Dual Phosphorylation of Suppressor of Fused (Sufu) by PKA and GSK3β Regulates Its Stability and Localization in the Primary Cilium

Abstract

Suppressor of fused (Sufu) is an essential negative regulator of the sonic hedgehog (Shh) pathway, but little is known about how Sufu itself is normally regulated. Here, we report that Sufu is phosphorylated at Ser-342 and Ser-346 by GSK3␤ and cAMPdependent protein kinase A (PKA), respectively, and phosphorylation at this dual site stabilizes Sufu against Shh signalinginduced degradation. We further show that localization of Sufu in the primary cilium is induced by Shh signaling and is required for the turnover of both phosphorylated and total Sufu. Perturbing Sufu phosphorylation with PKA inhibitors or replacing Ser-346 with alanine reduced the stay and replacing Ser-342 and Ser-346 with aspartic acid prolonged the stay of Sufu in the cilia. Finally, ciliary localization of Gli2/3 also required Smo and was similarly influenced by perturbations of PKA activity or mutations at the dual Sufu phosphorylation site. Thus, Shh likely induced trafficking of phospho-Sufu into the primary cilium in a complex with Gli2/3, and dephosphorylation triggered a retrograde export, allowing Sufu to be degraded by the ubiquitinproteasome system.

SCF (Fbxl17) ubiquitylation of Sufu regulates Hedgehog signaling and medulloblastoma development

“…Substitution of S342 and S346 to D (S342/6D) to mimic phosphorylation decreased recognition by Fbxl17 (Fig 3A). These data are consistent with the increased stabilization of Sufu detected upon the phosphorylation of these residues by PKA (protein kinase A) and GSK3β (glycogen synthase kinase 3β) (Chen et al , 2011). …”

“…The interaction between Fbxl17 and Sufu is hampered by the phosphorylation of Sufu on S342 and S346 residues, which were shown to be target sites for PKA and GSK3β, respectively (Chen et al , 2011) (Fig 8A). Phosphorylation of Sufu retains it in the primary cilium (Chen et al , 2011) and could represent a failsafe mechanism to avoid unscheduled pathway activation when the Hh ligands are not present. A similar but opposite mechanism of regulation is operated downstream of Sufu at the levels of Gli2 and Gli3 by the SCF βTrcp ubiquitin ligase.…”

Suppressor of Fused (Sufu) Mediates the Effect of Parathyroid Hormone-like Hormone (Pthlh) on Chondrocyte Differentiation in the Growth Plate

“…Phosphorylation of Sufu protein by PKA can positively regulate its stability in cultured fibroblasts (14), and we found that Pthlh treatment similarly increased Sufu phosphorylation in growth plate chondrocytes, increasing the stability of the protein. Intriguingly, Kif7 functions positively in the regulation of Hh pathway activity through regulating Sufu levels (12).…”

“…Immunoblotting was performed overnight at 4°C with the following primary antibodies: antiGli3 antibody (1:800; Santa Cruz Biotechnology), anti-actin antibody (1:10,000; Oncogene), anti-Sufu antibody (22), antiGli2 antibody (18), and anti-phospho-Sufu antibody (Signalway Antibody, Nanjing, China) (14).…”

“…PKA, the main effector of Pthlh in chondrocytes, can stimulate Sufu protein stability through phosphorylation (12,14). This raises the possibility that Pthlh and the Hh signaling intermediates Sufu and Kif7 interact in growth plate cells, regulating chondrocyte hypertrophic differentiation.…”