The strategy of this proposal is to use a retrograde approach by starting at the actin promoter and tracing the signaling pathway back to the stretch stimulus. Serum response factor (SRF) binds to serum response element1 (SRE1) in the chicken skeletal actin promoter. SRE1 is necessary and sufficient for the increase in actin promoter activity caused by stretch of the anterior latissimus dorsi (ALD) muscle of young chickens. A faster migration of the SRF-SRE binding complex occurs from nuclear extracts of 3- and 6-day stretched ALD muscles, which indicates a post-translational modification to SRF. Specific aim 1 is to determine whether the phosphorylation status of SRF is altered during the stretch of chicken primary myocytes. 32P incorporation will be measured in SRF from stretched cells. Times of initial increases among SRF phosphorylation, skeletal actin mRNA, actin promoter activity, and faster migration of SRE-SRF in non-denaturing gels will be compared to determine the sequence of events. Precedence for the hypotheses in this proposal is provided by the extensive literature about the growth factor transactivation of c-fos by phosphorylation of SRF. Specific aim 2A is to determine which serine(s)/threonine(s) in the SRF is phosphorylated during stretch by the methods of two-dimensional phosphopeptide mapping and phosphoamino analysis. Precedence for specific aim 2 is that growth factors phosphorylate SRF and its accessory protein during induction of the c-fos promoter. Specific aim 2B will determine which nuclear protein kinase is responsible for phosphorylating SRF during stretch in cultured cells. Preliminary data show that stretch is associated with a faster mobility of SRF from nuclear but not cell extracts. Specific aim 3 will use the GAL4 system to determine the function of SRF amino acids which are phosphorylated by stretch. GAL4-SRF fusion proteins will demonstrate the region of SRF which transmits the stretch signal to the actin promoter. A key experiment will then be whether a site-specific mutation to an amino acid that is phosphorylated by stretch in the functional region of SRF abolishes the stretch-induced activation of SRF in the GAL4 system. This experiment's results will then be extended to the ALD muscle of young chickens. Specific aim 4 will use a dominant negative mutant of the kinase which was identified as responsible for stretch-induced activation of SRF. The molecular basis of muscle hypertrophy will assist physical training and pharmaceutical strategies to build skeletal muscle mass in the elderly for the purpose of keeping elderly out of nursing homes by maintaining their physical strength and thus their independence.