Isolation and characterization of cortactin isoforms and a novel cortactin-binding protein, CBP90.
BACKGROUND
Cortactin is a major phosphotyrosyl protein in pp60v-src-transformed chicken embryo cells. Cortactin binds to actin filament (F-actin) through a unique region which consists of six tandem 37 amino acid repeats, named cortactin repeats. Furthermore cortactin has one src homology 3 (SH3) domain.


RESULTS
In this study we have isolated two new isoforms of cortactin from the rat brain using a polymerase chain reaction (PCR) method, and have named the original cortactin as cortactin-A and the newly isolated forms as cortactin-B and -C. Cortactin-A, -B and -C had six, five, and four cortactin repeats, respectively. All the isoforms were able to bind to F-actin, but only cortactin-A demonstrated an F-actin-crosslinking activity. In addition, cortactin-A was able to bind along the side of F-actin. Next, using a blot overlay assay with glutathione S-transferase (GST)-cortactin-A, we identified a cortactin-A-binding protein with an Mr of approximately 90 kDa in rat brain and named it CBP90 (cortactin-binding protein with an Mr of approximately 90 KDa). CBP90 was purified from rat brain and its cDNA was cloned from a rat brain cDNA library. The deduced amino acid sequence of CBP90 had no significant similarity to any other protein, but it had a proline-rich domain at the C-terminal region. CBP90 was able to bind to all the cortactin isoforms. A deletion mutant analysis of cortactin-A and CBP90 revealed that the SH3 domain of cortactin-A was able to bind to the proline-rich region of CBP90. A Western blot analysis with an anti-CBP90 antibody indicated that, among the rat tissues examined, CBP90 was exclusively expressed in brain. Furthermore, its subcellular distribution and developmental expression patterns were similar to those of cortactin.


CONCLUSIONS
These results suggest that cortactin interacts with CBP90 and plays a role in regulation of the actin cytoskeleton in brain.
Ohoka
Y
Y
Takai Biotimer Project, ERATO, Japan Science and Technology Corporation, 2-2-10 Murotani, Nishi-ku, Kobe 651-2241, Japan.
Takai
Y
Y
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D001923:Brain Chemistry; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D051356:Cortactin; D003598:Cytoskeletal Proteins; D018507:Gene Expression Regulation, Developmental; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D007536:Isomerism; D008840:Microfilament Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions; D014018:Tissue Distribution
D000199:Actins; D002352:Carrier Proteins; D051356:Cortactin; C495870:Cttn protein, rat; C115999:Cttnbp2 protein, rat; D003598:Cytoskeletal Proteins; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D011994:Recombinant Proteins; D005982:Glutathione Transferase