Regulation of Ci-SCFSlimb binding, Ci proteolysis, and hedgehog pathway activity by Ci phosphorylation

Area of Science:

• Cellular signaling
• Developmental biology
• Molecular mechanisms

Background:

• Hedgehog (Hh) proteins are crucial signaling molecules in development.
• Hh signaling regulates Ci/Gli transcription factors via proteolytic processing and activity modulation.
• In the absence of Hh, Ci/Gli undergoes phosphorylation, leading to SCF ubiquitin ligase complex binding and proteolysis.

Purpose of the Study:

• To elucidate the molecular mechanisms by which CK1 and GSK3 sites on Ci influence its interaction with SCF ubiquitin ligase complexes.
• To understand how these phosphorylation events contribute to a graded proteolytic response to Hh signaling.
• To investigate the role of Su(fu) in limiting Ci activity when proteolysis is impaired.

Main Methods:

• Biochemical assays to study the interaction between phosphorylated Ci and SCF complexes.
• Site-directed mutagenesis of CK1 and GSK3 phosphorylation sites on Ci.
• Analysis of Ci variants to assess proteolytic processing and transcriptional activity.
• Investigating the regulatory role of Su(fu) on Ci activity.

Main Results:

• Multiple successively phosphorylated CK1 sites on Ci form an extended binding site for SCF(Slimb).
• GSK3 enhances Ci binding to SCF complexes, potentially through interactions with other SCF components.
• Altered CK1 and GSK3 sites demonstrate that numerous phosphorylation sites confer a sensitive and graded proteolytic response to Hh.
• Su(fu) was identified as the principal regulator of Ci specific activity when proteolysis is compromised.

Conclusions:

• The extensive phosphorylation of Ci by CK1 and GSK3 is critical for mediating a graded proteolytic response to Hh signaling.
• This graded response, coupled with changes in Ci activity, orchestrates morphogenetic outcomes.
• Su(fu) plays a key role in regulating Ci activity, particularly under conditions of compromised proteolysis.