Evidence for the direct involvement of {beta}TrCP in Gli3 protein processing

Abstract

Hedgehog-regulated processing of the transcription factor cubitus interruptus (Ci) in Drosophila depends on phosphorylation of the C-terminal region of Ci by cAMP-dependent protein kinase and subsequently by casein kinase 1 and glycogen synthase kinase 3. Ci processing also requires Slimb, an F-box protein of SCF (Skp1/Cullin/F-box proteins) complex, and the proteasome, but the interplay between phosphorylation and the activity of Slimb and the proteasome remains unclear. Here we show that processing of the Gli3 protein, a homolog of Ci, also depends on phosphorylation of a set of four cAMP-dependent protein kinase sites that primes subsequent phosphorylation of adjacent casein kinase 1 and glycogen synthase kinase 3. Our gain- and loss-of-function analyses in cultured cells further reveal that betaTrCP, the vertebrate homolog of Slimb, is required for Gli3 processing, and we demonstrate that betaTrCP can bind phosphorylated Gli3 both in vitro and in vivo. We also find that the Gli3 protein is polyubiquitinated in the cell and that its processing depends on proteasome activity. Our findings provide evidence for a direct link between phosphorylation of Gli3/Ci proteins and betaTrCP/Slimb action, thus supporting the hypothesis that the processing of Gli3/Ci is affected by the proteasome.

Fig. 1.

Gli3 processing requires βTrCP. HEK293 cells were transfected with the expression constructs or together with either βTrCP siRNA or GFP siRNA control as shown above the blots and treated overnight with FSK as indicated. Gli3 protein was detected by immunoblotting with an anti-Gli3 antibody. Overexpression of βTrCP resulted in an increase in the Gli3–83 levels (A, compare lanes 5 and 7 with lane 3), whereas βTrCP RNAi blocks Gli3 processing (B, compare lane 6 with lanes 4 and 5).

Fig. 2.

PKA-primed phosphorylation of Gli3 protein by CK1 and GSK3. (A)A schematic drawing of hGli3 protein. Six dots stand for PKA sites, and a filled box represents the zinc finger DNA-binding domain. An arrow indicates the approximate cleavage site. Underlined is the consensus sequence for PKA phosphorylation with S residues shown in bold. Numbers below the lines correspond to the designated names of the Gli3 mutant constructs. PKA-primed phosphorylation sites for GSK3 and CK1 are circled and boxed, respectively. Overlined are the secondary S residues that may potentially become GSK3 and CK1 phosphorylation sites. (B) Immunoblot analysis revealed phosphorylation of Gli3 and its mutants as measured by mobility shift of the proteins in transfected chicken limb bud cells after treatment with dideoxy FSK (ddFSK) and FSK. Proteins were separated by 5% SDS/PAGE. (C) PKA-primed phosphorylation of GST-Gli3PR (839–920 aa) and its mutants by CK1 and GSK3 in vitro. Affinity-purified GST-Gli3PR and its mutant proteins were first incubated with PKA catalytic subunit in the presence of nonradioactive ATP and subsequently with CK1 or GSK3 in the presence of [γ-³²P]ATP. The phosphorylated proteins were resolved by SDS/PAGE and detected by autoradiography.

Fig. 3.

Putative PKA, CK1, and GSK3 phosphorylation sites in Gli3 protein are required for Gli3 processing. (A and B) Primary chick limb bud monolayer cultures were transfected with wild-type Gli3 or its mutant constructs with point mutations at PKA sites (A) or CK1 or GSK3 sites (B) (see Fig. 2 A for sites mutated). Cells were treated with FSK, dideoxy FSK (ddFSK), or DMSO vehicle for 16–18 h, and Gli3 processing was examined by immunoblotting with the anti-Gli3 antibody. Gli3–83 protein was not detected for Gli3P1–3, Gli3P4–6, and Gli3P1–6 (A, lanes 11–13) and for Gli3C1–4 and Gli3N2–4 (B, lanes 7 and 10).

Fig. 4.

Direct interaction of βTrCP with Gli3 protein. (A) Interaction between βTrCP and Gli3 in the cell. HEK293 cells were transfected with myc-mβTrCP alone or together with Gli3 or its mutant constructs and treated with FSK overnight as shown below the blots. The protein lysates from the cells were incubated with DNA-conjugated Sepharose beads containing specific Gli-binding consensus sequence (Gli-binding resin) or nonspecific sequence (control). The precipitated proteins were then resolved by 7% SDS/PAGE and transferred to nitrocellulose filters. The filters were horizontally cut into two halves. The upper half was immunoblotted with anti-Gli3 antibody (Upper), and the lower half was immunoblotted with anti-myc antibody to detect myc-mβTrCP (Lower). (B) Interaction between a phosphorylated Gli3 fragment and βTrCP. GST, GST-Gli3PR, or its mutants were first phosphorylated with the indicated kinases and were then used to pull down ³⁵S-labeled myc-mβTrCP or myc-mβTrCPΔWD proteins, which were detected by autoradiography.

Fig. 5.

Proteasome-dependent Gli3 processing. (A) An immunoblot showing that Gli3 processing was blocked by treatment of transfected HEK293 cells with the proteasome inhibitor MG115. (B) Gli3 is polyubiquitinated. HEK293 cells were transfected with expression constructs shown above the blots. After they were denatured, protein lysates from the cells were subjected to immunoprecipitation with either anti-Gli3 or anti-myc antibodies, followed by immunoblotting with reciprocal antibodies as indicated below the blots.