Fig. 1
The phosphorylation of SMTNL1 by PKA alters its tropomyosin-binding potential. a The SMTNL1 protein contains: a C-terminal calponin homology (CH) domain, Ser301 PKA-phosphorylation site, Ca-calmodulin (CaM)-binding domain (CBD1), apo-CaM-binding domain (CBD2), and tropomyosin (Tpm)-binding domain (indicated as the hatched area). b Purified recombinant SMTNL1-TMB was incubated with PKA as described in the Methods section. Samples were withdrawn at the indicated times and subjected to Phos-tag SDS-PAGE; two discrete bands representing unphosphorylated SMTNL1-TMB (0P, TMB) or SMTNL1-TMB phosphorylated at Ser301 (1P, pTMB) were detected by Coomassie stain. Samples subjected to Phos-tag SDS-PAGE in the absence of MnCl2 confirmed the shift in band migration to be a result of phosphorylation. c Unphosphorylated or phosphorylated SMTNL1-TMB (200 μg) was incubated with 40 μL of Tpm-Sepharose (α/β-heterodimer: Tpm1.4/Tpm2.1). Bound SMTNL1-TMB was eluted with boiling 0.1% SDS solution and detected with Coomassie staining of SDS-PAGE gels. d The SMTNL1-TMB bands were quantified by densitometry, and binding to Tpm-Sepharose was expressed as percentage of the SMTNL1-TMB binding found for the unphosphorylated state. All experiments are n = 3–5 and were analyzed by Student’s t-test. *- Significantly different from unphosphorylated SMTNL1-TMB, p < 0.05