Binding of smoothelin-like 1 to tropomyosin and calmodulin is mutually exclusive and regulated by phosphorylation
© The Author(s). 2017
Received: 19 October 2016
Accepted: 15 March 2017
Published: 21 March 2017
The smoothelin-like 1 protein (SMTNL1) can associate with tropomyosin (Tpm) and calmodulin (CaM), two proteins essential to the smooth muscle contractile process. SMTNL1 is phosphorylated at Ser301 by protein kinase A during calcium desensitization in smooth muscle, yet the effect of SMTNL1 phosphorylation on Tpm- and CaM-binding has yet to be investigated.
Using pull down studies with Tpm-Sepharose and CaM-Sepharose, we examined the interplay between Tpm binding, CaM binding, phosphorylation of SMTNL1 and calcium concentration. Phosphorylation greatly enhanced the ability of SMTNL1 to associate with Tpm in vitro; surface plasmon resonance yielded a 10-fold enhancement in K D value with phosphorylation. The effect on CaM binding is more complex and varies with the availability of calcium.
Combining both CaM and Tpm with SMTNL1 shows that the binding to both is mutually exclusive.
KeywordsSMTNL1 CHASM Smooth muscle Thin filament Protein binding
Smoothelin-like 1 protein (SMTNL1), also termed calponin homology-associated with smooth muscle (CHASM) protein, was originally identified as a protein phosphorylated in response to cyclic nucleotide-dependent protein kinase (i.e., protein kinase A (PKA) and protein kinase G (PKG)) activation during calcium desensitization of gastrointestinal smooth muscle . Additional studies have revealed a role for SMTNL1 in vascular smooth muscle contractile activity, as well as cardiovascular and skeletal muscle adaptation to exercise, development and pregnancy [2–6].
The ubiquitous calmodulin (CaM) is the primary cellular signal transducer that provides spatial and temporal responses to changes in intracellular calcium levels . CaM provides signaling by association with specific binding sites in target proteins, both in its calcium-saturated (Ca-CaM) or its calcium-free (apo-CaM) form [8, 9]. We have previously reported that SMTNL1 possesses two CaM-binding sites: a classic CaM-binding domain (CBD1, amino acids 310–325) in the center of the protein as well as an IQ-motif that serves as an apo-CaM-binding site (CDB2, amino acids 439–457) located in the carboxy-terminal calponin homology domain [10–12]. SMTNL1 was established as a bona fide CaM-binding protein within aortic A7r5 smooth muscle cells using the proximity ligation assay. In this regard, CBD1 is thought to provide the majority of CaM-binding in situ; however, CBD2 may contribute cooperatively to binding .
A second binding partner of SMTNL1 has also been identified in smooth muscle. Tropomyosin (Tpm), a muscle protein that aids in the stabilization of actin filaments, is incorporated as super-helical polymers along smooth muscle thin filaments and contribute to the regulation of actin-myosin cross bridge cycling during smooth muscle contraction [13–15]. In smooth muscle, two Tpm isoforms (Tpm1.4 and Tpm2.1; previously identified as Tmsm-α/Tm6 and Tmsm-β/Tm1β, respectively ) are predominantly expressed . SMTNL1 was shown to associate with smooth muscle Tpm α/β dimers with a binding surface comprising of its N-terminal intrinsically-disordered region and the C-terminal calponin homology domain .
Previous in vitro studies have defined SMTNL1 as a Tpm- and CaM-binding protein [10–12, 18, 19]. Moreover, the phosphorylation of SMTNL1 was identified during calcium desensitization of smooth muscle [1, 5]. However, the effect of phosphorylation on the ability of SMTNL1 to associate with Tpm and/or CaM has not been investigated. Intriguingly, the binding domains for CaM (CBD1 and CBD2) are localized in close vicinity to the Tpm-binding surface on the SMTNL1 protein. In addition, the Ser301 phosphorylation site targeted by cyclic nucleotide-dependent protein kinases (i.e., PKA and PKG) is located near CBD1 and within the Tpm-binding region [11, 18]. This led us to inquire about the interplay of SMTNL1 phosphorylation and calcium levels on the multi-functionality of Tpm- and CaM-binding. Herein, we demonstrate that phosphorylation of Ser301 greatly enhanced the ability of SMTNL1 to associate with smooth muscle Tpm in vitro. The effect of Ser301 phosphorylation on CaM binding was more complex and varied with the availability of calcium. Combining both CaM and Tpm revealed that binding with SMTNL1 was mutually exclusive.
PreScission Protease, glutathione-Sepharose and CNBr-activated Sepharose were from GE Healthcare (Piscataway, NJ). The Phos-tag acrylamide reagent was from NARD Chemicals (Kobe City, Japan). Smooth muscle Tpm was a purified α/β heterodimer of chicken gizzard Tpm1.4sm(a.a.b.d) (equivalent to Tm6 or Tmsm-α) and Tpm2.1sm(a.b.a.d) (equivalent to TM-1, βTm or Tmsm-β)  and was provided by Dr. Michael Walsh (University of Calgary). CaM was provided by Dr. Hans Vogel (University of Calgary). PKA was purified from bovine heart using the method described in .
Expression and Purification of SMTNL1
A clone comprising the Tpm-binding region of SMTNL1 (SMTNL1-TMB, amino acids 195–459; NP_077192) was described previously [18, 19]. SMTNL1-TMB was expressed as a glutathione S-transferase (GST)-fusion protein in E. coli and purified with glutathione-Sepharose. The fusion protein was cleaved with PreScission Protease, and GST was removed by an additional pass over glutathione-Sepharose along with a final clean-up using MonoQ anion exchange chromatography. In some cases, SMTNL1-TMB was phosphorylated with PKA at a molar ratio of 500:1 (SMTNL1: PKA) in a buffer consisting of 2 mM MgCl2, 0.2 mM ATP, 25 mM HEPES and 150 mM NaCl. The phosphorylated protein was buffer-exchanged and concentrated using a 3 kDa cut-off centrifugation filter unit (EMD Millipore, Billerica, MA).
Tpm-Sepharose binding assay
Purified smooth muscle Tpm or Tris-HCl (as control) was covalently bound to CNBr-activated Sepharose following the manufacturers’ instructions and as described in . SMTNL1-TMB (200 μg) was incubated with 40 μL of Tpm-Sepharose or Control-Sepharose in binding buffer (20 mM Tris-HCl, pH 7.5 with 0.1% (v/v) β-mercaptoethanol) for 2 h at 4 °C and then washed extensively with the same buffer supplemented with 150 mM NaCl. Bound protein was eluted with SDS-PAGE loading buffer, separated by gel electrophoresis, and detected by Coomassie stain.
Calmodulin-Sepharose binding assay
Calmodulin (CaM)-Sepharose pull-down experiments were completed as described previously . Briefly, SMTNL1-TMB (200 μg) was incubated with 40 μL of CaM-Sepharose in binding buffer (20 mM HEPES, pH 7.0 in the presence of 5 mM CaCl2 (Ca-CaM) or 1 mM EDTA (apo-CaM)). After incubation for 1 h at 4 °C, the CaM-Sepharose was washed extensively with the same buffer supplemented with 150 mM NaCl. Bound SMTNL1-TMB protein was eluted with SDS-PAGE loading buffer, separated by gel electrophoresis, and detected by Coomassie stain. In some experiments, SMTNL1-TMB was premixed with Tpm in different molar ratios, allowed to form complexes for 1 h at 4 °C and then added to the CaM-Sepharose.
Surface Plasmon Resonance (SPR)
The binding between Tpm and SMTNL1-TMB with or without phosphorylation was evaluated by SPR using a BIAcore X100 instrument (GE Healthcare). Purified Tpm was immobilized via amine-coupling onto a CM5 sensor chip (GE Healthcare). The running buffer contained 20 mM HEPES pH 7.5, 100 mM KCl, 1 mM DTT, and 0.005% (v/v) Tween-20. Five concentrations of SMTNL1-TMB from 0.12 to 10 μM were prepared by serial dilution and were injected at a flow rate of 30 μL/min with a contact time of 1 min at 25 °C. The chip was regenerated by injecting 1 M NaCl for 4 min followed by glycine-HCl (10 mM, pH 3.0) for 1 min. Each experiment was repeated three times (n = 3) to obtain a standard error (SE). The BIAevaluation software 2.0 (GE Healthcare) was used to analyze the SPR sensorgrams and to obtain the dissociation constants (Kd).
Values are presented as the mean ± S.E.M., with n indicating the number of independent experiments. Data were analyzed with two-tailed Student’s t test, or for comparison of multiple groups, with one-way analysis of variance (ANOVA) and Tukey’s post hoc test. Differences were considered to be statistically significant for p < 0.05. All statistical analyses were performed using the GraphPad Prism 6.0 program.
Phosphorylation of SMTNL1 by PKA enhances Tpm-binding potential
Phosphorylation of SMTNL1-TMB influences binding to CaM-Sepharose
Phosphorylation and calcium influence SMTNL1-TMB binding to CaM-Sepharose in the presence of Tpm
Equimolar amounts of SMTNL1-TMB and Tpm proteins were pre-incubated and then applied to CaM-Sepharose so that competition between CaM and Tpm for binding to SMTNL1-TMB could be assessed. Binding of SMTNL1-TMB to apo-CaM-Sepharose was reduced by approximately 60% by the addition of Tpm to SMTNL1-TMB prior to capture with the resin (Fig. 2b). These data indicate that Tpm could interfere with the apo-CaM binding properties of SMTNL1-TMB, likely due to the proximity of the two binding sites within the calponin homology (CH) domain. Furthermore, we can conclude that SMTNL1-TMB interacts with Tpm more strongly than with apo-CaM. This was not unexpected as the interaction of SMTNL1 with apo-CaM is weak (Kd ~10−6 M ). The results of the binding assays were not affected by the order of addition of proteins since pre-incubating SMTNL1-TMB with CaM-Sepharose followed by competition with Tpm showed the same reduction in binding (data not shown). The subsequent addition of Tpm to the mixture of phosphorylated SMTNL1 and apo-CaM-Sepharose, shown in Fig. 2b, further decreased binding potential to barely detectably levels. When investigating interactions with Ca-CaM, the binding of unphosphorylated SMTNL1-TMB to Ca-CaM-Sepharose was not significantly influenced by the addition of equimolar amounts of Tpm (Fig. 2a). However, the addition of Tpm did significantly reduce the recovery of phosphorylated SMTNL-TMB on the Ca-CaM-Sepharose resin.
SMTNL1 phosphorylation diminishes Ca-CaM-binding potential under conditions of high Tpm content
Phosphorylation events are known to regulate binding of CaM to target proteins. Several examples of phosphorylation within or near CaM-binding domains (CBDs) have been reported in the literature, including but not limited to smooth muscle myosin light chain kinase , MARKS protein , plasma membrane Ca2+-ATPase , endothelial nitric oxide synthase (eNOS) , and the Ca2+-dependent K+-channel . CaM binds to its targets by wrapping around a short amphipathic helix within the CBD [8, 9], thus the phosphorylation of residues within a CBD can disrupt CaM interactions even if the Ser/Thr residues subject to phosphorylation are not necessarily required for CaM binding . Herein, we describe a novel incidence where phosphorylation regulates CaM binding to a target protein. In this case, the PKA-dependent phosphorylation of SMTNL1 at Ser301 modulates both Ca- and apo-CaM binding. Ser301 lies well upstream of the core hydrophobic residues within CBD1 required for Ca-CaM-binding (i.e., 20 amino acids from Phe321 which is the critical contributor ). It is likely that the distance between Ser301 and the hydrophobic patch of CBD1 is too distant for phosphorylation to directly block Ca-CaM docking. This agrees with our observation that the rate of PKA-dependent phosphorylation at Ser301 was not influenced when apo- or Ca-CaM was included in the kinase assay (unpublished results). We have previously demonstrated that intramolecular interactions between the C-terminal IQ motif (localization of the apo-CaM binding site, CBD2) and the intrinsically disordered region (localization of the phosphorylation site) influence apo-CaM binding [11, 12]. Furthermore, a portion of the N-terminal intrinsically disordered region forms intramolecular contacts with the globular C-terminal calponin homology (CH) domain . Thus, it is conceivable that phosphorylation within the N-terminal region has far-reaching effects in the C-terminal domain and can attenuate both Ca- and apo-CaM binding potentials.
The phosphorylation of SMTNL1-TMB enhanced the Tpm-binding potential in both pull down studies and by confirming SPR assays. Inconsequential amounts of nonspecific Tpm-binding were found with CaM-Sepharose, and its binding was not influenced by the presence of SMTNL1-TMB, irrespective of phosphorylation or calcium. Thus, we conclude that a heterotrimeric complex of SMTNL1-TMB, CaM and Tpm was not formed under any of the binding conditions employed. Since no heterotrimer was generated, we suggest that Tpm and Ca-CaM compete for a similar binding site on SMTNL1-TMB. This conclusion is further supported by the fact that the binding surfaces for CaM and Tpm overlap on SMTNL1. Indeed, only very weak binding of Tpm is observed with the isolated CH domain (containing: CBD2; apo-CaM binding) in the absence of the intrinsically-disordered upstream sequence (containing: CBD1, Ca-CaM binding; and Ser301, phosphorylation consensus sequence) .
Our findings place SMTNL1 in a position to influence both Ca2+/CaM-regulated contractile events and Ca2+ desensitization pathways in smooth muscle. Depending on intracellular calcium concentration and cyclic nucleotide-dependent kinase activation, SMTNL1 is predicted to cycle between Tpm-bound and Ca-CaM-bound complexes. The impact of SMTNL1 on smooth muscle contractile processes may be via its binding partners and associations with the contractile filament. In its dephosphorylated state SMTNL1 has weaker affinity for Tpm and would be more likely to associate with CaM. With an increase in intracellular calcium due to a contractile signal, Ca-CaM could bind to SMTNL1 and initiate the dissociation of the complex from the thin filament. Unphosphorylated SMTNL1 can inhibit myosin light chain phosphatase (MLCP) activity in vitro , so the release of SMTNL1 could attenuate MLCP-dependent dephosphorylation of myosin regulatory light chain (LC20). Conversely, SMTNL1 phosphorylation during calcium desensitization of smooth muscle could provide enhanced binding to Tpm and restrict binding of CaM in the apo-state, securing phosphorylated SMTNL1 with Tpm on the thin filament. The weak binding modes of SMTNL1 with apo-CaM might not possess enough stability to occur in vivo . While the physiological significance of SMTNL1 association with the contractile filament in situ has not been investigated, we speculate that the protein may act in analogous manners to caldesmon or calponin on actin-myosin cross bridge cycling. Calponin interacts with many cytoskeleton and related proteins (including tropomyosin) and functions as an inhibitor of actin-activated myosin ATPase . The binding of Ca-CaM dissociates calponin from the actin filament to facilitate smooth muscle contraction . Calponin can also be phosphorylated in response to cyclic nucleotide signals to cause a reduced affinity for the thin filament . Caldesmon is another actin filament-associated regulatory protein; it also binds to Tpm and regulates cross-bridge cycling in a Ca-CaM dependent manner . A detailed physiological analysis of the CaM- and Tpm-interactions of SMTNL1 and their functional implications are required to provide a clear description of role of SMTNL1 in smooth muscle contractility. Further investigation will be needed to verify the role of SMTNL1 in fine-tuning thin filament dynamics.
Herein, we provide evidence that calcium availability increases CaM association with SMTNL1, and CaM and tropomyosin binding to SMTNL1 are mutually exclusive events. Moreover, SMTNL1 binding to CaM is reduced by phosphorylation of Ser301 while binding to tropomyosin is enhanced. We propose a model whereby SMTNL1 can exist in distinct complexes with either Tpm or CaM, depending on availability of calcium and activity of PKA. This switching mechanism could aid in the fine-tuning of smooth muscle contraction.
- CBD1 and CBD2:
Calmodulin binding domain 1 and 2, respectively
Protein kinase A
Protein kinase G
Tropomyosin-binding region of SMTNL1
This work was supported by the Canadian Institutes of Health Research (MOP-97931). JAM held an Alberta Innovates – Health Solutions (AIHS) Senior Scholar Award and was recipient of a Canada Research Chair (Tier 2) in Smooth Muscle Pathophysiology. AUL was a Heart & Stroke Foundation of Canada Postdoctoral Fellow.
Availability of data and materials
All data generated and/or analysed during this study are included in the published article or its Additional file 1.
AUL, HI and DHS designed, performed and analyzed the majority of experiments. AUL and JAM wrote the manuscript and drafted figures. JAM and HJV conceived and coordinated the study. All authors reviewed the results and approved the final version of the manuscript.
The authors declare that they have no competing interests.
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All experiments utilizing animal tissues were approved by the Health Sciences Animal Care Committee, University of Calgary and conform to the guidelines set by the Canadian Council on Animal Care.
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