Cloning and characterization of ectopically expressed transcripts for the actin-binding protein MIPP in mouse mammary
J N VanHouten1, H L Asch, B B Asch
1Division of Experimental Pathology, Roswell Park Cancer Institute Corporation, Buffalo, New York, NY 14263, USA. joshua.vanhouten@yale.edu
Abstract:
Mipp is a kelch-related, placental-specific gene that is ectopically expressed in many BALB/c mouse mammary carcinomas of various etiologies. The Kelch family encompasses proteins that are emerging as key links between microfilaments and a variety of cellular structures and functions. Mouse mammary tumors express two mipp transcripts (2.2 and 5.6 kb). We cloned the 2.2 kb mipp mRNA and analysed the product of its 1.7 kb ORF. The 584 residue MIPP protein has an N-terminal BTB domain and six C-terminal tandem Kelch repeats. Despite expression of two mipp RNAs, only a single MIPP protein is expressed in mammary tumors. MIPP protein binds to microfilaments in vitro and co-immunoprecipitates with actin. MIPP co-localized with concanavalin A at the endoplasmic reticulum, suggesting that MIPP might mediate interactions between microtubules and actin filaments. Because MIPP expression is widespread in mouse mammary tumors, it might contribute to tumorigenesis. Although MIPP had little effect on the growth rate of human breast cell lines following transfection, it greatly reduced the formation of duct-like structures on reconstituted basement membrane. Our results suggest that MIPP could contribute to malignant progression in the mouse mammary epithelial cells by perverting their response to cues from the extracellular matrix.
Insights
The MIPP protein, found in mouse mammary tumors, interacts with actin filaments and may drive cancer progression by altering cell responses to the extracellular matrix.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- The Kelch family proteins link microfilaments to cellular structures.
- Mipp is a placental-specific gene ectopically expressed in mouse mammary tumors.
- Two MIPP transcripts (2.2 and 5.6 kb) are found in mouse mammary tumors.
Purpose of the Study:
- To characterize the MIPP protein and its function in mammary tumors.
- To investigate MIPP's role in tumorigenesis and malignant progression.
Main Methods:
- Cloning and analysis of the 2.2 kb MIPP mRNA and its 1.7 kb open reading frame (ORF).
- In vitro binding assays to assess MIPP's interaction with microfilaments.
- Co-immunoprecipitation with actin to confirm protein interactions.
- Immunofluorescence microscopy to determine MIPP localization.
- Transfection studies in human breast cell lines and assessment of duct-like structure formation.
Main Results:
- The 584-residue MIPP protein possesses an N-terminal BTB domain and six C-terminal Kelch repeats.
- Only a single MIPP protein isoform is expressed in mammary tumors despite two transcripts.
- MIPP protein binds to microfilaments in vitro and co-immunoprecipitates with actin.
- MIPP localizes to the endoplasmic reticulum and may link microtubules and actin filaments.
- MIPP expression in mouse mammary tumors suggests a role in tumorigenesis.
- MIPP transfection reduced duct-like structure formation in human breast cells but had minimal impact on growth rate.
Conclusions:
- MIPP protein interacts with the actin cytoskeleton.
- MIPP may contribute to malignant progression in mouse mammary epithelial cells by disrupting their response to extracellular matrix cues.
