Research ArticleBiochemistry

Tandem phosphorylation within an intrinsically disordered region regulates ACTN4 function

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Science Signaling  26 May 2015:
Vol. 8, Issue 378, pp. ra51
DOI: 10.1126/scisignal.aaa1977
  • Fig. 1 Phosphorylation of Tyr31 in ACTN4 is necessary and sufficient to inhibit actin binding.

    (A) Multiple sequence alignment (right) and phylogenetic tree (left) of ACTN isoforms from various animal species. Only the disordered N-terminal regions are shown in the alignment. The column colored in red contains the highly conserved Tyr31 from human ACTN4 (HOMO_4), which is homologous to Tyr12 in human ACTN1 (HOMO_1). Columns containing the Tyr4 sites of ACTN4 and ACTN1 are colored in blue and green, respectively. Note that the ACTN4 sequences are characterized by the insertion of a hydrophobic linker between Tyr4 and Tyr31. Numbers to the right of the branch points in the phylogenetic tree represent bootstrap support based on 100 replicates. Branches within the ACTN1-4 clusters all have bootstrap support values of at least 0.98. Scale bar represents the number of substitutions per site. (B) Representative gel of actin binding assay for wild-type (WT), Y4E, Y31E, and Y4/31E constructs of ACTN4 after ultracentrifugation and separation of supernatant (S) and pellet (P). (C) Quantitation of three actin binding experiments for the fraction of bound ACTN4 in the pellet. Error bars indicate SEM. *P < 0.05 based on two-sided Student’s t test. NS, not significant. The asterisk applies to the following four pairwise comparisons: WT versus Y31E, WT versus Y4/31E, Y4E versus Y31E, and Y4E versus Y4/31E.

  • Fig. 2 MDS predict that ACTN4 Tyr31 is kinase-inaccessible without phosphorylated Tyr4.

    Three independent MDS were performed for N-terminal constructs of wild-type ACTN4, Y4E ACTN4, truncated ACTN4, and WT ACTN1. In each panel, a representative relative solvent-accessible surface area (SASA) plot (top) and a structure snapshot (bottom) are shown. Snapshots are displayed with rainbow coloring from dark blue to red in the N-to-C direction. (A) Tyr4 and Tyr31 (dark blue and yellow spheres) were mostly solvent-exposed and buried (relative SASA: 71 ± 9% and 11 ± 8%), respectively. Two helices adopted an antiparallel arrangement that buried Tyr31. The primary m-calpain cleavage site (Tyr13-Gly14 peptide bond; red arrow) was exposed. (B) Tyr31 is mostly solvent-exposed (relative SASA: 64 ± 13%) in Y4E ACTN4 because the additional negative charge flipped the helices into a parallel arrangement. (C) Removal of ACTN4 residues 1 to 13 led to a mostly helical region between Pro15 to Ala22 and kept Tyr31 buried (relative SASA: 17 ± 8%). (D) ACTN1 was more extended because of its hydrophilic sequence, and Tyr12 (green spheres) was mostly solvent-exposed (relative SASA: 73 ± 12%). Relative SASA values are given as means ± SD over the three MDS runs.

  • Fig. 3 Tyr31 phosphorylation in ACTN4 requires prior phosphorylation at Tyr4.

    (A) Representative gel of phosphorylation detection assay for WT, Y4E, and 14-911 ACTN4-GFP constructs in the presence (+) or absence (−) of EGF. IP:GFP, immunoprecipitation with an antibody recognizing GFP; IB:pY, immunoblot with an antibody recognizing phosphorylated tyrosines. Total ACTN4-eGFP was detected with Coomassie blue G-250. (B) Quantitation of three experiments for the ratio of density units between phosphorylated tyrosine band and total protein band for each ACTN4 construct. Error bars indicate SEM. *P < 0.05, two-sided Student’s t test. (C) Tandem phosphorylation model for the ACTN4 disordered N-terminal region. (i) Tyr4 and Tyr31 are initially accessible and inaccessible, respectively, for phosphorylation (Pi), and Tyr4 gets phosphorylated (pY4). (ii) Phosphorylation of Tyr4 triggers structural changes in the disordered N-terminal region that lead to the exposure of Tyr31 and its phosphorylation (pY31). (iii) Phosphorylated Tyr31 inhibits binding of ACTN4 to actin by latching the two CH domains of the ABD in a closed configuration. Wavy lines in the N-terminal region denote α-helical structures. Red arrow indicates the m-calpain cleavage site between Tyr13 and Gly14.

  • Fig. 4 Phosphorylation of ACTN4 Tyr4 is decreased upon mutation of the adjacent Asp3.

    (A) Representative plots (for one of three independent MDS) showing relative SASA of Tyr4 and Tyr31 from D3A (top) and D30A (bottom) ACTN4 constructs. These mutations lead to both Tyr4 and Tyr31 being mostly solvent-exposed (relative SASA of Tyr4 and Tyr31: 67 ± 14% and 66 ± 14% for D3A, 66 ± 12% and 64 ± 9% for D30A). Relative SASA values are given as means ± SD over the three runs. (B) Representative gel of phosphorylation detection assay for WT, D3A, D30A, D3/30A, Y4E, Y31E, and Y4/31E ACTN4-GFP constructs in the presence (+) or absence (−) of EGF. Total ACTN4-eGFP protein was detected with Coomassie blue G-250. (C) Quantitation of three experiments for the ratio of density units between phosphorylated tyrosine band over total protein band for each ACTN4 construct. Error bars indicate SEM. *P < 0.05 based on two-sided Student’s t test.

  • Fig. 5 TAM family members, but not FAK, may be involved in EGF-induced phosphorylation of ACTN4.

    (A) Representative gel of EGF-induced phosphorylation detection assay for ACTN4-GFP and ACTN1-GFP in the presence (+) or absence (−) of 10 μM FAK inhibitor (inh.) II. (B) Quantitation of three experiments for the ratio of density units between phosphorylated tyrosine band and total protein band for ACTN4 or ACTN1. Error bars indicate SEM. (C) Representative gel of phosphorylation detection assay for WT, Y4E, and Y31E ACTN4-GFP constructs in the presence of EGF alone or with one of the following: 10 μM EGF receptor (EGFR) inhibitor (PD153035), 15 μM p38 inhibitor (SB203580), or 15 μM TAM inhibitor (R428). (D) Quantitation of three experiments for the ratio of density units between phosphorylated tyrosine band and total protein band for each ACTN4 construct. Error bars indicate SEM. *P < 0.05 based on two-sided Student’s t test. Total ACTN4-eGFP protein was detected by staining with Coomassie blue G-250.

  • Fig. 6 Tuning the kinase affinity of Tyr4 is enough to design a switch.

    (A) Modeling results for phosphorylation in a single-site model (such as in ACTN1). Fraction of phosphorylated site is plotted against the ratio of kinase/phosphatase concentrations ([Kin]/[Pho]) and total ACTN1 concentration normalized by the kinase KM,K ([ACTN1]/ KM,KY12; plotted as log scale). The KM of the kinase and phosphatase for the site are set equal. (B) Results for phosphorylation of the functional site in a 1-kinase/1-phosphatase (1K1P) two-site tandem model (such as in ACTN4), where the kinase has a 50-fold weaker affinity for the switch site (KM,KY4) than the functional site (KM,KY31). Axes are similar as in (A). (C) Results for same model as in (B) but where the kinase has the same KM,K for both sites. Black curve shows the amount of phosphorylated Tyr31 as a function of [Kin]/[Pho] when [ACTN4]/ KM,KY31 = 10. (D) Results for the 1K1P two-site tandem model where the fraction of phosphorylated site is plotted against [Kin]/[Pho] and the kinase KM,K ratio for both sites (KM,KY31/ KM,KY4), with [ACTN4]/ KM,KY31 = 10. Black curve shows the amount of phosphorylated Tyr31 as a function of [Kin]/[Pho] when KM,KY31/ KM,KY4 = 1. All enzyme concentrations are assumed to be directly proportional to their activities.

Supplementary Materials

  • Supplementary Materials for:

    Tandem phosphorylation within an intrinsically disordered region regulates ACTN4 function

    Timothy Travers, Hanshuang Shao, Brian A. Joughin, Douglas A. Lauffenburger, Alan Wells, Carlos J. Camacho*

    *Corresponding author. E-mail: ccamacho{at}pitt.edu

    This PDF file includes:

    • Note S1. Reaction rules and parameter values for the simulations of single-site and two-site tandem phosphorylation models.

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    Citation: T. Travers, H. Shao, B. A. Joughin, D. A. Lauffenburger, A. Wells, C. J. Camacho, Tandem phosphorylation within an intrinsically disordered region regulates ACTN4 function. Sci. Signal. 8, ra51 (2015).

    © 2015 American Association for the Advancement of Science

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