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Cloning and characterization of Cbl-associated protein splicing isoforms.

ABSTRACT: Cbl-associated protein (CAP) is an adaptor protein that plays important roles in both signal transduction and cytoskeleton rearrangement. Alternative splicing of the gene SORBS1 results in multiple isoforms of CAP protein. We report here the cloning of 3 new CAP isoforms, CAP2, CAP3, and CAP4, from mouse adipose tissue. RT-PCR analyses reveal that the isoform mRNAs are differentially expressed. CAP2, CAP3, and CAP4 contain a coiled-coil domain. In addition, CAP4 contains a proline-rich region, part of which exists in CAP3. Coimmunoprecipitation experiments show that CAP4 forms a homodimeric complex. While these new isoforms similarly interact with Cbl, they exhibit varied binding specificity toward vinculin. In contrast to CAP1 and CAP2, CAP4 does not interact with vinculin, and CAP3 binds with low affinity. Immunofluorescence analysis demonstrates differential subcellular localization of Myc-tagged CAP isoforms in 3T3-L1 adipocytes. These results suggest that these new isoforms of CAP might play different signaling roles.

SUBMITTER: Zhang M

PROVIDER: S-EPMC1430382 | biostudies-literature | 2003 Jan-Feb

REPOSITORIES: biostudies-literature

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Cloning and characterization of Cbl-associated protein splicing isoforms.

Zhang Mei M Kimura Akiko A Saltiel Alan R AR

Molecular medicine (Cambridge, Mass.) 20030101 1-2

Cbl-associated protein (CAP) is an adaptor protein that plays important roles in both signal transduction and cytoskeleton rearrangement. Alternative splicing of the gene SORBS1 results in multiple isoforms of CAP protein. We report here the cloning of 3 new CAP isoforms, CAP2, CAP3, and CAP4, from mouse adipose tissue. RT-PCR analyses reveal that the isoform mRNAs are differentially expressed. CAP2, CAP3, and CAP4 contain a coiled-coil domain. In addition, CAP4 contains a proline-rich region, p ...[more]

PMID: 12765336

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Project description:BackgroundSperm binding proteins and their C-terminal peptides of the Sperm Associated Antigen 11 (SPAG11) family were found to play an important role in epididymal innate immunity in addition to their role in sperm maturation. However, the expression of Spag11 transcripts in rodents is not well documented.MethodsComputational analysis was employed to identify novel Spag11 isoforms in the rat. RT-PCR analyses were carried out on RNAs isolated from the male reproductive tract tissues of rat using gene specific primers for Spag11c and Spag11t. The identities of PCR products were confirmed by sequencing. Tissue distribution, developmental expression and androgen regulation of Spag11t and Spag11c were studied using RT-PCR. The antimicrobial activities of recombinant Spag11t and Spag11c were tested against E coli in a colony forming unit assay.ResultsIn this study, we identified two novel Spag11 transcripts, namely, Spag11t and Spag11c derived from the long arm of chromosome 16 in the rat (Rattus norvegicus), using both in silico and molecular biology approaches. Spag11c is expressed in all three regions of the epididymis, in testis and in ovary but is absent from the seminal vesicle. Spag11t expression is confined to the caput and it is not expressed in the testis, seminal vesicle or ovary. Age dependent expression of Spag11t and Spag11c was observed in the epididymides of rats (10-60 day old). Their expression was found to be most abundant in the adult rat (60 day) suggesting roles in mature reproductive function. Further, both Spag11t and Spag11c expression was down regulated in castrated rat epididymides and the expression was maintained in the testosterone replaced castrated rats. SPAG11C is a potent antibacterial agent. SPAG11T also displayed bactericidal capacity although weaker than SPAG11C and SPAG11E.ConclusionThe abundant expression of Spag11t and Spag11c in the male reproductive tract suggests an important role in male reproductive tract immunity. Their expression is developmentally regulated and androgen dependent. Characterization of novel SPAG11 isoforms will contribute to our understanding of the role of epididymal proteins in sperm maturation and innate immunity.

Project description:BackgroundIt is often the case that mammalian genes are alternatively spliced; the resulting alternate transcripts often encode protein isoforms that differ in amino acid sequences. Changes among the protein isoforms can alter the cellular properties of proteins. The effect can range from a subtle modulation to a complete loss of function.Results(i) We examined human splice-mediated protein isoforms (as extracted from a manually curated data set, and from a computationally predicted data set) for differences in the annotation for protein signatures (Pfam domains and PRINTS fingerprints) and we characterized the differences & their effects on protein functionalities. An important question addressed relates to the extent of protein isoforms that may lack any known function in the cell. (ii) We present a database that reports differences in protein signatures among human splice-mediated protein isoform sequences.Conclusion(i) Characterization: The work points to distinct sets of alternatively spliced genes with varying degrees of annotation for the splice-mediated protein isoforms. Protein molecular functions seen to be often affected are those that relate to: binding, catalytic, transcription regulation, structural molecule, transporter, motor, and antioxidant; and the processes that are often affected are nucleic acid binding, signal transduction, and protein-protein interactions. Signatures are often included/excluded and truncated in length among protein isoforms; truncation is seen as the predominant type of change. Analysis points to the following novel aspects: (a) Analysis using data from the manually curated Vega indicates that one in 8.9 genes can lead to a protein isoform of no "known" function; and one in 18 expressed protein isoforms can be such an "orphan" isoform; the corresponding numbers as seen with computationally predicted ASD data set are: one in 4.9 genes and one in 9.8 isoforms. (b) When swapping of signatures occurs, it is often between those of same functional classifications. (c) Pfam domains can occur in varying lengths, and PRINTS fingerprints can occur with varying number of constituent motifs among isoforms - since such a variation is seen in large number of genes, it could be a general mechanism to modulate protein function. (ii)DataThe reported resource (at http://www.bioinformatica.crs4.org/tools/dbs/splivap/) provides the community ability to access data on splice-mediated protein isoforms (with value-added annotation such as association with diseases) through changes in protein signatures.

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Cloning and characterization of Cbl-associated protein splicing isoforms.

Publications

Cloning and characterization of Cbl-associated protein splicing isoforms.

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