Figure 6.

An altered tyrosine-phosphorylated protein in zyxin-null cells is identified as the actomyosin regulator caldesmon. Indirect immunofluorescence of pY-containing proteins in wild-type (A) and zyxin-null (B) fibroblasts. (C) Immunoblot analysis of equivalent amounts of protein from wild-type and zyxin−/− cells using pY-specific antibody 4G10 detected a 75-kD signal (arrowheads) in wild-type cells, and an 80-kD signal (arrowhead) in zyxin−/− cells. (D) 2D gel electrophoresis and anti-pY immunoblot analysis identified a slower mobility series of pY spots from zyxin-null fibroblasts compared with wild-type fibroblasts. (E) Wild-type and zyxin-null pY-positive spots of interest were excised from 2D gels, partially digested with trypsin, and then subjected to LC-MS/MS for protein sequence analysis. Both sets of spots contained multiple peptide sequences for the actomyosin regulator caldesmon, which are shown aligned with l-caldesmon coding exons1′–13. Exons 3b and 4, which would be found in h-caldesmon, are shown above the l-caldesmon structure. (F) Immunoblot for caldesmon in wild-type and zyxin−/− cell lysates separated by 10% SDS-PAGE. Short exposure (left) showed nonmuscle caldesmon (l-caldesmon) migrating between 75–80 kD and longer exposure (right) identified the 140-kD smooth-muscle caldesmon (h-caldesmon). (G) Tissue extracts (lung, bladder, and spleen) from wild-type (+/+), zyxin-heterozygous (+/−), and zyxin-null (−/−) mice were electrophoresed, transferred to nitrocellulose, and probed for zyxin (B71 rabbit anti-serum; top) and caldesmon (bottom).