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. CONCLUSION:These results suggest that cortactin interacts with CBP90 and plays a role in regulation of the actin cytoskeleton in brain.

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