PMC

Mechanism of Akt-mediated Shh signaling. (A) Gli-luciferase activity in NIH 3T3 cells transiently transfected with WT Gli2 (WT), Gli2 GSM (clusters I–IV), and Gli2 PSM (clusters I, II, and IV), either alone or in combination with PKA or both PKA and GSK-3β expression vectors. Expression of the Gli2 variants was determined by Western blot. (B) Gli2 WT, Gli2 PSM, or Gli GSM were cotransfected with empty vector or dnAkt in NIH 3T3 cells and assayed for Gli-luciferase activity after 48 h. Expression levels of Gli2 variants in the presence of dnAkt were determined by Western blot. (C) Subcellular localization of Gli2 WT (Left) and Gli2 PSM (Right) in NIH 3T3 cells in the absence (Top) and the presence (Middle and Bottom) of dnAkt. Gli2 was detected with Xpress Ab (1:250)/Alexa Fluor 546 (red) and dnAkt with Akt Ab (1:120)/Alexa Fluor 488 (green). DAPI staining of the nuclei is in blue. Asterisks indicate cells coexpressing dnAkt and Gli2. (D) Model of Shh signaling showing the requirement PI3-kinase/Akt activation by Smo or other proliferative pathways in the regulation of Gli phosphorylation by PKA. All phosphorylation events are represented by ○p. Details are described in the text.

PI3-kinase signaling is required for Shh-induced neural patterning and chondrogenesis. (A Upper) Treatment of neural tube/notochord explants for 48 h with LY294002 (30 μM) blocked Akt phosphorylation, as assayed by Western blotting, and blocked Shh signaling, as assayed by whole-mount immunostaining for Pax7 and Islet1 expression. (A Lower) Explants were photographed dorsal side up for Pax7 and ventral side up for Islet1. High-magnification insets show nuclear localization. Lowest panel shows staining of Shh in the floor plate (fp). (Scale bar: 100 μm.) (B) Quantification of Pax7 and Islet1 expression, BrdUrd incorporation, and activated caspase-3 in control and LY294002-treated neural tube explants. (C) Effects of LY294002 (15 μM) on AP induction by 5 μg/ml N-Shh in 10T1/2 cells after 4 days. RU, relative units. (D) 10T1/2 cells stably transfected with empty vector or p-myr-Akt treated as in C. (E) Effect of dnAkt on Gli-luciferase activity in 10T1/2 cells transiently transfected with the indicated constructs. Data are the mean ± SEM of three experiments performed in triplicate. ∗, P < 0.01.

PI3-kinase signaling through Akt is essential for Shh signal transduction. (A) Gli-luciferase activity in LIGHT cells induced for 24 h by increasing concentrations of N-Shh in the absence or presence of 50 nM IGF-I. (B) Gli-luciferase activity in LIGHT cells after 24 h of treatment with IGF-I or N-Shh (5 μg/ml). Phosphorylation of Akt (Ser-473) after 15 min of treatment with increasing concentrations of IGF-I was determined by Western blot (phospho-Akt, 1:1,000; total Akt 1:2,000). (C) Effect of 5 μg/ml N-Shh on Akt phosphorylation at different time points, assessed as in B. Densitometry values representing the increase in P-Akt are indicated at the bottom. (D) Effect of LY294002 (15 μM) and KAAD-cyclopamine (100 nM) on Gli-luciferase induced by Shh-conditioned medium (Shh-CM) or N-Shh in LIGHT cells at 24 h. (E) LIGHT cells were transfected with Akt1 or control siRNA, and 48 h later they were stimulated with N-Shh for 24 h. (F) Effect of dnAkt on Gli-luciferase activity in NIH 3T3 cells transiently transfected with the indicated constructs. (G) LIGHT cells stably transfected with LacZ or myr-Akt were stimulated with 5 μg/ml N-Shh with or without LY294002 (15 μM) for 24 h and assayed for Gli-luciferase activity, normalized to the baseline in the absence of N-Shh. Data represent the mean ± SEM of three experiments. ∗, P < 0.01. RLU, relative luciferase units.

Gli2 has functional sites for PKA and GSK-3β phosphorylation. (A) NIH 3T3 cells were cotransfected with Gli2 and empty vector, PKA, GSK-3β or both and assayed for Gli-luciferase activity 48 h later. (B Upper) Immunoprecipitated myc-Gli2 was incubated in 40 mM Tris·HCl (pH 7.4), 20 mM magnesium acetate, and 0.2 mM [γ-³²P]ATP (1 μCi/μmol) (control), with 10 units of PKA catalytic subunit or with PKA and 10 units of protein kinase inhibitor (PKI) for 30 min at 30°C. Beads were washed with 50 mM Tris·HCl (pH 7.5) and subjected to 5% SDS/PAGE and autoradiography. (B Lower) myc-Gli2 was incubated in 20 mM Tris·HCl (pH 7.5), 10 mM MgCl₂, 5 mM DTT, and 200 μM [γ-³²P]ATP (1 μCi/μmol) without (control) or with 500 units of GSK-3β for 30 min at 30°C, or Gli2 was phosphorylated first with PKA and cold ATP, as described above, followed by 10 units of PKI, 500 units of GSK-3β, and 200 μM [γ-³²P]ATP and incubated for 30 min. Beads were processed as before. (C) Sequence of the Gli2 peptides corresponding to the four clusters with the conserved PKA and GSK-3β phosphorylation sites highlighted in boxes. (D) Phosphorylation of peptides shown in C and variant peptides in which each candidate residue was substituted by alanine. Peptides (1 mM) were treated with PKA and GSK-3β or both enzymes sequentially, as described for Gli2 in B. Reactions then were stopped by spotting onto P81 paper and washed with 50 mM H₃PO₄, and ³²P incorporation was determined by liquid scintillation. PKA phosphorylation of kemptide was taken as 100%. (E) Gli2 WT, Gli2 PSM, and Gli2 GSM were expressed in 293 cells, immunoprecipated with Xpress antibody, and used as substrates for phosphorylation by PKA and GSK-3β as described in B. Phosphorylation and expression were assayed by autoradiography (Autorad.) and Western blot (WB) (Xpress Ab 1:5,000). Densitometric analysis of three experiments is shown in the graph. (F) Intact 293 cells were transfected with Gli2 WT or Gli2 PSM, labeled with [³²P]orthophosphate, and stimulated with 10 μM forskolin for 2 h. Gli2 was immunoprecipitated with Xpress Ab, and ³²P incorporation was determined by autoradiography. Quantification by densitometry of three experiments was performed by using the basal level of phosphorylation of Gli2 WT as 100%.