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Identification and genetic analysis of human and mouse activated Cdc42 interacting protein-4 isoforms
Lin Wang1, William A Rudert, Anatoly Grishin
1Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Abstract:
By yeast two-hybrid screening with the Src kinase Lyn as bait, we identified a novel gene product with features of a scaffolding protein. Reported as Felic ( es-related, with homology to Ezrin, Lyn interactor with Cdc42), it is related to the CIP4 (Cdc42 Interacting Protein-4) gene. Southern blotting for CIP4/Felic of genomic DNA shows a single band, suggesting no gene duplication. Felic differs from CIP4 because of a 29 nucleotide sequence derived from the end of intron 13. Consequently, there is an out-of-frame translation that destroys an SH3 domain. Analysis of various tissues shows that the original CIP4 is the predominant transcript. Therefore, we propose to call that, CIP4a and Felic, CIP4b. During screening of the colorectal CaCo2 cell line, clones corresponding to a third CIP4-related transcript (CIP4c) were identified. CIP4c encodes a premature stop codon, resulting in the loss of the SH3 domain. A fourth, relatively abundant transcript (CIP4h) was isolated from heart, lung, and trachea tissue. CIP4h retains the SH3 domain. CIP4 levels are modified by all-trans-retinoic acid. The presence of alternative splice transcripts, with or without SH3 domains, suggests that CIP4 regulates cytoskeletal organization through structural-functional differences in a tissue-specific manner.
Insights
Researchers identified novel scaffolding protein variants, CIP4b (Felic) and CIP4c, related to Cdc42 Interacting Protein-4 (CIP4). These variants, differing in SH3 domain presence, suggest tissue-specific regulation of cytoskeletal organization.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The Src kinase Lyn interacts with scaffolding proteins.
- Cdc42 Interacting Protein-4 (CIP4) is involved in cellular processes.
- Alternative splicing generates protein diversity.
Purpose of the Study:
- Identify novel Lyn-interacting proteins.
- Characterize the CIP4 gene family and its splice variants.
- Investigate the functional implications of CIP4 splice variants.
Main Methods:
- Yeast two-hybrid screening.
- Southern blotting.
- RNA analysis and transcript identification.
- Tissue-specific expression analysis.
Main Results:
- A novel scaffolding protein, Felic (CIP4b), was identified.
- CIP4b contains a unique intronic sequence, leading to a non-functional SH3 domain.
- Two additional CIP4 variants, CIP4c (lacking SH3 domain) and CIP4h (with SH3 domain), were identified.
- CIP4 transcript levels are modulated by all-trans-retinoic acid.
- CIP4 expression is tissue-specific.
Conclusions:
- The CIP4 gene produces multiple splice variants (CIP4a, CIP4b, CIP4c, CIP4h) with distinct structural features.
- The presence or absence of the SH3 domain in CIP4 variants may dictate their role in cytoskeletal organization.
- CIP4 variants exhibit tissue-specific expression, suggesting specialized functions.