Identification of N-terminal residues of Sonic Hedgehog important for palmitoylation by Hedgehog acyltransferase

Abstract

Sonic Hedgehog (Shh) is a secreted morphogen that regulates embryonic development. After removal of the signal peptide, Shh is processed to the mature, active form through autocleavage and a series of lipid modifications, including the attachment of palmitate. Covalent attachment of palmitate to the N-terminal cysteine of Shh is catalyzed by Hedgehog acyltransferase (Hhat) and is critical for proper signaling. The sequences within Shh that are responsible for palmitoylation by Hhat are not known. Here we show that the first six amino acids of mature Shh (CGPGRG) are sufficient for Hhat-mediated palmitoylation. Alanine scanning mutagenesis was used to determine the role of each amino acid and the positional sequence requirement in a cell-based Shh palmitoylation assay. Mutation of residues in the GPGR sequence to Ala had no effect on palmitoylation, provided that a positively charged residue was present within the first seven residues. The N-terminal position exhibited a strong but not exclusive requirement for Cys. Constructs with an N-terminal Ala were not palmitoylated. However, an N-terminal Ser served as a substrate for Hhat, but not the Drosophila melanogaster ortholog Rasp, highlighting a critical difference between the mammalian and fly enzymes. These findings define residues and regions within Shh that are necessary for its recognition as a substrate for Hhat-mediated palmitoylation. Finally, we report the results of a bioinformatics screen to identify other potential Hhat substrates encoded in the human genome.

FIGURE 1.

Palmitoylation of Shh-EGFP fusion constructs. COS-1 cells were transfected with the indicated constructs and labeled for 4 h with [¹²⁵I]iodopalmitate. Cell lysates were immunoprecipitated with anti-GFP antibody and analyzed by SDS-PAGE and phosphorimaging ([¹²⁵I]iodopalmitate, top panels) and Western blotting with anti-GFP antibody (EGFP (IP/WB), middle panels). HA-tagged Hhat expression was determined by Western blotting of total cell lysates with anti-HA antibody (HA-Hhat (WB), bottom panels). A, analysis of EGFP and Shh(1–29) EGFP in the absence and presence of co-transfected HA-Hhat. B, analysis of Shh(1–29) EGFP and Shh(1–34) EGFP constructs with co-expressed Hhat. C, COS-1 cells were co-transfected with plasmids encoding Hhat and either full-length Shh or Shh(1–29)EGFP or with a plasmid encoding Fyn. Cells were labeled for 4 h with [¹²⁵I]iodopalmitate. Cell lysates were immunoprecipitated with either anti-Shh or anti-GFP or anti-Fyn antibody, as appropriate. Immunoprecipitates were treated with 1 m Tris, pH 7.0, or 1 m NH₂OH, pH 7.0, for 30 min at 20 °C, as indicated and then analyzed as described above. The 19-kDa ShhNp product is shown in the first lane. D, analysis of Shh(1–29) EGFP and longer fusion constructs.

FIGURE 2.

A single Cys following the signal sequence is not sufficient for Shh palmitoylation. COS-1 cells were transfected with the indicated constructs, labeled with [¹²⁵I]iodopalmitate and analyzed as described for Fig. 1. A, Shh(1–24) EGFP is not palmitoylated. B, substitution of Lys for Gly at position 25 prevents palmitoylation of Shh(1–29) EGFP. C, substitution of Ser at position 25 allows palmitoylation of Shh(1–29) EGFP but not Shh(1–24) EGFP. IP, immunoprecipitation; WB, Western blot.

FIGURE 3.

Palmitoylation is preserved when N-terminal conserved residues are mutated within Shh(1–29) EGFP. COS-1 cells were transfected with the indicated constructs, labeled with [¹²⁵I]iodopalmitate, and analyzed as described in Fig. 1. Levels of [¹²⁵I]iodopalmitate incorporation were divided by the expression level of each construct and normalized to that of Shh(1–29) EGFP (set at 1.0). Each bar represents the average of experiments that were performed in duplicate and repeated two or three times.

FIGURE 4.

Influence of positively charged residues within the Shh N-terminal region on palmitoylation. A, COS-1 cells were co-transfected with constructs encoding full-length Shh and Hhat and labeled for 4 h with [¹²⁵I]iodopalmitate. Cell lysates were immunoprecipitated with anti-Shh antibody and analyzed as described in Fig. 1. The levels of [¹²⁵I]iodopalmitate incorporation were divided by the expression level of each construct and normalized to that of WT Shh (set at 1.0). Each bar represents the average of experiments that were performed in duplicate and repeated two times. B, palmitoylation of Shh(1–29) EGFP constructs containing mutations of Arg and/or Lys in the N-terminal region was analyzed as described in Fig. 1. IP, immunoprecipitation; WB, Western blot.

FIGURE 5.

An N-terminal Ser in Shh can be palmitoylated by Hhat. A and B, palmitoylation of wild-type and C24S and C24A mutants of ShhNp (A) and Shh(1–197) EGFP (B) was analyzed in COS-1 cells co-transfected with Hhat. A doublet for palmitoylated ShhNp was observed occasionally and may represent degradation or additional processing. C, quantification of the data from A and B, from one to three experiments, each performed in duplicate or triplicate. IP, immunoprecipitation; WB, Western blot.

FIGURE 6.

Substrate specificity of Rasp. A, COS-1 cells were co-transfected with Rasp and the indicated Shh(1–29) EGFP constructs, labeled with [¹²⁵I]iodopalmitate, and analyzed as described for Fig. 1. B, palmitoylation of wild-type and mutant ShhNp was analyzed in COS-1 cells co-transfected with Rasp. C, palmitoylation of wild-type and mutant HhNp (expressed from a plasmid containing the signal sequence from Spitz) was analyzed in COS-1 cells co-transfected with Rasp. The migration positions of the 45-kDa Hh precursor and 19-kDa HhNp product are marked. D, palmitoylation of wild-type and mutant Spitz-EGFP constructs was analyzed in COS-1 cells co-transfected with Rasp. IP, immunoprecipitation; WB, Western blot.

FIGURE 7.

ShhNp containing Pro or Gln at position 2 are palmitoylated. COS-1 cells were transfected with constructs encoding full-length Shh, labeled with [¹²⁵I]iodopalmitate, and analyzed as described for Fig. 4. IP, immunoprecipitation; WB, Western blot.

FIGURE 8.

Schematic model for the Hhat palmitoylation motif within Shh. See “Discussion” for details.