Tyrosine phosphorylation of caldesmon is required for binding to the Shc center dot Grb2 complex

S3-v-erbB is a retroviral oncogene that encodes a ligand-independent, trans forming mutant of the epidermal growth factor receptor. This oncogene has b een shown to be sarcomagenic in vivo and to transform fibroblasts in vitro. Our previous studies (McManus, M.J., Lingle, W, L,, Salisbury, J, L,, and Maihle, N, J, (1997) Proc. Natl Acad. Sci, U.S.A, 94, 11351-11356) showed t hat expression of S3-v-erbB in primary fibroblasts results in the tyrosine phosphorylation of caldesmon (CaD), an actin- and calmodulin-binding protei n. This phosphorylation is transformation-associated, and the phosphorylate d form of CaD is associated with a signaling complex consisting of Shc, Grb 2, and Sos in transformed fibroblasts, To identify the tyrosine phosphoryla tion site(s) in the CaD molecule and to further elucidate the functional ro le of CaD tyrosine phosphorylation in S3-v-ErbB oncogenic signaling, we hav e generated a series of mutant CaDs in which one or more tyrosine residues have been replaced with phenylalanine. Using a CaD null cell line, DF1 cell s (an immortalized chicken embryo fibroblast cell line), and transient tran sfection assays, we demonstrated that Tyr-27 and Tyr-393 are the major site s of tyrosine phosphorylation on CaD, Interestingly, Tyr-27 is located with in the myosin binding domain of CaD, and Tyr-393 is adjacent to one of the major actin binding and actomyosin ATPase inhibitory domains. Our studies a lso show that the tyrosine phosphorylation of CaD enhances its binding to t he Shc.Grb2 complex. Specifically, replacement of Tyr-27, but not of Tyr-16 5 or Tyr-393, significantly reduces the ability of CaD to interact with the Shc Grb2 complex, Together, these studies demonstrate that the major sites of tyrosine phosphorylation on CaD are located in the myosin and actin bin ding domains of CaD and that Tyr-27 is the major tyrosine phosphorylation s ite through which CaD interacts with the Shc Grba complex.