Siah-1 N-terminal RING domain is required for proteolysis function, and C-terminal sequences regulate oligomerization and binding to target proteins.
The Drosophila seven in absentia (sina) gene was initially discovered because its inactivation leads to R7 photoreceptor defects. Recent data indicate that Sina binds to the Sevenless pathway protein Phyllopod, and together they mediate degradation of Tramtrack, a transcriptional repressor of R7 cell fate. Independent studies have shown that Sina and its highly related mammalian homologues Siah-1 and Siah-2 bind to the DCC (deleted in colorectal cancer) protein and promote its proteolysis via the ubiquitin-proteasome pathway. To determine the roles of mammalian Siahs in proteolysis and their interactions with target proteins, we sought to define Siah-1 domains critical for regulation of DCC. Mutant Siah-1 proteins, harboring missense mutations in the carboxy (C)-terminal domain analogous to those present in Drosophila sina loss-of-function alleles, failed to promote DCC degradation. Point mutations and deletion of the amino (N)-terminal RING finger domain of Siah-1 abrogated its ability to promote DCC proteolysis. In the course of defining Siah-1 sequences required for DCC degradation, we found that Siah-1 is itself rapidly degraded via the proteasome pathway, and RING domain mutations stabilized the Siah-1 protein. Siah-1 was found to oligomerize with itself and other Sina and Siah proteins via C-terminal sequences. Finally, evidence that endogenous Siah-1 regulates DCC proteolysis in cells was obtained through studies of an apparent dominant negative mutant of Siah-1, as well as via an antisense approach. The data indicate that the Siah-1 N-terminal RING domain is required for its proteolysis function, while the C-terminal sequences regulate oligomerization and binding to target proteins, such as DCC.
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Division of Molecular Medicine and Genetics, Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan.
Fearon
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D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002461:Cell Line, Transformed; D003546:Cysteine Endopeptidases / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D013329:Structure-Activity Relationship; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; D000098563:Seven in Absentia Proteins
D015815:Cell Adhesion Molecules; D009097:Multienzyme Complexes; D009687:Nuclear Proteins; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; D000098563:Seven in Absentia Proteins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex