Figure 1.

Schematic representation of the human CMS and protein sequence. (A) Alignment of human cDNA clones isolated in the yeast two-hybrid screen, 5′ rapid amplification of complementary DNA ends (RACE) and λgt11 cDNA library screen with respect to the full-length CMS cDNA. PR, proline-rich region. (B) Predicted protein sequence of CMS. Sequence numbers are shown on the left. The SH3 domains are underlined. Proline-rich sequences are marked in bold, the CC domain is marked in italics, and the putative actin binding sites are bold underlined. IP, immunoprecipitation. (C) In vitro interaction of p130^Cas and CMS. 293T cells were transiently transfected with the flag-tagged CMS yeast TH clone (CMS-TH) together with the CasSH3 domain or full-length Cas (both GST tagged) or the vector alone (GST control). Cell lysates were immunoprecipitated with anti-flag antibody or glutathione Sepharose beads, and precipitates were subjected to SDS/PAGE and probed with anti-GST antibody and anti-flag antibody, respectively. The upper blot was striped and reprobed with anti-flag antibody. (D) Direct interaction of CMS with p130^Cas. Five hundred micrograms of protein from 293T cells expressing the various CMS peptides or C3G used as a positive control was immunoprecipitated with the antibodies indicated above. Blots were probed for binding with ³²P-labeled GST-p130^CasSH3 domain.