the histograms above representative western blots demonstrating relative Nmyc1 protein levels. Notice that, 96 hours after infection, the proportion of cells in S phase in Nmyc1T50A- and Nmyc1S54A-infected cultures, but not wild-type Nmyc1-infected cultures, remained significantly higher than Shh-treated controls. Western blots below the graphs show relative Nmyc1 protein levels. (C) Nmyc1-, Nmyc1T50A- and Nmyc1S54A-infected cells all show upregulation of cyclins D1 and D2 at 48 hours and 96 hours after infection. Representative western blot autoradiographs are shown. ß-Tubulin immunoreactivity indicates equivalent loading of the lanes.

View larger version (43K): [in a new window]   Fig. 3. Nmyc1T50A and Nmyc1S54A show transactivation capacity equivalent to wild-type Nmyc1 but have enhanced stability. (A) Nmyc1, Nmyc1T50A and Nmyc1S54A were compared for their ability to activate target gene expression as determined by a luceriferase reporter assay. The graph depicts relative luciferase activity in HeLa cells co-transfected with Nmyc1 or Nmyc1 phosphorylation mutant retroviral constructs, and a reporter plasmid containing luciferase driven by either the human Cdk4 promoter (dark bars) or the same promotor lacking E boxes, Myc proteins' cognate DNA recognition sequence (light bars). Results shown are the average of three independent experiments±s.e.m. No significant differences in transactivation capacity between wild-type Nmyc1 or the phosphomutants were detected. (B) Nmyc1T50 and Nmyc1S54 are more stable than wild-type Nmyc1. Primary CGNP cultures were infected with retroviruses expressing wild-type Nmyc1, Nmyc1T50A or Nmyc1S54A. After 24 hours, the cells were pulsed with cycloheximide for 30-90 minutes (three trials per time point) to prevent the synthesis of new protein and to allow an assessment of remaining previously synthesized protein. Protein lysates were prepared and blotted for Nmyc1. A representative western blot is shown from a 90 minute cycloheximide pulse. By 90 minutes, levels of retrovirally expressed wild-type Nmyc1 had substantially declined. Levels of Nmyc1T50A and Nmyc1S54A proteins were not strongly affected by cycloheximide treatment. Relative to retrovirally expressed wild type Nmyc1, increased levels of Nmyc1T50A and Nmyc1S54A protein levels can be seen (left), despite similar levels of retroviral transcript expression, as determined by northern blotting for Nmyc1 (right), consistent with an inability to be phosphorylated leading to protein stabilization and accumulation. (C) Nmyc1T50A and Nmyc1S54A enhance proliferation in human neuroblastoma cells, in comparison with ectopically expressed wild-type Nmyc1. SK-N-SH neuroblastoma cells were infected as indicated and proliferation was assessed after 48 hours using flow cytometry. The graph shows levels of cells in S phase, relative to GFP-infected cells. (D) Mutation of T50 or S54 to aspartic acid has different effects on Nmyc1 turnover. Cycloheximide chase assay was performed as described, and a representative western blot from a 90-minute cycloheximide chase is shown. Nmyc1S54E shows similar turnover characteristics to wild-type Nmyc1, whereas Nmyc1T50E is more stable than wild-type Nmyc1. These results indicate that the effect of phosphorylation at that site requires the presence of the phosphate group and does not depend merely upon the negative charge transferred by phosphorylation.

View larger version (28K): [in a new window]   Fig. 4. Nmyc1 phosphorylation is regulated by GSK3 and the PI3K pathway in CGNPs, independent of Shh signaling. (A) CGNP cultures were treated with Shh, cyclopamine and/or the proteosome inhibitor lactacystin, which effectively blocks ongoing turnover of endogenous Nmyc1 (compare lanes 3 and 5 to lanes 2 and 4). Lysates were blotted to determine levels of T50-phosphorylated Nmyc1 relative to total Nmyc1 and results representative of three independent experiments are shown. Tubulin immunoreactivity indicates equivalent loading of the lanes. The level of T50-phosphorylated Nmyc1 relative to total Nmyc1 was comparable regardless of treatment with Shh or cyclopamine, indicating that Shh signaling has neither positive (destabilizing) nor negative (stabilizing) effects on endogenous Nmyc1 phosphorylation. (B) GSK3 activity is required for endogenous Nmyc1 phosphorylation. Shh-treated CGNP cultures were exposed to LiCl (left) or a commercially available GSK3 inhibitor (right) for the indicated length of time. Both methods for blocking endogenous GSK3 activity effectively reduced levels of Nmyc1 phosphorylation. (C) Activity of PI3K is required for stabilizing Nmyc1. CGNP cultures were infected as indicated. After 24 hours, the cells were treated (3 hours) with wortmannin, a PI3K inhibitor. In the presence of wortmannin, levels of wild-type retroviral Nmyc1 were sharply reduced. By contrast, levels of Nmyc1T50A and Nmyc1S54A were unaffected, ruling out a general effect of wortmannin treatment on protein synthesis. These results also show that the non-phosphorylatable mutants do not depend on the PI3K pathway for stabilization. (D) IGF, a prominent activator of PI3K in CGNPs, stabilizes endogenous Nmyc1. CGNPs cultured in N2 (insulin)-containing medium were treated with (lanes 2-5) or without Shh (lane 1) for 24 hours. At this time, medium in lanes 2-4 was withdrawn and cells were washed extensively and treated with fresh medium containing Shh (lanes 2-5), N2 (lanes 2, 5), no N2 or IGF1 (lane 3), IGF1 (20 ng ml–1, lane 4), or LY294002 (10 µM, lane 5). After 3 hours, protein lysates were prepared and western blotted for Nmyc1, phsophorylated Akt, and phosphorylated GSK3. Lack of N2 or IGF1 resulted in downregulation of the PI3K pathway, and decreased Nmyc1 protein. N2 and IGF1 were each capable of supporting PI3K pathway activity and Nmyc1 protein levels. LY294002 inhibited the PI3K pathway and promoted destabilization of endogenous Nmyc1.

View larger version (17K): [in a new window]   Fig. 5. Model for combined effects of Shh signaling and PI3K on cell cycle progression through Nmyc1 regulation in CGNP cultures. Previous work indicates that Shh signaling induces expression of the proto-oncogene transcription factor Nmyc1. Nmyc1 is necessary and sufficient for maintaining CGNP proliferation. The present findings indicate that maintenance of Nmyc1 protein levels in vitro is regulated in a Shh-independent manner. Our data support a role for IGFR signaling in activation of the PI3K pathway, which inhibits GSK3-mediated phosphorylation of Nmyc1 T50 and its subsequent degradation. The kinase priming Nmyc1 for GSK3 phosphorylation, by acting at S54, remains to be identified. Other growth factors (e.g. SDF-1) or cell-cell interactions (e.g. via integrins) are candidates for PI3K regulation and inhibition of GSK3 activity in vivo. The combined effect of concerted Shh and PI3K activity is to promote G1 progression and precisely control the timing of cell cycle exit.

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