Binding of smoothelin-like 1 to tropomyosin and calmodulin is mutually exclusive and regulated by phosphorylation

Figures

• 

  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 Samples were withdrawn at the indicated times and subjected to Phos-tag SDS-PAGE; two discrete bands representing unphosphorylated SMTNL1-TMB (0P, TMB) or SMTNL1-TMB phosphorylated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…

• 

  Fig. 2. Interdependency of SMTNL1 phosphorylation, calcium and tropomyosin on calmodulin-binding. SMTNL1-TMB (200 μg, 7 nmol) was incubated with an equimolar amount of Tpm. The mixture was then incubated with CaM-Sepharose (40 μL; ligand density of ~10–14 μmol/mL) in the presence (a; Ca-CaM, 5 mM CaCl2) or absence (b; apo-CaM, 1 mM EDTA) of calcium. Some experiments were completed with SMTNL1-TMB that had been previously phosphorylated with PKA. After washing, the retention of SMTNL1-TMB or phosphorylated SMTNL1-TMB was analyzed. The band densities were quantified and binding to CaM-Sepharose expressed as percentage of SMTNL1-TMB recovered under maximal binding conditions (i.e., Ca-CaM in the absence of Tpm). All experiments are n = 3–5 and were analyzed by one-way ANOVA with Tukey’s post hoc analysis. Different letters indicate significant differences among groups (a,b,c: comparison among all Ca-CaM conditions; d,e,f: comparison among all apo-CaM conditions; p < 0.05)

• 

  Fig. 3. SMTNL1 phosphorylation precludes Ca-CaM-binding in favour of Tpm-binding under conditions of high Tpm content. SMTNL1-TMB or phosphorylated SMTNL1-TMB (concentrations as described in Fig. 2) was pre-incubated with the indicated molar ratio of purified α/β Tpm dimer. The mixtures were then added to CaM-Sepharose in the absence (a, c; apo-CaM, 1 mM EDTA), or presence (b, d; Ca-CaM, 5 mM CaCl2) of calcium. After extensive washing, bound SMTNL1-TMB was eluted and detected with Coomassie staining of SDS-PAGE gels. The band density was quantified and binding expressed as percentage of maximum binding within each condition. All experiments are n = 3 and were analyzed by one-way ANOVA with Tukey’s post hoc analysis. Different letters indicate significant differences among groups (p < 0.05)