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[Preprint]. 2023 Jul 9:2023.06.30.547189.
doi: 10.1101/2023.06.30.547189.

Distinct functional constraints driving conservation of the cofilin N-terminal regulatory tail

Joel A Sexton  1 , Tony Potchernikov  2 , Jeffrey P Bibeau  2 , Gabriela Casanova-Sepúlveda  2 , Wenxiang Cao  2 , Hua Jane Lou  1 , Titus J Boggon  1   2 , Enrique M De La Cruz  2 , Benjamin E Turk  1
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Distinct functional constraints driving conservation of the cofilin N-terminal regulatory tail

Joel A Sexton et al. bioRxiv. .

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Abstract

Cofilin family proteins have essential roles in remodeling the cytoskeleton through filamentous actin depolymerization and severing. The short unstructured N-terminal region of cofilin is critical for actin binding and harbors the major site of inhibitory phosphorylation. Atypically for a disordered sequence, the N-terminal region is highly conserved, but the aspects of cofilin functionality driving this conservation are not clear. Here, we screened a library of 16,000 human cofilin N-terminal sequence variants for their capacity to support growth in S. cerevisiae in the presence or absence of the upstream regulator LIM kinase. Results from the screen and subsequent biochemical analysis of individual variants revealed distinct sequence requirements for actin binding and regulation by LIM kinase. While the presence of a serine, rather than threonine, phosphoacceptor residue was essential for phosphorylation by LIM kinase, the native cofilin N-terminus was otherwise a suboptimal LIM kinase substrate. This circumstance was not due to sequence requirements for actin binding and severing, but rather appeared primarily to maintain the capacity for phosphorylation to inactivate cofilin. Overall, the individual sequence requirements for cofilin function and regulation were remarkably loose when examined separately, but collectively restricted the N-terminus to sequences found in natural cofilins. Our results illustrate how a regulatory phosphorylation site can balance potentially competing sequence requirements for function and regulation.

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Figures

Figure 1.
Figure 1.. A dual-functionality screen of a cofilin-1 N-terminal mutagenesis library.
(A) Growth of TeTO7-COF1 yeast exogenously expressing the indicated cofilin in the presence or absence of doxycycline. Image representative of n = 2. (B) Yeast harboring the indicated expression plasmids were grown on either glucose or galactose to induce LIMK1 expression in the presence or absence of doxycycline. Image representative of n = 7. (C) Design of cofilin N-terminal combinatorial mutagenesis library. X indicates any of the 20 natural amino acids. (D) Schematic of the cofilin library dual-functionality screen. (E) Graphs show change in relative representation of the indicated cofilin variants over time during culture in either glucose (no LIMK1) or galactose (LIMK1 induction). Data are from three replicate screens performed independently. Blue circles indicate timepoints used to identify functional sequences. Orange circles indicate timepoints used to identify sequences inhibited by LIMK1. Time zero is after de-repression of PGAL-LIMK1 over the course of 1 – 2 population doublings in raffinose + DOX media.
Figure 2.
Figure 2.. Cofilin library screen results for yeast growth rescue.
(A) Waterfall plot depicting enrichment or depletion of all 16,000 cofilin library sequences expressed in TeTO7-COF1 yeast grown in doxycycline. Sequences are ordered left to right from highest to lowest average enrichment from three independent screens. (B) Probability logo of cofilin N-terminal library sequences enriched in the screen. (C) Heat map of cofilin N-terminal library sequence enrichment or depletion. Color scale indicates the mean enrichment score of the 800 (positions 2, 4 and 5) or 8000 (position 3) library sequences containing the indicated residue across 3 independent replicates. (D) Distribution of cofilin sequences by enrichment scores according to identity of residues at the indicated positions. Boxes indicate 2nd and 3rd quartile of distribution with median line. Bars indicate sequences in the 10 – 90 percentile range.
Figure 3.
Figure 3.. Impact of cofilin N-terminal mutations on yeast growth and actin binding.
(A,B) Growth assay comparing cofilin mutants containing substitutions of residues Ala2, Gly4, or Val5. Images representative of n = 3. (C-E) Pyrene quenching assays comparing cofilinWT to the indicated cofilin mutants. Actin concentration pre-polymerization was 1 μM. Replicates for binding assays are as follows: cofilinWT (n = 4), cofilinA2L (n = 3), cofilinA2D (n = 4), cofilinS3D (n = 3), phospho-cofilinWT (n = 2), cofilinG4A (n = 3), cofilinG4F (n = 2), cofilinG4E (n = 2), cofilinG4K (n = 3), cofilinV5M (n = 3), and cofilinA2L,V5M (n = 2).
Figure 4.
Figure 4.. Cofilin library screen results for yeast growth rescue with and without LIMK1.
(A) Scatter plot depicting change in representation of Ser3 cofilin library sequences with and without LIMK1 expression. Data points show average enrichment in glucose and galactose across three replicates. (B) Logo of sequences supporting growth in yeast but are selectively inhibited by LIMK1 (lower right quadrant in panel A) on the background of all sequences that support yeast growth in the absence of LIMK1 (upper and lower right quadrants in panel A). (C) Distribution of cofilin sequences by enrichment scores with and without LIMK1 expression according to identity of the phosphoacceptor residue. Boxes indicate 2nd and 3rd quartiles, and bars indicate sequences with 10- and 90-percentiles of distribution with median line.
Figure 5.
Figure 5.. Effects of cofilin Ser3 substitutions on phosphorylation by LIMK1.
(A) TeTO7-COF1 yeast expressing the indicated human cofilin-1 mutants were grown in the presence of doxycycline to repress endogenous Cof1 expression and/or galactose to induce LIMK1 expression. Images are representative of n = 3 independent experiments. (B) Immunoblots with the indicated antibodies of lysates corresponding to yeast plated in panel A. Samples in the cofilin blot were separated on Phos-tag SDS-PAGE to resolve phosphorylated (upper band) from unphosphorylated protein (lower band). Kss1 serves as a loading control. The chart shows the fraction of cofilin phosphorylated, calculated from quantified intensities of the upper and lower bands. Error bars, SD for n = 3 independent experiments. (C) In vitro radiolabel kinase assay comparing phosphorylation of purified cofilin (2 μM) by LIMK1CAT (2 nM) for 10 min at 30 °C. Catalytically inactive LIMK1D460N kinase domain serves as a negative control. The chart shows quantification of phosphorylation rates of cofilin mutants normalized to cofilinWT. Error bars, SD for n = 4 independent experiments. (F) N-terminal sequences of mammalian and yeast isoforms of cofilin and twinfilin. (G) In vitro radiolabel kinase assay showing phosphorylation of the indicated forms of cofilin-1 or twinfilin (2 μM) by LIMK1 kinase domain (2 nM) for 10 min at 30 °C. LIMK1D460N kinase domain serves as a negative control. The graph shows quantification of phosphorylation rates normalized to cofilinWT. Error bars, SD for n = 3 independent experiments.
Figure 6.
Figure 6.. Effects of cofilin Glycine 4 substitutions on LIMK1 substrate suitability.
(A) Yeast growth assay with the TeTO7-COF1 strain expressing the indicated cofilin variants with or without LIMK1 expression. Representative of n = 3 independent experiments. (B) Immunoblots with the indicated antibodies of lysates corresponding to yeast plated in panel A separated by standard or Phos-tag PAGE. (C) Quantification of immunoblots measuring the fraction of total cofilin phosphorylated by LIMK1. Error bars show SD for n = 3 independent experiments. (D) In vitro radiolabel kinase assay comparing phosphorylation of purified 2 μM cofilin by 2 nM LIMK1 kinase domain for 10 min at 30 °C. Catalytically inactive LIMK1D460N kinase domain serves as a negative control. (E) Quantification of phosphorylation rates of cofilin mutants normalized to cofilinWT. Error bars show SD for n = 6 independent experiments. (F) In vitro radiolabel kinase assay comparing phosphorylation of cofilinWT and cofilinG4F by 2 nM LIMK1 kinase domain across a range of concentrations. Reactions performed for 10 min at 30 °C. (G) Quantification of phosphorylation rates of cofilin mutants. Error bars show SD for n = 3 independent experiments. Kinetic parameters include SE for kcat and Km.
Figure 7.
Figure 7.. Cofilin G4F mutation counters the inhibitory effects of phosphomimetic S3D substitution.
(A) Yeast growth assay with TeTO7-COF1 strain expressing the indicated cofilin variants in the presence or absence of doxycycline. (B) Immunoblots of cofilin protein levels corresponding to yeast plated in panel A. Kss1 serves as a loading control. Images are representative of n = 3 independent experiments. (C) Pyrene quenching assays comparing cofilinWT to cofilin variants with a phosphomimetic substitution or phosphorylation modification at residue Ser3. Actin concentration pre-polymerization was 1 μM. WT and G4F single mutant data are the same as shown in Fig 3 and are included here for comparison. Binding parameters are listed in Table 1. Replicates for binding assays are as follows: cofilinWT (n = 4), cofilinS3D (n = 3), phosphor-cofilinWT (n = 2), cofilinG4F (n = 2), and cofilinS3D,G4F (n = 3). (D) Overlapping constraints promoting conservation of the cofilin phosphosite motif sequence.
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